enhancing the resilience of bt cowpea vigna …eprints.qut.edu.au/96968/4/bosibori bett...

ENHANCING THE RESILIENCE OF BT COWPEA [VIGNA UNGUICULATA (L.) WALP] FOR INSECT

RESISTANCE MANAGEMENT

Bosibori Bwari Bett

(BSc., MSc.)

School of Earth, Environmental and Biological Sciences

Science and Engineering Faculty

SUBMITTED IN FULFILMENT OF THE DEGREE OF DOCTOR OF PHILOSOPHY

QUEENSLAND UNIVERSITY OF TECHNOLOGY

BRISBANE, AUSTRALIA

2016

ii

Keywords

Cowpeas, Bacillus thuringiensis, Bt, Vegetative insecticidal proteins (vips), vip3 genes,

Maruca, Maruca pod borer (MPB), Insect resistance management, Agrobacterium‐

mediated transformation, Insect bioassays

iii

ABSTRACT

Cowpea [Vigna unguiculata (L.) Walp.] is an important food security crop that is grown

in all tropical areas. Africa is the main producer with annual production of over 5 million

tonnes. Cowpea is a drought‐tolerant crop that plays a major role in the diets of people

living in the developing world especially in West Africa where it is used as food for

human consumption and as animal feed. The dry seeds are used as a grain and are high

in protein, amounting to 25 % of dry weight. It is also a major source of essential

vitamins and minerals.

Despite its importance as a food security crop, the production of cowpea is constrained

by many factors including insect pests and diseases. The insect pests are of major

concern causing considerable economic losses. One such pest is the legume pod borer

(Maruca vitrata) (Lepidoptera), also known as the Maruca Pod Borer (MPB), which is

responsible for yield losses of up to 50 % in the field. Although breeding for host plant

resistance is currently the main way of controlling the problem of pests and diseases in

cowpea, this approach is not yet possible for MPB as no genes for resistance to Maruca

have been identified in cowpea germplasm. Therefore, control strategies have focussed

on biological control using strains of the bacterium Bacillus thuringiensis (Bt) which

contains insecticidal toxins such as the crystal (Cry) proteins and vegetative insecticidal

proteins (Vips) encoded by the cry and vip genes, respectively.

A cry gene has previously been used to develop cowpeas resistant to Maruca by

researchers at CSIRO, Canberra. This line contains a cry1Ab gene and was shown to be

effective against Maruca in the field in West Africa. However, since insects can develop

resistance to a single Bt protein, the generation of transgenic plants containing two or

more Bt genes is a common strategy used to reduce the likelihood of resistance

occurring and thus improve insect resistance management (IRM). Since the Cry and Vip

proteins have different mechanisms of action, it has been hypothesised that the genes

encoding these proteins would represent suitable targets for gene stacking in the

generation of transgenic cowpea. The main aim of this study, therefore, was to identify a

vip gene with toxicity to MPB and to generate MPB‐resistant transgenic cowpea. Such a

iv

gene could be used to complement the current cry transgene and ultimately enhance

the long term sustainability of a Maruca‐resistant transgenic cowpea.

The initial part of the research project involved the identification of vip gene(s) from

Australian Bt isolates that encode toxins active against MPB. Considering the abundance

of vip3 genes in the database, a bioinformatics approach was initially used to identify a

diverse range of Vip3 proteins as candidates for expression and testing for insecticidal

activity against MPB. Based on the amino acid sequence analyses of published Vip

sequences, five diverse vip3 genes were selected for further analysis, namely vip3Aa35,

vip3Af1, vip3Ag, vip3Ca2 and vip3Ba1. A collection of 224 Bt isolates was screened with

gene‐specific primers to identify those containing the target genes. Out of these, 78

strains were confirmed to contain vip3A genes. Out of 165 strains screened at random, 8

were found to contain vip3B genes. No Bt strain(s) in the collection were found to

contain vip3C genes. Primers were designed to isolate the coding sequences of

vip3Aa35, vip3Af1, vip3Ag, vip3Ca2 and vip3Ba1 by PCR. This method was successful for

three out of five of these genes, while vip3Af1 and vip3Ca2 were chemically synthesized.

The coding sequences of the vip3 genes were cloned and over‐expressed in Escherichia

coli to produce Vip3 protein. The soluble and insoluble fractions of the Vip3 proteins

were analysed by SDS‐PAGE. The Vip3A and Vip3C proteins were present in the insoluble

fraction while Vip3B protein was present in the soluble and insoluble fractions. An

MS/MS analysis was carried out on the Vip3 protein bands and showed that they were

indeed the target Vips. The respective Vip3 proteins were incorporated into Maruca

artificial diets at 0, 3, 10, 30 and 100 µg toxin per g for use in insect bioassays with MPB

larvae to screen for toxicity. Of these, Vip3Ba1 protein was found to inhibit larval growth

by over 90 % and was therefore selected as the candidate gene for cowpea

transformation.

The second component of this study focussed on reconstructing the vip3Ba gene for

plant expression and then transforming cowpea using Agrobacterium tumefaciens. The

coding region of the vip3Ba gene was optimized by increasing the GC content from

approximately 30 % to 50 %, and deleting the eight potential polyadenylation sites and

twelve mRNA destabilizing sequence motifs. The codons were also optimized to enhance

translation in dicots. A modified version of a previous protocol developed at CSIRO was

v

used to transform cowpea. The modifications included omitting certain key plant

hormones in the growth media, a stringent antibiotic selection regime involving two

different antibiotics, and employing sonication techniques to permeate the plant cell

wall to facilitate Agrobacterium‐mediated T‐DNA transfer. Out of 6696 cowpea explants

transformed with the reconstructed vip3Ba gene, a total of 73 independent transgenic

lines were generated (a transformation frequency of just over 1 %). Further analysis of

T1 progeny revealed that nine transgenic lines expressed the vip3Ba gene at varying

levels. Of these, four lines were selected for further experiments based on the fact that

variation in Vip3Ba expression between the four lines could serve to determine whether

insect mortality was dependent on toxin dosage. There was 100 % mortality of larvae

feeding on transgenic cowpea leaves.

The results of this study demonstrate for the first time that the growth of MPB is

severely inhibited by low levels of Vip3Ba protein. Additionally, transgenic lines

developed in this work were completely protected from MPB, confirming that Vip3Ba is

a very effective toxin for the control of MPB larvae at least in the lab. The outcome of

this study identified vip3Ba gene as a potential candidate to complement cry genes in

the development of Bt cowpeas resistant to MPB, thus contributing to an effective

strategy for insect resistance management. The introgression of this additional

resistance trait into the existing cry‐1Ab cowpea would considerably reduce the

likelihood or greatly delay the development of resistance in MPB, help to increase yield

and income, and reduce the dependency on pesticides.

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TABLE OF CONTENTS

Keywords……………………………………………………………………………………………………………...

ii

Abstract ……………………………………………………………………………………………………………….

iii

Table of Contents …………………………………………………………………………………………………

vi

List of Figures ……………………………………………………………………………………………………….

xi

List of Tables ……………………………………………………………………………………………………….

xiv

List of Abbreviations ……………………………………………………………………………………………

xvi

Declaration ………………………………………………………………………………………………………….

xix

Acknowledgements …………………………………………………………………………………………….

xx

Dedication ……………………………………………………………………………………………………………

xxiv

Chapter One: Introduction and Literature review …………………………………………………

1

1.1 Cowpea …………………………………………………………………………………………………….

1

1.2 Cowpea taxonomy and morphology …………………………………………………………

1

1.3 Cowpea cultivation ………………………………………………………………………………….

3

1.4 Cowpea production constraints ……………………………………………………………….

3

1.4.1 Diseases …………………………………………………………………………………………

3

1.4.2 Weeds ……………………………………………………………………………………………

5

1.4.3 Insect pests ……………………………………………………………………………………

6

1.4.3.1 Storage pests …………………………………………………………………….

6

1.4.3.2 Pre‐flowering pests …………………………………………………………….

6

1.4.3.3 Post‐flowering pests …………………………………………………………….

7

1.5 The legume pod borer ………………………………………………………………………………

7

1.6 Justification for transgenic approaches …………………………………………………….

8

1.7 The use of Bacillus thuringiensis (Bt) as an insect control strategy …………... 11

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1.7.1 Bt insecticidal proteins ……………………………………………………………………..

13

1.7.1.1 Crystal (Cry) proteins …………………………………………………………..

13

1.7.1.2 Vegetative insecticidal proteins (Vips) ………………………………….

13

1.7.2 Bt transgenic crops ………………………………………………………………………..

15

1.7.3 Insect Resistance Management ………………………………………………………..

16

1.8 Research Hypothesis …………………………………………………………………………………

19

1.9 Research Objectives ………………………………………………………………………………….

19

2.0 Expected output .........................................................................................

20

Chapter Two: General Materials and Methods .......................................................

21

2.1 Preparation of bacterial cultures ................................................................

21

2.1.1 Bacillus thuringiensis (Bt) cultures .......................................................

21

2.1.2 Preparation of Mannitol Glutamate Luria (MGL) culture medium for Agrobacterium tumefaciens .............................................................................

21

2.2 Isolation of bacterial DNA ...........................................................................

22

2.2.1 Isolation of genomic DNA from Bacillus thuringiensis .........................

22

2.2.2 Isolation of plasmid DNA from Escherichia coli and Agrobacterium tumefaciens .....................................................................................................

22

2.3 Purification of PCR products .......................................................................

22

2.4 Cloning of vip genes ....................................................................................

22

2.4.1 Expresso™ T7 Cloning and Expression System .................................................

23

2.4.2 The Gibson assembly method of DNA cloning .................................................

23

2.5 Sequencing of vip3 genes cloned in E. coli ...................................................

24

2.6 Preparation of electrocompetent Agrobacterium (AGL1) cells ....................

24

2.7 Transformation of Agrobacterium tumefaciens (AGL1) cells .......................

25

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2.8 Transformation of E. coli cells .....................................................................

25

2.9 Protein expression and analysis ..................................................................

26

2.10 Identification of Vip protein using Mass Spectrometry …………………………....

29

2.11 Maintenance of a Maruca vitrata insect colony in vitro .............................

29

2.11.1 Preparation of Maruca artificial diet ...................................................

29

2.11.2 Preparation of Honey solution for Maruca adults ...............................

31

2.11.3 Preparation of honey pots for feeding adults whilst in cages ..............

31

2.11.4 Procedures for egg collection ..............................................................

32

2.11.5 Rearing of Maruca larvae .....................................................................

32

2.12 Preparation of plant tissue culture media ...................................................

35

2.12.1 Preparation of MS Stock solutions .......................................................

35

2.12.2 Preparation of hormones, antibiotics and other agents ......................

36

2.12.3 Cowpea suspension medium (for co‐cultivation) ................................

36

2.12.4 Cowpea shoot induction medium …………………………………………………….

36

2.12.5 Cowpea shoot elongation and rooting medium ……………………………….

37

2.13 Isolation of plant genomic DNA ..................................................................

37

2.14 Protocol for PCR analysis of transgenic cowpea lines ..................................

38

2.15 Detection of Vip3Ba protein by western blotting ........................................

38

Chapter Three: Toxicity of Vip3 proteins to the legume pod borer Maruca vitrata (Lepidoptera) .........................................................................................................

40

3.1 Introduction ...............................................................................................

40

3.2 Materials and Methods ..............................................................................

41

3.2.1 Bacterial strains ...................................................................................

41

3.2.2 PCR ....................................................................................................... 41

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3.2.3 Cloning of vip3 and cry2Aa genes in E. coli ..........................................

43

3.2.4 Protein expression and analysis ...........................................................

44

3.2.5 Insect bioassays ....................................................................................

44

3.3 Results .......................................................................................................

45

3.3.1 Selection of candidate vip3 genes ........................................................

45

3.3.2 Confirmation of Bt strains ....................................................................

46

3.3.3 Identification of Bt strains containing candidate vip3 genes ...............

48

3.3.4 Expression of vip3 genes in E. coli ........................................................

50

3.3.5 Insect bioassays ....................................................................................

52

3.4 Discussion ..................................................................................................

59

Chapter Four: Transgenic cowpeas (Vigna unguiculata L. Walp) expressing Bacillus thuringiensis (Bt) Vip3Ba protein to protect against the Maruca pod borer (Maruca vitrata) ....................................................................................................

63

4.1 Introduction ...............................................................................................

63

4.2 Materials and methods ..............................................................................

65

4.2.1 Construction of a Vip3Ba gene for plant expression ............................

65

4.2.2 Transformation of cowpea ...................................................................

66

4.2.2.1 Preparation of Agrobacterium culture .....................................

66

4.2.2.2 Preparation of cowpea explants and Agrobacterium‐mediated transformation .....................................................................

66

4.2.2.3 Regeneration and maintenance of cowpea cultures ...............

68

4.2.3 Detection of vip3Ba and nptII genes in the transgenic plants .............

69

4.2.4 Detection and estimation of Vip3Ba expression in transgenic plants ...........................................................................................................................

70

4.2.5 Insect bioassays using transgenic leaf material ...................................

70

4.3 Results ....................................................................................................... 71

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4.3.1 Reconstruction of the vip3Ba gene for cowpea transformation ..........

71

4.3.2 Transformation and regeneration of cowpea with the optimized vip3Ba gene ......................................................................................................

75

4.3.3 Characterization of transgenic lines by PCR .........................................

78

4.3.4 Expression of Vip3Ba in T1 and T2 generations .....................................

78

4.3.5 Efficacy of transgenic lines expressing Vip3Ba protein on MPB larvae ...........................................................................................................................

87

4.4. Discussion ...................................................................................................

89

Chapter Five: General Discussion and Conclusions .................................................

93

Chapter Six: References .........................................................................................

98

APPENDICES ...........................................................................................................

123

APPENDIX I: SUMMARY OF vip3 GENES SEQUENCING RESULTS ..................................

123

APPENDIX II: The DNA sequence of the vip3Ba gene modified for expression in plants ............................................................................................................................

143

APPENDIX III: Sequence alignment of vip3Ba gene (optimized cowpea version vs the original bacterial version)

145

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LIST OF FIGURES

Fig. 1.1: Cowpea dry seed used as a grain (Photo credit: Carl Davies) …………………….

2

Fig. 1.2: Cowpea field showing fresh leaf and pods (Photo credit: Jeff Ehlers) .………………………………………………………………………………………………………………………….......... 4

Fig. 1.3: Cowpea infestation by Maruca pod borer. (A) Larvae attacking cowpea flower and (B) damaged cowpea pod..............................................................................

9

Fig. 1.4: Mode of action of Bt crystal toxins …………………………………………………………….

12

Fig. 1.5: Classification of vegetative insecticidal proteins believed to share a common three domain structure …………………………………………………………………………….

14

Fig. 2.1: Protein extraction flow chart …………………………………………………………………….

28

Fig. 2.2: General procedure for Maruca rearing and maintenance ………………………......

33

Fig. 2.3: Balconies in artificial diet placed in a larger lunch box ……………………………....

34

Fig. 2.4: Preparation of cages for emerging adults. (A) Set up of vermiculite and honey pots. (B) cage covered with nappy liners and lid for egg collection ………………….

34

Fig. 3.1: Identification of putative Bt strains by PCR using PI‐PLC primers. Lane M: DNA Molecular marker; Lanes 1 to 10: DNA template from ten putative Bt strains; N: water (negative control); P: DNA from Bt strain kurstaki (positive control) …………........

47

Fig. 3.2: Identification of Bt strains carrying vip3A genes by PCR. Lane M: DNA Molecular marker; Lanes 1 to 10: DNA template from 10 Bt strains; N: water (negative control); P: bacterial DNA harbouring a vip3Aa gene (positive control) ……...

49

Fig. 3.3: Identification of Bt strains carrying vip3B genes by PCR. Lane M: DNA Molecular marker; Lanes 108 to 117: DNA template from 10 Bt strains; N: water (negative) control and P: plasmid DNA containing the vip3Bb2 gene (positive control) …………………………………………………………………………………………………………………….................

49

Fig. 3.4: SDS‐PAGE analysis of Vip3 and Cry2Aa proteins expressed in E. coli. Panels A‐F are stained gels from studies involving the expression of Vip3Aa35, Vip3Af1, Vip3Ag, Vip3Ca2, Vip3Ba1 and Cry2Aa, respectively. Lane M: Protein molecular weight marker (kDa). Lane 1: Soluble protein fraction of untransformed E. coli. Lane 2: Soluble protein fraction of E. coli transformed with its respective vip or cry gene. Lane 3: Insoluble protein fraction of untransformed E. coli. Lane 4: Insoluble protein fraction of E. coli transformed with its respective vip or cry gene .........................................................................................................................................

54

Fig. 3.5: The full Vip3Ba amino acid sequence. The highly conserved cysteine regions

xii

are highlighted in yellow, while the predicted disulphide bonding is highlighted in green (Ceroni et al., 2006)...............................................................................................

56

Fig. 3.6: Maruca larvae after 10 days feeding on artificial diet with and without Bt toxin. (A): Larvae on a diet containing no Bt toxin (0 µg toxin per g diet), (B) Larvae on a diet containing 3 µg of Vip3Ba toxin per g of diet ..................................................

57

Fig. 3.7: Effect of Vip and Cry2Aa proteins on the average weight of surviving MPB larvae after 10 days on artificial diets. (A): Group 1 (Vip3Aa35) and Cry2Aa proteins, (B): Group 2 (Vip3Af1) and Cry2Aa proteins, (C): Group 3 (Vip3Ag) and Cry2Aa proteins, (D): Group 4 (Vip3Ca2) and Cry2Aa proteins and (E): Group 5 (Vip3Ba1) and Cry2Aa proteins...............................................................................................................

58

Fig. 4.1: Schematic diagram of the Agrobacterium binary vector T‐DNA; LB, RB: left and right borders of Agrobacterium T‐DNA, respectively. The antibiotic selection gene neomycin phosphotransferase II (nptII) from E. coli was flanked by the S1 promoter derived from segment 1 of the subterranean clover stunt virus (SCSV) genome and segment 3 (S3) 3′ end while the op mized coding region of the vip3Ba gene was flanked by the Arabidopsis thaliana small subunit (AraSSU) promoter and Nicotiana tabacum small subunit (TobSSU) 3′ end ……………………………………………………...................................................................................

72

Fig. 4.2: Plasmid map showing unique restriction sites in the pVip3Ba construct used in the transformation of cowpea.......................................................................

73

Fig. 4.3: Confirmation of the Vip3Ba cassette in the Agrobacterium binary vector by restriction digestion. Lane M: DNA Molecular ladder; Lanes 1 and 2: Agrobacterium DNA digested with the enzymes EcoRV and PvuII resulting in the expected four fragments of 1.5, 2.7, 4.4 and 6.4 kb, respectively …………………………………………………….

74

Fig. 4.4: Cowpea explants used for transformation. (A) Cotyledons with attached axes that had their radicle tips removed (arrows) ready for infection with Agrobacterium and (B) after co‐cultivation with Agrobacterium for 3 days ……………....

76

Fig. 4.5: In vitro regeneration of cowpea following co‐cultivation with Agrobacterium. (A) Cowpea explant with cotyledon and primary shoots on shoot induction medium with selection at 2 weeks (B) trimmed explant with cotyledon and primary shoot removed at 4 weeks (C) multiple shoots formed on callus (D) single shoots formed on shoot induction medium with 30 mg/L geneticin at 8 weeks (E) shoots grown on medium with 30 mg/L geneticin at 10 weeks, (F) individual shoots in elongation and rooting medium at 14 weeks and (G) rooted plants in the soil at 16 weeks ………………………………………………………………………………………………………....................

77

Fig. 4.6: PCR analysis of 10 putative transgenic cowpea lines using (A) vip3Ba‐specific primers designed to amplify a 187 bp product and (B) nptII‐specific primers designed to amplify a 970 bp product. Lane M: DNA Molecular marker; Lane N: water (negative control); Lane P: Plasmid DNA of the optimized gene construct

xiii

(positive control); numbers above other lanes represent the line number of randomly selected transgenic cowpeas used for PCR analysis ……………………………...........................................................................................................

79

Fig. 4.7: Western blots using a monoclonal antibody to Vip3Ba showing the levels of Vip3Ba in seven cowpea lines using varying levels of Vip3Ba expressed in E. coli as standards. In both blots, Lane M is the protein molecular weight ladder. Lanes L1 to L5 represent E. coli expressed Vip3Ba loaded at 100, 50, 25, 12.5 and 6.25 ng per lane, respectively. Blot A includes protein (40µg/lane) from cowpea lines 9, 24, 25 and 43 while Blot B includes protein (40µg/lane) from lines 56, 87 and 107..................

82

Fig. 4.8: Western blot analysis to determine Vip3Ba expression in transgenic cowpea lines using a monoclonal antibody to Vip3Ba. Blot A: Line V24: Lane M: Protein precision markers; Lanes 1 to 6: TSP (40 µg) extract from six T2 plants; Lanes 7 to 9: 30 ng, 100 ng and 300 ng protein from E. coli expressing Vip3Ba protein (positive control). Blot B: Line V25: Lanes 1 to 8: TSP (40 µg) extract from eight T1 plants; Lanes 9 to 10: 10 ng and 30 ng protein from E. coli expressing Vip3Ba protein (positive control). Blot C: Line V43: Lane M: Protein precision markers; Lanes 1 to 6: TSP (40 µg) from six T2 plants; Lanes 7 to 9: 30 ng, 100 ng and 300 ng protein from E. coli expressing Vip3Ba protein (positive control). Blot D: Line V87: Lane M: Protein precision markers; Lanes 1 to 5: TSP (40 µg) from five T2 plants; Lanes 6 to 9: 10 ng, 30 ng, 100 ng and 300 ng Vip protein from E. coli expressing Vip3Ba protein (positive control). Vip3Ba band migrates at ~75 kDa ………………………............................................

84

Fig. 4.9: Phenotypes of selected transgenic T1 cowpea lines expressing Vip3Ba protein at different ages. (A) Line V24‐2 at 3 weeks after sowing; (B) Line V25‐8 at 2 weeks after sowing; (C) Line V43‐11 at 2 ½ months after sowing; (D) Line V87‐3 at 3 weeks after sowing and; (E) 3‐week old negative segregants of lines V43 and V87 .........................................................................................................................................

85

Fig. 4.10: Leaf damage after 10 days of feeding by Maruca larvae on non‐transgenic line IT86D and transgenic lines expressing Cry 1Ab or Vip3Ba protein. A: Line 709A (expressing Cry 1Ab), B: Line V24, C: Line V25, D: Line V43, E: Line V87, F, G, H: Line IT86D ...............................................................................................................................

88

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LIST OF TABLES

Table 2.1: Ingredients for bacterial MGL medium …………………………………………………

21

Table 2.2: Ingredients for YMB medium ……………………………………………………………….

25

Table 2.3: Ingredients for Auto‐induction medium, pH 6.9 (for 500 mL) ……………….

27

Table 2.4: Constituents of 1000 X Trace minerals (for 500 mL) ...............................

27

Table 2.5: Ingredients for Maruca artificial diet .....................................................

30

Table 2.6: Ingredients to make 100 g Wesson salt mixture .....................................

30

Table 2.7: Ingredients to make 100 mL Vitamin solution ........................................

31

Table 2.8: Ingredients for the preparation of honey solution .................................

31

Table 2.9: Components to prepare MS Macro stock solutions ................................

35

Table 2.10: Components to prepare cowpea suspension medium ..........................

36

Table 2.11: Components to prepare cowpea shoot induction medium (SIM) and cowpea shoot elongation and rooting medium (SEM) ............................................

37

Table 3.1: Primers used in this study: The underlined nucleotides in primer pairs of PCR set C define 6 His codons of the vector sequence ........................................

42

Table 3.2: Grouping of Vip3 proteins on the basis of amino acid sequence identity with reference protein Vip3Aa35 ...........................................................................

46

Table 3.3: Examples of Bt strains harbouring target vip3 genes ..............................

51

Table 3.4: DNA sequence analysis of target vip3 genes from groups 1 to 5 (with the His tag sequences deleted) ..............................................................................

51

Table 3.5: MS/MS identification of the putative Vip3 proteins expressed in E. coli ...............................................................................................................................

55

Table 4.1: Modifications of the transformation system used for cowpea ...............

67

Table 4.2: Outline of steps in cowpea transformation with the plant‐optimized vip3Ba gene ...........................................................................................................

69

Table 4.3: Primer sequences for the detection of nptII and vip3Ba genes in transgenic plants ...................................................................................................

70

xv

Table 4.4: A comparison of the vip3Ba gene from Bt with the version optimized for expression in plants .........................................................................................

72

Table 4.5: Summary of the molecular analysis of transgenic cowpea lines .............

80

Table 4.6: Summary of average Vip3Ba protein levels in the 4 selected independent transgenic cowpea lines (1st or 2nd generation progeny) ……………..................................................................................................................

80

Table 4.7: Levels of Vip3Ba toxin in the T1 progeny of seven cowpea lines..............

83

Table 4.8: Summary of average Vip3Ba protein levels in the 4 selected independent transgenic cowpea lines (1st or 2nd generation progeny) ....................

87

xvi

LIST OF ABBREVIATIONS

ADP Adenosine diphosphate

AIRS Aminoimidazole ribonucleotide synthetase

AraSSU Arabidopsis small subunit

AT Adenine, Thymine

BAP Benzylaminopurine

BCIP 5‐bromo‐4‐chloro‐3‐indolyl phosphate

BICMV Blackeye cowpea mosaic virus

BLAST Basic Local Alignment Search Tool

bp Base pair(s)

Bt Bacillus thuringiensis

CaCl2.2H2O Calcium chloride dihydrate

CaMV Cauliflower mosaic virus

CDS Coding Sequence

CG Cytosine, Guanine

CoCl2 Cobalt (II) chloride

CoCl2.6H2O Cobalt (II) chloride hexahydrate

Cry Crystal

CTCB Centre for Tropical Crops and Biocommodities

CuSO4 Copper (II) sulfate

CuCl2.2H2O Copper (II) chloride dihydrate

CMV Cucumber mosaic virus

CSIRO Commonwealth Scientific and Industrial Research Organisation

CYMV Cowpea yellow mosaic virus

dATP Deoxyadenosine triphosphate

dCTP Deoxycytidine triphosphate

dH2O Distilled water

dGTP Deoxyguanosine triphosphate

DNA Deoxyribunucleic acid

dNTP(s) Deoxyribonucleotide triphosphate(s)

DTT Dithiothreitol

dTTP Deoxythymidine triphosphate

dH2O Distilled water

EBI European Bioinformatics Institute

E. coli Escherichia coli

EDTA Ethylene diamine tetra acetic acid

EMBOSS European Molecular Biology Open Software Suite

FASTA Fast‐All

FAOSTAT Food and Agricultural Organisation statistics

FeCl3 Iron (III) chloride

g Gram(s)

GA3 Gibberelic acid

GC Guanine Cytosine

GM Genetic modification/Genetically modified

xvii

GUS β‐glucuronidase

HDR Higher Degree Research

Hr(s) Hour(s)

H3BO3 Boric acid

H3BO4 Perboric acid

IAA indole‐3‐acetic acid

IITA International Institute of Tropical Agriculture

IRM Insect Resistance Management

K2HPO4 Dipotassium phosphate

kb kilo base

kbp kilo basepairs

kDa kilo Dalton

KH2PO4 Monopotassium phosphate

KI Potassium iodide

KNO3 Potassium nitrate

KOH Potassium hydroxide

L Litre(s)

LA Luria Agar

LB Luria Bertani

mL Millilitres

MES Morpholine‐4‐ethanesulfonic acid

MGL Mannitol Glutamate Luria

MgSO4 Magnesium sulfate

MgSO4.7H20 Magnesium sulphate heptahydrate

Min(s) Minute(s)

MnCl2 Manganese (II) chloride

MOPS (3‐(N‐morpholino) propanesulfonic acid)

MPB Maruca Pod Borer

mRNA Messenger RNA

MS Murashige and Skoog

MS/MS Tandem mass spectrometry

MW Molecular Weight

NaCl Sodium chloride

NAD Nicotinamide adenine dinucleotide

Na2EDTA Disodium ethylenediaminetetraacetic acid

Na2MoO4 Sodium molybdate

Na2MoO4.2H20 Molybdic acid sodium salt dihydrate

NaOH Sodium hydroxide

NaSeO3 Sodium selenite

Na2SO4 Sodium sulfate

NBF(s) National Biosafety Framework(s)

NBT Nitro blue tetrazolium chloride

NCBI National Centre for Biotechnology Information

NH4Cl Ammonium chloride

NH4NO3 Ammonium nitrate

xviii

NiCl2.6H2O Nickel (II) chloride hexahydrate

Npt Neomycin transferase

OD Optical Density

ORF(s) Open Reading Frame(s)

PCR Polymerase chain reaction

pH Power of hydrogen ion concentration

PI‐PLC Phosphatidylinositol‐specific phospholipase C

PMSF Phenylmethanesulfonyl fluoride

PTGS Post transcriptional gene silencing

QTL(s) Quantitative Trait Loci

QUT Queensland University of Technology

RNA Ribonucleic acid

RO Reverse Osmosis

RPM Revolutions per minute

S Seconds

SCSV Subterranean clover stunt virus

SDS‐PAGE Sodium‐dodecyl sulphate polyacrylamide gel electrophoresis

SOC Super Optimal broth Catabolite repression

SYBR N',N'‐dimethyl‐N‐[4‐[(E)‐(3‐methyl‐1,3‐benzothiazol‐2‐ylidene)methyl]‐1‐phenylquinolin‐1‐ium‐2‐yl]‐N‐propylpropane‐1,3‐diamine

TBS Tris‐buffered saline

TE Tris EDTA

TES N‐[Tris(hydroxymethyl)methyl]‐2‐aminoethanesulfonic acid

TGS Transcriptional gene silencing

TobSSU Tobacco small subunit

TTBS Tris‐Buffered Saline and Tween 20

Vip(s) Vegetative Insecticidal Protein(s)

YMB Yeast Mannitol Broth

ZnSO4.7H2O Zinc sulphate, Heptahydrate

µg Microgram(s)

µL Microlitre(s)

xix

Declaration

The work contained within this thesis has not been previously submitted for a

degree or diploma at any other higher education institution. To the best of my

knowledge and belief, this thesis contains no material previously published or

written by another person except where due reference is made.

Signed: QUT Verified Signature

Date: July 2016

xx

Acknowledgements

First I thank the almighty God for granting me good health and peace. I thank Him for

taking me this far.

I thank my dear family – my husband Mohammed Bett Soi, my sons Andrew Bett and

Samuel Bett, and my daughter Abigail Bett, for their unending support. Dear Abigail,

thank you for realising that mummy had to write her thesis by spending most of your

time asleep, what a wonderful baby you are. Dear Andrew and Samuel, thank you for

being obedient and for knowing that mummy had to finish quickly, hence the reason I

could not accompany you to the park sometimes. This kept mummy going. Dear

Mohammed, thank you for your love, support, encouragement and prayers. Thanks for

stepping in for me when I was in the lab. I will forever cherish this. I also thank my

parents, Andrew & Rose Michieka and Elijah & Eunice Soi for their constant phone calls,

prayers and encouragement. I thank Yvette and Yvonne for the endless support.

I would like to thank the Australians, particularly their government, for offering me the

prestigious AusAID scholarship to pursue my PhD studies. To the ILRI/BecA hub and

CSIRO staff – Santie, Leena, Segenet, Jagger, Rob, Appolinnaire and Bruce Pengelley

thank you for paving the way for my success.

I would like to appreciate my supervisors Prof. TJ Higgins of CSIRO and Prof. Rob

Harding. Dear TJ and dear Rob: First, when I decided to take up the scholarship, I knew

that I needed an expert in legume (cowpea) technology at CSIRO and, also, I was keen to

get enrolment at QUT. With the constant communications we had, I was certain that my

PhD studies would commence, and you therefore made it possible for me to integrate

into both organizations. Thank you for accepting me as your student, for all the

administrative procedures and, for mentoring me. Secondly, when I began my studies, I

thought I knew how to write. Only to realize that with your supervision, there was a

perfect way of doing it. Thanks for opening my eyes by constantly guiding me and

correcting the many mistakes I made in my presentations and in the whole writing

process. Dear TJ, I used to wonder how I would complete one experiment after another

without mental stress, only to realize that you provided ALL funds for my research

project. I therefore lacked NOTHING and was able to accomplish the objectives of my

xxi

work. I am fully indebted to you. Dear Rob, thank you for toiling with me during my last

days in Australia when I was writing up, by going out of your way to secure a QUT/CTCB

contribution to research program scholarship and a HDR tuition fee sponsorship to

facilitate my writing. This was very touching and I am indebted to you.

Dear Steve Hoover, thank you for the emotional support and overwhelming assistance

that you gave us towards the end of the scholarship period and thereafter.

To the cowpea research group – Andy, Steph and Lisa, thank you for making it happen.

Dear Andy, when I made up my mind to transform cowpea with a novel Bt gene, I knew

that I had to employ molecular biology techniques to accomplish this. The truth was that

I had no idea of how to go about this. But with your simple explanation of how to isolate

the genes, make several constructs, conduct experiments and come up with a suitable

construct for cowpea, it became so clear, and I was able to commence my work. Thank

you also for orientating me in the lab and for helping me familiarise myself with what

goes where. I am forever grateful.

Dear Steph, well, what can I say? First, when I was leaving for Canberra (CSIRO) to work

on cowpea, I was a little bit apprehensive, as I was made to understand that cowpea was

a difficult crop to transform, due to its recalcitrant nature. But when we started the

transformation experiments, you took me through a simple and straightforward system

that I could repeatedly carryout on my own, transforming explants en masse in a short

time. Thank you too for sharing your hood with me and for showing me how we reared

“our bugs”. Secondly, thank you for being my big sister and confidant in the lab. You

always offered quick solutions to my many personal questions and problems. Your

encouragement and laughter in the lab kept me going when and before I carried

Matilda. I am forever grateful too.

Dear Lisa, thank you for your support in ensuring that the chemicals and reagents I

required everyday were in stock and within reach. I appreciate.

To my other friends in the lab, thank you for walking through this journey with me. Dear

Phil and Vince, thanks for welcoming me into the crèche and for making sure that I was

never cold in there. Nayana, Steph B and Rash, thanks for being my sisters and sharing

xxii

crazy ideas, laughter that kept us going, and tears of joy both in and out of the lab. I am

grateful to plant industry lab members ‐ Wendy, Mandy, Jenny, Anna, Kim, Xiaodi, Holly,

Terese, Maud, Kareena W, Christoph, Samba, Craig W, Wendelin, John Moore, Dhara,

Smitha, Sumita, Soma, Hannerlie Botha, the list is endless– you all played a role in one

way or another to see that I succeeded in my research studies were a success. To the

lab members at Entomology– Bill James, Tom Walsh, Joel and Tek, thanks for granting

me access to the Bt strains and for providing a wonderful place for “my bugs”. To Benard

Mware and Betty Namukwaya we started this journey together and we have come a

long way. Thanks for your encouragement.

I would like to express my thanks to those that made me feel at home away from home:

Dear Leanne Wohoro and Evans Lagudah, thank you for connecting me to the Kenyan

community. I thank especially Jane Boke, Flo Muthinja, Willie Bico and their families for

continuous support by orientating me around Canberra and ensured that I settled well.

Thanking Abdul Kibet, Emily Sang, Victor Valla, Purity Goj and Benard Mutai, Gladys and

Ken Omari, Kristeen Riungu and Charles Njora, Faith and Shallo, Carmen and Joseph

Kaburu, Jackie and Papy, Norah and Anthony Thompson, Flo and George Kiogora, Milkah

and Caxton Kirimi, Esther and Jeff, Susan Chumo, Joyce Waweru, Andrew and Phibian

Lang’at, Irene and Pete Wiencke, Joshua and Orpha, Mary Onsarigo, Hellen, Mumbi

Kamau, Maggie Sikulu, and many others. To Amanda Jukes and Maree, Carolyne and

John Bourke, Sandi and Brent Chamberlain, Emma and Shane, Joan and Ian Campbell,

Pete and Caren Curtis, thank you for supporting my family while in Canberra.

I am indebted to the CCH family. Dear Brian and Mandy Johnstone, Damian and Renee

Lowes, Jace Sargent and Jacqui Day, Brendan and Danielle Jordan, Swani and Sandra,

Bonnita and the rest of the CCH family, I thank you for your kindness, for your

understanding and for the enormous support you gave to my family – You rock!

Dear Kerubo (K.B.), thank you for your advice and prayers and for the counsel you

offered, which prepared me in advance. Thank you for making me understand that I’ve

got to do what I’ve got to do, by helping me with a formula of how to juggle with the

writing up and a newborn – it worked incredibly well. God bless you!

xxiii

My sincere thanks go to the then KARI director, Dr. E. Mukisira and Mr. Martin Kivui for

granting me study leave. Dear Dr. Mukisira and Martin, when I sought an extension of

my study leave while in Australia, you replied my emails promptly amidst your busy

schedules. You were keen to endorse my request and facilitated my extension within the

shortest time possible. Thank you for this. To Drs. Nang’ayo, Gicheru, Lusike, Gichuki,

Soi, Miano and Taracha, I thank you for your support.

To my Aunty Grace and Uncle Richard, Musi, Ohmms, Kwamboka, Gedi, Nyaboe,

Armeline, Meg, Joyce, Emmanuel, Mark, Kemunto Arasa, Sammy Omwenga, Efelina

Boyani, Gina Ouattara, Stella Wafukho, Evans Nyaboga, Maureen Miruka and Jemimah

Njuki, I thank you for your support and the logistics that you played in seeing that I

settled back home well and accomplish my writing – I owe you so much.

Finally, I thank everyone else who supported me in different ways towards the

completion of my studies. May the Lord bless you all.

xxiv

Dedication

I dedicate this thesis to Abigail Matilda Bett

1

CHAPTER ONE

Introduction and Literature review

1.1 Cowpea

Cowpea [Vigna unguiculata (L.) Walp.] is an annual herbaceous savannah species known to

originate from Africa (Ehlers and Hall, 1997; Padulosi and Ng, 1997), where it was

domesticated from its wild ancestor V. unguiculata subsp. unguiculata var spontanea

(Pasquet, 1999). It is commonly referred to as the blackeye pea or the pinkeye pea in the

United States (Fery, 2002). Cowpea is a food legume grown in many tropical areas of Africa

and Asia with an annual production of 5 million tonnes from Africa alone, which is the main

producer (FAOSTAT, 2012; Timko and Singh, 2008; Andrea et al., 2007). Nigeria and Niger

are the largest cowpea‐producing countries in Africa with tonnages of 2.5 million and about

1 million tonnes, respectively (FAOSTAT, 2012). In Africa, cowpea is grown by resource‐poor

farmers and is regarded as a woman’s crop (Murdock et al., 2013; Nkongolo et al., 2009).

The crop is drought‐tolerant and plays an important role in the diets of many people in

Africa and in other parts of the developing world (Labuschagne et al., 2008; Murdock et al.,

2008). The grain (Fig 1.1) has a protein content of 25 % and is consumed boiled in the form

of curry, roasted, deep‐fried as a snack (“akara” balls) or ground into flour for bread and

soup‐making (Singh and Singh, 1992; Andrea et al., 2007). The young leaves and green pods

are used as a vegetable alongside a staple dish and are an excellent source of essential

minerals and vitamins (Singh et al., 2003; Nielsen et al., 1997). Following the harvest, the

cowpea residues (haulms) are cut and dried for use as animal feed. In the Western part of

Africa, improved cowpea varieties are also cultivated for animal feed (Nielsen et al., 1997;

Singh et al., 2003). Figure 1.2 shows young cowpea plants growing in the field.

1.2 Cowpea taxonomy and morphology

Cowpea is a dicotyledonous plant belonging to the family Leguminosae and genus Vigna.

The genus Vigna comprises seven sub‐genera namely Haydonia, Lasiospron, Macrorhyncha

(also known as Wajira), Plectotropis, Sigmoidotropis, Ceratotropis and Vigna. The subgenus

Vigna has the most species (38) among them unguiculata (Tomooka et al., 2011; Pasquet,

2

Fig. 1.1: Cowpea dry seed used as a grain (Photo credit: Carl Davies)

3

2000). Further, there are 11 different subspecies which include baoulensis, burundiensis,

letouzeyi, aduensis, pawekiae, alba, pubescens, tenuis, stenophylla, dekindtiana and

unguiculata. The subspecies unguiculata is annual, comprising four cultivar groups namely,

sesquipedalis, biflora, textilis, and unguiculata (Pasquet, 2000). Among the subspecies,

variations have been observed on the growth habit, and the vegetative and reproductive

organs, making it easy to classify them. Wild (var. spontanea) and cultivated cowpea (var.

unguiculata) belong to the cultivar group unguiculata (Padulosi and Ng, 1997; Pasquet,

2000; Tomooka et al., 2011).

1.3 Cowpea cultivation

Cowpea grows well in well‐drained sandy loam soils, thriving at temperatures between 28

and 30 0C and rainfall between 500 and 1200 mm per year (Fig 1.2). Spraying the field with a

herbicide such as glyphosate is recommended while preparing land, which is ploughed,

harrowed and ridges made prior to sowing the seeds. Application of fertilizer, in particular

phosphorous, is usually recommended. Harvesting is done when pods are fully mature and

dry, and these must be stored appropriately to avoid infestation by storage pests (Dugje et

al., 2009).

1.4 Cowpea production constraints

Although cowpea is considered to be an important food legume, its production is

constrained by abiotic and biotic factors. Biotic factors include diseases and pests, which

cause considerable economic loss.

1.4.1 Diseases

Cowpea diseases are caused by viruses, bacteria and fungi. Viral diseases can cause serious

yield losses in cowpea (Hampton et al., 1997). The crop is susceptible to 12 major diseases

including cowpea stunt disease, caused by a dual interaction of cucumber mosaic virus

(CMV) and blackeye cowpea mosaic virus (BICMV). Cowpea plants affected by this disease

are characterized by leaf distortions, mottles and blisters and it is responsible for significant

yield losses. It is also a problem in seed production and germplasm distribution (Gillaspie,

2001). Cowpea yellow mosaic virus (CYMV) infection is characterised by light‐green mottle

4

Fig. 1.2: Cowpea field showing fresh leaf and pods (Photo credit: Jeff Ehlers)

5

and leaf distortion while BICMV‐infected plants exhibit stunting and vein necrosis. Both

diseases are prevalent in Africa and can be responsible for reducing yields by up to 100 % in

some years (Thottappilly and Rossel, 1992).

Other diseases include bacterial pustule and cowpea bacterial blight caused by

Xanthomonas campestris pv. vignicola (Burkholder). These diseases can also cause yield

losses of considerable economic importance. Bacterial blight is reported to reduce yields by

up to 71 % of the cowpea crop (Okechukwu and Ekpo, 2004). Symptoms shown by plants

infected with bacterial blight are necrosis and cracked stems (Williams, 1975). Leaf curl

disease, caused by Mungbean yellow mosaic India virus, is characterized by severe leaf

distortion, yellow mosaic and vein swellings (Rouhibakhsh and Malathi, 2005). The major

fungal diseases of cowpea include Anthracnose, Ascochyta blight, Black leaf spot (leaf smut),

Cercospora leaf spot, and powdery mildew, among others (Emechebe and Florini, 1997).

These diseases can sometimes be responsible for yield reductions between 30 and 50 % of

grain yield (Williams, 1975). For instance, Anthracnose affects the seed resulting in rotting

and seedling mortality causing yield losses of up to 40 % while Ascochyta blight causes

defoliation and lesions on the stems and pod (Emechebe and Florini, 1997).

1.4.2 Weeds

Cowpea is a susceptible host to two important parasitic weeds namely Alectra vogelii

[Benth.] and Striga gesnerioides [Wild.] Vatke (Visser et al., 1977; Visser et al., 1990; Parker

and Polniaszek, 1990; Lane et al., 1991; Emechebe and Lagoke, 2002; Boukar and Fatokun,

2007). Uncontrolled growth of these weeds causes considerable yield losses of economic

importance (Boukar and Fatokun, 2007). Upon infestation of Vigna species, Alectra vogelii

develops lateral roots that penetrate into the root of the host causing disarrangement and

loosening of host tissue cells (Visser et al., 1977) while for Striga, the surface of the host

roots split causing the roots of the parasite to penetrate into the endodermis (Lane et al.,

1991). In other cases, weeds act as sources of virus inoculum. These invasive plants harbor

viruses (Begomoviruses) that are transmitted to and infect cowpea causing significant

losses. Such weeds include Sida rhombifolia, Herissantia crispa, Waltheria indica among

others (Asssunção et al., 2006). Other weeds reported are the annual grasses and those

common in cowpea include Brachiaria deflexa Robyns, Brachiaria lata C.E. Hubbard,

6

Digitaria horizontalis Wild, goose grass, ricegrass and, Paspalum. These are capable of

causing yield reductions of up to 80 % if uncontrolled (Akobundu, 1982). Other weed species

have been found in Asian cowpea fields. These include Trianthema monogyna and Cyperus

rotundus which have been shown to negatively affect cowpea yields (Chattha, et al., 2007).

In Nigeria, yield losses estimated between 50 % and 80 % were reported due to the

presence of weed species belonging to the Asteraceae, Euphorbiaceae and Poaceae (Takim

and Uddin, 2010).

1.4.3 Insect pests

Insect pests are generally considered to be the most important threat to cowpea production

and can lead to significant yield losses if no control measures are used (Jackai, 1995; Ngakou

et al., 2008). They attack every developmental stage of the crop (Murdock et al., 2008).

Cowpea is a host for a number of insect pests that are classified into storage, pre‐ and post‐

flowering pests. These include weevils, pod borers, flower bud thrips, leaf beetles, pod

suckers, aphids, and leafhoppers (Singh and van Emden, 1979; Singh and Allen, 1979; Jackai

and Daoust, 1986).

1.4.3.1 Storage pests

The cowpea weevil (Callosobruchus maculatus) and C. chinensis are pests known to infest

cowpea during storage (Singh and Allen, 1979; Jackai and Daoust, 1986). C. maculatus is

responsible for grain losses of between 30 and 100 % (Singh and Allen, 1979; Jackai and

Daoust, 1986). The weevil can attack the seed while in the field. The adults lay their eggs on

the seed surface and the resulting larvae penetrate into the seed. The adult subsequently

emerges onto the surface of the seed through the holes bored by the larvae (Singh and

Allen, 1979). These pests can be controlled by fumigating the grain with insecticide,

application of ash or sand, use of natural plant products such as chillies, peppermint, garlic

(Tiroesele et al., 2015) and various botanical extracts and essential oils (Balachandra, et al.,

2012).

1.4.3.2 Pre‐flowering pests

The cowpea pre‐flowering pests include aphids, leafhoppers and the foliage beetle (Singh

and Allen, 1979). The cowpea aphid (Aphis craccivora) is a major pest in cowpea growing

7

areas. The aphid mostly feeds on the under‐surface of young leaves, stem tissue and pods of

mature plants causing stunting, leaf distortion, premature defoliation and death of seedlings

(Singh and Allen, 1979). This pest also plays a role in transmitting cowpea aphid‐borne

mosaic virus (Singh and Allen, 1979). Controlling the aphid is achieved though cultural

practices, physical, biological and chemical methods (Ofuya, 1997). A few cowpea varieties

are known to be resistant to the cowpea aphid (Huynh et al., 2015). The leafhoppers attack

cowpea during the seedling stage. The leaves infested by leafhoppers are characterized by

discoloration and cupping and there is subsequent stunted growth (Singh and Allen, 1979).

Yield losses of up to 40 % have been reported as a result of leafhopper damage (Raman et

al., 1978). The beetle (Ootheca mutabilis) feeds on foliage of cowpea seedlings causing

defoliation of leaves. This pest is also a vector of cowpea mosaic virus (Singh and Allen,

1979).

1.4.3.3 Post‐flowering pests

The post‐flowering pests include thrips, flower beetles, the pod sucking bugs and pod borers

(Singh and Allen, 1979). Thrips (Megalurothrips sjostedti) can cause yield losses of up to 70

% if uncontrolled. The damage caused by thrips is characterized by necrosis and abscission

of flowers and flower buds (Ngakou et al., 2008). Besides the use of conventional

insecticides, thrips can be managed using host plant resistance approaches (Olabi et al.,

2003; Olabi et al., 2004).

1.5 The legume pod borer

Among the post‐flowering pests, the Maruca pod borer (MPB), Maruca vitrata, a

lepidopteran, causes significant grain losses of up to 80 % if no control measures are

employed (Jackai, 1995; Singh and van Emden, 1979). It is found within the tropic and sub‐

tropic regions including Northern Australia, East Asia and sub‐Saharan Africa (Margam et al.,

2011). Cowpea infestation occurs at about 21 days after sowing. The adult moths lay single

eggs (6 to about 180 eggs) mostly on flower and leaf buds and flowers. The eggs are a

translucent and shiny light yellow colour and hatch after 5 days. Larval development takes 8

to 14 days. During this time, the larvae undergo various instar stages (first to fifth). The larva

has a dark head and is spotted from the thorax onwards, thereby referred to as the spotted

caterpillar (Sharma et al., 1999). The larva is the destructive stage of this pest, feeding on

8

flowers, flower buds, peduncles, tender parts and green pods of cowpea as well as several

other leguminous crops (Singh and van Emden, 1979; Ba et al., 2009; Fig. 1.3). In cowpea,

the larvae roll and web the leaves and feed on them. They also web the flowers, buds and

pods in order to protect themselves from natural enemies and other adverse effects during

the daytime, and emerge at night on the plant surface (Singh and van Emden, 1979). Upon

maturity, the larvae migrate underneath the leaves and develop to pre‐pupae, which last for

2 days. Thereafter, pupation sets in lasting between 6 and 11 days after which the adults

emerge and live for 7 days. Hence the lifecycle of Maruca takes 27 days on average, with

optimum temperatures between 22 0C and 28 0C (Sharma, 1998).

1.6 Justification for transgenic approaches

Several approaches have been used to address the production constraints in cowpea.

Breeding for host plant resistance is currently the main way of controlling the problem of

pests and diseases. There have been remarkable efforts in cowpea collaborative breeding

programs between Africa and USA towards breeding cowpea varieties for biotic and abiotic

stress. Breeding for resistance has been successful for bacterial and viral diseases, parasitic

weeds and insect pests including thrips, aphids and beetles (Hall et al., 1997; Ehlers and Hall,

1997; Hall et al., 2003). Sources of resistance to bacterial diseases were discovered in the

cowpea genome and cowpea cultivars resistant to bacterial blight and bacterial pustule

were developed at the International Institute of Tropical Agriculture (IITA) (Hall et al., 1997).

Additionally, genes for resistance to virus diseases were identified and cowpea cultivars

resistant to cowpea mosaic virus, cowpea aphid‐borne mosaic virus, cucumber mosaic virus

among others were also developed at IITA (Hall et al., 1997). Genes for resistance to Striga

have been identified in the germplasm collection and used to develop a Striga‐resistant

cultivar, IT90K‐76 released in Nigeria (Hall et al., 1997). There has been also success in

breeding cowpea lines resistant to nematodes (Meloidogyne incognita), (Kirkpatrick and

Morelock, 1987; Singh et al., 2002).

9

Fig. 1.3: Cowpea infestation by Maruca pod borer. (A) Larvae attacking cowpea flower and (B) damaged cowpea pod (Photo Credit: Venu Margam)

10

In West Africa, two cowpea cultivars, ‘Mouride’ and ‘Melakh’ resistant to bacterial blight

and cowpea aphid‐borne mosaic virus were also developed. ‘Mouride’ has good resistance

to several biotypes of the parasitic weed Striga, while ‘Melakh’ is also resistant to the

cowpea aphid and partially resistant to thrips (Ehlers and Hall, 1997; Hall et al., 2003). These

cultivars were released and distributed to farmers in the Sahelian region of Africa including

Ghana (Hall et al., 2003). There has been a very significant effort to breed cowpeas resistant

to insect pests. Genes for resistance to aphids and thrips have been incorporated into new

cultivars. Examples include ISRA‐2065, a breeding line with partial resistance to flower thrips

(Hall et al., 2003) and ‘Bettergro Black‐eye’ developed in the USA with a high level of

resistance to the cowpea curculio beetle (Ehlers and Hall, 1997). Other lines developed at

IITA that are resistant to certain aphids such as TVu 86, TVu 801 and TVu 3000 have been

used in national cowpea breeding programmes for the development of aphid‐resistant

cultivars (Hall et al., 2003). Although significant progress has been made in breeding

cowpeas with resistance to several insect pests, the identification of germplasm with

resistance to pod borer and pod sucking bugs remains a challenge (Fatokun, 2002). Genes

conferring resistance to the MPB are present in the genome of a wild Vigna species, Vigna

vexillata. Although this seems encouraging, V. unguiculata and V. vexillata are sexually

incompatible and hence crossing is not feasible (Fatokun, 2002). Breeding for host plant

resistance to MPB has therefore not been successful.

Control of insect pests in cowpea through the use of insecticidal sprays has been

spectacularly successful (Ajeigbe and Singh, 2006; Kawuki et al., 2005). While technically

feasible, this approach is not viable due to the low incomes of small‐holder farmers and

limited knowledge of the use of insecticides. Besides, the use of chemicals on cowpea leaves

is not adopted as the leaves are utilized as a vegetable (Okeyo‐Owuor et al., 1983;

http://www.aatf‐africa.org/userfiles/CowpeaFAQ.pdf; Isman, 2010). Furthermore,

excessive use of insecticides can be detrimental to human health and the environment, if

the correct dosage is not used (Pimentel et al., 1992; Garry, 2004). As such, alternative

control strategies are needed, for example biological control using the entomopathogenic

Bacillus thuringiensis (Bt). MPB can be partially controlled through the use of well‐timed

insecticidal sprays derived from microbial formulations (Taylor, 1968). Alternatively,

11

cowpeas expressing insecticidal proteins derived from Bt genes through genetic

manipulation are in an advanced stage of development (Mohammed et al., 2014).

1.7 The use of Bacillus thuringiensis as an insect control strategy

Bacillus thuringiensis (Bt) produces spores which contain insecticidal proteins, known as δ‐

endotoxins or crystal (Cry) proteins (Schnepf et al., 1998; de Maagd et al., 2003). The

ingestion of the Cry proteins by target insects causes the crystalline inclusion to solubilise in

the alkaline condition of the insect midgut, thereby yielding the active protein. The

activated protein binds to specific receptors on the epithelial cells in the midgut, causing

pore formation and disruption of the osmotic balance. The cells lyse and the larva starves

and dies (Whalon and Wingerd, 2003; Fig. 1.4).

Bt has been used in agriculture to control pests thereby reducing the need for chemical

sprays and sometimes improving crop yields. Bt is an aerobic, gram positive, spore‐forming

entomopathogenic bacterium belonging to the Bacillus cereus group. This bacterium was

discovered over a century ago and since then it has been used in agriculture to control

insect pests (Bravo et al., 2005). Bt contains toxins that have been used as biocontrol agents

in the form of biopesticides (Taylor, 1968). These commercial bio‐pesticidal formulations

such as Thuricide® Dipel™ and Delfin™ are trade names of products based on Bt var. kurstaki

and are specific to lepidopteran pests including Maruca (Taylor 1968; Rodgers, 1993;

Whalon and Wingerd, 2003; Sanchis and Bourguet, 2008; Kaur, 2000; Yule and Srinivasan,

2013). Other biopesticides based on recombinant Bt strains and specific to lepidopteran

insects include Maatch™ and M‐Peril™ both derived from Bt kurstaki strains that contain

Cry1A/Cry1C and Cry1Ac, respectively, (Rodgers, 1993; Kaur, 2000). Potential benefits of

these biopesticides include their low risk to the environment and health. They are applied in

small doses and they readily decompose resulting in minimal environmental pollution

(Thakore, 2006).

12

Fig. 1.4: Mode of action of Bt crystal toxins (Watkins et al., 2011)

13

1.7.1 Bt insecticidal proteins

1.7.1.1 Crystal (Cry) proteins

Different Cry proteins have proven to be toxic to different insects and can be highly specific

(van Frankenhuyzen, 2009). Based on their specificity to different insect orders, the Cry

proteins have been divided into four major classes (Cry1 to Cry4) which are Lepidoptera‐

specific, Lepidoptera‐ and Diptera‐specific, Coleoptera‐specific and Diptera‐specific,

respectively. These Cry protein classes are encoded by cry1, cry2, cry3 and cry4 genes,

respectively (Hofte and Whiteley, 1989; Crickmore et al., 1998; van Frankenhuyzen, 2009).

Within the classes, the Cry proteins are ranked based on their amino acid identity, and

further assigned unique names comprising an uppercase and a lowercase letter (eg. Cry1Ac)

based on their sequence identities (Crickmore et al., 1998; Crickmore et al., 2012).

1.7.1.2 Vegetative insecticidal proteins (Vips)

The Vips are an additional family of proteins which are synthesized by Bt, in this case during

the vegetative growth phase (Estruch et al., 1996). The Vips bear no amino acid sequence

similarity to Cry proteins (Estruch et al., 1996) and have different mechanisms of action (Lee

et al., 2003). Upon ingestion by insects, Vips are solubilised by gut proteases that activate

the protein. The activated Vip then interacts with specific receptors found in the midgut

epithelial tissue. This leads to formation of pores and the rupture of the epithelial cells,

resulting in cell death (Singh et al., 2010). Symptoms develop within a period of 48 to 72

hours following the ingestion of Vip3 toxins (Yu et al., 1997). Vips exhibit insecticidal activity

towards a range of lepidopteran (Estruch et al., 1996) and coleopteran insects (Warren,

1997) and are classified into three different groups, Vip1, Vip2 and Vip3. This classification

was proposed by the Bacillus thuringiensis toxin nomenclature committee (Crickmore et al.,

2012). Figure 1.5 is a dendrogram based on amino acid sequence, illustrating the

relatedness of the Vip toxins believed to share a common three‐domain structure

(Crickmore et al., 2012). Although the sequence of Vip4 protein has been released to

GenBank (http://www.ncbi.nlm.nih.gov/nuccore/328833560), there is no publicly available

information on the host range of Vip4.

14

Fig. 1.5: Classification of vegetative insecticidal proteins believed to share a common three domain structure. Source: http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/

15

The Vip1 and Vip2 proteins are active against certain Coleoptera (Warren, 1997) and

Homoptera (Yu et al., 2011b). Vip1 and Vip2 form a binary toxin of the A‐B type where Vip2

is the cytotoxic A‐domain for enzymatic reaction and Vip1 contains the receptor‐binding B‐

domain (de Maagd, et al., 2003). The mechanism of action of the binary toxins involves the

binding of Vip1 protein to specific receptors in the midgut cells of the susceptible insect.

This forms a channel that provides a pathway for Vip2 to pass through the pore and into the

cytoplasm of the target cells. Based on its homology to ADP‐ribosyltransferases, Vip2

modifies actin inhibiting its polymerization, thereby disrupting the integrity of the

cytoskeleton (Leuber et al., 2006; Aktories et al., 2011) which leads to death.

The Vip3 toxins are active against some Lepidoptera (Estruch et al., 1996) and have become

an important class of insecticidal proteins because of their insecticidal spectrum within the

Lepidopteran pests (Lee et al. 2003; Liu et al., 2007). There are more than 70 different vip3

gene sequences in the Crickmore database

(http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/vip.html).

The Vip3 proteins are classified into 3 families, namely 3A, 3B and 3C, and further grouped

into eight 3A sub‐families (Vip3Aa to Ah), two 3B sub‐families (Vip3Ba and Bb) and one 3C

sub‐family (Vip3Ca) based on their DNA sequence similarities (Crickmore et al., 2012). Gene

products of the vip3A family such as vip3Aa19 have been shown to be toxic to the beet

armyworm (Spodoptera exigua), cotton bollworm (Helicoverpa armigera) and the

diamondback moth (Plutella xylostella) (Liu et al., 2007) while vip3Bb2 is active against the

Australian bollworm (Helicoverpa punctigera) (Beard et al., 2008).

1.7.2 Bt transgenic crops

Bt genes have been used to develop transgenic crops expressing insecticidal proteins

(Shelton et al., 2002). Nearly 30 million hectares of Bt crops were grown globally in 2014 and

the area continues to grow (James, 2014). Bt genes have been transferred to and expressed

in major crops including corn, cotton, soybean, canola among others to provide host plant

resistance against major insect pests. Several of these Bt crops have been commercialized in

both developing and developed countries (James, 2014; Qaim and Kouser, 2013).

16

Bt cotton expressing Cry1Ac insecticidal protein against Helicoverpa (Lepidoptera) was

among the first transgenic crops to be developed and commercialized. It was available in the

market as BollGard® (USA) or INGARD® (Australia) and provided control of the pest,

resulting in reduced insecticide use (Fitt, 2003;

https://www.icac.org/cotton_info/tis/biotech/documents/recorderdocs/december_03.pdf

Fitt, 2008) and sometimes an increase in yield depending on the farming practice. The Bt

cotton was also commercialized and adopted by farmers in developing countries such as

South Africa, which realized yields nearly double those of conventional cotton (Toenniessen,

et al., 2003; Qaim and Kouser, 2013). Other transgenic Bt crops developed with insect

resistance traits are vegetables including Bt broccoli and Bt eggplant/aubergine, both

expressing Cry1Ac protein targeting lepidopteran pests particularly Plutella xylostella. The Bt

eggplant was developed in India, where it is known as Bt brinjal and was first

commercialized in Bangladesh in 2014. It is likely to be commercialized in India in the near

future (James, 2014).

1.7.3 Insect Resistance Management

Despite the dramatic achievements of these Bt crops, there is a concern that natural insect

resistance to Bt crops will develop over time (Gould, 1998; Sumerford et al., 2013). In a

laboratory‐based study (Akhurst et al., 2003), the BX strain of the cotton bollworm,

Helicoverpa armigera (Hubner), fed on an artificial diet containing various Cry toxins

including Cry1Ac, Cry1Ab, and Cry2Ab for several generations, developed resistance which

allowed the lepidopteran pest to survive on transgenic cotton expressing Cry1Ac protein.

Another lab‐based experiment showed that two strains (Loxahatchee and Cry1C‐Sel) of the

diamondback moth (Plutella xylostella), developed resistance to Cry1Ac and Cry1C,

respectively. They were able to survive and complete larval development on two transgenic

Bt broccoli expressing Cry1Ac and Cry1C, respectively (Tabashnik et al., 2003). Resistance to

a commercial Bt‐transgenic cotton cultivar expressing Cry1Ac was also demonstrated for

two laboratory‐derived strains of the pink bollworm, Pectinophora gossypiella (Tabashnik et

al., 2002).

Incidences of field‐evolved insect resistance to Bt crops carrying a single cry gene have been

reported. In the United States, Tabashnik et al. (2008) monitored fields of Bt transgenic

17

cotton producing Cry1Ac toxin. Data collected over a 2‐year period showed field‐evolved

resistance in one (Helicoverpa zea) out of five Lepidopteran pests. Alvi et al. (2012)

investigated the resistance of H. armigera to Cry1Ac Bt‐cotton in Pakistan. They showed

that field‐collected populations of H. armigera survived on Bt cotton and resistance to

Cry1Ac increased significantly after six generations of selection in the lab. In India, Dhurua

and Gujar (2011) sampled a population of pink bollworm from non‐Bt cotton fields and used

it to demonstrate field‐evolved resistance to Bt cotton expressing a single Bt toxin (Cry1Ac).

There was a high incidence and survival of the pest where Bt cotton had been cultivated for

several years. Ferre et al. (1991) obtained a field population of diamondback moth larvae

and exposed them to five different Bt crystal proteins. The surviving larvae were resistant to

the Cry1Ab toxin and appeared to have lost the ability to bind the toxin (Ferre et al., 1991).

The above cases present evidence that insect resistance to Bt crops can develop in the field.

One way of addressing the development of resistance to Bt toxins is to use more than one

Bt gene (gene pyramiding) to develop a crop variety expressing two or more insecticidal

proteins with different mechanisms of action (Roush, 1998; Bates et al., 2005). Laboratory

bioassays were carried out by Stewart et al. (2001) to compare the toxicity of single‐ and

dual‐toxin Bt cotton cultivars expressing Cry1Ac alone and Cry1Ac and Cry2Ab, respectively.

Fresh tissues were fed to larvae of three lepidopteran pests namely tobacco budworm, fall

and beet armyworm. The larvae of the three pests were more susceptible to leaves and

flowers of the dual toxin Bt cotton than the single‐Bt cotton or non‐Bt cotton cultivars. Cao

et al. (2002) pyramided cry1Ac and cry1C genes in broccoli by sexually crossing plants

containing the two cry genes. They obtained a cry1Ac + cry1C hybrid expressing both cry

proteins. When the leaves of this hybrid were fed to two different populations of

diamondback moths (one was Cry1A‐resistant and the other was Cry1C‐resistant) there was

high mortality to both diamondback moth populations. In other reports, a population of H.

armigera (strain SP15) was shown to be resistant to Cry2Ab toxin in laboratory assays

(Mahon et al., 2007). When the SP15 strain was tested using leaves of a pyramided

transgenic Bt cotton expressing both Cry1Ac and Cry2Ab it proved to be susceptible to

Cry1Ac, the additional toxin found in this dual‐Bt cotton cultivar (Mahon et al., 2007). This

demonstrates the efficacy of stacking two different cry genes in one transgenic crop.

18

In addition to the use of twin cry proteins, a Vip protein has been utilized to complement

one or more cry proteins for insect resistance management. Several Bt pyramided corn lines

developed using a combination of cry genes and a vip gene were tested for their efficacy in

the control of the fall armyworm (S. frugiperda), an important pest of corn. These Bt

pyramids included the Genuity®VT Double Pro™ producing Cry1A.105 and Cry2Ab2 and

commercialized in USA. A field population of S. frugiperda was confirmed to be resistant to

Cry1F protein (Huang et al., 2014; Niu et al., 2014) and Cry1Ab (Yang et al., 2013). In

greenhouse trials the resistant populations caused as much damage to the leaves of two

single‐Bt corn hybrids with Cry1F and Cry1Ab, respectively, as occurred on non‐Bt corn

leaves. However, the insect populations did not survive on the pyramided Bt corn (Niu et al.,

2014). VipCot™ is a cotton cultivar developed by stacking Vip3A with Cry1Ab to control

major lepidopteran cotton pests. Adamczyk and Mahaffey (2008) demonstrated the efficacy

of Vip3A, Cry1Ab or a combination of these two proteins on four lepidopteran pests (fall and

beet armyworms, bollworms and tobacco budworms). In 2004, cotton containing Vip3A had

less tobacco budworms after 5 days in leaf and floral bioassays when compared to non‐GM

cotton. The larval weights of the four pests were significantly less than the larvae on non‐

GM cotton. In 2005, cotton lines stacked with Cry1Ab + Vip3A traits caused a significantly

higher mortality in fall and beet armyworms and the bollworms, than cotton with just the

Vip3A trait. A similar scenario was observed by Bommireddy and Leonard (2008) when

testing the survival of two lepidopteran cotton pests (H. zea and H. virescens) on a cotton

line having both Cry and Vip traits (VipCot). Survival levels as low as 1 % were observed on

H. zea after 96 hours of exposure to terminal leaves and reproductive parts, when

compared to conventional cotton. In H. virescens, survival was 4 % and 2 % following 96

hours exposure on terminal leaves and reproductive parts, respectively. Kurtz et al. (2007)

and Palekar et al. (2011) confirmed this high mortality when bioassays were carried out

using neonate and fourth instar larvae of H. zea or H. virescens on lyophilized tissues or in

VipCot fields artificially infested with the pests. In both cases, larval mortality in H. zea or H.

virescens ranged between 97 and 100 %. Stacking genes with different modes of action can

therefore offer a possible solution to control resistant pests and reduce the chances of

developing insects resistant to Bt proteins. Furthermore, transgenic rice expressing a fusion

protein (Cry1Ab/Vip3Ah) caused 100 % mortality (in all developmental stages of the rice

plants) to the Asiatic rice borer (C. suppressalis) in a 7‐day bioassay (Chen et al., 2010).

19

Several studies have shown that Maruca is susceptible to Bt insecticidal proteins (cry toxins)

either incorporated in artificial diets (Srinivasan, 2008) or in a Bt cowpea containing a

cry1Ab gene in the field (Higgins et al., 2012; Mohammed et al., 2014). As discussed above

for other pests, there is a chance that MPB will develop resistance to the single‐gene Bt

cowpea. To avoid this or at least greatly delay the development of resistance, the addition

of a second Bt (vip) gene to complement the cry1Ab gene seems prudent. However, it was

unknown whether Vips were toxic to MPB.

1.8 Research Hypothesis

The Vips of Bt are a class of insecticidal proteins currently being utilized commercially to

complement the Cry1 and 2 δ‐endotoxins in corn and cotton (Whitehouse et al., 2007;

Castagnola and Jurat‐Fuentes, 2012). Vip proteins bind to different target sites than Cry

proteins in several tested Lepidopteran species. Since combining two or more insecticidal

genes into one crop variety reduces the risk of insect resistance in Bt crops, it is envisaged

that Vip proteins could be used together with Cry proteins for Insect Resistance

Management (IRM) in cowpea. The hypothesis therefore states that the vip gene encodes a

toxin with insecticidal activity against the Maruca pod borer. Thus, in the longterm, insect

resistance in cowpea will be better managed by using a combination of a cry1 and a vip

gene.

1.9 Research Objectives

This study sought to identify suitable vip gene(s) in Bt that will provide resistance to MPB

and thus complement the Cry1Ab‐transgenic cowpea already developed. The main aim of

the research therefore was to develop a Bt cowpea carrying a vip gene. This was achieved

through the following objectives:

1. Screen Bt strains for genes encoding Vip toxins expected to be active against

Lepidoptera

2. Overexpress selected Vip toxins in Escherichia coli

3. Conduct insect bioassays using MPB larvae on single Vip toxins

4. Re‐construct a vip gene for cowpea transformation

5. Transform cowpea with the re‐constructed vip gene.

20

6. Regenerate and characterize transgenic plants

7. Conduct insect feeding studies on transgenic cowpea using Maruca larvae to

determine resistance

2.0 Expected output

From this work, it was expected that a vip‐transgenic cowpea line resistant to the MPB

would be developed. In due course, this trait could be introgressed by cowpea breeders into

existing Bt cowpea lines carrying a cry1Ab gene. This approach would avoid, or at least

greatly delay, the resistance of Maruca to Bt toxins thus providing an enhanced strategy to

manage insect resistance in this important food legume.

21

CHAPTER TWO

General Materials and Methods

2.1 Preparation of bacterial cultures

2.1.1 Bacillus thuringiensis (Bt) cultures

To establish Bt cultures, Luria agar (LA) medium consisting of tryptone (10 g/L), NaCl (5 g/L),

yeast extract (5 g/L) and bacteriological agar (15 g/L) was poured into sterile Petri dishes

and allowed to cool. Luria Broth (LB) was prepared using the above ingredients but without

agar. The medium was inoculated with bacteria using the dilution streak method and

incubated overnight at 28 0C. For each strain, a single bacterial colony was picked and used

to inoculate 5 mL of LB in sterile test tubes. The bacterial cultures were incubated overnight

at 28 0C on a shaker at 230 revolutions per min (rpm). To archive the Bt strains, a 500 μL

aliquot of the bacterial culture was added to a 1.5 mL tube containing 500 μL of sterile 30%

glycerol. The tubes were shaken gently and allowed to stand for 1 h at room temperature.

They were stored in a ‐800C freezer.

2.1.2 Preparation of Mannitol Glutamate Luria (MGL) culture medium for Agrobacterium

tumefaciens

To make 1 L of MGL medium, the ingredients in Table 2.1 were mixed thoroughly in about

800 mL of sterile RO water. The pH was adjusted to 7 using 1N NaOH and the volume

adjusted to 1 L. The solution was dispensed into 100 mL bottles and autoclaved. For MGL

plates, 15 g of Bacto Agar was added to 1 L of medium prior to autoclaving.

Table 2.1: Ingredients for bacterial MGL medium

Chemicals Quantities to make 1L medium

Mannitol 5.0 g L‐glutamic acid 1.0 g KH2PO4 0.25 g NaCl 0.1 g MgSO4.7H2O 0.1 g Tryptone 5.0 g Yeast extract 2.5 g Biotin (from 0.1 mg/mL stock) 10 µL

22

2.2 Isolation of bacterial DNA

2.2.1 Isolation of genomic DNA from Bacillus thuringiensis

The Bacterial Genomic DNA isolation kit from Sigma‐Aldrich (St. Louis, MO 63103, USA) was

used to extract DNA from bacterial cultures. The following modifications were made to the

manufacturer’s instructions: A final volume of 200 µL lysozyme solution was prepared

foreach DNA isolation and used to suspend the bacterial cells. A volume of 20 µL of

Proteinase K solution was added to the sample followed by 200 µL of Lysis Solution C. A 500

µL volume of column preparation solution was added to each GenElute Miniprep Binding

Column. The lysate was centrifuged for 12000 g for 5 min. For the first wash, 500 µL of wash

solution was added and centrifuged for 1 min at 12000 g. For the second wash, 500 µL of

wash solution concentrate was added. For DNA elution, 200 µL of elution solution was

added and the column was centrifuged for 1 min at 6500 g. The eluted DNA was stored at 4

0C for further analysis such as PCR.

2.2.2 Isolation of plasmid DNA from Escherichia coli and Agrobacterium tumefaciens

A single colony of E. coli was grown in 10 mL of LB medium with selection overnight at 37 0C.

Unless otherwise stated, plasmid DNA was isolated from the bacterial culture following the

protocol described in the Bioline (Alexandria, NSW 1435 Australia) Plasmid Mini Kit. The

preparation of reagents and isolation procedures followed the manufacturer’s instructions.

Plasmid DNA was isolated from Agrobacterium cultures using the Bioline kit described

above.

2.3 Purification of PCR products

Unless otherwise stated, the purification of PCR products was carried out following the

QIAquick Gel Extraction Kit protocol (Qiagen 2010).

2.4 Cloning of vip genes

Cloning of vip genes was carried out following the procedures previously described in the

Expresso™ T7 Cloning and Expression System (Lucigen Corporation, Middleton, WI 53562

USA) and/or the Gibson assembly method (Gibson et al., 2009; Gibson, 2011). In both cases,

23

primers designed to be compatible with the expression vector were used to amplify the

coding regions of the vip genes.

2.4.1 Expresso™ T7 Cloning and Expression System

The PCR products were purified and mixed with the pETite vector for direct transformation

into E. coli strain 10G. Cells were cultured on LB plates containing 30 mg/L kanamycin

overnight at 37 0C at 250 rpm. To confirm the presence of the insert and expression vector,

plasmid DNA was isolated (as described in section 2.2.2) and either used as a template for

PCR or for restriction mapping. Subsequently, plasmid DNA of the positive clones was used

to transform E. coli strain BL21 for protein expression following the instructions supplied in

the Expresso™ T7 Cloning and Expression System. The transformed colonies were selected

on LB plates containing kanamycin and a single colony was grown in LB at 37 0C on a shaker

(250 rpm).

2.4.2 The Gibson assembly method of DNA cloning

Gibson assembly is a method of joining two or more DNA fragments (also known as bricks)

without the use of restriction enzymes. The bricks are joined by their overlapping end

sequences of approximately 40 bp in a PCR reaction with the aid of three enzymes – an

exonuclease, DNA polymerase and DNA ligase. The exonuclease creates a 3’ overhang to

facilitate joining the bricks complementary to each other on one end, DNA polymerase fills

gaps within the joined bricks and DNA ligase seals the nicks of the joined bricks

(https://www.addgene.org/plasmid‐protocols/gibson‐assembly/).

Forward and reverse primers were designed for the DNA bricks (8 kbp or less) to be

assembled. For PCR amplification, 1 µL of forward primer (10 µM stock), 1 µL reverse primer

(10 µM), 10 µL 5x Phusion High Fidelity Buffer, 1 µL dNTPs (10 mM), 0.5 µL template DNA,

0.5 µL Phusion DNA polymerase (2 U/µL) and 36 µL nuclease‐free water were mixed in a

single reaction tube. The PCR cycling conditions were 98 0C for 30 s, 30 cycles of 98 0C for 10

s, 65 0C for 20 s and 72 0C for 1 min and 30 s, a final extension of 72 0C for 5 min and hold at

4 0C. The products were separated by electrophoresis through a 1 % agarose gel and the

selected DNA bands were purified as in section 2.3. To carry out the assembly reaction, a

total of 5 µL of DNA was added to 15 µL aliquot of 1.33x Gibson Master Mix stock [100 µL of

24

5X isothermal buffer, 2 µL T5 exonuclease (1.0 U/µL), 6.25 µL Phusion DNA pol (2U/µL), 50

µL Taq DNA ligase (40 U/µL) and 216.75 µL water to make up to 375 µL final volume]. The 5

X isothermal buffer comprised 25 % polyethylene glycol (PEG)‐8000; 500 mM Tris‐HCL pH

7.5; 50 mM dithiothreitol (DTT), 1 mM dATP, 1 mM dTTP, 1 Mm dCTP, 1 mM dGTP, 5 mM

dGTP, 5 mM nicotinamide adenine dinucleotide (NAD). For each brick, 10 to 100 ng of DNA

was added and the reaction incubated at 50 0 C for 1 h. E. coli cells were transformed with 1

µL of the assembly reaction without purification and incubated at 37 0 C. Plasmid DNA was

extracted from E. coli following procedures in section 2.2.2 and the construct was confirmed

by restriction mapping.

2.5 Sequencing of vip3 genes cloned in E. coli

Plasmid DNA (150 – 300 ng) extracted from E. coli was used as a template for sequencing.

For one reaction, an aliquot of 1 µL of Big Dye® Terminator (Applied Biosystems Cat #

4337454) containing Taq DNA polymerase, standard dNTPs and labelled ddNTPs, 3.5 µL 5x

sequencing buffer (Applied Biosystems Cat # 4305605) optimized for use with Big Dye®

Terminator, 3.2 pmol of primer (0.3 µL of 10 pmol/ µL stock) and MilliQ water to make a

final volume of 20 µL. Each sequencing reaction was replicated three times. The PCR cycles

were as follows: 96 0C for 1 min, and 25 cycles of 96 0C for 10 s, 50 0C for 5 s and 60 0C for 4

min. To purify the PCR products, each reaction was transferred to a 1.5 mL Eppendorf tube.

An aliquot of 5 µL of 125 mM EDTA, 60 µL of 100 % cold ethanol was added to 20 µL of the

PCR reaction and mixed well. The sample was incubated at room temperature for 15 min

then centrifuged at 12000 g for 10 min. The supernatant was discarded and 60 µL of 70 %

ethanol was added, centrifuged at 12000 g for 5 min and the supernatant removed. The

pellet was dried in a Speedivac for 5 min.

2.6 Preparation of electrocompetent Agrobacterium (AGL1) cells

Sterile YMB medium (Table 2.2) was prepared and 100 mL of this inoculated (in a 500 mL

flask) with a loop of AGL1 cells and grown at 28 0 C until the OD600 reached between 0.7 and

1.0. The culture was placed in the cold room for 30 min and centrifuged at 4000 g for 15 min

at 4 0C. The supernatant was discarded and pellet resuspended gently with 35 mL of cold

sterile MilliQ water in an ice bucket on an orbital shaker. The cells were centrifuged again at

4000 g for 15 min at 4 0 C using a swing‐out rotor. The supernatant was discarded and pellet

25

gently resuspended in 5 mL of cold sterile 10 % glycerol solution. Aliquots of 40 µL of cells

were dispensed into sterile 1.5 mL Eppendorf tubes, frozen in liquid nitrogen and stored at ‐

80 0C.

Table 2.2: Ingredients for YMB medium

Chemicals Quantity to make 1 L

Yeast extract 400 mg Mannitol 10 gNaCl 100 mg MgSO4.7H2O 200 mg K2HPO4 200 mg Yeast extract 2.5 g

2.7 Transformation of Agrobacterium tumefaciens (AGL1) cells

An aliquot (40 µL) of AGL1 cells (see section 2.6) was thawed on ice and added to a pre‐

chilled 1.5 mL eppendorf tube. An aliquot of 2 µL of DNA was added and stirred gently with

a pipette tip. The mixture was allowed to stand in ice for 2 min and then transferred to a

chilled electroporation cuvette (2 mm gap) and subjected to a pulse of 2.5 kV. The cuvette

was returned to ice and 1 mL of chilled SOC medium added. This mixture was transferred to

a sterile eppendorf tube and incubated on a shaker at 28 0C and 100 rpm for 90 min. An

aliquot of 100 µL was plated on MGL medium with 0.2 mg/L Rifampicin, other appropriate

antibiotics for selection. The cultures were grown at 28 0C for 2 days. Single colonies were

selected and grown in 10 mL of liquid MGL medium with appropriate antibiotics for 2 days.

An aliquot (500 µL) was stored as a glycerol stock and the remainder used to isolate DNA.

The DNA was used to confirm the construct by restriction mapping.

2.8 Transformation of E. coli cells

Transformation of competent E. coli cells with the plasmid containing the genes of interest

was conducted using the High Efficiency Transformation protocol (C2987) described by the

New England Biolabs (UK). Briefly, a tube of E. coli DH5α cells was thawed on ice for 10 min

and 100 ng of plasmid DNA added. The cells were mixed with the DNA by flicking the tube 4‐

5 times and incubated on ice for 30 min. The cells were heat‐shocked at 42 0C for 30 s and

immediately placed on ice for 5 min. An aliquot of 950 µL of SOC medium (room

26

temperature) was added and cells incubated at 37 0 C for 1 h with vigorous rotation at 250

rpm. Cells were mixed thoroughly by inverting the tube and diluted in several 10‐fold serial

dilutions in SOC medium. Aliquots of 50 to 100 µL of each dilution were spread onto warm

(37 0C) LB plates with selection agent and incubated overnight at 37 0C.

2.9 Protein expression and analysis

Protein was extracted from bacterial cultures as described by Studier (2005) as summarized

in Fig. 2.1. E. coli BL21 transformed with vip3 genes or cry2Aa was grown overnight at 37 0C.

Single colonies were grown in 2 mL of LB overnight on a shaker (250 rpm) at 37 0C. An

aliquot (500 µL) was used to inoculate 100 mL of auto‐induction medium (Tables 2.3 and

2.4) containing 30 mg/L kanamycin and incubated overnight at 37 0C with vigorous shaking

at 255 rpm. At an OD of 4 to 10, the cells were centrifuged at 4000 g for 10 min at 4 0 C and

the supernatant discarded. A volume of 20 mL of a Tris‐buffered protein extraction reagent,

BugBuster™ (Novagen®) and 200 µL of lysozyme (10 mg/mL) was added to the pellet and

incubated at room temperature on an orbital shaker (60 rpm) for 2 h. An aliquot of 20 µL of

DNase 1 solution (2000 U/mL) was added to the lysed suspension, incubated at 37 0 C for 15

min and centrifuged at 8000 g for 30 min at 4 0C and the supernatant (soluble fraction)

stored on ice. The pellet was re‐suspended with 10 mL of protein extraction reagent, 200 µL

of lysozyme (10 mg/mL) added and incubated at room temperature for 15 min. The

suspension was centrifuged at 6000 g for 15 min at 4 0C insoluble fraction stored on ice. To

wash the pellet, 10 mL of 1 in 10 diluted protein extraction reagent was added to the pellet,

vortexed and centrifuged at 6000 g, 4 0C for 15 min and the supernatant was discarded.

These washing steps were repeated 3 times with the last centrifugation at 8000 g for 15 min

at 4 0 C. The pellet was suspended in 2 mL of distilled water. An aliquot of 25 µL of SDS gel‐

loading buffer was added to 100 µL of the resulting suspension and solubilized by boiling for

5 min. The protein concentrations were determined by Bradford (1976) assay and samples

were prepared for sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE)

analysis by the addition of SDS gel loading buffer (422 mM Tris HCl, 563 mM Tris base, 0.8 %

lauryl dodecyl sulfate, 1.6 mM EDTA, 0.2 mM DTT). The 10 % Bis‐Tris precast NuPage gels

were obtained from Life Technologies (Mulgrave, Victoria 3170 Australia), samples were

loaded (30 µg of protein per lane) and the samples electrophoresed at 100 V for 90 min.

27

Protein from an untransformed E. coli (BL21) was used as a negative control while E. coli

transformed with cry2Aa was used as a positive control (Srinivasan 2008).

Table 2.3: Ingredients for Auto‐induction medium, pH 6.9 (for 500 mL)

Chemicals Final concentration

NZ‐Amine 1 % Na2HPO4 25 mMNH4Cl 50 mM MgSO4 2 mM K2HPO4 25 mM Yeast extract 0.5 % Na2SO4 5 mM Glycerol 0.5 % Glucose 0.05 %α‐lactose monohydrate 0.2 % 1000 X Trace minerals (Table 2.4) 0.2 X

Table 2.4: Constituents of 1000 X Trace minerals (for 500 mL)

Chemicals Final concentration

FeCl3 60% solution 50 mM CaCl2.2H20 20 mMMnCl2 10 mM ZnSO4.7H20 10 mMCoCl2.6H20 2 mM CuCl2.2H20 2 mMNiCl2.6H20 2 mM Na2MoO4.2H20 2 mMNaSeO3 2 mM H3BO4 or H3BO3 2 mM

28

Fig. 2.1: Protein extraction flow chart

29

2.10 Identification of Vip protein using Mass Spectrometry

A mass spectrometry (MS) analysis was carried out to identify the expressed Vip3 proteins.

To prepare the samples, protein SDS‐PAGE gels were transferred onto a glass plate and the

protein bands excised. The bands were forwarded to the Australian Proteome Analysis

Facility (Macquarie University, Sydney NSW 2109 Australia) for mass spectrometry. Briefly,

the gels were destained with ammonium bicarbonate: acetonitrile, and then reduced (25

mM DTT in 25 mM ammonium bicarbonate) at 56 0C for 30 min. They were then alkylated

(55 mM IAA in 25 mM ammonium bicarbonate) in the dark for 20 min. Following incubation

the gel bands were washed, dried and digested with trypsin (150 ng) overnight. The

peptides were extracted from the gel using acetonitrile: formic acid, and then concentrated

prior to analysis. They were directed into a mass spectrometer (Triple TOF 5600 AB Sciex)

and then fragmented for a second mass analysis. The resulting data was generated using

Analyst 2.0 MASCOT script searched by Mascot (Matrix science, 2014) and subsequently

subjected to database matching (Cottrell, 2011). The protein identification program used for

the analysis assigns a score for peptides that match the predicted fragments based on the

peptide sequences in the database (Matrix science, 2014).

2.11 Maintenance of a Maruca vitrata insect colony in vitro

2.11.1 Preparation of Maruca artificial diet

The preparation of Maruca artificial diet was based on the method of Jackai & Raulston

(1988). To make 1 L of artificial diet, the ingredients in Table 2.5 [cowpea flour, dried leaf

powder, ascorbic acid, nipagen, sorbic acid, sucrose, wheat germ and Wesson salts (Table

2.6)] were mixed in a blender. Water (500 mL) was heated for 1 min in a microwave on 1000

watts, and added to the blender and mixed. KOH, choline chloride, acetic acid, vitamin

solution (Table 2.7) and aureomycin were then added followed by pulsing for 5 s after each

ingredient. The water (for agar) and agar was combined and heated in microwave for 3 min

on high (1000 watts), stirred and heated for another 1.5 min on high. The agar solution was

added to the blender and mixed well, carefully pulsing twice for 2 s. The mixture was poured

into eight rectangular Chinese food containers (15 x 10 x 3.5cm) to a depth of 1 cm and

allowed to cool and set.

30

Table 2.5: Ingredients for Maruca artificial diet

Ingredients Quantities to make 1L Diet

Hot water (for initial blending) 500 mL Cowpea seed flour 100 gCowpea leaf (freeze dried)* 12.5 g Ascorbic acid 6.25 gNipagen 1.58 g Sorbic acid 0.835 g Sucrose 15 g Wheatgerm 31.8 g Wesson salt mix (Table 2.6) 10.6 gKOH (4M) 5.5 mL Choline chloride (15 %) 7.4 mLAcetic acid (25 %) 12.5 mL Vitamin solution (Table 2.7) 7.5 mL Aureomycin (5 % active ingredient) (Chlortetracycline hydrochloride)

3.5 mL

Agar 14.8 g Water (for adding agar) Enough to make final volume to 1 L

(~450ml)

*The cowpea leaf is 4‐week old cowpea leaves freeze dried and crushed to a fine powder.

Table 2.6: Ingredients to make 100 g Wesson salt mixture

Ingredients Quantities

Calcium carbonate 21 g Copper sulphate (5H2O) 39 mgFerric phosphate/Iron (III) phosphate 1.47 g Manganous sulphate (anhydrous) 20 mgMagnesium sulphate (anhydrous) 9 g Potassium aluminium sulphate 9 mg Potassium chloride 12 gPotassium dihydrogen phosphate 31 g Potassium iodide 5 mgSodium chloride 10.5 g Sodium fluoride 57 mgTri‐calcium phosphate 14.9 g

31

Table 2.7: Ingredients to make 100 mL Vitamin solution


Calcium pantothenate 1.2 g Niacin 0.6 gRiboflavin 0.3 g Folic acid 0.3 gThiamine HCl 150 mg Pyridoxine HCl 150 mg Biotin 12 mg B12 (Cyanocobalamine) 6 mg

2.11.2 Preparation of Honey solution for Maruca adults

To prepare 1 L of honey solution for the emerging adult insects, all ingredients in Table 2.8

(except for ascorbic acid) were combined in a 1 L Schott bottle. The solution was

microwaved on high for 2 min, mixed well and autoclaved. The solution was allowed to cool

to 60 0C and then the ascorbic acid was added. The honey solution was stored at 4 0 C until

used.

Table 2.8: Ingredients for the preparation of honey solution


Sugar 40 gHoney* 40 g Sorbic acid 1 gAscorbic acid 2 g Water Up to 1 L

* Capilano 100 % Natural Australian Honey (Richlands, QLD 4077 Australia)

2.11.3 Preparation of honey pots for feeding adults whilst in cages:

Honey pots were prepared by boring a 1 cm hole in the lids of 60 mL urine specimen plastic

containers. An 8 cm length of cotton dental wick was inserted in the hole. Honey solution

(40 mL) (section 2.10.2) was poured into the plastic container and covered with the lid

containing the wick. Honey pots were placed in the cage (step 4, Fig. 2.2) for rearing the

adult Maruca.

32

2.11.4 Procedures for egg collection

The artificial diet (2.10.1) was divided into 32 cubes per container. Nappy liners cut in half

(“balconies”) containing eggs (see 2.10.5) were placed onto the surface of the diet, and

transferred to a larger lunch box (25 x 19 x 6 cm) and covered with a lid. A window (21 x 14

cm) was cut in the lid and a very fine wire mesh was attached for ventilation (Fig. 2.3). After

closing the box with the ventilated lid, it was covered with cling film and 5 to 6 holes were

punctured for ventilation using sharp forceps.

2.11.5 Rearing of Maruca larvae

The procedures for the maintenance of a Maruca colony are shown in Fig. 2.2. To start the

insect colony, a 5 L bucket (“the cage”) was prepared with two honey pots and a Petri dish

filled with wet vermiculite to increase humidity (Fig. 2.4 A). Two “balconies” were placed

over the edge of the cage (Fig. 2.4 A) as a source of egg collection as the adults lay eggs on

these. A Petri dish with the pupae (from Step 4 in Fig 2.2) was added to the bucket. The

bucket was then covered with a nappy liner and lid. The bucket lid had a 10 cm‐diameter

hole for ventilation (Fig. 2.4 B). The cages were placed in a growth room set at 25 0C and 50

% humidity. A humidifier was placed on a timer for 15 min on/15 min off over 3 h between

12 am and 3 am to increase humidity to 50 % for optimal mating conditions. The first eggs

were collected 3 days after the adults emerged and subsequent egg collections were carried

out every 3 days until no more eggs were laid. The eggs were placed on artificial diets to

hatch (Fig. 2.3). Second and third instar larvae were fed on fresh beans and re‐fed after a

further 5 days until the pupation stage.

33

Fig. 2.2: General procedure for Maruca rearing and maintenance

34

Fig. 2.3: Balconies in artificial diet placed in a larger lunch box

Fig. 2.4: Preparation of cages for emerging adults. (A) Set up of vermiculite and honey pots.

(B) cage covered with nappy liners and lid for egg collection

35

2.12 Preparation of plant tissue culture media

2.12.1 Preparation of MS Stock solutions

To make up the MS Macro stock solutions, the components in Table 2.9 were dissolved in

Reverse Osmosis (RO) water and made up to 1 L.

Table 2.9: Components to prepare MS Macro stock solutions

Chemicals Quantities to make 1 L

20 X MS Macro NH4NO3 33 gCaCl2.2H2O 8.8 g KNO3 38 g MgSO4.7H2O 7.4 gKH2PO4 3.4 g 200 X MS Micro H3BO3 1.245 gMgSO4 4.46 g ZnSO4.7H2O 1.72 gKI 166 mg NaMoO4 (1 mg/mL) 50 mL CuSO4 (1 mg/mL) 5 mLCoCl2 (1 mg/mL) 5 mL 200 X MS Iron 60 % FeCl3 solution 5.4 mL 200 X MS Na2EDTA Na2EDTA 6.71 g 100 X MS Vitamins thiamine HCl 10 mg nicotinic acid 50 mgpyridoxine HCl 50 mg Glycine 200 mg

36

2.12.2 Preparation of hormones, antibiotics and other agents

The following hormones and antibiotics were aseptically prepared by filter sterilization: 200

mM acetosyringone (0.3924 g dissolved in 10 mL dimethyl sulfoxide), 5 mg/mL asparagine, 1

mg/mL benzylaminopurine (BAP), 154 mg/mL dithiothreitol (DTT), 50 mg/mL geneticin (G418),

2 mg/mL gibberellic acid (GA3), 1 mg/mL indole‐3‐acetic acid (IAA), 100 mg/mL kanamycin, 25

mg/L meropenem trihydrate (Wilmimgton, DE, USA), and 1 mM sodium thiosulphate. These

were stored at 40 C until required.

2.12.3 Cowpea suspension medium (for co‐cultivation)

To make 1 L of cowpea suspension medium, the components in Table 2.10 were added to 800

mL of sterile RO water. The pH was adjusted to 5.6 using 1N NaOH and the volume adjusted to

1 L. The suspension was dispensed into 2 x 500‐mL bottles and autoclaved. The following were

added when the medium had cooled to 50 0C: 125 µL of GA3 (2 mg/mL), 1 mL of acetosyringone

(200 mM), 1.7 mL of BAP (1 mg/mL stock) and 1 mL of sodium thiosulphate (1 mM).

Table 2.10: Components to prepare cowpea suspension medium

Chemicals Quantities to make 1 L

200 X MS Iron 0.5 mL 200 X MS EDTA 0.5 mL200 X MS Micro 0.5 mL 20 X MS Macro 50 mL100 X MS Vitamins 10 mL Sucrose 30 gMyo‐inositol 0.1 g MES Hydrate 3.9 gGranulated agar (Difco™). 8 g

2.12.4 Cowpea shoot induction medium (SIM)

To make 1 L of shoot induction medium, the components in Table 2.11 were added to 800 mL

sterile RO water. The pH was adjusted to 5.6 using 1N NaOH and volume adjusted to 1 L. The

suspension was dispensed into 2 x 500‐mL bottles and autoclaved. The medium was allowed to

cool to 500C, after which 1.7 mL of BAP (1 mg/mL stock), 25 mg of meropenem trihydrate, and

1.5 mL of kanamycin (100 mg/mL stock) or 600 µL geneticin (50 mg/mL stock) were added.

37

2.12.5 Cowpea shoot elongation and rooting medium (SEM)

To make 1 L of shoot elongation medium the components in Table 2.11 were added to 800 mL

sterile RO water. The pH was adjusted to 5.6 using 1N NaOH and volume adjusted to 1 L. The

suspension was dispensed into 2 x 500‐mL bottles and autoclaved. The medium was allowed to

cool to 500C and 250 µL of GA3 (2 mg/mL), 10 mL of asparagine (5 mg/mL), 100 µL of IAA (1

mg/mL), 25 mg of meropenem trihydrate and 600 µL geneticin (50 mg/mL) were added.

Table 2.11: Components to prepare cowpea shoot induction medium (SIM) and cowpea shoot elongation and rooting medium (SEM)

Quantities to make 1 L

Chemicals SIM SEM

200 X MS Iron 5 mL 5 mL 200 X MS EDTA 5 mL 5 mL 200 X MS Micro 5 mL 5 mL 20 X MS Macro 50 mL 50 mL 100 X MS Vitamins 10 mL 10 mL Sucrose 30 g 30 g Myo‐inositol 0.1 g 0.1 g MES Hydrate 3.9 g 3.9 g Granulated agar (Difco™) 8 g 8 g 6‐Benzyl‐aminopurine (BAP) 1.67 mg ‐ Gibberellic acid (GA3) ‐ 0.5 mg Asparagine ‐ 50 mg Indole‐3‐acetic acid (IAA) ‐ 0.1 mg

2.13 Isolation of plant genomic DNA

Plant genomic DNA was isolated using a nucleic acid isolation kit (Puregene, USA). To make 500

mL of cell lysis solution, 5 mL of 1 M Tris (pH 8) and 1 mL 0.5 M EDTA (pH 8) were added to 450

mL of RO water and autoclaved. A volume of 50 mL of 10 % SDS was added after the solution

cooled to 500C. Young leaves were collected and frozen in liquid nitrogen. Approximately 20‐40

mg of frozen leaf was crushed and 800 L of cell lysis solution added. The samples were mixed

by vortexing and incubated at 65oC for 90 min. The samples were centrifuged at 12000 g for 5

min and the supernatant transferred to a fresh tube. An aliquot of 1 L of 10 mg/mL RNAse

38

solution was added, mixed and incubated at 37oC for 30 min. The samples were centrifuged at

12000 g for 3 min to pellet the debris, and the supernatant collected. The samples were cooled

at room temperature, 250 L of 6 M ammonium acetate added and vortexed for 20 s, after

which they were centrifuged at 13000 g for 5 min. The supernatant was transferred to a new

tube and 800 L of cold isopropanol added, mixed thoroughly and centrifuged at 13000 g for 5

min. The supernatant was discarded and the pellet washed using 70% ethanol, centrifuged at

12000 g for 3 min and the supernatant discarded. A second centrifugation step was carried out

at 12000 g for 1 min and supernatant discarded. An aliquot of 50 L of TE buffer (10 mM Tris

pH8, 0.25 mM EDTA) was added to the pellet and air dried for 5 min. An aliquot of 1 L was

resolved by electrophoresis through a 0.8% agarose gel together with 50, 150 and 500 ng of

lambda DNA to estimate the DNA yield.

2.14 Protocol for PCR analysis of transgenic cowpea lines

A PCR analysis was carried out to confirm the presence of the vip3Ba gene in the transgenic

lines. For a 10 L PCR reaction, 100 ng genomic DNA of transgenic lines was used as a template.

In a PCR tube, 5 L of Phusion® Hot Start Flex 2X master mix (Cat # M0536S, New England

Biolabs), forward and reverse Vip3Ba primers (2 L each) or nptII primers and 10 L dH2O were

added. Genomic DNA was added and samples loaded for PCR under the following conditions:

980C for 30 s, 980C for 10 s, 600C for 20 s and 720C for 90 s (30 cycles), followed by 720C for 10

min and 250C for 1 min. The PCR products were fractionated by electrophoresis through a 1.8 %

agarose gel at 60 mA.

2.15 Detection of Vip3Ba protein by western blotting

Vip3Ba protein was detected in leaf extracts of transgenic plants by western blotting using

vip3Ba monoclonal antibodies commercially produced by Abmart Inc. (Juke Bio‐tech park,

200233, Shanghai, China). To raise the antibodies, the Vip3Ba protein sequence was subjected

to analysis to identify fragments that would most likely yield monoclonal antibodies. Eight

peptide epitopes were identified at various positions within the sequences. Eight peptide

antigens were designed and optimized for immunization of mouse to produce specific

antibodies against Vip3Ba (Abmart Inc., Juke Bio‐tech park, 200233, Shanghai, China).

39

Protein from transgenic plants was extracted by macerating 80‐110 mg of young fully expanded

cowpea leaf in 500 µL of protein extraction buffer (0.1 M TES pH7.6, 0.2 M NaCl, 1 mM PMSF, 1

mM EDTA) in a 1.5 mL Eppendorf tube. The slurry was centrifuged at 12000 g for 5 min and the

protein concentration determined (Bradford 1976). For SDS PAGE, 40 g of total soluble protein

was mixed together with 8 l of loading dye and 2 l of DTT. Bacterial protein (1 g) containing

Vip3Ba was used as the positive control while a sample prepared from the non‐transgenic

parent line (IT86D) was used as a negative control. The samples were incubated at 65 0 C for 10

min and loaded onto a 10‐well NuPage 10% bis‐tris 1.5 mm precast gel (Invitrogen Cat#NP0315)

and electrophoresed at 150 V for 2 h on NuPage MOPS running buffer (50 mM MOPS, 50 mM

Tris‐base, 3.5 mM SDS, 1 mM EDTA). The polypeptides were transferred to nitrocellulose

membrane using the semi‐dry method of transfer (Thermo Scientific Pierce G2 Fast Blotter) at

25 V, 2 amps for 10 min. A post‐transfer check was carried out by staining the membrane with

1X Amido Black 10B stain for approximately 5 min. The membrane was blocked in 5 % low‐fat

milk in TBS buffer (20 mM Tris pH7.5, 0.5M NaCl,) either for at least 1 h on a shaker at 100 rpm

after which it was rinsed twice with TBS buffer for 5 min each, followed by a third rinse in TTBS

buffer (TBS buffer and 0.1 % Tween 20) for 5 min. The membrane was incubated (by shaking)

with the primary antibody (anti‐Vip3Ba from mouse) in a 1 in 500 dilution in 6 mL of TTBS

buffer and 6 mg of milk powder for 1 h. The membrane was rinsed three times with TTBS for 5

min each and incubated with the secondary antibody (anti‐mouse alkaline phosphatase

conjugate, Bio‐Rad, Gladesville, NSW 2111 Australia) in a 1 in 2000 dilution in 6 mL and 6 mg of

milk powder. The membrane was incubated for an hour and rinsed twice with TTBS for 5 min

each, and a final rinse with TBS for 5 min. The membrane was incubated with the substrate

(BCIP/NBT tablet (Sigma Cat. #B5655) dissolved in 12 mL of distilled water) on a shaking

platform (230 rpm), for 20 to 30 min for color development. Once the immunoreactive protein

bands were visible, the membrane was thoroughly rinsed in water to stop the reaction.

40

CHAPTER THREE

Toxicity of Vip3 proteins to the legume pod borer Maruca vitrata (Lepidoptera)

3.1 Introduction

The Maruca pod borer (MPB), Maruca vitrata (Lepidoptera), also known as legume pod borer,

is a pest that causes large losses in cowpea yields (Jackai, 1995; Singh & van Emden, 1979). MPB

occurs throughout tropical and sub‐tropical areas, but is most devastating in sub‐Saharan Africa

(Margam et al., 2011). The larvae feed on flower parts, green pods and seeds of cowpea and

several other leguminous crops (Singh & van Emden, 1979; Ba et al., 2009). Although MPB can

be controlled using insecticides (Ajeigbe & Singh, 2006; Kawuki et al., 2005), farmers in

developing countries do not spray as they can rarely afford the chemicals (Harwood, 1979).

Furthermore, inappropriate or excessive use of insecticides can be detrimental to human health

and the environment (Pimentel et al., 1992; Garry, 2004). Thus, alternative control strategies

for MPB are needed. One such strategy is based on the deployment of insect‐resistant

transgenic crops producing highly specific insecticidal proteins (cry toxins) from the soil

bacterium Bacillus thuringiensis (Bt), so called Bt crops. Genes conferring resistance against

other pests have been isolated from Bt and transformed into crops, such as Bt corn and Bt

cotton carrying cry1Ab and cry1Ac genes, respectively. It has been shown that these Bt crops

are effectively protected against targeted insect pests, resulting in increased yields and reduced

insecticide applications (Schuler et al., 1998; Bravo et al., 2005; Qaim, 2009). Although Bt crops

have been very successful, studies have shown that insects can develop resistance to specific Bt

toxins, particularly in those carrying a single cry gene (Wilson et al., 1992; Shelton et al., 2002).

Stacking two resistance genes with differing mechanisms of action can substantially reduce the

likelihood of development of resistance (Roush, 1998). The vegetative insecticidal proteins

(Vips) of Bt, which are synthesized during the vegetative growth phase of the bacterium

(Estruch et al., 1996), bear no amino acid sequence similarity to Cry proteins and have a

different mechanism of action (Lee et al., 2003, Gouffon et al., 2011). Hence, they have been

used to complement Cry proteins for insect resistance management (IRM) in cotton

(Whitehouse et al., 2007).

41

Vip toxins with insecticidal activity are grouped according to their specificity for target insects.

The Vips have been shown to be active against a range of insect pests. The Vip1 and Vip2 toxins

are binary proteins active against coleopteran (Warren, 1997) and homopteran pests (Yu et al.,

2011a), respectively. Vip3 toxins are active against lepidopteran insects (Estruch et al., 1996;

Lee et al. 2003; Liu et al., 2007) making them potential candidates for MPB management. Vips

are activated by insect gut proteases upon ingestion and subsequently interact with specific

receptors found in the midgut epithelial tissue. This leads to formation of pores, rupture of the

epithelial cells and eventually cell death (Lee et al., 2003).

There are more than 70 vip genes encoding different Vip3 proteins listed in the Crickmore

database (http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/). These genes are

classified into 3 families, namely 3A, 3B and 3C, and further grouped into nine sub‐families in

3A (Vip3Aa to Ai), two 3B sub‐families (Vip3Ba and Bb) and one 3C family member (Vip3Ca)

based on nucleotide sequence similarities (Crickmore et al., 2014).

The work contained in this chapter was aimed at the identification of a vip3 gene product with

activity against MPB and potential to improve IRM in cowpea. Representatives of five vip3 gene

groups were isolated, cloned and expressed in Escherichia coli to produce Vip3 protein for

insect bioassays. A candidate gene encoding a Vip3Ba toxin was selected as a potential source

for MPB management in cowpea.

3.2 Materials and Methods

3.2.1 Bacterial strains

A collection of 267 putative Bt strains, previously collected throughout Australia (Beard et al.,

2001, 2008), were used in this study. The strains were cultured on LB medium as previously

described (section 2.1.1). E. coli strain BL21 (DE3) was used for protein expression as described

in section 2.4. All bacterial strains were stored in 30 % glycerol at ‐80 ºC.

3.2.2 PCR

A variety of different primer pairs were used in this study (Table 3.1) all of which were

synthesised by Sigma‐Aldrich (St. Louis, USA). The primers were designed to (i) screen the

42

Table 3.1: Primers used in this study: The underlined nucleotides in primer pairs of PCR set C define 6 His codons of the vector sequence.

PCR set Primer name

Sequence (5´ to 3´) Product length (bp)

PI‐PLC

F: CGCTATCAAATGGACCATGGR: GGACTATTCCATGCTGTACC 569

Vip3A group F:GATTATTTTAATGGCATTTATGGATTTGCCACTGG R:CGGCATGTTGTTAAAGTAAGAAAAGCTTTTGCTTG 827

A Vip3B group F:TGGCATTTATGGATTTGCCACTGGTATCAAAGAC R:CATAAAAGTGTAGACATTACCAATTTCACTTCCC

766

Vip3Ca F:TCTAAGAGCAAAACAAGGAATTTTTAAC R:GTTCAGATTCTAAAATAACTTTTGCATAATTG

500

Vip3Aa35

F:CCGCCAAGTGGTTTTATTAAA R:ATCGGAAAACTTGACAATAGGG

746

B

Vip3Af1 F:CCTTATCGGAGGTTATTTATGGTGATAC R:TAGTTGAAGACATAAGGTTGCTTCG 1090

Vip3Ag

F:CTTTTGAAAGATGAAAAAAATGGTGR:TTTTTCAAATAATACTTCACGGGG

390

Vip3Ba1

F:GGATGCCATGGTGGGATTTAGR:CTAATTATACCTTCGCCGTATAAGCG

587

V3Aa35_cds

F:CATCATCACCACCATCACAACAAGAATAATACTAAATTAAGCACAAG R:GTGGCGGCCGCTCTATTACTTAATAGAGACATCGGAAAACTTG

2,364

V3Af_cds

F:CATCATCACCACCATCACAATATGAATAATACTAAATTAAACGCAAG R:GTGGCGGCCGCTCTATTATTTAATAGAAACGTTTTCAAATGATATATG

2,367

C V3Ag_cds

F:CATCATCACCACCATCACAACAAGAATAATACTAAATTAAGCGCAAG R:GTGGCGGCCGCTCTATTACTTAATTGAAAAATCTCGGAAATTTATAGC

2,358

V3Ca2_cds

F:CATCATCACCACCATCACAACATGAATAATACTAAATTAAACGCAAGGG R:GTGGCGGCCGCTCTATTATTCAATCTTTTCCTTAATAGAAATATCACGAA

2,406

V3Ba1_cds

F:CATCATCACCACCATCACAACATGAATAATACTAAATTAAACGCAAGGGCC R:GTGGCGGCCGCTCTATTATTCTTTAACTATAGATACATCCGAAAAAATAACCCC

2,406

43

microbial collection for the presence of a phosphatidylinositol‐specific phospholipase C (PI‐PLC)

gene unique to Bacillus thuringiensis (PI‐PLC F/R; Lechner et al., 1989), and (ii) screen the Bt

strains for the presence of vip3 genes to enable the amplification of selected ORFs for

subsequent protein expression. Primer sequences for the latter purpose were designed using

DNA sequences of vip3 genes obtained from the National Centre for Biotechnology Information

(NCBI) database (www.ncbi.nlm.nih.gov).

To identify vip3 family‐specific genes, sequences were subjected to a multiple sequence

alignment in FASTA format using the Clustal W version 2.0 program hosted at the European

Bioinformatics Institute (EBI) (Larkin et al., 2007) with default settings to identify conserved

regions for primer design. To design primers for specific vip3 gene fragments and to amplify the

coding regions, the target gene sequences were retrieved from the NCBI database.

Genomic bacterial DNA was isolated from cultures using a Bacterial Genomic DNA isolation kit

as in section 2.2. The DNA was used as a template in PCR to test for the presence of the PI‐PLC

and vip3 genes using specific primers designed above. For each primer set, the following were

added to each tube: Distilled water (5.5 μl); forward primer (0.5 μl); reverse primer (0.5 μl) and;

mastermix (7.5 μl). The total volume (14 μl) was added into the well of a PCR plate followed by

1 μl (~100 ng) of bacterial DNA for each Bt strain to give a total of 15 μl in each well for PCR. A

negative control (distilled water) and a known positive control from a known Bt strain (serovar

kurstaki) were included. For the vip3A genes, bacterial DNA harbouring a vip3Aa gene, kindly

provided by Dan Llewellyn at CSIRO Plant Industry was used as a positive control while for the

vip3B gene, plasmid DNA from E. coli carrying a characterized vip3Bb2 gene cloned from Bt

strain C81 (Beard et al., 2008), was used as a positive control. The samples were subjected to

the following PCR conditions: 95 0C for 15 min; 30 cycles of 95 0C for 30 s, an annealing

temperature of 60 0C for 30 s and 72 0C for 2 min; an extension of 72 0C for 10 min and; 25 0C

for 1 min. PCR products were analyzed by electrophoresis through 1.8 % agarose gels in TBE

buffer. The amplicons were visualized under a transilluminator and gel documentation system.

3.2.3 Cloning of vip3 and cry2Aa genes in E. coli

The full coding regions of vip3 genes, vip3Aa35, vip3Ag and vip3Ba1, were isolated from Bt DNA

by PCR amplification while the full ORFs of vip3Af1 and vip3Ca2 were synthesized by GeneArt

44

synthesis, Life Technologies Australia Pty Ltd (Mulgrave VIC 3170 Australia), based on accession

numbers AJ872070 and JF916462, respectively. These were inserted into a pMk‐RQ (kanR)

vector flanked by a kanamycin resistance gene and an origin of replication site, Col E1 (Life

Technologies, Australia), and were subsequently amplified and isolated from the plasmids.

The PCR products of all five genes were purified (section 2.3) and transferred into pETite

vectors (section 2.4). The cells were cultured on LB plates containing 30 mg/L kanamycin and

incubated overnight at 37 0C. Single colonies were selected and grown overnight in LB liquid

medium containing 30 mg/L kanamycin for selection at 37 ºC on a shaker at 250 rpm. To

confirm the presence of the insert in the expression vector, plasmid DNA was extracted and

either used as a template for PCR analysis or for restriction mapping and finally, DNA

sequencing. The plasmid DNA of the positive clones was used to transform E. coli strain BL21

(DE3) for protein expression (section 2.4). The cry2Aa gene was previously cloned at CSIRO

Plant Industry lab by Dan Pikler and subsequently used in transforming E. coli strain BL21 to

express Cry2Aa protein.

3.2.4 Protein expression and analysis

E. coli transformed with the vip3 gene of interest or cry2Aa was plated on selection medium

and grown overnight at 37 ºC. Protein was extracted from bacterial cultures as described by

Studier (2005) (section 2.9). The protein concentrations of the supernatant and pellet fractions

were determined according to Bradford (1976) and 30 µg of protein was loaded on 10 % Bis‐Tris

precast NuPage gels (Invitrogen) for SDS‐PAGE analysis (section 2.16). Untransformed E. coli

(BL21) cells were used as a negative control and E. coli transformed with cry2Aa was used as a

positive control (Srinivasan 2008). Mass spectrometry of selected protein bands was used to

confirm that the specified protein bands were Vips (section 2.10).

3.2.5 Insect bioassays

A replicated series of bioassays using MPB larvae was carried out with proteins encoded by the

gene representing each of the five groups. Earlier experiments with Cry1A or 2A toxins at Plant

Industry indicated that levels in the range of 1‐20 µg/g diet of Cry1A or 2A were toxic to MPB

larvae (data not shown). Since there was no data available for Vip toxins, a wider range of toxin

concentration was used initially. Five concentrations of each Vip3 protein (unpurified) were

45

therefore used in preparing artificial MPB diets (Jackai and Raulston, 1988): 0, 3, 10, 30 and 100

µg of Vip3 protein per gram of diet. Diets with Cry2Aa protein (at 0, 3, 10 and 30 µg/g diet) and

without Bt protein represented positive and negative controls in the bioassays, respectively. To

calculate the amount of Vip or Cry protein to be incorporated in the artificial diet, the amount

of Vip or Cry toxin present was first estimated based on the Vip protein bands as seen on the

coomassie stained gels, and then calculated accordingly. For instance, if the Vip3Ba or Cry2Aa

protein band represented 10 % of the total bacterial protein in the gel, then this figure was

used calculate the quantity of freeze‐dried total protein to be added to the artificial diet, to

obtain concentrations of 3, 10, 30, or 100 µg per g of diet. The bacterial proteins were dissolved

in 50 mM sodium carbonate (pH 11.2) and incorporated into 50 ml of diet. For Vip3Ba protein,

both the soluble and insoluble fractions were used. The diet was dispensed into Petri plates and

allowed to solidify at room temperature. Prior to commencing the assays, the diet was further

divided into 16 cubes of ~2.5 g each, and placed in 16 individual wells of a plastic feeding tray.

For each protein concentration, one first‐instar larva was introduced into each well containing

one cube of diet (i.e., N = 16 larvae per concentration) and sealed with a perforated film using a

hot sealer. These were incubated in a climate chamber at 26 oC and 50 % RH. After 10 days,

when larvae in the negative control experiments reached their fifth instar, mortality was scored

and the weight of all surviving larvae was recorded using a digital microbalance. There were

three separate bioassays conducted for each protein. Mortality and average weight was

calculated from the pooled data of all bioassays.

3.3 Results

3.3.1 Selection of candidate vip3 genes

A bioinformatics approach was initially used to identify a diverse range of Vip3 proteins as

candidates for subsequent expression and testing for insecticidal activity against MPB. This was

done by comparing the amino acid sequences of vip3 genes from all three families (3A, 3B and

3C) in the Bt nomenclature database

(http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/vip.html). The majority (93/102) of

the vip3 genes in the database belong to the 3A family with 61 of the 3A sequences belonging

to the “Aa” subfamily. Analysis of these 61 sequences using a pair‐wise sequence alignment

tool (EMBOSS Water) (www.ebi.ac.uk) revealed they were more than 95 % identical. Based on

this analysis, protein Vip3Aa35 was arbitrarily selected as the point of reference and was

46

designated to represent Group 1. Alignment of the amino acid sequence of Vip3Aa35 with all

other Vip3 proteins was subsequently carried out revealing that the other Vip3 proteins

clustered into four groups (designated 2‐5) with between 90 and 60 %, identity with Vip3Aa35,

respectively (Table 3.2). Based on these sequence comparisons, the proteins Vip3Aa35 (Group

1), Vip3Af1 (Group 2), Vip3Ag (Group 3), Vip3Ca2 (Group 4) and Vip3Ba1 (Group 5) were

selected as the candidate proteins for further analysis.

Table 3.2: Grouping of Vip3 proteins on the basis of percentage amino acid sequence identity

with reference protein Vip3Aa35

vip3 protein

Group

Aa35

Aa35 1 100 Af1 2 89.2Ag 3 82.2 Ca2 4 70.1Ba1 5 61.8

3.3.2 Confirmation of Bt strains

The Bt strains provided for use in this study had been collected throughout Australia from

previous work. To confirm their identity as Bacillus thuringiensis, DNA was extracted from the

bacterial colonies and used as a template in PCR with Bt‐specific primer pair, PI‐PLC F/R,

designed to amplify a 569 bp fragment of the PI‐PLC gene. Of the 267 strains tested, a product

of the expected size was amplified from 224 strains confirming that they were indeed Bacillus

thuringiensis (Fig. 3.1).

47

Fig. 3.1: Identification of putative Bt strains by PCR using PI‐PLC primers. Lane M: DNA

Molecular marker; Lanes 1 to 10: DNA template from ten putative Bt strains; N: water (negative

control); P: DNA from Bt strain kurstaki (positive control).

48

3.3.3 Identification of Bt strains containing candidate vip3 genes

The strategy to identify Bt strains containing the candidate vip3 genes initially involved the

identification of Bt strains containing vip3A, vip3B and vip3C sequences. The sequences of vip3

genes were aligned and conserved regions were identified from which Vip3A, 3B and 3C family‐

specific primers were designed (Table 3.1; PCR Set A). Since the sequence of only one member

of the Vip3C family, gene vip3Ca2, was available at the commencement of this study, primers

were designed to specifically amplify this sequence (Table 3.1).

When the 224 Bt strains were tested for the presence of vip3A genes using the vip3A family‐

specific primer set, an amplicon of the expected size (827 bp) was present in 78 strains (35 %)

(Fig. 3.2). Using a random selection of 165 Bt strains, amplicons of the expected size (766 bp)

were obtained from eight strains using primers to detect the presence of vip3B genes (Fig. 3.3).

No amplicons were obtained using the Vip3Ca primer set from 224 strains tested.

Following the identification of Bt strains containing vip3A and vip3B genes, primers were

designed which would specifically amplify fragments of the four candidate vip3 genes, namely

vip3Aa35, vip3Af1, vip3Ag and vip3Ba (Table 3.1; PCR Set B). When the Bt strains identified

above were tested by PCR using these primers, PCR products of the expected sizes for

vip3Aa35, vip3Af1, vip3Ag and vip3Ba1 were amplified from a number of strains (Table 3.3).

The DNA of these strains was subsequently used as a template to amplify the full coding

sequences (CDS) of the four genes for subsequent cloning into a protein expression vector.

Primer pairs were designed to amplify the entire open reading frames (ORFs) of each of the

four genes. In order for the primers in this set to be compatible with the expression vector, a

His tag sequence (18 nucleotides long) was added to each ORF after the start codon for protein

purification (Table 3.1; Primer Set C). Amplicons of the expected size (~2.3 to 2.4 kb) were

obtained using primers designed to amplify the CDS of genes vip3Aa35, vip3Ag and vip3Ba but

no amplification was observed using the V3Af_cds primer pair. Therefore, the coding sequence

of vip3Af was synthesized as was the coding sequence of vip3Ca2 since no Bt strains containing

vip3C gene were identified.

49

Fig. 3.2: Identification of Bt strains carrying vip3A genes by PCR. Lane M: DNA Molecular

marker; Lanes 1 to 10: DNA template from 10 Bt strains; N: water (negative control); P:

bacterial DNA harbouring a vip3Aa gene (positive control).

Fig. 3.3: Identification of Bt strains carrying vip3B genes by PCR. Lane M: DNA Molecular

marker; Lanes 108 to 117: DNA template from 10 Bt strains; N: water (negative) control and P:

plasmid DNA containing the vip3Bb2 gene (positive control).

50

3.3.4 Expression of vip3 genes in E. coli

The coding regions of the five genes were cloned in pETite vectors for subsequent protein

expression. To confirm the presence of the genes in the expression vectors, plasmid DNA was

extracted and either used as a template for PCR analysis or for restriction mapping and finally,

DNA sequencing. Sequence analysis (based on global alignment) of the five cloned candidate

vip3 gene sequences revealed they were identical (taking into account the His tag sequences) to

their respective counterparts in the Crickmore database (Table 3.4; Appendix I).

The plasmids containing the five vip3 genes (vip3Aa35, vip3Af1, vip3Ag, vip3Ba1 and vip3Ca2)

were subsequently transformed into E. coli strain BL21 for protein expression. The cry2Aa had

been previously cloned and was also transformed into E. coli strain BL21 for Cry2Aa expression.

Soluble and insoluble protein fractions were extracted from each of the five vip3‐transformed

cultures and the cry2Aa‐transformed culture and these were analysed for the presence of the

Vip and Cry2Aa proteins using SDS‐PAGE and MS. Equivalent fractions from untransformed E.

coli were also extracted as controls. When the extracts were analysed by SDS‐PAGE, no bands

of the expected size (~90 kDa for Vip3 proteins) were seen in the either the soluble or insoluble

fractions derived from any vip3‐transformed cells. However, a major band of about 75 kDa, was

present in extracts from E. coli transformed with vip3Aa35, vip3Af1, vip3Ag, vip3Ba1 and

vip3Ca2, with some lower MW bands also observed in some extracts These bands, which were

absent from the untransformed control extracts, were mostly in the insoluble fraction for

Vip3Aa35, Vip3Af1, Vip3Ag and Vip3Ca2 (Fig 3.4 A‐D), although the 75 kDa band was present in

both the soluble and insoluble fractions from E. coli transformed with vip3Ba1 (Fig. 3.4 E). High

molecular weight bands (greater than 250 kDa) were also observed in the soluble fractions

extracted from E. coli transformed with vip3Aa35, vip3Af1, vip3Ag and vip3Ca2 (Fig. 3.4, lanes 2

in A‐D). When extracts from Cry2Aa‐transformed and non‐transformed E. coli were analysed, a

major band of the size expected for the Cry2Aa protein (~65 kDa) was present in the insoluble

fraction from the Cry2Aa‐transformed cells (Fig. 3.4 F). In addition to the ~65 kDa band, several

additional lower MW bands were also present in the insoluble fraction.

51

Table 3.3: Examples of Bt strains harbouring target vip3 genes

Group vip3 gene Strain(s)

1 vip3Aa35 45, 47, 49 and 117

2 vip3Af1 99, 100, 107, 113, 121, 122 and 130

3 vip3Ag 100

4 vip3Ca2 None identified

5 vip3Ba1 7 and 46

Table 3.4: DNA sequence analysis of target vip3 genes from groups 1 to 5 (with the His tag sequences deleted)

Group Target gene Reference gene in database

% Identity to reference gene

No. of amino acids encoded

1 vip3Aa35 GU733921.1 100 789

2 vip3Af1 AJ872070.1 100 788

3 vip3Ag FJ556803.2 100 787

4 vip3Ca2 JF916462.1 100 803

5 vip3Ba1 AY823271.1 100 803

52

To determine whether the protein bands migrating at ~75 kDa in the insoluble fractions derived

from the five vip3‐transformed cultures were the target Vips, the bands were excised and

analysed by MS/MS. Subsequent interpretation of the MS/MS data using the Mascot database

revealed that the highest Mascot scores were with Vip sequences. The protein coverage of

Vip3A proteins ranged between 55 and 70%, Vip3Ca was 70 % and Vip3Ba protein coverage was

76 % (Table 3.5). Based on this data, it was considered highly likely that the 75 kDa bands were

indeed the target Vips. When one of the high molecular weight bands (>250 kDa) from the

soluble fraction from Group 2 was subjected to mass spectrometry analysis it was found to be

composed of Vip3Af1 sequences with a Mascot score of 1347 and 39% coverage (Table 3.5). On

close examination of the vip3 proteins, the difference lies at the C‐terminal end of the proteins

with two highly conserved proteolytic cleavage sites repeated four times

(DCCEEDDCCEEDDCCEEDDCCEED) in Vip3Ba1. This generates a sequence rich in negatively

charged cysteines (Fig. 3.5).

3.3.5 Insect bioassays

To test the insecticidal activity of the bacterially‐expressed Vip3Aa35, Vip3Af1, Vip3Ag,

Vip3Ba1, Vip3Ca2 and Cry2Aa proteins, a replicated series of bioassays using MPB larvae was

carried out. In preliminary bioassays, the incorporation of unpurified protein extracts from non‐

transformed E. coli incorporated into an artificial MPB diet was found to have no effect on

mortality of MPB larvae and the insects grew normally to pupation (data not shown). As such,

although a His tag was included to assist in purification of the Vip3 proteins, purification was

not considered necessary and unpurified Vip proteins were incorporated into the artificial diets

in all bioassays. Five concentrations (0, 3, 10, 30 and 100 µg) of each freeze‐dried, unpurified

Vip3 protein and four concentrations (0, 3, 10 and 30 µg) of Cry2Aa protein were incorporated

per gram into an artificial MPB diet (see 3.2.6) and the effect on MPB larvae was investigated

over a 10‐day period.

The average weight of negative control larvae fed on diets without Vips or Cry2Aa protein

consistently ranged between 70 and 80 mg after 10 days growth. Further, all larvae fed on the

control diet developed into the fifth‐instar stage of their life cycle. For larvae fed on Cry2Aa

diets, the average mortality at 3, 10 and 30 µg per g was 13, 10.6 and 36 %, respectively. On

53

average, the growth of surviving larvae on this diet at 3, 10 and 30 µg per g diet was reduced by

91, 98 and 99%, respectively (Fig. 3.6) and they only developed to their second instar (data not

shown). In all diets containing Vip3Aa35, Vip3Af1, Vip3Ag and Vip3Ca2, apart from one bioassay

with Vip3Af1, there was little or no effect observed on the growth or development of MPB

larvae and all larvae developed to fifth instars (Fig. 3.7).

54

Fig. 3.4: SDS‐PAGE analysis of Vip3 and Cry2Aa proteins expressed in E. coli. Panels A‐F are stained gels from studies involving the expression of Vip3Aa35, Vip3Af1, Vip3Ag, Vip3Ca2, Vip3Ba1 and Cry2Aa, respectively. Lane M: Protein molecular weight marker (kDa). Lane 1: Soluble protein fraction of untransformed E. coli. Lane 2: Soluble protein fraction of E. coli transformed with its respective vip or cry gene. Lane 3: Insoluble protein fraction of untransformed E. coli. Lane 4: Insoluble protein fraction of E. coli transformed with its respective vip or cry gene. * Denotes bands which are only present in the protein extracts derived from transformed E. coli.

55

Table 3.5: MS/MS identification of the putative Vip3 proteins expressed in E. coli

Group Identification Mascot score Protein coverage

1 Vip3Aa 10118 55% 2 Vip3Af1 5324 68% 3 Vip3Ag 9379 70% 4 Vip3Ca 4634 71% 5 Vip3Ba 8861 76%

In contrast, the growth of larvae fed on a diet containing Vip3Ba1 was reduced by 83.8, 84.2,

71.8 and 89.2 % at concentrations of 3, 10, 30 and 100 µg per g of diet, respectively (Fig 3.6).

The mortality at 3 µg per g diet was 13 %. Most surviving larvae on this diet only developed to

their third instar stage (Fig. 3.6).

56

Results for Vip3Ba

.........10........20........30........40........50........60........70........ AA MNMNNTKLNARALPSFIDYFNGIYGFATGIKDIMNMIFKTDTGGGNLTLDEILKNQDLLNQISDKLDGINGDLGDLIAQ DB_state DB_conf 80........90........100.......110.......120.......130.......140.......150...... AA GNLNSELTKELLKIANEQNLMLNNVNAQLNSINATLNTYLPKITSMLNEVMKQNYVLSLQIEFLSKQLQEISDKLDIIN DB_state DB_conf .160.......170.......180.......190.......200.......210.......220.......230..... AA LNVLINSTLTEITPAYQRIKYVNDKFDELTSTVEKNPKINQDNFTEDVIDNLTDLTELARSVTRNDMDSFEFYIKTFHD DB_state DB_conf ..240.......250.......260.......270.......280.......290.......300.......310.... AA VMIGNNLFSRSALKTASELIAKENIHTRGSEIGNVYTFMIVLTSLQAKAFLTLTTCRKLLGLADIDYTQIMNENLDREK DB_state 0 DB_conf 8 ...320.......330.......340.......350.......360.......370.......380.......390... AA EEFRLNILPTLSNDFSNPNYTETLGSDLVDPIVTLEAEPGYALIGFEILNDPLPVLKVYQAKLKPNYQVDKESIMENIY DB_state DB_conf ....400.......410.......420.......430.......440.......450.......460.......470.. AA GNIHKLLCPKQREQKYYIKDMTFPEGYVITKIVFEKKLNLLGYEVTANLYDPFTGSIDLNKTILESWKEDCCEEDCCEE DB_state 0 00 00 DB_conf 8 99 98 .....480.......490.......500.......510.......520.......530.......540.......550. AA DCCEEDCCEELYKIIEADTNGVYMPLGVISETFLTPIYSFKLIIDEKTKKISLAGKSYLRESLLATDLVNKETNLIPSP DB_state 00 00 DB_conf 88 88 ......560.......570.......580.......590.......600.......610.......620.......630 AA NGFISSIVQNWHITSDNIEPWKANNKNAYVDKTDAMVGFSSLYTHKDGEFLQFIGAKLKAKTEYIIQYTVKGNPEVYLK DB_state DB_conf .......640.......650.......660.......670.......680.......690.......700.......710 AA NNKDICYEDKTNNFDTFQTITKKFNSGVDPSEIYLVFKNQIGYEAWGNNFIILEIKSLETLPQILKPENWIPLGNAEIK DB_state 0 DB_conf 8 ........720.......730.......740.......750.......760.......770.......780.......790AA EDGKIEISGNGSMNQYIQLEQNSKYHLRFSVKGKGRVTMQAQTSHINVPATNEEVSIMIETTRLYGEGIISLLNDEVEN DB_state DB_conf .........800. AA SGVIFSDVSIVKE DB_state DB_conf

Key: AA ‐ amino acid sequence DB state ‐ predicted disulfide bonding state (1+disulfide bonded, 0=not disulfide bonded) DB conf ‐ confidence of disulfide state prediction (0=low to 9=high)

Fig. 3.5: The full Vip3Ba amino acid sequence. The highly conserved cysteine regions are

highlighted in yellow, while the predicted disulphide bonding is highlighted in green (Ceroni et

al., 2006).

57

Fig. 3.6: Maruca larvae after 10 days feeding on artificial diet with and without Bt toxin. (A): Larvae on a diet containing no Bt toxin (0 µg toxin per g diet), (B): Larvae on a diet containing 3 µg of Vip3Ba toxin per g of diet.

58

B

C

Fig. 3.7: Effect of Vip and Cry2Aa proteins on the average weight of surviving MPB larvae after 10 days on artificial diets containing different levels of toxins. Assays were repeated three times. (A): Group 1 (Vip3Aa35) and Cry2Aa proteins, (B): Group 2 (Vip3Af1) and Cry2Aa proteins, (C): Group 3 (Vip3Ag) and Cry2Aa proteins, (D): Group 4 (Vip3Ca2) and Cry2Aa proteins and (E): Group 5 (Vip3Ba1) and Cry2Aa proteins.

E

A

D

59

3.4 Discussion The aim of this work was to identify a vip3 gene in a collection of Australian Bt strains that

encoded a toxin active against the Maruca pod borer (Lepidoptera). These genes were

targeted as they are known to have insecticidal activity against certain lepidopteran pests

(Estruch et al., 1996; Beard et al., 2008). To increase the probability of identifying a vip3 gene

product with activity against MPB, a diverse range of Vip3‐encoded proteins were targeted.

The selection of the target proteins was based on sequence analysis of Vip3 amino acid

sequences in the Bt database. Since approximately 60 % of sequences in the Bt database

encoded Vip3Aa proteins with 95 % amino acid identity, Vip3Aa35 was arbitrarily chosen as a

target. Using Vip3Aa35 as a reference, four additional Vip3 target proteins, namely Vip3Af1,

Vip3Ag, Vip3Ca2 and Vip3Ba1, were selected whose amino acid sequences showed between

90‐60 % identity to vip3Aa35.

The screening of the Australian Bt collection for genes encoding the target Vip3 proteins

revealed that it was dominated by vip3A genes (35 %), with a small number of vip3B genes (5

%) and no vip3C genes. The percentage of Bt strains containing vip3A genes in this study was in

general agreement to that reported in several other studies where the incidence of vip3A

genes ranged from 18 to 87 % (Doss et al., 2002; Bhalla et al., 2005; Liu et al., 2007; Beard et

al., 2008; Hernandez‐Rodriguez et al., 2009; Yu et al., 2011b). The abundance of vip3 genes

observed in the current study was also generally in accordance with the Bt database

(Crickmore et al., 2014) where the majority of vip3 genes belong to the A family, with lesser

members in the B family and very few members in family C. One of the Bt strains that was

found to contain a vip3 gene in the present study was previously found to contain cry1 and

cry2 genes by Beard et al. (2001), indicating that both vip and cry genes can exist in one Bt

strain. A similar situation was also reported by Seifinejad et al. (2008) and Hernandez‐

Rodriguez et al. (2009). In the current study, one Bt strain (strain 100) was also found to harbor

two vip3 genes belonging to family A, namely vip3Af1 and vip3Ag. The presence of two or

more vip3A genes in a single Bt strain has also been reported by others (Hernandez‐Rodriguez

et al., 2009; Li et al., 2012; Sauka et al., 2013), indicating the random distribution of various

vip3A‐type genes among B. thuringiensis (Yu et al., 2011b). This observation shows the

60

diversity of vip3‐type genes represented in the Bt pool and indicates that these genes are

abundant in nature.

Using PCR with primers designed to the target gene sequences, vip3Aa35, vip3Af1, vip3Ag and

vip3Ba1 were identified in the Bt collection whereas no Bt isolate containing vip3Ca2 was

identified. Primers were subsequently designed to amplify the full coding sequence of

vip3Aa35, vip3Af1, vip3Ag and vip3Ba1 and the CDS of three of the five target vip genes

(vip3Aa35, vip3Ag and vip3Ba1) was amplified for subsequent cloning into a protein expression

vector. It was necessary to chemically synthesise the coding sequences of vip3Af1 and vip3Ca2

because they were either unable to be cloned (vip3Af1) or were not found in the Bt collection

(vip3Ca2). Following cloning, the five vip3 genes were expressed in E. coli. Based on the amino

acid sequence of the encoded proteins, the expected sizes of the proteins were around 90 kDa.

This was consistent with the sizes of Vip3 proteins reported by others (Yu et al., 2011b; Estruch

et al., 1996; Hernandez et al., 2013; Palma et al., 2012, Beard et al., 2008).

SDS‐PAGE analysis of the expressed target proteins revealed the presence of a major band of

approximately 75 kDa which was mainly present in the insoluble fraction for Vip3Aa35,

Vip3Af1, Vip3Ag and Vip3Ca2 but in both soluble and insoluble fractions for Vip3Ba1. High

molecular weight bands and some lower bands were also found in some of the Vip3 protein

extracts. The high molecular bands are possibly aggregates, a phenomenon common reported

during the overexpression of recombinant protein in E. coli (Lebendiker and Danieli, 2014). This

can sometimes be overcome by the use of fusion proteins and buffers or solvents to obtain

stable proteins and proper protein folding (Sorensen and Mortensen, 2005; Bondos and

Bicknell, 2003). It is possible that the low molecular weight bands reflect degradation products

of the recombinant protein. Andberg et al. (2007) reported that proteins with purification tags

were easily cleaved in the presence of a buffer and metal salts. This is likely with the Vip

proteins in this work as they contained His tags and were combined with a buffer that

contained Tris‐HCl and sodium salts.

Although the sizes of the five expressed Vips were predicted to be approximately 90 kDa, the

major band observed in the protein extracts in this study was approximately 75 kDa. This was

not entirely unexpected as it is known that the migration of proteins electrophoresed through

61

SDS‐PAGE is influenced by many numerous factors and does not always provide an accurate

reflection of their molecular weight (Jong et al., 1978). For example, one possible reason why

the protein extracts resolved at 75 kDa is the fact that an increased pH in the Tris‐glycine

buffer system accelerates the rate of protein migration on SDS‐PAGE (Schagger and von Jagow,

1987; Liu and Chang, 2010). To provide definitive evidence that the 75 kDa bands were indeed

the target vips, the protein bands were excised and analysed by MS/MS. The analysis revealed

that the bands were indeed the target proteins and, based on this evidence, the proteins were

used for insect bioassays.

A protein solubility prediction (Wilkinson and Harrison 1991) carried out for the Vip3 protein

sequences revealed that the chances of full solubility of most of these proteins when over‐

expressed in E. coli were low. For example, the solubility scores for Vip3Aa35, Vip3Af, Vip3Ag

and Vip3Ca2 ranged from 37 to 48 %. In contrast, Vip3Ba was predicted to have a 59.1 %

chance of being soluble. Further, when Rang et al. (2005) and Beard et al. (2008) over‐

expressed Vip3Ba1 and Vip3Bb2 proteins in E. coli, they were shown to be present in the

soluble and insoluble fractions, respectively. In another study, Palma et al. (2013) reported that

Vip3Aa45 and Vip3Ag4 were present in both the soluble and insoluble fractions, while

Escudero et al. (2014) and Hernandez‐Martinez et al. (2013) extracted soluble forms of Vip3Aa,

Vip3Ab, Vip3Ad, Vip3Ae and Vip3Af. Based on this information, both the soluble and insoluble

fractions of the protein extract derived from the five targeted vips were analysed by SDS‐PAGE.

To assess the effect of the Vips on larvae of MPB, bioassays were conducted where various

concentrations of the Vips were incorporated into an artificial MPB diet and the effect on MPB

larvae was investigated over a 10‐day period. The results from these bioassays showed that Vip

proteins from the 3A and 3C families (Vip3Aa35, Vip3Af1, Vip3Ag, Vip3Ca2) had no effect on

caterpillar growth, with the MPB larvae fed on diets containing these proteins developing into

their fifth and final instar stages. Palma et al. (2012) also reported that Vip3C toxin had little or

no effect on seven other lepidopteran pests. However, several studies have reported growth‐

inhibiting effects of five different Vip3A proteins on selected lepidopteran pests (Estruch et al.,

1996; Chakroun et al., 2012; Hernandez‐Martinez et al., 2013). In the current study, Vip3Ba

toxin was shown to inhibit the growth of MPB larvae by 90 % when exposed to 3 ppm of the

toxin (soluble and insoluble fractions). These results are consistent with, and extend the data

62

of Rang et al. (2005), who demonstrated that Vip3Ba impaired the larval growth of two other

lepidopteran species (Ostrinia nubilalis and P. xylostella).

The results presented in this chapter demonstrate for the first time that the growth of MPB

larvae is severely inhibited by low levels of Vip3Ba protein. Thus, it is proposed that the vip3Ba

gene could complement cry genes in the development of Bt cowpeas resistant to MPB, thus

contributing to an effective strategy for insect resistance management. This combination

would considerably reduce the likelihood of development of resistance in MPB, help to

increase yield and income, and reduce the dependency on pesticides.

63

CHAPTER FOUR

Transgenic cowpeas (Vigna unguiculata L. Walp) expressing Bacillus

thuringiensis (Bt) Vip3Ba protein to protect against the Maruca pod borer

(Maruca vitrata)

4.1 Introduction

Cowpea is an important grain legume in the developing world. It is cultivated in the tropics

with West Africa producing close to 5 million tonnes of dry grain (FAOSTAT, 2013). It is adapted

to the savannah region because of its drought tolerance (Boukar et al., 2013) and is grown by

resource‐poor farmers for multiple uses including food and fodder (Nkongolo et al., 2009;

Labuschagne et al., 2008; Murdock et al., 2008).

Cowpea production is constrained by diseases and pests, with insects causing significant

economic losses. The insect pests that attack cowpea include pod sucking bugs, weevils, flower

bud thrips and pod borers, and are responsible for cowpea losses of increasing economic

significance (Singh and van Emden, 1979). The Lepidoptera pests, which include the cowpea

seed moth, Cydia ptychora, and the pod borers, among others, are of major concern and can

account for losses up to 100 % (Jackai and Daoust, 1986). MPB is the most devastating insect

pest responsible for losses of up to 80 % if no control measures are employed. The larva is the

most destructive stage of this pest (Jackai and Daoust, 1986; Ngakou et al., 2008; Singh & van

Emden, 1979).

The development of host plant resistance has been very successful in controlling certain insect

pests. Cowpea lines with resistance to the cowpea curculio beetle, aphids and flower thrips

have been developed in national breeding programmes and adopted in several countries (Hall

et al., 2003). Breeding for host plant resistance, however, has not proved to be practical for

MPB. Although wild Vigna species containing genes for resistance to MPB exist, these genes

could not be crossed into cowpea due to sexual incompatibility (Fatokun, 2002). As such,

controlling infestations of MPB has been achieved through the use of insecticides and

microbial biopesticide formulations (Taylor, 1968). However, farmers in the tropics can rarely

afford them (Isman, 2010).

64

Genetic engineering has been proposed as an option to improve cowpea yields (Machuka et

al., 2000). Incorporating useful genes into cowpea germplasm using genetic engineering

depends on the availability of a reproducible transformation system to generate transgenic

plants (Popelka et al., 2006; Aragao and Campos, 2007; Citadin et al., 2013). In vitro plant

regeneration techniques have been applied to grain legumes via direct shoot organogenesis

(Raveendar et al., 2009; Le et al., 2002) and indirect organogenesis comprising the

establishment of callus cultures, somatic embryogenesis or embryogenic cell suspension

cultures (Aasim et al., 2010; Ramakrishan et al., 2005; Anand et al., 2000). In these techniques,

various workers have utilized different organs or tissues as the starting material including

mature seeds (Raveendar et al., 2009), cotyledonary nodal cuttings (Le et al., 2002; Popelka et

al., 2006; Chaudhury et al., 2007) mature embryos (Penza et al., 1991), plumules (Aasim et al.,

2010) or shoot apices (Mao et al., 2006). Despite several reports over nearly three decades, the

genetic engineering of cowpea is still challenging, consistent with the generally recalcitrant

nature of legumes to in vitro manipulation (Somers et al., 2003). This is well illustrated by the

many methods that have been trialled using cowpea regeneration via somatic embryogenesis

and direct multiple shoot organogenesis (Garcia et al., 1987; Brar et al., 1999; Popelka et al.,

2006; Chaudhury et al., 2007; Raveendar et al., 2009; Behura et al., 2015).

Garcia et al. (1987) were among the first to attempt transformation experiments in cowpeas

using Agrobacterium. Although transgene expression in callus cultures was reported, no

regenerated plants were obtained. More recently, Agrobacterium‐mediated and biolistic

transformation have been used successfully to introduce genes conferring traits of potential

agronomic importance into cowpea. These traits include insect resistance, herbicide resistance

and virus resistance (Popelka et al., 2006; Adesoye et al., 2008; Solleti et al., 2008; Higgins et

al., 2012; Citadin et al., 2013; Cruz and Aragao, 2014).

A cry transgene has been recently used to generate many cowpea lines expressing Cry 1Ab

protein and one line was selected as a breeding parent following several years of field trialling

against Maruca in the field in West Africa (Higgins et al., 2012; Mohammed et al., 2014). It has

been shown, however, that insects can develop field‐evolved resistance to a single Bt gene

(Mahon, 2007). As such, it is possible that Maruca could eventually develop resistance to the

Cry 1Ab protein. To manage this possible resistance to the Bt cowpea, a second Bt gene with a

65

different mechanism of action could be combined with the cry gene in the existing cowpea

line. Stacking of a vip3 and the cry1Ab gene is proposed here as a long‐term solution for

resistance management.

In the previous chapter, a vip3Ba gene was isolated and its product shown to strongly inhibit

the growth and development of MPB larvae. This gene was therefore selected as a candidate

to transform cowpea. This chapter describes the transformation of cowpea with the vip3Ba

gene optimized for expression in plants and using Agrobacterium tumefaciens for transfer. The

transgenic lines were shown to express the vip3Ba gene at varying levels. Laboratory‐based

bioassays with MPB feeding on cowpea leaf material from four different cowpea lines with

varying concentrations of Vip3Ba confirmed that Vip3Ba was a very effective toxin for the

control of MPB larvae. Therefore, stacking the vip3Ba gene with cry genes such as cry1Ab is

proposed for sustainable MPB management.

4.2 Materials and methods

4.2.1 Construction of a Vip3Ba gene for plant expression

To enhance the expression of the Bt vip3Ba gene in cowpea, the coding region was modified

according to Perlak et al. (1991). The GC content was increased, polyadenylation signals and

mRNA destabilizing sequence motifs were deleted and plant‐preferred codons were used. The

re‐designed gene was synthesized by GeneArt and inserted into an Agrobacterium

tumefaciens‐ binary vector based on pART27 (Gleave, 1992) but in which the Nos‐NptII was

replaced with an S1‐Npt II from pPLEX 502 (Schunmann et al., 2003) using the assembly

method described by Gibson (2009) and Gibson et al. (2011) in section 2.4.2. The expression of

the vip3Ba gene was under the control of the Arabidopsis small subunit promoter and

Nicotiana tabacum small subunit 3’ end derived from pSF 12 (Tabe et al., 1995). Electro‐

competent A. tumefaciens AGL1 cells were transformed with the binary vector by

electroporation (see section 2.7). Plasmid DNA was extracted from the AGL1 cells as described

in section 2.2.3 and used to re‐transform E. coli (section 2.8).

66

4.2.2 Transformation of cowpea

4.2.2.1 Preparation of Agrobacterium culture

An aliquot of 400 µL of glycerol stock of the Agrobacterium tumefaciens strain AGL1 (Lazo et

al., 1991) containing the Vip3Ba construct was added to 100 mL of MGL liquid medium (section

2.1.2) in a sterile 250 mL flask. Spectinomycin (50 mg/L) was added for selection. The culture

was grown overnight in an orbital shaker at 28 ºC at 200 rpm and centrifuged for 15 min at

7500 g at room temperature. The pellet was re‐suspended in 100 mL of cowpea suspension

liquid medium (section 2.12.3) by placing on an orbital shaker at 200 rpm for a minimum of 1 h

at 28 ºC. An additional 100 mL of cowpea suspension liquid medium was added to the

Agrobacterium culture prior to infection of explants, making a total of 200 mL, sufficient to

immerse 400 explants in eight 250 mL flasks.

4.2.2.2 Preparation of cowpea explants and Agrobacterium‐mediated transformation

The transformation of cowpea was a modified version of a protocol by Popelka et al. (2006)

(See Table 4.1 for details of the modifications made to the original protocol). Dry cowpea seed

(30 g) was weighed into a 250 mL Schott bottle and sterilized by adding approximately 50 mL of

70 % ethanol for 1 min. The bottle was then shaken vigorously for 30 s and the ethanol poured

off. A volume of 50 mL of 20 % commercial bleach was added and allowed to stand for 30 min

at room temperature. The seeds were rinsed 5 times in sterile reverse osmosis (RO) purified

water and seeds were allowed to imbibe in 50 mL of sterile RO water overnight.

The excess water was poured from the imbibed seed and the seed coat removed aseptically.

The seed was split in two by separating the cotyledons. Using the cotyledon with the attached

embryonic axis, the lower ⅔ of the radicle was excised. This is referred to as the explant.

Approximately 50 explants were placed in sterile 250 mL flasks containing 10 mL of cowpea

suspension liquid medium containing no L‐cysteine. For one experiment, 400 explants were

placed in eight flasks.

The cowpea suspension liquid medium was poured off the explants. Approximately 25 mL of

the re‐suspended Agrobacterium culture was added to submerge the 50 explants. The explants

were sonicated for 30 s and incubated for a minimum of 1 h on a rotary shaker at 28 ºC and

200 rpm. The Agrobacterium culture was poured off and discarded and the explants placed

67

Table 4.1: Modifications of the transformation system used for cowpea

Transformation protocol according

to Popelka et al. (2006)

Modifications made in the

current study

Seed sterilization ‐ Dry seed sterilized for 45 minutes

in 50 % bleach.

‐ Dry seed sterilized in 70 %

ethanol for 1 minute, followed by

20 % commercial bleach for 30

minutes.

Agro‐infection stage ‐ Wounded the explants using a

scalpel blade.

‐ Explants sonicated for 30 s while

submerged in Agro infection

culture.

Co‐cultivation stage ‐ Infected explants co‐cultured for

6 days.

‐ Incorporated 1 g/L of L‐cysteine in

the co‐cultivation media.

‐ Infected explants co‐cultured for

3 days.

‐ Absence of L‐cysteine in co‐

cultivation media.

Shoot induction stage ‐ Incorporated 250 mg/L sodium

thiosulfate in the shoot induction

medium.

‐ Absence of sodium thiosulphate

in the shoot induction medium.

Selection Conditions ‐ Explants on shoot induction

medium without selection for 12

days i.e. Days 4 – 16 (Table 4.2).

‐ The explants were then

transferred to selection media

(phosphinothricin) at a constant

level for the next eight sub‐cultures

including rooting stage.

‐ Explants were immediately

transferred to selection

(kanamycin at 100 mg/L) on

shoot induction medium for days

4 – 16 (Table 4.2).

‐ The explants were then

transferred to a higher level of

selection (150 mg/L kanamycin)

for days 17 – 44 (Table 4.2).

‐ The selection agent was then

changed to geneticin at 30 mg/L

for the remaining cycles including

rooting i.e days 45 – 129 (Table

4.2).

68

onto new sterile filter papers to blot the excess culture medium. Co‐cultivation plates were

prepared by placing sterile filter paper on solid cowpea suspension medium. The blotted

explants were placed on the filter paper plates and co‐cultivated for 3 days (fourth

modification) at 28 ºC, 16/8 h (light/dark).

4.2.2.3 Regeneration and maintenance of cowpea cultures

Following co‐cultivation, the explants were placed with the growing shoot facing down onto

solidified shoot induction medium containing kanamycin (100 mg/L) for selection and

meropenem (Merrem) (25 mg/L) to prevent the growth of Agrobacterium (section 2.12.4). This

medium contained no sodium thiosulphate. The cultures were incubated for 12 to 14 days in a

growth chamber at 28 ºC, 16/8 h light/dark regime. In the second transfer, the cotyledons

were excised and discarded and the remaining portion of the explants transferred to solidified

shoot induction medium with kanamycin (150 mg/L) and Merrem for 12 to 14 days. At the

third transfer, the primary shoots were excised and the excess or brown callus trimmed off and

remaining callus was again placed onto solidified shoot induction medium with kanamycin (150

mg/L) selection and Merrem. The callus was subsequently trimmed off followed by a fourth

transfer. At the fifth transfer, any multiple shoots were transferred to shoot induction medium

with more stringent selection using geneticin (30 mg/L) and Merrem otherwise a further cycle

with kanamycin (150 mg/L) was repeated. Following this, one more cycle of transfer onto

shoot induction medium with 30 mg/L geneticin was carried out. Large clumps consisting of

many shoots were separated into smaller clumps or as singles depending on the vigour and the

callus was trimmed. By the seventh transfer, multiple shoots were separated and at the eighth

transfer, single shoots were placed onto shoot elongation medium with geneticin (30 mg/L)

and Merrem for elongation and rooting (section 2.12.5). Depending on their height, shoots

were either placed in Petri plates or 250 mL pots. The shoots that developed healthy roots

(plantlets) were transferred into small pots (50 cm diameter x 10 cm height) containing a light

sandy soil mix (25 % perlite, 25 % vermiculite, 30 % river sand and 20 % commercial potting

mix), and subsequently maintained in the culture room for up to 4 weeks to establish a healthy

root system and acclimatize prior to transfer to the greenhouse. During this stage the putative

transgenics were screened by PCR (see 4.2.3) after which positive plants were transferred to

soil (70 % commercial potting mix, 10 % perlite, 10 % river sand, 10 % peat, 1.5 g/L Osmocote,

69

0.5 g/L Nitram and 3 g/L Carb lime) in 20 cm pots in the glasshouse. A brief summary of the

steps in the transformation and regeneration of cowpea is outlined in Table 4.2.

Table 4.2: Outline of steps in cowpea transformation with the plant‐optimized vip3Ba gene

Step Procedure Time required

1 Dry seed sterilization 1 h

2 Seed imbibitions Overnight

3 Explant preparation 400 explants in 3 h

4 Sonication of explants in presence of Agrobacterium 30 s

5 Infection with Agrobacterium 1 h

6 Co‐cultivation with Agrobacterium Days 1 – 3

7 First round of shoot induction (in presence of cotyledons) Days 4 – 16

8 Cotyledon excision Day 17

9 Second round of shoot induction Days 17 – 28

10 Third round of shoot induction Days 29 – 44

11 Fourth round of shoot induction Days 45 – 55

12 Fifth round of shoot induction Days 56 ‐ 69

13 Sixth round of shoot induction Days 70 ‐ 84

14 Seventh round of shoot induction Days 85 ‐ 99

15 First round of shoot elongation and rooting Days 100 ‐ 114

16 Second round of shoot elongation and rooting Days 115 ‐ 129

16 Acclimatization of rooted plantlets in sandy soil mix Days 130 ‐ 145

17 Transfer of established plants to soil in greenhouse Days 115 ‐ 145

4.2.3 Detection of vip3Ba and nptII genes in the transgenic plants

To test for the presence of the transgenes (vip3Ba and nptII) in putatively transgenic cowpea

plants, genomic DNA was extracted using the lysis method described in section 2.13.

Approximately 100 ng of genomic DNA was used as a template for a PCR as previously

described in section 2.14 using the primers described in Table 4.3. Water and/or DNA from the

70

non‐transformed cowpea line IT86D was used as a negative control while plasmid DNA with

vip3Ba and nptII genes was used as a positive control. The PCR products were resolved by

electrophoresis through 1.8 % agarose gels and made visible with a transilluminator gel

documentation system (GelDoc 2000 software, BioRad).

Table 4.3: Primer sequences for the detection of nptII and vip3Ba genes in transgenic plants

Primer name Primer sequence Product size

nptII F: GCTTGGGTGGAGAGGCTATTR: TCATTTCGAACCCCAGAGTC

970 bp

Vip3Ba_set1 F: GAACGCTCAGCTCAACTCCA R: GGTGGAGTTGATGAGCACGT

187 bp

4.2.4 Detection and estimation of Vip3Ba expression in transgenic plants

Seeds from the T0 transgenic lines were harvested and sown to obtain T1 plants for DNA and

protein isolation. Young fully expanded leaves of T1 plants (7‐days old) were harvested for PCR

and expression of Vip3Ba protein was detected using western blot analysis following the

procedures described in section 2.16. The amount of Vip3Ba protein was estimated based on a

standard curve derived using protein standards from E. coli expressing Vip3Ba protein at 1 ng,

3 ng, 10 ng and 30 ng per lane. The data were expressed as ng Vip3Ba per mg of total soluble

protein.

4.2.5 Insect bioassays using transgenic leaf material

Leaves of transgenic cowpea lines expressing Vip3Ba were fed to Maruca larvae. Bt cowpea

line 709A expressing Cry1Ab previously developed at CSIRO (Higgins et al., 2012) was used as a

positive control while the non‐transgenic parent line IT86D was used as a negative control.

Feeding trays consisted of 32 wells (1 cm diameter x 1 cm height). For each line, sixteen wells

were partially filled with 5 mL of 1 % water agar containing 0.1 % (w/v) sorbic acid to avoid

fungal contamination (Acharjee et al., 2010). Approximately 20 mg of fresh leaf material was

placed on the agar in each well.

One 6‐day old larva (2.2 ± 0.1 mg) was introduced onto each leaf and the trays sealed with a

perforated film using a hot sealer. The assays were done in a climate chamber at 26 ºC and 50

71

% relative humidity. Surviving larvae were re‐fed with fresh leaf every other day. After 10 days,

the bioassay results were scored; mortality and final weights of surviving larvae were recorded.

Three separate bioassay experiments were conducted for each cowpea line, resulting in a

minimum number of replications of 48 per treatment.

4.3 Results

4.3.1 Reconstruction of the vip3Ba gene for cowpea transformation

Prior to transformation into cowpea, the coding region of the vip3Ba gene was optimized for

plant expression by increasing the GC content from approximately 30 % to 50 %, and deleting

the eight potential polyadenylation sites and twelve mRNA destabilizing sequence motifs that

were present. The codons were also optimized to enhance translation in dicots. These changes

are summarized in Table 4.4.

The reconstructed vip3Ba gene (the full DNA sequence of the open reading frame is shown in

Appendix II and its sequence comparison with the bacterial vip3Ba gene in Appendix III) was

cloned into the binary vector, pART27, and electroporated into A. tumefaciens AGL1 (Fig. 4.1

and 4.2). To confirm the presence of the construct in A. tumefaciens, plasmid DNA was

extracted from bacterial cultures and digested with EcoRV and PvuII. Four fragments of the

expected sizes (1.5, 2.7, 4.4, 6.4 kb) were observed confirming the presence of the construct

(Fig. 4.3).

72

Table 4.4: A comparison of the vip3Ba gene from Bt with the version optimized for expression in plants

Parameter Original vip3Ba

Optimized vip3Ba

G+C content 29.8% 50.5% PolyA sites (AATAAA and/or AATAAT) 8 0 RNA destabilizing sequences (ATTTA) 12 0 Examples of codon usage change

‐ Isoleucine ATT ATC ‐ Leucine TTA CTC ‐ Proline CCA CCT ‐ Arginine AGA/CGT CGA ‐ Valine GTA GTT/GTC ‐ Tyrosine TAT TAC

Fig. 4.1: Schematic diagram of the Agrobacterium binary vector T‐DNA; LB, RB: left and right borders of Agrobacterium T‐DNA, respectively. The antibiotic selection gene neomycin phosphotransferase II (nptII) from E. coli was flanked by the S1 promoter derived from segment 1 of the subterranean clover stunt virus (SCSV) genome and segment 3 (S3) 3′ end while the optimized coding region of the vip3Ba gene was flanked by the Arabidopsis thaliana small subunit (AraSSU) promoter and Nicotiana tabacum small subunit (TobSSU) 3′ end.

73

Fig. 4.2: Plasmid map showing unique restriction sites in the pVip3Ba construct used in the

transformation of cowpea

74

Fig. 4.3: Confirmation of the Vip3Ba cassette in the Agrobacterium binary vector by restriction digestion. Lane M: DNA Molecular ladder; Lanes 1 and 2: Agrobacterium DNA digested with the enzymes EcoRV and PvuII resulting in the expected four fragments of 1.5, 2.7, 4.4 and 6.4 kb, respectively.

75

4.3.2 Transformation and regeneration of cowpea with the optimized vip3Ba gene

Sterilized dry cowpea seed was imbibed with water and explants were prepared for

transformation by excising approximately ⅔ of the radicle from the cotyledon containing the

embryonic axis (Fig. 4.4 A). The explants were subsequently co‐cultivated with Agrobacterium

containing the optimized vip3Ba gene for 3 days (Fig. 4.4 B) before being incubated on

selection media (Fig. 4.5 A) for a maximum of 14 days. The cotyledons and primary shoots were

subsequently excised (see 4.2.2.3) (Fig. 4.5 B) to obtain callus. Subsequent cycles of transfer of

calli to fresh shoot induction medium led to the formation of multiple shoots (Fig. 4.5 C) which

were separated to obtain single shoots (Fig. 4.5 D, E) which further developed into rooted

plantlets (Fig. 4.5 F) that were first acclimatized prior to transfer to the glasshouse (Fig. 4.5 G).

76

Fig. 4.4: Cowpea explants used for transformation. (A) Cotyledons with attached axes that had their radicle tips removed (arrows) ready for infection with Agrobacterium and (B) after co‐cultivation with Agrobacterium for 3 days.

77

Fig. 4.5: In vitro regeneration of cowpea following co‐cultivation with Agrobacterium. (A) Cowpea explant with cotyledon and primary shoots on shoot induction medium with selection at 2 weeks (B) trimmed explant with cotyledon and primary shoot removed at 4 weeks (C) multiple shoots formed on callus (D) single shoots formed on shoot induction medium with 30 mg/L geneticin at 8 weeks (E) shoots grown on medium with 30 mg/L geneticin at 10 weeks, (F) individual shoots in elongation and rooting medium at 14 weeks and (G) rooted plants in the soil at 16 weeks.

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4.3.3 Characterization of transgenic lines by PCR

A total of 6696 explants were co‐cultivated with Agrobacterium containing the vip3Ba

binary vector. Following selection, 77 putative independent transgenic lines were obtained,

with all plants that regenerated from one explant considered as clones (siblings) belonging

to one transgenic event. To assay for the presence of the transgene in these lines, genomic

DNA was extracted and used as a template in PCR using primers designed to amplify a 970

bp region of the nptII gene and a 187 bp fragment of the vip3Ba gene (Fig. 4.6). Of the 77

plants, 73 were positive for both the vip3Ba and nptII genes (Table 4.5), resulting in an

average transformation frequency of 1.1 %. While the copy number of inserts was not

determined, transgenic lines with a single locus were selected based on segregation

analysis. Lines V24, V25, V43 and V87 showed Mendelian inheritance of the vip3Ba gene

with a segregation ratio of 3:1 following a Chi square analysis (Table 4.6).

4.3.4 Expression of Vip3Ba in T1 generation

All the 73 T0 plants were fertile and seed from 42 lines was collected for further work. A

minimum of 8 seeds from each of the 42 T0 lines were germinated to produce T1 plants. The

non‐transgenic parent line (IT86D) was propagated as a negative control. Total soluble

protein (TSP) was extracted from leaf samples from a pool of 6‐8 T1 plants generated from

each of the 42 transgenic lines. A 40 µg aliquot of TSP was then subjected to SDS‐PAGE,

blotted to membranes and analysed for expression of Vip3Ba by western blot using a

Vip3Ba‐specific monoclonal antibody. Of the 6‐8 T1 plants derived from each of the 42 T0

lines that were analysed, a band of the expected size for Vip3Ba ( ̴75 kDa) was detected in

extracts from 9 of the 42 lines (ie, lines V9, V24, V25, V43, V56, V87, V107, V176 and V191)

(Table 4.5). An additional, non‐specific band of 2̴2 kDa was also observed in protein extracts

from all cowpea lines including the non‐tranformed negative control. A representative blot

of TSP extracts from T1 progeny derived from lines V9, V24, V25, V43, V56, V87 and V107

showing average expression in these lines is displayed in Fig. 4.7. The concentration of

Vip3Ba protein in the extracts was estimated by visual comparison with known levels (1 ng,

3 ng, 10 ng, 30 ng and 100 ng) of E. coli‐expressed Vip3Ba protein. The limit of detection of

Vip3Ba protein was found to be 1.5 ng, which is equivalent to 37.5 ng/mg TSP (data not

79

shown). Based on this, the levels of Vip3Ba protein expression in the 7 lines ranged between

0.25 and 5.0 µg/mg TSP (Table 4.7).

Fig. 4.6: PCR analysis of 10 putative transgenic cowpea lines using (A) vip3Ba‐specific primers designed to amplify a 187 bp product and (B) nptII‐specific primers designed to amplify a 970 bp product. Lane M: DNA Molecular marker; Lane N: water (negative control); Lane P: Plasmid DNA of the optimized gene construct (positive control); numbers above other lanes represent the line number of randomly selected transgenic cowpeas used for PCR analysis.

80

Table 4.5: Summary of the molecular analysis of transgenic cowpea lines

No. of primary transgenics

No. of lines PCR positive for nptII

No. of lines PCR positive for vip3Ba

No. of lines tested by western blots

No. of lines with detectable levels of Vip3Ba protein

77

73

73

42

9

Table 4.6: Segregation analysis of transgenic cowpea T1 plants

Vip3Ba lines

No. of

plants

tested

Positive

(vip3Ba)

Negative

(vip3Ba) Ratio X2

V24 13 10 3 3.3:1 1.83

V25 8 7 1 7:1 0.11

V43 25 16 9 1.8:1 1.61

V87 36 26 10 2.6:1 0.147

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T1 progeny from four lines, namely V24‐9, V25‐8, V43‐3 and V87‐2, were selected for further

analysis. The selection was based on the fact that variation in Vip3Ba expression between

the four lines could serve to determine whether insect mortality was dependent on toxin

dosage. The selected T1 plants were grown on to produce T2 seed, and at least five T2 plants

from each of these four lines were further subjected to Western blot analysis. Figure 4.8

shows blots of extracts from progeny of lines V25, V24, V43 and V87, indicating expression

in the segregating progeny of the four selected lines.

In the four selected T1 transgenic lines (V24, V25, V43 and V87), plants expressing Vip3Ba

showed abnormal phenotype. The phenotype was characterized by stunted growth (lines 24

and 43), deformed and or wrinkled leaves (lines 43 and 87). The non‐transformed parent

line IT86D and the null segregants appeared phenotypically normal (Fig. 4.9).

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Fig. 4.7: Western blots using a monoclonal antibody to Vip3Ba showing the levels of Vip3Ba in seven cowpea lines using varying levels of

Vip3Ba expressed in E. coli as standards. In both blots, Lane M is the protein molecular weight ladder. Lanes L1 to L5 represent E. coli

expressed Vip3Ba loaded at 100, 50, 25, 12.5 and 6.25 ng per lane, respectively. Blot A includes protein (40µg/lane) from cowpea lines 9, 24,

25 and 43 while Blot B includes protein (40µg/lane) from lines 56, 87 and 107.

83

Table 4.7: Levels of Vip3Ba toxin in the T1 progeny of seven cowpea lines.

Line Level (in ng) of Vip3Ba toxin in 40 ug TSP Vip 3Ba (µg/mg TSP)

V9 50 1.25 V24 50 1.25 V25 50 1.25 V43 75 1.9 V56 10 0.25 V87 200 5.0 V107 25 0.63

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Fig. 4.8: Western blot analysis to determine Vip3Ba expression in transgenic cowpea lines using a monoclonal antibody to Vip3Ba. Blot A: Line V24: Lane M: Protein precision markers; Lanes 1 to 6: TSP (40 µg) extract from six T2 plants; Lanes 7 to 9: 30 ng, 100 ng and 300 ng protein from E. coli expressing Vip3Ba protein (positive control). Blot B: Line V25: Lanes 1 to 8: TSP (40 µg) extract from eight T1 plants; Lanes 9 to 10: 10 ng and 30 ng protein from E. coli expressing Vip3Ba protein (positive control). Blot C: Line V43: Lane M: Protein precision markers; Lanes 1 to 6: TSP (40 µg) from six T2 plants; Lanes 7 to 9: 30 ng, 100 ng and 300 ng protein from E. coli expressing Vip3Ba protein (positive control). Blot D: Line V87: Lane M: Protein precision markers; Lanes 1 to 5: TSP (40 µg) from five T2 plants; Lanes 6 to 9: 10 ng, 30 ng, 100 ng and 300 ng Vip protein from E. coli expressing Vip3Ba protein (positive control). Vip3Ba band migrates at ~75 kDa.

85

Fig. 4.9: Phenotypes of selected transgenic T1 cowpea lines expressing Vip3Ba protein at different ages. (A) Line V24 at 3 weeks after sowing; (B) Line V25 at 2 weeks after sowing; (C) Line V43 at 2 ½ months after sowing; (D) Line V87 at 3 weeks after sowing and; (E) 3‐week old negative segregants of lines V43 and V87.

86

4.3.5 Efficacy of transgenic lines expressing Vip3Ba protein on MPB larvae

To test the effect of the selected transgenic lines expressing the Vip3Ba toxin on the

mortality of MPB larvae, a replicated series of bioassays was carried out. Sixteen replicate

leaf samples from each of the four selected Vip3Ba‐expressing cowpea lines were fed to

MPB larvae and the effect of the toxins was investigated over a 10‐day period by measuring

MPB mortality and the weight of any surviving larvae. As controls, samples were also taken

from leaves of the positive control Cry1Ab‐line (709A) and from the non‐transgenic negative

control line (IT86D).

The average weight of larvae fed on leaf samples from IT86D ranged between 23 and 26 mg

after 10 days’ growth and no mortality was observed. Most of the larvae fed on IT86D

leaves had progressed to the fifth and final instar stage. When larvae were fed on leaves

from the positive control line 709A expressing Cry1Ab, the mortality was 100 % as expected.

There was also 100 % mortality of larvae feeding on leaves of all four lines expressing

Vip3Ba protein (Table 4.9). Leaf damage on the Vip3Ba‐transgenic lines, as well as the

positive control, was negligible with most of the leaf discs left fully intact (Fig. 4.9 A‐E). In

contrast, leaf damage on line IT86D was severe with abundant frass observed on the leaf

disks indicating that the insects feeding on these plants were developing normally (Fig 4.9 F‐

H).

87

Table 4.8: Mortality of Maruca larvae feeding on transgenic cowpea leaves in four separate and replicated bioassays (N = 16 larvae per replication).

Linea

Level of toxin (ng/mg TSP)

Mortality of Maruca larvae (%)

1st Bioassay 2nd Bioassay 3rd Bioassay 4th Bioassay

IT86D 0 0 0 0 0709A 260 100 100 100 100 V24 425 100 100 100 100V25 155 100 100 100 100 V43 895 100 100 100 100 V87 365 100 100 100 100

a Lines V24 to V87 are four independent transgenic lines expressing Vip3Ba protein between 155 and 895 ng/mg TSP, respectively. Line IT86D is the untransformed parent line (i.e. the negative control). Line 709A is a cowpea line transformed with the cry1Ab gene (positive control).

88

Fig. 4.10: Leaf damage after 10 days of feeding by Maruca larvae on non‐transgenic line IT86D and transgenic lines expressing Cry 1Ab or Vip3Ba protein. A: Line 709A (expressing Cry 1Ab), B: Line V24, C: Line V25, D: Line V43, E: Line V87, F, G, H: Line IT86D

89

4.4. Discussion

Cowpea production worldwide is constrained by several insect pests, however, the Maruca

pod borer (MPB) is considered one of the most devastating. The absence of genes for

resistance to Maruca in cowpea germplasm has precluded the use of conventional plant

breeding as a means to control this pest (Fatokun, 2002). As such, genetic engineering

appears to be the most viable control option. The aim of this chapter, therefore, was to

generate transgenic cowpeas expressing Vip3Ba and determine whether they were in fact

resistant to MPB.

In general, insecticidal proteins need to be expressed at relatively high levels in their plant

hosts in order to effectively control the insect target (Gatehouse, 2008). One limitation of

using native Bt genes is that they are expressed poorly in higher eukaryotes (Schuler et al.

1998; Perlak et al. 2001). This is most likely due to the fact these bacterial genes are AT‐rich

and contain polyadenylation signals and mRNA destabilizing sequence motifs;

charactersitics that can negatively affect mRNA processing in plants (Estruch et al., 1997;

Jouanin et al., 1998). In order to maximise vip3Ba gene expression in transgenic cowpea, the

coding sequence of the bacterial‐derived vip3Ba gene was modified using the strategy

described by Perlak et al. (1991). This involved increasing the GC content of the coding

sequence and deleting the polyadenylation and mRNA destabilizing sequences without

altering the amino acid sequence. Also, the codon usage was optimized to enhance

translation in dicotyledons by using plant‐preferred codons. Using a similar strategy, high

level expression of optimized cry1Ac, cry1Ab and cry1C genes have been reported for

engineered resistance to lepidopteran insect pests in transgenic cotton, tomato and

tobacco, respectively (Perlak et al., 1990; Perlak et al., 1991; van der Salm et al., 1994).

In order to generate cowpea lines expressing the optimised vip3Ba gene, a reliable

transformation and regeneration system for cowpea had to be first established in house. It

is well documented that transformation of many plant species is dependent on the

genotype, the type of explant used, the antibiotic or herbicide selection regime and plant

growth media composition (Manman et al., 2013). In this study, significant modifications

were made to a published cowpea transformation protocol (Popelka et al., 2006) to improve

its efficiency. These modifications included using a growth media in the absence of certain

90

key plant hormones, a different antibiotic selection regime, and employing sonication to

permeate the plant cell wall and thereby facilitate Agrobacterium‐mediated T‐DNA transfer

into the host plant cell. These technical changes resulted in a cowpea transformation system

with an efficiency of just over 1 %, with 73 independent transgenic lines obtained. This rate

is comparable to other cowpea transformation studies which ranged from 0.15 % to 3.9 %

(Chaudhury et al., 2007; Ivo et al., 2008; Raveendar and Ignacimuthu, 2010; Solleti et al.,

2008; Popelka et al., 2006; Behura et al., 2015; Adesoye et al., 2010).

Independent lines were assayed for the presence of the transgene in T0. The progeny of nine

lines were assayed for Vip protein in the T1 generation by western blot. It is highly likely that

the 9 lines were independently transformed as we expect that there was random

integration of the transgene in the genome. Consequently, protein expression is expected to

be dependent on the locus where the transgene has integrated, either in a transcriptionally

active or inactive locus (Chen et al., 1998; Kohli et al., 2010). Consequently, further analysis

to determine the transgene loci for these lines is therefore recommended.

The levels of Vip3Ba protein were measured in a number of transgenic cowpea lines using

western blots. The amount of Vip3Ba varied and ranged between 155 ng/mg TSP and 895

ng/mg TSP. Variable transgene expression is a common phenomenon in genetically modified

plants and levels can vary widely between independent transgenic lines (Matzke & Matzke,

1998; Schuler et al., 1998). This variation is primarily the result of (i) the position effect i.e.

where in the genome the transgene cassette has integrated, and (ii) complex integration

events which can trigger post transcriptional gene silencing (PTGS) and/or transcriptional

gene silencing (TGS) (Matzke & Matzke, 1998; Schuler et al., 1998; Stam et al., 1997). The

latter of these phenomena are of particular importance as both PTGS and TGS can result in

the complete shutdown of a transgene’s expression.

The levels of Vip3Ba measured in this study are within the range of those observed in

cowpea lines expressing Cry 1Ab (av. 260 ng/mg TSP), but higher than those reported in

chickpea expressing Cry 1Ac (0.5 to 23.5 ng/mg TSP) (Kar et al., 1997; Sanyal et al., 2005;

Indurker et al., 2010; Mehrotra et al., 2011; Ganguly et al., 2014). In contrast, very high

levels of Bt toxin expression have been reported in other transgenic crops. In these cases,

the Bt toxin gene product was targeted to the chloroplast using a plastid transit peptide

91

fused to its N‐terminus. Using this strategy, Cry1Ac accumulated up to 6000 ng/mg TSP in

transgenic soybean (Miklos et al., 2007) and Vip3A levels reached between (5950 to 6120

ng/mg TSP) in transgenic cotton (Wu et al., 2011). It is likely intracellular targeting of these

proteins acts to stabilise and protect the product from degradation by cytosolic proteases or

protein turn‐over resulting in abundant accumulation of the target protein.

Four lines expressing different levels of Vip3Ba (ranging from 155 to 895 ng/mg TSP) were

selected for insect bioassays. These lines were selected in order to study a toxin dose

response i.e. the optimal level of toxicity at which insect mortality occurs. Despite varied

levels of Vip3Ba between plants, 100 % mortality rates of Maruca vitrata larvae were

observed in feeding trials with all transgenic lines tested. This would suggest Vip3Ba levels

lower than 155 ng/mg TSP are sufficient to cause Maruca larvae death, and that this toxic

product is highly effective at low doses.

A high toxin dose is preferred for robust insect resistance in transgenic crops carrying a

single Bt toxin gene (Gryspeirt and Gregoire, 2012). However, over‐expression of the vip3Ba

gene alone caused abornmal phenotypes in transgenic cowpea, including stunting,

deformed and/or wrinkled leaves, chlorosis, and the development of fewer flowers and

pods. This effect positively correlated with the level of Vip3Ba gene expression as high

expressing lines exhibited more severe phenotypic effects compared to low‐expressing lines

which showed little to none. This suggests high levels of Vip3Ba accumulation may be

phytotoxic in cowpea. Such a phenomenon was also observed in chickpea over‐expressing

the cry2Aa genes (Acharjee et al., 2010) which displayed stunting, abnormal root

development, and abnormal flower and leaf formation (Deineko et al., 2007; Acharjee et al.,

2010). Considering this, it would be of benefit to screen other cowpea lines expressing levels

of Vip3Ba that are lower than 150 ng/mg TSP in insect bioassays in order to determine the

minimal toxin dose required for MPB protection while producing plants with a fully normal

phenotype. Other than leaf infestation, Maruca larvae mostly feed on the floral parts in

cowpea. Further research on testing for protein expression in the flowers should therefore

be considered.

92

A Cry1Ab‐transgenic cowpea line has previously been developed with resistance to MPB in

the field (Higgins et al., 2012). However, it has been shown that some insect pests can build

up a tolerance to a single Bt toxin over time (Liu et al., 2010) and it is likely MPB will do the

same. It has been suggested that the best strategy to overcome this is to pyramid Bt genes

such that a second toxin gene is also expressed in the plant, but at lower titre (Gryspeirt and

Gregoire, 2012). This study has proven the Vip3Ba is a lethal control agent against MPB and

is active at relatively low concentrations. Together, this would suggest vip3Ba is an

attractive gene candidate for stacked resistance to MPB in cry1Ab‐transgenic cowpea. If a

phenotypically normal, low Vip3Ba‐expressing cowpea line with robust resistance to MPB

can be identified from the pool of cowpea lines developed in this study, it may be useful for

traditional backcrossing into cry1Ab‐containing cowpea cultivar for IRM. If such a line

cannot be found then new ways of regulating Vip3Ba expression and minimising unwanted

phenotypical changes now need to be explored, for example using a weaker promoter to

provide low‐level constitutive expression of Vip3Ba or targeting the protein to the

chloroplast.

93

CHAPTER FIVE

General Discussion and Conclusions

Grain legumes play a significant role in contributing to the livelihoods of farmers in Africa.

They provide human nutrition and generate income through sale of surplus grain. These

legumes improve soil fertility thereby leading to sustainable soil productivity (Odendo et al.,

2011). In sub‐Saharan Africa, legumes integrate with cereals and livestock thus contributing

to food security (Odendo et al., 2011). Cowpea is one such grain legume that is important in

the diets of people living in sub‐Saharan Africa. It has a high protein and mineral content in

the grains and leaves and it is cultivated for both human consumption and as an animal

feed.

One of the most serious problems affecting cowpea production is damage caused by the

lepidopteran pest, Maruca pod borer (MPB). This insect attacks the pods and leaves of this

crop, is a major insect pest of other grain legumes and is responsible for substantial yield

losses in cowpea (Suh and Simbi, 1983). The current approaches used to control this pest

have mainly focussed on the use of chemical sprays. However, spraying can be harmful to

the environment and human health, and is also costly. Although conventional plant

breeding has been used to successfully control some pests and diseases affecting cowpea

(Huynh et al., 2015), the lack of resistance to MPB in the primary genepool as well as the

sexual incompatibility of cowpea with its wild relatives precludes the use of this approach

for controlling MPB. Therefore, an alternative strategy is needed to control this pest in

cowpea.

Genetic engineering offers such a solution and there are numerous examples of the

generation of insect‐resistant crops expressing Bt endotoxin genes (Ishida et al., 2007;

Acharjee et al., 2010). Although a Bt‐encoded cry 1Ab gene has been transformed into

cowpea (Higgins et al., 2012) and shown to confer resistance to MPB in the field

(Mohammed et al., 2014), there is a likelihood that this pest could eventually evolve

resistance to a single Cry protein. As such, this project sought to develop transgenic

cowpeas with resistance to MPB using a Bt‐encoded vip as transgene. The vip3 gene

products were considered excellent targets as they are toxic to a number of lepidopteran

94

pests (Estruch et al., 1996). Further, the toxins expressed by the vip and cry genes have

different target sites in the insect midgut (Lee et al., 2003), thereby presenting a way of

delaying resistance to one type of Bt protein.

Bt genes are abundant in nature and a large group of sequences are deposited in the Bt

database (Crickmore et al., 2014). Their nomenclature is based on DNA sequence

similarities. In this study, in order to target the diversity of the 3 groups (A, B and C) of vip

genes for screening, the genes were subjected to further phylogenetic analyses based on

the amino acid sequences of the proteins they encode. As a result, five groups of vip genes

were identified and a representative gene selected from each group. When a collection of

Australian Bt isolates were screened for these target sequences, four of the five vip3 genes

were found to be present. Primers were subsequently designed to amplify the coding

sequences to enable the target proteins to be expressed in E. coli and to assess their toxicity

to MPB in feeding trials. Only three of the target sequences were able to be amplified from

the Bt collection, so the remaining two target genes were chemically synthesized prior to

their cloning. Expression of all five genes in E. coli resulted in in high levels of protein as

expected (Frommer and Ninnemann, 1995). Bioassays were carried out with MPB larvae

using different concentrations of each of the Vips incorporated into an artificial diet.

Artificial diets are commonly used to test for insecticidal activity of Bt toxins against insect

pests (Estruch et al., 1996; Beard et al., 2008). The bioassays revealed that one of the five

Vip3 proteins tested (Vip3Ba) inhibited the growth of MPB larvae. The was a significant

outcome and was the first to report on the toxicity of Vip proteins to this particular pest.

Based on this result, VipBa was chosen as the candidate gene to express in transgenic

cowpea.

Cowpea is generally recalcitrant to transformation and regeneration and a considerable

effort has been directed towards improving the low transformation frequencies and

regeneration efficiencies reported earlier. For example, different regeneration pathways

have been examined such as the establishment of callus and direct or indirect

organogenesis (via somatic embryogenesis) through cell suspension cultures or on agar‐

solidified media. Attempts to increase the efficiency of both transformation and

regeneration have also included the use of different types of explants (leaf discs,

cotyledonary nodes, embryonic axes), subjecting explants to several selection methods and

95

optimizing culture conditions, but ultimately, the regeneration potential is dependent on

the genotype (Somers et al., 2003; Citadin et al., 2011; Atif et al., 2013). With this in mind, a

considerable effort was made in the current study to modify an existing transformation and

regeneration protocol (Popelka et al., 2006). These modifications included transforming

many (about 400) explants simultaneously in one experiment and using a sonication

procedure to aid in the transfer of the transgene into the plant cell. Sonication was used to

disrupt the cell wall of many explants concurrently thus facilitating Agrobacterium‐mediated

transformation. The protocol used here yielded a transformation efficiency that was close to

8‐fold higher than that reported by Popelka et al. (2006).

To enhance expression of a bacterial gene in a plant host, modifications are made without

altering the sequence of the encoded protein. Perlak et al. (1991) were the first to use this

approach to greatly enhance the expression levels of insecticidal proteins in tobacco.

Accordingly, in order to maximise the expression of Vip3Ba in transgenic cowpea, the vip3B

gene was also modified prior to Agrobacterium‐mediated transformation. The Arabidopsis

small subunit (AraSSU) promoter and Nicotiana tabacum small subunit (TobSSU) terminator

were selected to direct expression of the vip3Ba gene in planta. The AraSSU promoter has

been used by other researchers to express Bt insecticidal proteins (Acharjee et al., 2010)

and was preferred as it is strongly expressed in leaves and flowers and has been shown to

greatly enhance expression levels compared to other promoters such as CaMV 35S (Wong et

al., 1992; Tabe et al., 1995). Following Agrobacterium‐mediated transformation of 6696

cowpea explants with the vip3Ba‐expressing construct, 77 potential transgenic lines were

regenerated of which 73 were confirmed to be transgenic following molecular

characterization. Western blot analyses revealed that several lines expressed Vip3Ba protein

at varying levels ranging from 155 to 895 ng/mg total soluble protein. It was noted that

some lines exhibited abnormal phenotypes which generally correlated with higher levels of

toxin expression. Targeting protein expression to the chloroplast has been previously used

by researchers to overcome this problem. For example, chloroplast‐targeting of insecticidal

proteins in transgenic tobacco yielded normal phenotypes as compared to the non‐

organelle targeted controls which exhibited sterility (Corbin et al., 2001). In transgenic

cotton, chloroplast‐targeted Vip3A protein resulted in plants with normal agronomic traits

96

(Wu et al., 2011). Such an approach may therefore be needed in future studies with

cowpea.

Four transgenic cowpea lines were subsequently selected for insect bioassays to determine

the toxicity to MPB and all four lines were shown to be completely protected from MPB

damage. This was a significant and important outcome, and clearly demonstrated that

vip3Ba genes can be utilized in the development of cowpeas that are resistant to pod

borers. Importantly, this gene can be used to complement other Bt genes in tackling the

possibility of insect resistance development. Stacking cry1Ab and vip3Ba genes by

introgressing vip3Ba into cry 1Ab cowpea through back crossing will increase yields for the

small‐holder farmers in Africa and allow them to reduce the need for chemical sprays. The

pyramided Bt cowpea will reduce the likelihood of resistance developing in Maruca and thus

contribute to insect resistance management in cowpea.

Although the development of transgenic cowpea with resistance to MPB is now a reality,

the adoption of the technology is another issue that will need to be addressed. Since the

initial commercialization of biotech (GM) crops in 1996, there has been acceptance of the

technology in many but not all countries including developing nations (James, 2014). Most

African countries rely on agriculture as a source of income and livelihood, however the

sector is faced with many challenges. These include production constraints at farm level and

trade barriers to name but a few (Wafula and Guerre, 2013). Increasing population and

escalating food prices make it difficult to sustain food security at a household level.

Although GM technology has the potential to help address food security, the adoption of

agricultural biotechnology (GM crops) has been slower than expected in the African

continent (Chambers, 2013). The slow adoption of these crops and their products is

attributed to human and environmental safety concerns, ethics, and trade‐related aspects

of GM products (Chambers, 2013). Despite these issues, four African countries have been

actively involved in the commercialization of GM crops and are part of the 10 countries that

have fully functional National Biosafety Frameworks (NBFs) in place while many others

either have interim NBFs or are in the process of developing an NBF (Falck‐Zepeda, et al.,

2013).

97

The future direction of the current study is to screen for an elite line, which exhibits normal

phenotypic characteristics, and one that expresses levels of Vip3Ba protein that are less

than 100 ng/mg TSP but still confer 100 % mortality on Maruca. This line would

subsequently be tested for efficacy in insect bioassays. Should there be no such line, then a

construct that targets the expression of Vip3Ba protein to the chloroplast will be designed

and transformed into cowpea. Following bioassay studies with several independent

transgenic lines expressing Vip3Ba protein, a suitable candidate line will be selected for

further work. This work will involve the backcrossing of the elite line into the existing Bt

cowpea variety with the aim of developing a robust dual‐Bt toxin cowpea for long‐term

sustainability thereby enhancing its resilience against MPB resistance. Such a gene stack in a

farmer‐preferred variety will yield an improved cultivar for subsequent release under the

regulatory framework and adoption by smallholder farmers. This will offer a solution

towards the plight of farmers within the region who depend on cowpea as a staple food

crop.

98

CHAPTER SIX

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APPENDICES

APPENDIX I: SUMMARY OF vip3 GENES SEQUENCING RESULTS

Group 1: vip3Aa35

>vip3Aa35_nucleotide ATGCATCATCACCACCATCACAACAAGAATAATACTAAATTAAGCACAAGAGCCTTACCAAGTTTTATTGATTATTTTAATGGCATTTATGGATTTGCCACTGGTATCAAAGACATTATGAACATGATTTTTAAAACGGATACAGGTGGTGATCTAACCCTAGACGAAATTTTAAAGAATCAGCAGTTACTAAATGATATTTCTGGTAAATTGGATGGGGTGAATGGAAGCTTAAATGATCTTATCGCACAGGGAAACTTAAATACAGAATTATCTAAAGAAATATTAAAAATTGCAAATGAACAAAATCAAGTTTTAAATGATGTTAATAACAAACTCGATGCGATAAATACGATGCTTCGGGTATATCTACCTAAAATTACCTCTATGTTGAGTGATGTAATGAAACAAAATTATGCGCTAAGTCTGCAAATAGAATACTTAAGTAAACAATTGCAAGAGATTTCTGATAAGTTGGATATTATTAATGTAAATGTACTTATTAACTCTACACTTACTGAAATTACACCTGCGTATCAAAGGATTAAATATGTGAACGAAAAATTTGAGGAATTAACTTTTGCTACAGAAACTAGTTCAAAAGTAAAAAAGGATGGCTCTCCTGCAGATATTCTTGATGAGTTAACTGAGTTAACTGAACTAGCGAAAAGTGTAACAAAAAATGATGTGGATGGTTTTGAATTTTACCTTAATACATTCCACGATGTAATGGTAGGAAATAATTTATTCGGGCGTTCAGCTTTAAAAACTGCATCGGAATTAATTACTAAAGAAAATGTGAAAACAAGTGGCAGTGAGGTCGGAAATGTTTATAACTTCTTAATTGTATTAACAGCTCTGCAAGCAAAAGCTTTTCTTACTTTAACAACATGCCGAAAATTATTAGGCTTAGCAGATATTGATTATACTTCTATTATGAATGAACATTTAAATAAGGAAAAAGAGGAATTTAGAGTAAACATCCTCCCTACACTTTCTAATACTTTTTCTAATCCTAATTATGCAAAAGTTAAAGGAAGTGATGAAGATGCAAAGATGATTGTGGAAGCTAAACCAGGACATGCATTGGTTGGGTTTGAAATTAGTAATGATTCAATTACAGTATTAAAAGTATATGAGGCTAAGCTAAAACAAAATTATCAAGTTGATAAGGATTCCTTATCGGAAGTTATTTATGGTGATATGGATAAATTATTGTGCCCAGATCAATCTGAACAAATCTATTATACAAATAACATAGTATTTCCAAATGAATATGTAATTACTAAAATTGATTTTACTAAAAAAATGAAAACTTTAAGATATGAGGTAACAGCGAATTTTTATGATTCTTCTACAGGAGAAATTGACTTAAATAAGAAAAAAGTAGAATCAAGTGAAGCGGAGTATAGAACGTTAAGTGCTAATGATGATGGAGTGTATATGCCGTTAGGTGTCATCAGTGAAACATTTTTGACTCCGATTAATGGGTTTGGCCTCCAAGCTGATGAAAATTCAAGATTAATTACTTTAACATGTAAATCATATTTAAGAGAACTACTGCTAGCAACAGACTTAAGCAATAAAGAAACTAAATTGATCGTCCCGCCAAGTGGTTTTATTAAAAATATTGTAGAGAACGGGTCCATAGAAGAGGACAATTTAGAGCCGTGGAAAGCAAATAATAAGAATGCGTATGTAGATCATACAGGCGGAGTGAATGGAACTAAAGCTTTATATGTTCATAAcGACGGAGGAATTTCACAATTTATTGGAGATAAGTTAAAACCGAAAACTGAGTATGTAATCCAATATACTGTTAAAGGAAAACCTTCTATTCATTTAAAAGATGAAAATACTGGATATATTCATTATGAAGATACAAATAATAATTTAGAAGATTATCAAACTATTACTAAACGTTTTACTACAGGAACTGATTTAAAGGGAGTGTATTTAATTTTAAAAAGTCAAAATGGAGATGAAGCTTGGGGAGATAACTTTATTATTTTGGAAATTAGTCCTTCTGAAAAGTTATTAAGTCCAGAATTAATTAATACAAATAATTGGACGAGTACGGGATCAACTAATATTAGCGGTAATACACTCACTCTTTATCAGGGAGGACGAGGAATTCTAAAACAAAACCTTCAATTAGATAGTTTTTCAACTTATAGAGTGTATTTTTCTGTGTCCGGAGATGCTAATGTAAGGATTAGAAATTcTAGGGAAGTGTTATTTGAAAAAAGATATATGAGCGGTGCTAAAGATGTTTCTGAAATTTTCACTACAAAATTGGGAAAAGATAACTTTTATATAGAGCTTTCTCAAGGGAATAATTTAAATGGTGGCCCTATTGTCAAGTTTTCCGATGTCTCTATTAAGTAA Alignment of vip3Aa35 with reference GU733921.1 my_vip3Aa35 1 ATGCATCATCACCACCATCACAACAAGAATAATACTAAATTAAGCACAAG 50 ||| ||||||||||||||||||||||||||||| GU733921.1 1 ATG------------------AACAAGAATAATACTAAATTAAGCACAAG 32 my_vip3Aa35 51 AGCCTTACCAAGTTTTATTGATTATTTTAATGGCATTTATGGATTTGCCA 100 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 33 AGCCTTACCAAGTTTTATTGATTATTTTAATGGCATTTATGGATTTGCCA 82 my_vip3Aa35 101 CTGGTATCAAAGACATTATGAACATGATTTTTAAAACGGATACAGGTGGT 150 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 83 CTGGTATCAAAGACATTATGAACATGATTTTTAAAACGGATACAGGTGGT 132 my_vip3Aa35 151 GATCTAACCCTAGACGAAATTTTAAAGAATCAGCAGTTACTAAATGATAT 200

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|||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 133 GATCTAACCCTAGACGAAATTTTAAAGAATCAGCAGTTACTAAATGATAT 182 my_vip3Aa35 201 TTCTGGTAAATTGGATGGGGTGAATGGAAGCTTAAATGATCTTATCGCAC 250 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 183 TTCTGGTAAATTGGATGGGGTGAATGGAAGCTTAAATGATCTTATCGCAC 232 my_vip3Aa35 251 AGGGAAACTTAAATACAGAATTATCTAAAGAAATATTAAAAATTGCAAAT 300 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 233 AGGGAAACTTAAATACAGAATTATCTAAAGAAATATTAAAAATTGCAAAT 282 my_vip3Aa35 301 GAACAAAATCAAGTTTTAAATGATGTTAATAACAAACTCGATGCGATAAA 350 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 283 GAACAAAATCAAGTTTTAAATGATGTTAATAACAAACTCGATGCGATAAA 332 my_vip3Aa35 351 TACGATGCTTCGGGTATATCTACCTAAAATTACCTCTATGTTGAGTGATG 400 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 333 TACGATGCTTCGGGTATATCTACCTAAAATTACCTCTATGTTGAGTGATG 382 my_vip3Aa35 401 TAATGAAACAAAATTATGCGCTAAGTCTGCAAATAGAATACTTAAGTAAA 450 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 383 TAATGAAACAAAATTATGCGCTAAGTCTGCAAATAGAATACTTAAGTAAA 432 my_vip3Aa35 451 CAATTGCAAGAGATTTCTGATAAGTTGGATATTATTAATGTAAATGTACT 500 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 433 CAATTGCAAGAGATTTCTGATAAGTTGGATATTATTAATGTAAATGTACT 482 my_vip3Aa35 501 TATTAACTCTACACTTACTGAAATTACACCTGCGTATCAAAGGATTAAAT 550 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 483 TATTAACTCTACACTTACTGAAATTACACCTGCGTATCAAAGGATTAAAT 532 my_vip3Aa35 551 ATGTGAACGAAAAATTTGAGGAATTAACTTTTGCTACAGAAACTAGTTCA 600 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 533 ATGTGAACGAAAAATTTGAGGAATTAACTTTTGCTACAGAAACTAGTTCA 582 my_vip3Aa35 601 AAAGTAAAAAAGGATGGCTCTCCTGCAGATATTCTTGATGAGTTAACTGA 650 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 583 AAAGTAAAAAAGGATGGCTCTCCTGCAGATATTCTTGATGAGTTAACTGA 632 my_vip3Aa35 651 GTTAACTGAACTAGCGAAAAGTGTAACAAAAAATGATGTGGATGGTTTTG 700 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 633 GTTAACTGAACTAGCGAAAAGTGTAACAAAAAATGATGTGGATGGTTTTG 682 my_vip3Aa35 701 AATTTTACCTTAATACATTCCACGATGTAATGGTAGGAAATAATTTATTC 750 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 683 AATTTTACCTTAATACATTCCACGATGTAATGGTAGGAAATAATTTATTC 732 my_vip3Aa35 751 GGGCGTTCAGCTTTAAAAACTGCATCGGAATTAATTACTAAAGAAAATGT 800 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 733 GGGCGTTCAGCTTTAAAAACTGCATCGGAATTAATTACTAAAGAAAATGT 782 my_vip3Aa35 801 GAAAACAAGTGGCAGTGAGGTCGGAAATGTTTATAACTTCTTAATTGTAT 850 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 783 GAAAACAAGTGGCAGTGAGGTCGGAAATGTTTATAACTTCTTAATTGTAT 832 my_vip3Aa35 851 TAACAGCTCTGCAAGCAAAAGCTTTTCTTACTTTAACAACATGCCGAAAA 900 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 833 TAACAGCTCTGCAAGCAAAAGCTTTTCTTACTTTAACAACATGCCGAAAA 882 my_vip3Aa35 901 TTATTAGGCTTAGCAGATATTGATTATACTTCTATTATGAATGAACATTT 950 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 883 TTATTAGGCTTAGCAGATATTGATTATACTTCTATTATGAATGAACATTT 932 my_vip3Aa35 951 AAATAAGGAAAAAGAGGAATTTAGAGTAAACATCCTCCCTACACTTTCTA 1000 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 933 AAATAAGGAAAAAGAGGAATTTAGAGTAAACATCCTCCCTACACTTTCTA 982 my_vip3Aa35 1001 ATACTTTTTCTAATCCTAATTATGCAAAAGTTAAAGGAAGTGATGAAGAT 1050

125

|||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 983 ATACTTTTTCTAATCCTAATTATGCAAAAGTTAAAGGAAGTGATGAAGAT 1032 my_vip3Aa35 1051 GCAAAGATGATTGTGGAAGCTAAACCAGGACATGCATTGGTTGGGTTTGA 1100 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 1033 GCAAAGATGATTGTGGAAGCTAAACCAGGACATGCATTGGTTGGGTTTGA 1082 my_vip3Aa35 1101 AATTAGTAATGATTCAATTACAGTATTAAAAGTATATGAGGCTAAGCTAA 1150 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 1083 AATTAGTAATGATTCAATTACAGTATTAAAAGTATATGAGGCTAAGCTAA 1132 my_vip3Aa35 1151 AACAAAATTATCAAGTTGATAAGGATTCCTTATCGGAAGTTATTTATGGT 1200 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 1133 AACAAAATTATCAAGTTGATAAGGATTCCTTATCGGAAGTTATTTATGGT 1182 my_vip3Aa35 1201 GATATGGATAAATTATTGTGCCCAGATCAATCTGAACAAATCTATTATAC 1250 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 1183 GATATGGATAAATTATTGTGCCCAGATCAATCTGAACAAATCTATTATAC 1232 my_vip3Aa35 1251 AAATAACATAGTATTTCCAAATGAATATGTAATTACTAAAATTGATTTTA 1300 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 1233 AAATAACATAGTATTTCCAAATGAATATGTAATTACTAAAATTGATTTTA 1282 my_vip3Aa35 1301 CTAAAAAAATGAAAACTTTAAGATATGAGGTAACAGCGAATTTTTATGAT 1350 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 1283 CTAAAAAAATGAAAACTTTAAGATATGAGGTAACAGCGAATTTTTATGAT 1332 my_vip3Aa35 1351 TCTTCTACAGGAGAAATTGACTTAAATAAGAAAAAAGTAGAATCAAGTGA 1400 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 1333 TCTTCTACAGGAGAAATTGACTTAAATAAGAAAAAAGTAGAATCAAGTGA 1382 my_vip3Aa35 1401 AGCGGAGTATAGAACGTTAAGTGCTAATGATGATGGAGTGTATATGCCGT 1450 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 1383 AGCGGAGTATAGAACGTTAAGTGCTAATGATGATGGAGTGTATATGCCGT 1432 my_vip3Aa35 1451 TAGGTGTCATCAGTGAAACATTTTTGACTCCGATTAATGGGTTTGGCCTC 1500 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 1433 TAGGTGTCATCAGTGAAACATTTTTGACTCCGATTAATGGGTTTGGCCTC 1482 my_vip3Aa35 1501 CAAGCTGATGAAAATTCAAGATTAATTACTTTAACATGTAAATCATATTT 1550 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 1483 CAAGCTGATGAAAATTCAAGATTAATTACTTTAACATGTAAATCATATTT 1532 my_vip3Aa35 1551 AAGAGAACTACTGCTAGCAACAGACTTAAGCAATAAAGAAACTAAATTGA 1600 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 1533 AAGAGAACTACTGCTAGCAACAGACTTAAGCAATAAAGAAACTAAATTGA 1582 my_vip3Aa35 1601 TCGTCCCGCCAAGTGGTTTTATTAAAAATATTGTAGAGAACGGGTCCATA 1650 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 1583 TCGTCCCGCCAAGTGGTTTTATTAAAAATATTGTAGAGAACGGGTCCATA 1632 my_vip3Aa35 1651 GAAGAGGACAATTTAGAGCCGTGGAAAGCAAATAATAAGAATGCGTATGT 1700 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 1633 GAAGAGGACAATTTAGAGCCGTGGAAAGCAAATAATAAGAATGCGTATGT 1682 my_vip3Aa35 1701 AGATCATACAGGCGGAGTGAATGGAACTAAAGCTTTATATGTTCATAAcG 1750 ||||||||||||||||||||||||||||||||||||||||||||||||.| GU733921.1 1683 AGATCATACAGGCGGAGTGAATGGAACTAAAGCTTTATATGTTCATAAGG 1732 my_vip3Aa35 1751 ACGGAGGAATTTCACAATTTATTGGAGATAAGTTAAAACCGAAAACTGAG 1800 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 1733 ACGGAGGAATTTCACAATTTATTGGAGATAAGTTAAAACCGAAAACTGAG 1782 my_vip3Aa35 1801 TATGTAATCCAATATACTGTTAAAGGAAAACCTTCTATTCATTTAAAAGA 1850 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 1783 TATGTAATCCAATATACTGTTAAAGGAAAACCTTCTATTCATTTAAAAGA 1832 my_vip3Aa35 1851 TGAAAATACTGGATATATTCATTATGAAGATACAAATAATAATTTAGAAG 1900

126

|||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 1833 TGAAAATACTGGATATATTCATTATGAAGATACAAATAATAATTTAGAAG 1882 my_vip3Aa35 1901 ATTATCAAACTATTACTAAACGTTTTACTACAGGAACTGATTTAAAGGGA 1950 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 1883 ATTATCAAACTATTACTAAACGTTTTACTACAGGAACTGATTTAAAGGGA 1932 my_vip3Aa35 1951 GTGTATTTAATTTTAAAAAGTCAAAATGGAGATGAAGCTTGGGGAGATAA 2000 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 1933 GTGTATTTAATTTTAAAAAGTCAAAATGGAGATGAAGCTTGGGGAGATAA 1982 my_vip3Aa35 2001 CTTTATTATTTTGGAAATTAGTCCTTCTGAAAAGTTATTAAGTCCAGAAT 2050 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 1983 CTTTATTATTTTGGAAATTAGTCCTTCTGAAAAGTTATTAAGTCCAGAAT 2032 my_vip3Aa35 2051 TAATTAATACAAATAATTGGACGAGTACGGGATCAACTAATATTAGCGGT 2100 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 2033 TAATTAATACAAATAATTGGACGAGTACGGGATCAACTAATATTAGCGGT 2082 my_vip3Aa35 2101 AATACACTCACTCTTTATCAGGGAGGACGAGGAATTCTAAAACAAAACCT 2150 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 2083 AATACACTCACTCTTTATCAGGGAGGACGAGGAATTCTAAAACAAAACCT 2132 my_vip3Aa35 2151 TCAATTAGATAGTTTTTCAACTTATAGAGTGTATTTTTCTGTGTCCGGAG 2200 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 2133 TCAATTAGATAGTTTTTCAACTTATAGAGTGTATTTTTCTGTGTCCGGAG 2182 my_vip3Aa35 2201 ATGCTAATGTAAGGATTAGAAATTcTAGGGAAGTGTTATTTGAAAAAAGA 2250 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 2183 ATGCTAATGTAAGGATTAGAAATTCTAGGGAAGTGTTATTTGAAAAAAGA 2232 my_vip3Aa35 2251 TATATGAGCGGTGCTAAAGATGTTTCTGAAATTTTCACTACAAAATTGGG 2300 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 2233 TATATGAGCGGTGCTAAAGATGTTTCTGAAATTTTCACTACAAAATTGGG 2282 my_vip3Aa35 2301 AAAAGATAACTTTTATATAGAGCTTTCTCAAGGGAATAATTTAAATGGTG 2350 |||||||||||||||||||||||||||||||||||||||||||||||||| GU733921.1 2283 AAAAGATAACTTTTATATAGAGCTTTCTCAAGGGAATAATTTAAATGGTG 2332 my_vip3Aa35 2351 GCCCTATTGTCAAGTTTTCCGATGTCTCTATTAAGTAA 2388 |||||||||||||||||||||||||||||||||||||| GU733921.1 2333 GCCCTATTGTCAAGTTTTCCGATGTCTCTATTAAGTAA 2370

The derived amino acid sequence from vip3Aa35 >vip3Aa35_amino acid (795 amino acids) MHHHHHHNKNNTKLSTRALPSFIDYFNGIYGFATGIKDIMNMIFKTDTGGDLTLDEILKNQQLLNDISGKLDGVNGSLNDLIAQGNLNTELSKEILKIANEQNQVLNDVNNKLDAINTMLRVYLPKITSMLSDVMKQNYALSLQIEYLSKQLQEISDKLDIINVNVLINSTLTEITPAYQRIKYVNEKFEELTFATETSSKVKKDGSPADILDELTELTELAKSVTKNDVDGFEFYLNTFHDVMVGNNLFGRSALKTASELITKENVKTSGSEVGNVYNFLIVLTALQAKAFLTLTTCRKLLGLADIDYTSIMNEHLNKEKEEFRVNILPTLSNTFSNPNYAKVKGSDEDAKMIVEAKPGHALVGFEISNDSITVLKVYEAKLKQNYQVDKDSLSEVIYGDMDKLLCPDQSEQIYYTNNIVFPNEYVITKIDFTKKMKTLRYEVTANFYDSSTGEIDLNKKKVESSEAEYRTLSANDDGVYMPLGVISETFLTPINGFGLQADENSRLITLTCKSYLRELLLATDLSNKETKLIVPPSGFIKNIVENGSIEEDNLEPWKANNKNAYVDHTGGVNGTKALYVHNDGGISQFIGDKLKPKTEYVIQYTVKGKPSIHLKDENTGYIHYEDTNNNLEDYQTITKRFTTGTDLKGVYLILKSQNGDEAWGDNFIILEISPSEKLLSPELINTNNWTSTGSTNISGNTLTLYQGGRGILKQNLQLDSFSTYRVYFSVSGDANVRIRNSREVLFEKRYMSGAKDVSEIFTTKLGKDNFYIELSQGNNLNGGPIVKFSDVSIK*

127

Group 2: vip3Af

>vip3Af_nucleotide ATGCATCATCACCACCATCACAATATGAATAATACTAAATTAAACGCAAGGGCCCTACCGAGTTTTATTGATTATTTTAATGGCATTTATGGATTTGCCACTGGTATCAAAGACATTATGAATATGATTTTTAAAACGGATACAGGTGGTAATCTAACCTTAGACGAAATCCTAAAGAATCAGCAGTTACTAAATGAGATTTCTGGTAAATTGGATGGGGTAAATGGGAGCTTAAATGATCTTATCGCACAGGGAAACTTAAATACAGAATTATCTAAGGAAATCTTAAAAATTGCAAATGAACAGAATCAAGTCTTAAATGATGTTAATAACAAACTCGATGCGATAAATACGATGCTTCATATATATCTACCTAAAATTACATCTATGTTAAGTGATGTAATGAAGCAAAATTATGCGCTAAGTCTGCAAATAGAATACTTAAGTAAGCAATTGCAAGAAATTTCTGATAAATTAGATATTATTAACGTAAATGTTCTTATTAACTCTACACTTACTGAAATTACACCTGCATATCAACGGATTAAATATGTGAATGAAAAATTTGAAGAATTAACTTTTGCTACAGAAACCACTTTAAAAGTAAAAAAGGATAGCTCGCCTGCTGATATTCTTGATGAGTTAACTGAATTAACTGAACTAGCGAAAAGTGTTACAAAAAATGACGTTGATGGTTTTGAATTTTACCTTAATACATTCCACGATGTAATGGTAGGAAATAATTTATTCGGGCGTTCAGCTTTAAAAACTGCTTCAGAATTAATTGCTAAAGAAAATGTGAAAACAAGTGGCAGTGAAGTAGGAAATGTTTATAATTTCTTAATTGTATTAACAGCTCTACAAGCAAAAGCTTTTCTTACTTTAACAACATGCCGAAAATTATTAGGCTTAGCAGATATTGATTATACTTCTATTATGAATGAACATTTAAATAAGGAAAAAGAGGAATTTAGAGTAAACATCCTTCCTACACTTTCTAATACTTTTTCTAATCCTAATTATGCAAAAGTTAAAGGAAGTGATGAAGATGCAAAGATGATTGTGGAAGCTAAACCAGGACATGCATTGGTTGGGTTTGAAATGAGCAATGATTCAATCACAGTATTAAAAGTATATGAGGCTAAGCTAAAACAAAATTATCAAGTTGATAAGGATTCCTTATCGGAGGTTATTTATGGTGATACGGATAAATTATTTTGTCCAGATCAATCTGAACAAATATATTATACAAATAACATAGTATTCCCAAATGAATATGTAATTACTAAAATTGATTTCACTAAAAAAATGAAAACTTTAAGATATGAGGTAACAGCGAATTTTTATGATTCTTCTACAGGAGAAATTGACTTAAATAAGAAAAAAGTAGAATCAAGTGAAGCGGAGTATAGAACGTTAAGTGCTAATGATGATGGAGTGTATATGCCATTAGGTGTCATCAGTGAAACATTTTTGACTCCGATAAATGGGTTTGGCCTCCAAGCTGATGAAAATTCAAGATTAATTACTTTAACATGTAAATCATATTTAAGAGAACTACTGCTAGCAACAGACTTAAGCAATAAAGAAACTAAATTGATCGTCCCACCAAGTGGTTTTATTAGCAATATTGTAGAGAACGGGTCCATAGAAGAGGACAATTTAGAGCCGTGGAAAGCAAATAATAAGAATGCGTATGTAGATCATACAGGCGGAGTGAATGGAACTAAAGCTTTATATGTTCATAAGGACGGAGGATTTTCACAATTTATTGGAGATAAGTTAAAACCGAAAACTGAGTATGTAATCCAATATACTGTTAAAGGAAAACCTTCTATTCATTTAAAAGATGAAAATACTGGATATATTCATTATGAAGATACAAATAATAATTTAAAAGATTATCAAACTATTACTAAACGTTTTACTACAGGAACTGATTTAAAGGGAGTGTATTTAATTTTAAAAAGTCAAAATGGAGATGAAGCTTGGGGAGATAAATTTACAATTTTAGAAATTAAGCCTGCGGAGGATTTATTAAGCCCAGAATTAATTAATCCGAATTCTTGGATTACGACTCCAGGGGCTAGCATTTCAGGAAATAAACTTTTCATTAACTTGGGGACAAATGGGACCTTTAGACAAAGTCTTTCATTAAACAGTTATTCAACTTATAGTATAAGCTTTACTGCATCAGGACCATTTAATGTGACGGTAAGAAATTCTAGGGAAGTATTATTTGAACGAAGCAACCTTATGTCTTCAACTAGTCATATTTCTGGGACATTCAAAACTGAATCCAATAATACCGGATTATATGTAGAACTTTCCCGTCGCTCTGGTGGTGGTGGTCATATATCATTTGAAAACGTTTCTATTAAATAA Alignment of vip3Af with reference AJ872070.1 my_vip3Af 1 ATGCATCATCACCACCATCACAATATGAATAATACTAAATTAAACGCAAG 50 ||| ||||||||||||||||||||||||||||| AJ872070.1 1 ATG------------------AATATGAATAATACTAAATTAAACGCAAG 32 my_vip3Af 51 GGCCCTACCGAGTTTTATTGATTATTTTAATGGCATTTATGGATTTGCCA 100 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 33 GGCCCTACCGAGTTTTATTGATTATTTTAATGGCATTTATGGATTTGCCA 82 my_vip3Af 101 CTGGTATCAAAGACATTATGAATATGATTTTTAAAACGGATACAGGTGGT 150 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 83 CTGGTATCAAAGACATTATGAATATGATTTTTAAAACGGATACAGGTGGT 132 my_vip3Af 151 AATCTAACCTTAGACGAAATCCTAAAGAATCAGCAGTTACTAAATGAGAT 200 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 133 AATCTAACCTTAGACGAAATCCTAAAGAATCAGCAGTTACTAAATGAGAT 182 my_vip3Af 201 TTCTGGTAAATTGGATGGGGTAAATGGGAGCTTAAATGATCTTATCGCAC 250 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 183 TTCTGGTAAATTGGATGGGGTAAATGGGAGCTTAAATGATCTTATCGCAC 232 my_vip3Af 251 AGGGAAACTTAAATACAGAATTATCTAAGGAAATCTTAAAAATTGCAAAT 300 ||||||||||||||||||||||||||||||||||||||||||||||||||

128

AJ872070.1 233 AGGGAAACTTAAATACAGAATTATCTAAGGAAATCTTAAAAATTGCAAAT 282 my_vip3Af 301 GAACAGAATCAAGTCTTAAATGATGTTAATAACAAACTCGATGCGATAAA 350 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 283 GAACAGAATCAAGTCTTAAATGATGTTAATAACAAACTCGATGCGATAAA 332 my_vip3Af 351 TACGATGCTTCATATATATCTACCTAAAATTACATCTATGTTAAGTGATG 400 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 333 TACGATGCTTCATATATATCTACCTAAAATTACATCTATGTTAAGTGATG 382 my_vip3Af 401 TAATGAAGCAAAATTATGCGCTAAGTCTGCAAATAGAATACTTAAGTAAG 450 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 383 TAATGAAGCAAAATTATGCGCTAAGTCTGCAAATAGAATACTTAAGTAAG 432 my_vip3Af 451 CAATTGCAAGAAATTTCTGATAAATTAGATATTATTAACGTAAATGTTCT 500 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 433 CAATTGCAAGAAATTTCTGATAAATTAGATATTATTAACGTAAATGTTCT 482 my_vip3Af 501 TATTAACTCTACACTTACTGAAATTACACCTGCATATCAACGGATTAAAT 550 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 483 TATTAACTCTACACTTACTGAAATTACACCTGCATATCAACGGATTAAAT 532 my_vip3Af 551 ATGTGAATGAAAAATTTGAAGAATTAACTTTTGCTACAGAAACCACTTTA 600 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 533 ATGTGAATGAAAAATTTGAAGAATTAACTTTTGCTACAGAAACCACTTTA 582 my_vip3Af 601 AAAGTAAAAAAGGATAGCTCGCCTGCTGATATTCTTGATGAGTTAACTGA 650 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 583 AAAGTAAAAAAGGATAGCTCGCCTGCTGATATTCTTGATGAGTTAACTGA 632 my_vip3Af 651 ATTAACTGAACTAGCGAAAAGTGTTACAAAAAATGACGTTGATGGTTTTG 700 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 633 ATTAACTGAACTAGCGAAAAGTGTTACAAAAAATGACGTTGATGGTTTTG 682 my_vip3Af 701 AATTTTACCTTAATACATTCCACGATGTAATGGTAGGAAATAATTTATTC 750 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 683 AATTTTACCTTAATACATTCCACGATGTAATGGTAGGAAATAATTTATTC 732 my_vip3Af 751 GGGCGTTCAGCTTTAAAAACTGCTTCAGAATTAATTGCTAAAGAAAATGT 800 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 733 GGGCGTTCAGCTTTAAAAACTGCTTCAGAATTAATTGCTAAAGAAAATGT 782 my_vip3Af 801 GAAAACAAGTGGCAGTGAAGTAGGAAATGTTTATAATTTCTTAATTGTAT 850 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 783 GAAAACAAGTGGCAGTGAAGTAGGAAATGTTTATAATTTCTTAATTGTAT 832 my_vip3Af 851 TAACAGCTCTACAAGCAAAAGCTTTTCTTACTTTAACAACATGCCGAAAA 900 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 833 TAACAGCTCTACAAGCAAAAGCTTTTCTTACTTTAACAACATGCCGAAAA 882 my_vip3Af 901 TTATTAGGCTTAGCAGATATTGATTATACTTCTATTATGAATGAACATTT 950 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 883 TTATTAGGCTTAGCAGATATTGATTATACTTCTATTATGAATGAACATTT 932 my_vip3Af 951 AAATAAGGAAAAAGAGGAATTTAGAGTAAACATCCTTCCTACACTTTCTA 1000 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 933 AAATAAGGAAAAAGAGGAATTTAGAGTAAACATCCTTCCTACACTTTCTA 982 my_vip3Af 1001 ATACTTTTTCTAATCCTAATTATGCAAAAGTTAAAGGAAGTGATGAAGAT 1050 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 983 ATACTTTTTCTAATCCTAATTATGCAAAAGTTAAAGGAAGTGATGAAGAT 1032 my_vip3Af 1051 GCAAAGATGATTGTGGAAGCTAAACCAGGACATGCATTGGTTGGGTTTGA 1100 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 1033 GCAAAGATGATTGTGGAAGCTAAACCAGGACATGCATTGGTTGGGTTTGA 1082 my_vip3Af 1101 AATGAGCAATGATTCAATCACAGTATTAAAAGTATATGAGGCTAAGCTAA 1150 ||||||||||||||||||||||||||||||||||||||||||||||||||

129

AJ872070.1 1083 AATGAGCAATGATTCAATCACAGTATTAAAAGTATATGAGGCTAAGCTAA 1132 my_vip3Af 1151 AACAAAATTATCAAGTTGATAAGGATTCCTTATCGGAGGTTATTTATGGT 1200 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 1133 AACAAAATTATCAAGTTGATAAGGATTCCTTATCGGAGGTTATTTATGGT 1182 my_vip3Af 1201 GATACGGATAAATTATTTTGTCCAGATCAATCTGAACAAATATATTATAC 1250 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 1183 GATACGGATAAATTATTTTGTCCAGATCAATCTGAACAAATATATTATAC 1232 my_vip3Af 1251 AAATAACATAGTATTCCCAAATGAATATGTAATTACTAAAATTGATTTCA 1300 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 1233 AAATAACATAGTATTCCCAAATGAATATGTAATTACTAAAATTGATTTCA 1282 my_vip3Af 1301 CTAAAAAAATGAAAACTTTAAGATATGAGGTAACAGCGAATTTTTATGAT 1350 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 1283 CTAAAAAAATGAAAACTTTAAGATATGAGGTAACAGCGAATTTTTATGAT 1332 my_vip3Af 1351 TCTTCTACAGGAGAAATTGACTTAAATAAGAAAAAAGTAGAATCAAGTGA 1400 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 1333 TCTTCTACAGGAGAAATTGACTTAAATAAGAAAAAAGTAGAATCAAGTGA 1382 my_vip3Af 1401 AGCGGAGTATAGAACGTTAAGTGCTAATGATGATGGAGTGTATATGCCAT 1450 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 1383 AGCGGAGTATAGAACGTTAAGTGCTAATGATGATGGAGTGTATATGCCAT 1432 my_vip3Af 1451 TAGGTGTCATCAGTGAAACATTTTTGACTCCGATAAATGGGTTTGGCCTC 1500 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 1433 TAGGTGTCATCAGTGAAACATTTTTGACTCCGATAAATGGGTTTGGCCTC 1482 my_vip3Af 1501 CAAGCTGATGAAAATTCAAGATTAATTACTTTAACATGTAAATCATATTT 1550 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 1483 CAAGCTGATGAAAATTCAAGATTAATTACTTTAACATGTAAATCATATTT 1532 my_vip3Af 1551 AAGAGAACTACTGCTAGCAACAGACTTAAGCAATAAAGAAACTAAATTGA 1600 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 1533 AAGAGAACTACTGCTAGCAACAGACTTAAGCAATAAAGAAACTAAATTGA 1582 my_vip3Af 1601 TCGTCCCACCAAGTGGTTTTATTAGCAATATTGTAGAGAACGGGTCCATA 1650 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 1583 TCGTCCCACCAAGTGGTTTTATTAGCAATATTGTAGAGAACGGGTCCATA 1632 my_vip3Af 1651 GAAGAGGACAATTTAGAGCCGTGGAAAGCAAATAATAAGAATGCGTATGT 1700 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 1633 GAAGAGGACAATTTAGAGCCGTGGAAAGCAAATAATAAGAATGCGTATGT 1682 my_vip3Af 1701 AGATCATACAGGCGGAGTGAATGGAACTAAAGCTTTATATGTTCATAAGG 1750 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 1683 AGATCATACAGGCGGAGTGAATGGAACTAAAGCTTTATATGTTCATAAGG 1732 my_vip3Af 1751 ACGGAGGATTTTCACAATTTATTGGAGATAAGTTAAAACCGAAAACTGAG 1800 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 1733 ACGGAGGATTTTCACAATTTATTGGAGATAAGTTAAAACCGAAAACTGAG 1782 my_vip3Af 1801 TATGTAATCCAATATACTGTTAAAGGAAAACCTTCTATTCATTTAAAAGA 1850 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 1783 TATGTAATCCAATATACTGTTAAAGGAAAACCTTCTATTCATTTAAAAGA 1832 my_vip3Af 1851 TGAAAATACTGGATATATTCATTATGAAGATACAAATAATAATTTAAAAG 1900 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 1833 TGAAAATACTGGATATATTCATTATGAAGATACAAATAATAATTTAAAAG 1882 my_vip3Af 1901 ATTATCAAACTATTACTAAACGTTTTACTACAGGAACTGATTTAAAGGGA 1950 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 1883 ATTATCAAACTATTACTAAACGTTTTACTACAGGAACTGATTTAAAGGGA 1932 my_vip3Af 1951 GTGTATTTAATTTTAAAAAGTCAAAATGGAGATGAAGCTTGGGGAGATAA 2000 ||||||||||||||||||||||||||||||||||||||||||||||||||

130

AJ872070.1 1933 GTGTATTTAATTTTAAAAAGTCAAAATGGAGATGAAGCTTGGGGAGATAA 1982 my_vip3Af 2001 ATTTACAATTTTAGAAATTAAGCCTGCGGAGGATTTATTAAGCCCAGAAT 2050 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 1983 ATTTACAATTTTAGAAATTAAGCCTGCGGAGGATTTATTAAGCCCAGAAT 2032 my_vip3Af 2051 TAATTAATCCGAATTCTTGGATTACGACTCCAGGGGCTAGCATTTCAGGA 2100 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 2033 TAATTAATCCGAATTCTTGGATTACGACTCCAGGGGCTAGCATTTCAGGA 2082 my_vip3Af 2101 AATAAACTTTTCATTAACTTGGGGACAAATGGGACCTTTAGACAAAGTCT 2150 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 2083 AATAAACTTTTCATTAACTTGGGGACAAATGGGACCTTTAGACAAAGTCT 2132 my_vip3Af 2151 TTCATTAAACAGTTATTCAACTTATAGTATAAGCTTTACTGCATCAGGAC 2200 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 2133 TTCATTAAACAGTTATTCAACTTATAGTATAAGCTTTACTGCATCAGGAC 2182 my_vip3Af 2201 CATTTAATGTGACGGTAAGAAATTCTAGGGAAGTATTATTTGAACGAAGC 2250 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 2183 CATTTAATGTGACGGTAAGAAATTCTAGGGAAGTATTATTTGAACGAAGC 2232 my_vip3Af 2251 AACCTTATGTCTTCAACTAGTCATATTTCTGGGACATTCAAAACTGAATC 2300 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 2233 AACCTTATGTCTTCAACTAGTCATATTTCTGGGACATTCAAAACTGAATC 2282 my_vip3Af 2301 CAATAATACCGGATTATATGTAGAACTTTCCCGTCGCTCTGGTGGTGGTG 2350 |||||||||||||||||||||||||||||||||||||||||||||||||| AJ872070.1 2283 CAATAATACCGGATTATATGTAGAACTTTCCCGTCGCTCTGGTGGTGGTG 2332 my_vip3Af 2351 GTCATATATCATTTGAAAACGTTTCTATTAAATAA 2385 ||||||||||||||||||||||||||||||||||| AJ872070.1 2333 GTCATATATCATTTGAAAACGTTTCTATTAAATAA 2367

The derived amino acid sequence from vip3Af >vip3Af_amino acid (794 amino acids) MHHHHHHNMNNTKLNARALPSFIDYFNGIYGFATGIKDIMNMIFKTDTGGNLTLDEILKNQQLLNEISGKLDGVNGSLNDLIAQGNLNTELSKEILKIANEQNQVLNDVNNKLDAINTMLHIYLPKITSMLSDVMKQNYALSLQIEYLSKQLQEISDKLDIINVNVLINSTLTEITPAYQRIKYVNEKFEELTFATETTLKVKKDSSPADILDELTELTELAKSVTKNDVDGFEFYLNTFHDVMVGNNLFGRSALKTASELIAKENVKTSGSEVGNVYNFLIVLTALQAKAFLTLTTCRKLLGLADIDYTSIMNEHLNKEKEEFRVNILPTLSNTFSNPNYAKVKGSDEDAKMIVEAKPGHALVGFEMSNDSITVLKVYEAKLKQNYQVDKDSLSEVIYGDTDKLFCPDQSEQIYYTNNIVFPNEYVITKIDFTKKMKTLRYEVTANFYDSSTGEIDLNKKKVESSEAEYRTLSANDDGVYMPLGVISETFLTPINGFGLQADENSRLITLTCKSYLRELLLATDLSNKETKLIVPPSGFISNIVENGSIEEDNLEPWKANNKNAYVDHTGGVNGTKALYVHKDGGFSQFIGDKLKPKTEYVIQYTVKGKPSIHLKDENTGYIHYEDTNNNLKDYQTITKRFTTGTDLKGVYLILKSQNGDEAWGDKFTILEIKPAEDLLSPELINPNSWITTPGASISGNKLFINLGTNGTFRQSLSLNSYSTYSISFTASGPFNVTVRNSREVLFERSNLMSSTSHISGTFKTESNNTGLYVELSRRSGGGGHISFENVSIK*

Group 3: vip3Ag

>vip3Ag_nucleotide ATGCATCATCACCACCATCACAACAAGAATAATACTAAATTAAGCGCAAGGGCCTTACCGAGTTTTATTGATTATTTTAATGGCATTTATGGATTTGCCACTGGTATCAAAGACATTATGAACATGATTTTTAAAACGGATACAGGTGGAAATCTAACCCTAGACGAAATTTTAAAAAATCAGCAGTTATTAAATGAGATTTCTGGTAAATTGGATGGGGTAAATGGGAGCTTAAACGATCTTATCGCACAGGGAAACTTAAATACAGAATTATCTAAGGAAATCTTAAAAATTGCAAATGAGCAGAATCAAGTCTTAAATGATGTTAATAACAAACTTAATGCGATAAATACAATGCTTCACATATATCTACCTAAAATTACATCTATGTTAAATGATGTAATGAAACAAAATTATGCACTAAGTCTGCAAATAGAATACCTAAGTAAACAATTGCAAGAAATTTCCGACAAGTTAGATGTCATTAACGTGAATGTACTTATTAACTCTACACTTACTGAAATTACACCTGCGTATCAACGGATGAAATATGTAAATGAAAAATTTGAAGATTTAACTTTTGCTACAGAAACCACTTTA

131

AAAGTAAAAAAGAATAGCTCCCCTGCAGATATTCTTGATGAGTTAACTGAGTTAACTGAACTAGCGAAAAGTGTAACAAAAAATGACGTGGATGGTTTTGAATTTTACCTTAATACATTCCACGATGTAATGGTAGGAAACAATTTATTCGGGCGTTCAGCTTTAAAAACTGCTTCGGAATTAATCGCTAAAGAAAATGTGAAAACAAGTGGCAGTGAGGTAGGAAATGTTTATAATTTCTTAATTGTATTAACAGCTCTGCAAGCAAAAGCTTTTCTTACTTTAACAACATGCCGGAAATTATTAGGCTTAGCAGATATTGATTATACTTTCATTATGAATGAACATTTAGATAAGGAAAAAGAGGAATTTAGAGTAAATATCCTTCCTACACTTTCTAATACTTTTTCTAATCCTAACTATGCAAAAGCTAAAGGAAGCAATGAAGATGCAAAGATAATTGTGGAAGCTAAACCAGGATATGCTTTGGTTGGATTTGAAATGAGCAATGATTCAATCACAGTATTAAAAGCATATCAGGCTAAGCTAAAACAAGATTATCAAGTTGATAAAGATTCGTTATCAGAAATTGTCTATGGTGATATGGATAAATTATTGTGCCCGGATCAATCTGAACAAATATATTATACAAATAACATTGCTTTTCCCAATGAATATGTAATTACTAAAATTACTTTTACTAAAAAAATGAATAGTTTAAGATATGAGGCAACAGCTAATTTTTATGATTCTTCTACAGGGGATATTGATCTAAATAAGACAAAAGTAGAATCAAGTGAAGCAGAGTATAGTACGCTAAGTGCTAGTACTGATGGAGTCTATATGCCGTTAGGTATTATCAGTGAAACATTTTTGACTCCAATTAATGGGTTTGGAATCGTAGTCGATGAAAATTCAAAATTAGTAAATTTAACATGTAAATCATATTTAAGAGAGGTATTATTAGCAACAGACTTAAGTAATAAAGAAACTAAATTGATTGTCCCACCTATTGGTTTTATTAGCAATATTGTAGAAAATGGGAACTTAGAGGGAGAAAACTTAGAGCCGTGGAAAGCAAATAACAAAAATGCGTATGTAGATCATACAGGCGGCGTAAATGGAACTAAAGCTTTATATGTTCATAAGGATGGTGAGTTTTCACAATTTATTGGAGATAAGTTGAAATCGAAAACAGAATATGTAATTCAATATATTGTAAAGGGAAAAGCTTCTATTCTTTTGAAAGATGAAAAAAATGGTGATTGCATTTATGAAGATACAAATAATGGTTTAGAAGATTTTCAAACCATTACTAAAAGTTTTATTACAGGAACGGATTCTTCAGGAGTTCATTTAATATTTAATAGTCAAAATGGCGATGAAGCATTTGGGGAAAACTTTACTATTTCAGAAATTAGGCTTTCCGAAGATTTATTAAGTCCAGAATTGATAAATTCAGATGCTTGGGTTGGATCTCAGGGAACTTGGATCTCAGGAAATTCACTCACTATTAATAGTAATGTGAATGGAACTTTTCGACAAAACCTTTCGTTAGAAAGCTATTCAACTTATAGTATGAACTTTAATGTGAATGGATTTGCCAAGGTGACAGTAAGAAATTCCCGTGAAGTATTATTTGAAAAAAATTATCCGCAGCTTTCACCTAAAGATATTTCTGAAAAATTCACAACTGCAGCCAATAATACCGGGTTGTATGTAGAGCTTTCTCGTTTTACATCGGGTGGCGCTATAAATTTCCGAGATTTTTCAATTAAGTAA Alignment of vip3Ag with reference FJ556803.2 my_vip3Ag 1 ATGCATCATCACCACCATCACAACAAGAATAATACTAAATTAAGCGCAAG 50 ||| ||||||||||||||||||||||||||||| FJ556803.2 1 ATG------------------AACAAGAATAATACTAAATTAAGCGCAAG 32 my_vip3Ag 51 GGCCTTACCGAGTTTTATTGATTATTTTAATGGCATTTATGGATTTGCCA 100 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 33 GGCCTTACCGAGTTTTATTGATTATTTTAATGGCATTTATGGATTTGCCA 82 my_vip3Ag 101 CTGGTATCAAAGACATTATGAACATGATTTTTAAAACGGATACAGGTGGA 150 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 83 CTGGTATCAAAGACATTATGAACATGATTTTTAAAACGGATACAGGTGGA 132 my_vip3Ag 151 AATCTAACCCTAGACGAAATTTTAAAAAATCAGCAGTTATTAAATGAGAT 200 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 133 AATCTAACCCTAGACGAAATTTTAAAAAATCAGCAGTTATTAAATGAGAT 182 my_vip3Ag 201 TTCTGGTAAATTGGATGGGGTAAATGGGAGCTTAAACGATCTTATCGCAC 250 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 183 TTCTGGTAAATTGGATGGGGTAAATGGGAGCTTAAACGATCTTATCGCAC 232 my_vip3Ag 251 AGGGAAACTTAAATACAGAATTATCTAAGGAAATCTTAAAAATTGCAAAT 300 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 233 AGGGAAACTTAAATACAGAATTATCTAAGGAAATCTTAAAAATTGCAAAT 282 my_vip3Ag 301 GAGCAGAATCAAGTCTTAAATGATGTTAATAACAAACTTAATGCGATAAA 350 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 283 GAGCAGAATCAAGTCTTAAATGATGTTAATAACAAACTTAATGCGATAAA 332 my_vip3Ag 351 TACAATGCTTCACATATATCTACCTAAAATTACATCTATGTTAAATGATG 400 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 333 TACAATGCTTCACATATATCTACCTAAAATTACATCTATGTTAAATGATG 382 my_vip3Ag 401 TAATGAAACAAAATTATGCACTAAGTCTGCAAATAGAATACCTAAGTAAA 450 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 383 TAATGAAACAAAATTATGCACTAAGTCTGCAAATAGAATACCTAAGTAAA 432

132

my_vip3Ag 451 CAATTGCAAGAAATTTCCGACAAGTTAGATGTCATTAACGTGAATGTACT 500 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 433 CAATTGCAAGAAATTTCCGACAAGTTAGATGTCATTAACGTGAATGTACT 482 my_vip3Ag 501 TATTAACTCTACACTTACTGAAATTACACCTGCGTATCAACGGATGAAAT 550 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 483 TATTAACTCTACACTTACTGAAATTACACCTGCGTATCAACGGATGAAAT 532 my_vip3Ag 551 ATGTAAATGAAAAATTTGAAGATTTAACTTTTGCTACAGAAACCACTTTA 600 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 533 ATGTAAATGAAAAATTTGAAGATTTAACTTTTGCTACAGAAACCACTTTA 582 my_vip3Ag 601 AAAGTAAAAAAGAATAGCTCCCCTGCAGATATTCTTGATGAGTTAACTGA 650 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 583 AAAGTAAAAAAGAATAGCTCCCCTGCAGATATTCTTGATGAGTTAACTGA 632 my_vip3Ag 651 GTTAACTGAACTAGCGAAAAGTGTAACAAAAAATGACGTGGATGGTTTTG 700 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 633 GTTAACTGAACTAGCGAAAAGTGTAACAAAAAATGACGTGGATGGTTTTG 682 my_vip3Ag 701 AATTTTACCTTAATACATTCCACGATGTAATGGTAGGAAACAATTTATTC 750 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 683 AATTTTACCTTAATACATTCCACGATGTAATGGTAGGAAACAATTTATTC 732 my_vip3Ag 751 GGGCGTTCAGCTTTAAAAACTGCTTCGGAATTAATCGCTAAAGAAAATGT 800 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 733 GGGCGTTCAGCTTTAAAAACTGCTTCGGAATTAATCGCTAAAGAAAATGT 782 my_vip3Ag 801 GAAAACAAGTGGCAGTGAGGTAGGAAATGTTTATAATTTCTTAATTGTAT 850 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 783 GAAAACAAGTGGCAGTGAGGTAGGAAATGTTTATAATTTCTTAATTGTAT 832 my_vip3Ag 851 TAACAGCTCTGCAAGCAAAAGCTTTTCTTACTTTAACAACATGCCGGAAA 900 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 833 TAACAGCTCTGCAAGCAAAAGCTTTTCTTACTTTAACAACATGCCGGAAA 882 my_vip3Ag 901 TTATTAGGCTTAGCAGATATTGATTATACTTTCATTATGAATGAACATTT 950 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 883 TTATTAGGCTTAGCAGATATTGATTATACTTTCATTATGAATGAACATTT 932 my_vip3Ag 951 AGATAAGGAAAAAGAGGAATTTAGAGTAAATATCCTTCCTACACTTTCTA 1000 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 933 AGATAAGGAAAAAGAGGAATTTAGAGTAAATATCCTTCCTACACTTTCTA 982 my_vip3Ag 1001 ATACTTTTTCTAATCCTAACTATGCAAAAGCTAAAGGAAGCAATGAAGAT 1050 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 983 ATACTTTTTCTAATCCTAACTATGCAAAAGCTAAAGGAAGCAATGAAGAT 1032 my_vip3Ag 1051 GCAAAGATAATTGTGGAAGCTAAACCAGGATATGCTTTGGTTGGATTTGA 1100 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 1033 GCAAAGATAATTGTGGAAGCTAAACCAGGATATGCTTTGGTTGGATTTGA 1082 my_vip3Ag 1101 AATGAGCAATGATTCAATCACAGTATTAAAAGCATATCAGGCTAAGCTAA 1150 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 1083 AATGAGCAATGATTCAATCACAGTATTAAAAGCATATCAGGCTAAGCTAA 1132 my_vip3Ag 1151 AACAAGATTATCAAGTTGATAAAGATTCGTTATCAGAAATTGTCTATGGT 1200 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 1133 AACAAGATTATCAAGTTGATAAAGATTCGTTATCAGAAATTGTCTATGGT 1182 my_vip3Ag 1201 GATATGGATAAATTATTGTGCCCGGATCAATCTGAACAAATATATTATAC 1250 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 1183 GATATGGATAAATTATTGTGCCCGGATCAATCTGAACAAATATATTATAC 1232 my_vip3Ag 1251 AAATAACATTGCTTTTCCCAATGAATATGTAATTACTAAAATTACTTTTA 1300 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 1233 AAATAACATTGCTTTTCCCAATGAATATGTAATTACTAAAATTACTTTTA 1282

133

my_vip3Ag 1301 CTAAAAAAATGAATAGTTTAAGATATGAGGCAACAGCTAATTTTTATGAT 1350 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 1283 CTAAAAAAATGAATAGTTTAAGATATGAGGCAACAGCTAATTTTTATGAT 1332 my_vip3Ag 1351 TCTTCTACAGGGGATATTGATCTAAATAAGACAAAAGTAGAATCAAGTGA 1400 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 1333 TCTTCTACAGGGGATATTGATCTAAATAAGACAAAAGTAGAATCAAGTGA 1382 my_vip3Ag 1401 AGCAGAGTATAGTACGCTAAGTGCTAGTACTGATGGAGTCTATATGCCGT 1450 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 1383 AGCAGAGTATAGTACGCTAAGTGCTAGTACTGATGGAGTCTATATGCCGT 1432 my_vip3Ag 1451 TAGGTATTATCAGTGAAACATTTTTGACTCCAATTAATGGGTTTGGAATC 1500 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 1433 TAGGTATTATCAGTGAAACATTTTTGACTCCAATTAATGGGTTTGGAATC 1482 my_vip3Ag 1501 GTAGTCGATGAAAATTCAAAATTAGTAAATTTAACATGTAAATCATATTT 1550 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 1483 GTAGTCGATGAAAATTCAAAATTAGTAAATTTAACATGTAAATCATATTT 1532 my_vip3Ag 1551 AAGAGAGGTATTATTAGCAACAGACTTAAGTAATAAAGAAACTAAATTGA 1600 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 1533 AAGAGAGGTATTATTAGCAACAGACTTAAGTAATAAAGAAACTAAATTGA 1582 my_vip3Ag 1601 TTGTCCCACCTATTGGTTTTATTAGCAATATTGTAGAAAATGGGAACTTA 1650 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 1583 TTGTCCCACCTATTGGTTTTATTAGCAATATTGTAGAAAATGGGAACTTA 1632 my_vip3Ag 1651 GAGGGAGAAAACTTAGAGCCGTGGAAAGCAAATAACAAAAATGCGTATGT 1700 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 1633 GAGGGAGAAAACTTAGAGCCGTGGAAAGCAAATAACAAAAATGCGTATGT 1682 my_vip3Ag 1701 AGATCATACAGGCGGCGTAAATGGAACTAAAGCTTTATATGTTCATAAGG 1750 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 1683 AGATCATACAGGCGGCGTAAATGGAACTAAAGCTTTATATGTTCATAAGG 1732 my_vip3Ag 1751 ATGGTGAGTTTTCACAATTTATTGGAGATAAGTTGAAATCGAAAACAGAA 1800 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 1733 ATGGTGAGTTTTCACAATTTATTGGAGATAAGTTGAAATCGAAAACAGAA 1782 my_vip3Ag 1801 TATGTAATTCAATATATTGTAAAGGGAAAAGCTTCTATTCTTTTGAAAGA 1850 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 1783 TATGTAATTCAATATATTGTAAAGGGAAAAGCTTCTATTCTTTTGAAAGA 1832 my_vip3Ag 1851 TGAAAAAAATGGTGATTGCATTTATGAAGATACAAATAATGGTTTAGAAG 1900 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 1833 TGAAAAAAATGGTGATTGCATTTATGAAGATACAAATAATGGTTTAGAAG 1882 my_vip3Ag 1901 ATTTTCAAACCATTACTAAAAGTTTTATTACAGGAACGGATTCTTCAGGA 1950 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 1883 ATTTTCAAACCATTACTAAAAGTTTTATTACAGGAACGGATTCTTCAGGA 1932 my_vip3Ag 1951 GTTCATTTAATATTTAATAGTCAAAATGGCGATGAAGCATTTGGGGAAAA 2000 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 1933 GTTCATTTAATATTTAATAGTCAAAATGGCGATGAAGCATTTGGGGAAAA 1982 my_vip3Ag 2001 CTTTACTATTTCAGAAATTAGGCTTTCCGAAGATTTATTAAGTCCAGAAT 2050 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 1983 CTTTACTATTTCAGAAATTAGGCTTTCCGAAGATTTATTAAGTCCAGAAT 2032 my_vip3Ag 2051 TGATAAATTCAGATGCTTGGGTTGGATCTCAGGGAACTTGGATCTCAGGA 2100 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 2033 TGATAAATTCAGATGCTTGGGTTGGATCTCAGGGAACTTGGATCTCAGGA 2082 my_vip3Ag 2101 AATTCACTCACTATTAATAGTAATGTGAATGGAACTTTTCGACAAAACCT 2150 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 2083 AATTCACTCACTATTAATAGTAATGTGAATGGAACTTTTCGACAAAACCT 2132

134

my_vip3Ag 2151 TTCGTTAGAAAGCTATTCAACTTATAGTATGAACTTTAATGTGAATGGAT 2200 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 2133 TTCGTTAGAAAGCTATTCAACTTATAGTATGAACTTTAATGTGAATGGAT 2182 my_vip3Ag 2201 TTGCCAAGGTGACAGTAAGAAATTCCCGTGAAGTATTATTTGAAAAAAAT 2250 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 2183 TTGCCAAGGTGACAGTAAGAAATTCCCGTGAAGTATTATTTGAAAAAAAT 2232 my_vip3Ag 2251 TATCCGCAGCTTTCACCTAAAGATATTTCTGAAAAATTCACAACTGCAGC 2300 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 2233 TATCCGCAGCTTTCACCTAAAGATATTTCTGAAAAATTCACAACTGCAGC 2282 my_vip3Ag 2301 CAATAATACCGGGTTGTATGTAGAGCTTTCTCGTTTTACATCGGGTGGCG 2350 |||||||||||||||||||||||||||||||||||||||||||||||||| FJ556803.2 2283 CAATAATACCGGGTTGTATGTAGAGCTTTCTCGTTTTACATCGGGTGGCG 2332 my_vip3Ag 2351 CTATAAATTTCCGAGATTTTTCAATTAAGTAA 2382 |||||||||||||||||||||||||||||||| FJ556803.2 2333 CTATAAATTTCCGAGATTTTTCAATTAAGTAA 2364

The derived amino acid sequence from vip3Ag >vip3Ag_amino acid (793 amino acids) MHHHHHHNKNNTKLSARALPSFIDYFNGIYGFATGIKDIMNMIFKTDTGGNLTLDEILKNQQLLNEISGKLDGVNGSLNDLIAQGNLNTELSKEILKIANEQNQVLNDVNNKLNAINTMLHIYLPKITSMLNDVMKQNYALSLQIEYLSKQLQEISDKLDVINVNVLINSTLTEITPAYQRMKYVNEKFEDLTFATETTLKVKKNSSPADILDELTELTELAKSVTKNDVDGFEFYLNTFHDVMVGNNLFGRSALKTASELIAKENVKTSGSEVGNVYNFLIVLTALQAKAFLTLTTCRKLLGLADIDYTFIMNEHLDKEKEEFRVNILPTLSNTFSNPNYAKAKGSNEDAKIIVEAKPGYALVGFEMSNDSITVLKAYQAKLKQDYQVDKDSLSEIVYGDMDKLLCPDQSEQIYYTNNIAFPNEYVITKITFTKKMNSLRYEATANFYDSSTGDIDLNKTKVESSEAEYSTLSASTDGVYMPLGIISETFLTPINGFGIVVDENSKLVNLTCKSYLREVLLATDLSNKETKLIVPPIGFISNIVENGNLEGENLEPWKANNKNAYVDHTGGVNGTKALYVHKDGEFSQFIGDKLKSKTEYVIQYIVKGKASILLKDEKNGDCIYEDTNNGLEDFQTITKSFITGTDSSGVHLIFNSQNGDEAFGENFTISEIRLSEDLLSPELINSDAWVGSQGTWISGNSLTINSNVNGTFRQNLSLESYSTYSMNFNVNGFAKVTVRNSREVLFEKNYPQLSPKDISEKFTTAANNTGLYVELSRFTSGGAINFRDFSIK*

Group 4: vip3Ca2

vip3Ca2_nucleotide ATGCATCATCACCACCATCACAACATGAATAATACTAAATTAAACGCAAGGGCCTTACCAAGTTTTATTGATTATTTTAATGGCATTTATGGATTTGCCACTGGTATCAAAGACATTATGAACATGATTTTTAAAACGGATACAGGTGGTGATCTAACCCTAGACGAAATTTTAAAGAATCAGCAGTTACTAAATGAAATTTCTGGTAAATTGGATGGGGTAAATGGGAGCTTAAATGATCTTATCGCACAGGGAAACTTAAATACAGAATTATCTAAGGAAATCTTAAAAATTGCAAATGAACAAAATCAAGTTTTAAATGATGTTAATAACAAACTAGATGCGATAAATACGATGCTTAACATATATCTACCTAAAATTACATCTATGTTAAGCGATGTAATGAAACAAAATTATGCGCTAAGTCTGCAGATAGAATACCTAAGTAGACAGTTACAGGAAATTTCAGATAAGTTAGATGTTATTAATTTAAATGTTTTAATTAATTCTACTCTTACTGAAATCACACCTTCATATCAACGTATTAAATATGTAAATGAGAAATTTGATAAATTAACATTTGCCACAGAAAGCACTCTAAGAGCAAAACAAGGAATTTTTAACGAGGATAGTTTTGATAATAATACTCTTGAAAATTTAACTGATCTAGCTGAACTAGCTAAAAGTATAACCAAAAATGATGTGGATAGTTTCGAATTTTATCTTCACACATTTCATGATGTATTAATTGGAAATAATTTATTTGGACGATCGGCTTTAAAAACAGCTTCAGAATTAATTACTAAAGACGAGATAAAGACAAGTGGAAGTGAGATTGGAAAAGTTTATAGTTTCTTAATTGTGCTAACTTCTTTACAAGCTAAAGCCTTTCTCACTTTAACAACATGTAGAAAATTATTGGGCTTATCAGATATTGATTATACTTCTATTATGAATGAACATCTAAATAATGAAAAAAATGAATTTCGTGATAACATACTTCCTGCACTGTCGAATAAGTTTTCTAACCCTAGCTACGCAAAAACTATAGGTAGTGACAATTATGCAAAAGTTATTTTAGAATCTGAACCAGGTTATGCTTTAGTCGGATTCGAAATTATTAATGACCCAATCCCGGTATTAAAAGCGTATAAAGCAAAACTAAAACAAAATTATCAAGTTGATAATCAGTCGTTATCAGAGATTGTTTATTTAGATATCGATAAACTATTTTGTCCGGAAAATTCAGAACAAAAATATTATACTAAAAATCTGACATTTCCTGATGGCTATGTTATTACTAAAATTACCTTTGAAAAAAAACTGAACAACCTAATTTATGAAGCAACA

135

GCAAATTTTTATGACCCATCTACAGGAGATATTGACTTAAATAAGAAGCAAGTGGAATCTACTTTTCCTCAAACGGATTATATTACTATGGATATAGGTGATGATGATGGTATTTATATGCCGTTAGGCGTTATCAGTGAAACGTTTTTGACTCCTATTAATAGTTTTGGATTAGAAGTTGACGCGAAATCGAAAACATTAACCTTAAAATGTAAATCTTACCTGCGAGAATATTTATTAGAGTCCGATCTAAAAAATAAAGAAACAGGCTTGATTGCCCCGCCAAATGTTTTTATTAGTAATGTTGTGAAAAATTGGGATATAGAGGAAGATAGTCTAGAACCATGGGTAGCAAATAACAAAAATGCCTATGTTGATAATACAGGCGGTATAGAAAGATCTAAAGCCCTCTTTACTCAAGGTGACGGGGAATTCTCACAATTTATTGGTGATAAATTAAAACCTAATACAGATTATATTATTCAATATACTGTAAAAGGAAAACCAGCCATTTATTTAAAAAATAAAAGTACTGGATATATTACGTACGAGGATACAAACGGTAATTCTGAAGAATTTCAAACTATAGCTGTAAAATTCACTTCAGAAACTGATCTTTCACAAACTCATTTAGTTTTTAAAAGTCAAAATGGTTATGAGGCTTGGGGAGACAATTTTATTATTTTAGAAGCTAAGCTATTTGAAACTCCTGAAAGTCCAGAATTGATAAAATTTAATGATTGGGAAAGATTTGGTACTACTTACATTACAGGAAATGAGCTTAGAATCGATCATAGTCGTGGAGGTTATTTTAGACAATCTCTTAATATAGACAGTTATTCAACTTATGATTTGAGCTTTTCTTTTAGTGGATTATGGGCTAAGGTTATTGTGAAAAATTCCCGAGGGGTAGTATTGTTTGAAAAAGTGAAAAACAACGGTTCATCATATGAAGATATTAGTGAAAGTTTTACTACCGCATCAAATAAGGATGGATTTTTTATAGAACTAACAGCCGAAAGAACGAGCTCAACTTTCCATAGCTTTCGTGATATTTCTATTAAGGAAAAGATTGAATAA

Alignment of vip3Ca2 with reference JF916462.1 my_vip3Ca2 1 ATGCATCATCACCACCATCACAACATGAATAATACTAAATTAAACGCAAG 50 ||| ||||||||||||||||||||||||||||| JF916462.1 1 ATG------------------AACATGAATAATACTAAATTAAACGCAAG 32 my_vip3Ca2 51 GGCCTTACCAAGTTTTATTGATTATTTTAATGGCATTTATGGATTTGCCA 100 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 33 GGCCTTACCAAGTTTTATTGATTATTTTAATGGCATTTATGGATTTGCCA 82 my_vip3Ca2 101 CTGGTATCAAAGACATTATGAACATGATTTTTAAAACGGATACAGGTGGT 150 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 83 CTGGTATCAAAGACATTATGAACATGATTTTTAAAACGGATACAGGTGGT 132 my_vip3Ca2 151 GATCTAACCCTAGACGAAATTTTAAAGAATCAGCAGTTACTAAATGAAAT 200 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 133 GATCTAACCCTAGACGAAATTTTAAAGAATCAGCAGTTACTAAATGAAAT 182 my_vip3Ca2 201 TTCTGGTAAATTGGATGGGGTAAATGGGAGCTTAAATGATCTTATCGCAC 250 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 183 TTCTGGTAAATTGGATGGGGTAAATGGGAGCTTAAATGATCTTATCGCAC 232 my_vip3Ca2 251 AGGGAAACTTAAATACAGAATTATCTAAGGAAATCTTAAAAATTGCAAAT 300 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 233 AGGGAAACTTAAATACAGAATTATCTAAGGAAATCTTAAAAATTGCAAAT 282 my_vip3Ca2 301 GAACAAAATCAAGTTTTAAATGATGTTAATAACAAACTAGATGCGATAAA 350 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 283 GAACAAAATCAAGTTTTAAATGATGTTAATAACAAACTAGATGCGATAAA 332 my_vip3Ca2 351 TACGATGCTTAACATATATCTACCTAAAATTACATCTATGTTAAGCGATG 400 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 333 TACGATGCTTAACATATATCTACCTAAAATTACATCTATGTTAAGCGATG 382 my_vip3Ca2 401 TAATGAAACAAAATTATGCGCTAAGTCTGCAGATAGAATACCTAAGTAGA 450 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 383 TAATGAAACAAAATTATGCGCTAAGTCTGCAGATAGAATACCTAAGTAGA 432 my_vip3Ca2 451 CAGTTACAGGAAATTTCAGATAAGTTAGATGTTATTAATTTAAATGTTTT 500 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 433 CAGTTACAGGAAATTTCAGATAAGTTAGATGTTATTAATTTAAATGTTTT 482 my_vip3Ca2 501 AATTAATTCTACTCTTACTGAAATCACACCTTCATATCAACGTATTAAAT 550 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 483 AATTAATTCTACTCTTACTGAAATCACACCTTCATATCAACGTATTAAAT 532

136

my_vip3Ca2 551 ATGTAAATGAGAAATTTGATAAATTAACATTTGCCACAGAAAGCACTCTA 600 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 533 ATGTAAATGAGAAATTTGATAAATTAACATTTGCCACAGAAAGCACTCTA 582 my_vip3Ca2 601 AGAGCAAAACAAGGAATTTTTAACGAGGATAGTTTTGATAATAATACTCT 650 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 583 AGAGCAAAACAAGGAATTTTTAACGAGGATAGTTTTGATAATAATACTCT 632 my_vip3Ca2 651 TGAAAATTTAACTGATCTAGCTGAACTAGCTAAAAGTATAACCAAAAATG 700 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 633 TGAAAATTTAACTGATCTAGCTGAACTAGCTAAAAGTATAACCAAAAATG 682 my_vip3Ca2 701 ATGTGGATAGTTTCGAATTTTATCTTCACACATTTCATGATGTATTAATT 750 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 683 ATGTGGATAGTTTCGAATTTTATCTTCACACATTTCATGATGTATTAATT 732 my_vip3Ca2 751 GGAAATAATTTATTTGGACGATCGGCTTTAAAAACAGCTTCAGAATTAAT 800 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 733 GGAAATAATTTATTTGGACGATCGGCTTTAAAAACAGCTTCAGAATTAAT 782 my_vip3Ca2 801 TACTAAAGACGAGATAAAGACAAGTGGAAGTGAGATTGGAAAAGTTTATA 850 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 783 TACTAAAGACGAGATAAAGACAAGTGGAAGTGAGATTGGAAAAGTTTATA 832 my_vip3Ca2 851 GTTTCTTAATTGTGCTAACTTCTTTACAAGCTAAAGCCTTTCTCACTTTA 900 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 833 GTTTCTTAATTGTGCTAACTTCTTTACAAGCTAAAGCCTTTCTCACTTTA 882 my_vip3Ca2 901 ACAACATGTAGAAAATTATTGGGCTTATCAGATATTGATTATACTTCTAT 950 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 883 ACAACATGTAGAAAATTATTGGGCTTATCAGATATTGATTATACTTCTAT 932 my_vip3Ca2 951 TATGAATGAACATCTAAATAATGAAAAAAATGAATTTCGTGATAACATAC 1000 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 933 TATGAATGAACATCTAAATAATGAAAAAAATGAATTTCGTGATAACATAC 982 my_vip3Ca2 1001 TTCCTGCACTGTCGAATAAGTTTTCTAACCCTAGCTACGCAAAAACTATA 1050 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 983 TTCCTGCACTGTCGAATAAGTTTTCTAACCCTAGCTACGCAAAAACTATA 1032 my_vip3Ca2 1051 GGTAGTGACAATTATGCAAAAGTTATTTTAGAATCTGAACCAGGTTATGC 1100 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 1033 GGTAGTGACAATTATGCAAAAGTTATTTTAGAATCTGAACCAGGTTATGC 1082 my_vip3Ca2 1101 TTTAGTCGGATTCGAAATTATTAATGACCCAATCCCGGTATTAAAAGCGT 1150 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 1083 TTTAGTCGGATTCGAAATTATTAATGACCCAATCCCGGTATTAAAAGCGT 1132 my_vip3Ca2 1151 ATAAAGCAAAACTAAAACAAAATTATCAAGTTGATAATCAGTCGTTATCA 1200 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 1133 ATAAAGCAAAACTAAAACAAAATTATCAAGTTGATAATCAGTCGTTATCA 1182 my_vip3Ca2 1201 GAGATTGTTTATTTAGATATCGATAAACTATTTTGTCCGGAAAATTCAGA 1250 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 1183 GAGATTGTTTATTTAGATATCGATAAACTATTTTGTCCGGAAAATTCAGA 1232 my_vip3Ca2 1251 ACAAAAATATTATACTAAAAATCTGACATTTCCTGATGGCTATGTTATTA 1300 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 1233 ACAAAAATATTATACTAAAAATCTGACATTTCCTGATGGCTATGTTATTA 1282 my_vip3Ca2 1301 CTAAAATTACCTTTGAAAAAAAACTGAACAACCTAATTTATGAAGCAACA 1350 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 1283 CTAAAATTACCTTTGAAAAAAAACTGAACAACCTAATTTATGAAGCAACA 1332 my_vip3Ca2 1351 GCAAATTTTTATGACCCATCTACAGGAGATATTGACTTAAATAAGAAGCA 1400 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 1333 GCAAATTTTTATGACCCATCTACAGGAGATATTGACTTAAATAAGAAGCA 1382

137

my_vip3Ca2 1401 AGTGGAATCTACTTTTCCTCAAACGGATTATATTACTATGGATATAGGTG 1450 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 1383 AGTGGAATCTACTTTTCCTCAAACGGATTATATTACTATGGATATAGGTG 1432 my_vip3Ca2 1451 ATGATGATGGTATTTATATGCCGTTAGGCGTTATCAGTGAAACGTTTTTG 1500 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 1433 ATGATGATGGTATTTATATGCCGTTAGGCGTTATCAGTGAAACGTTTTTG 1482 my_vip3Ca2 1501 ACTCCTATTAATAGTTTTGGATTAGAAGTTGACGCGAAATCGAAAACATT 1550 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 1483 ACTCCTATTAATAGTTTTGGATTAGAAGTTGACGCGAAATCGAAAACATT 1532 my_vip3Ca2 1551 AACCTTAAAATGTAAATCTTACCTGCGAGAATATTTATTAGAGTCCGATC 1600 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 1533 AACCTTAAAATGTAAATCTTACCTGCGAGAATATTTATTAGAGTCCGATC 1582 my_vip3Ca2 1601 TAAAAAATAAAGAAACAGGCTTGATTGCCCCGCCAAATGTTTTTATTAGT 1650 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 1583 TAAAAAATAAAGAAACAGGCTTGATTGCCCCGCCAAATGTTTTTATTAGT 1632 my_vip3Ca2 1651 AATGTTGTGAAAAATTGGGATATAGAGGAAGATAGTCTAGAACCATGGGT 1700 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 1633 AATGTTGTGAAAAATTGGGATATAGAGGAAGATAGTCTAGAACCATGGGT 1682 my_vip3Ca2 1701 AGCAAATAACAAAAATGCCTATGTTGATAATACAGGCGGTATAGAAAGAT 1750 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 1683 AGCAAATAACAAAAATGCCTATGTTGATAATACAGGCGGTATAGAAAGAT 1732 my_vip3Ca2 1751 CTAAAGCCCTCTTTACTCAAGGTGACGGGGAATTCTCACAATTTATTGGT 1800 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 1733 CTAAAGCCCTCTTTACTCAAGGTGACGGGGAATTCTCACAATTTATTGGT 1782 my_vip3Ca2 1801 GATAAATTAAAACCTAATACAGATTATATTATTCAATATACTGTAAAAGG 1850 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 1783 GATAAATTAAAACCTAATACAGATTATATTATTCAATATACTGTAAAAGG 1832 my_vip3Ca2 1851 AAAACCAGCCATTTATTTAAAAAATAAAAGTACTGGATATATTACGTACG 1900 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 1833 AAAACCAGCCATTTATTTAAAAAATAAAAGTACTGGATATATTACGTACG 1882 my_vip3Ca2 1901 AGGATACAAACGGTAATTCTGAAGAATTTCAAACTATAGCTGTAAAATTC 1950 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 1883 AGGATACAAACGGTAATTCTGAAGAATTTCAAACTATAGCTGTAAAATTC 1932 my_vip3Ca2 1951 ACTTCAGAAACTGATCTTTCACAAACTCATTTAGTTTTTAAAAGTCAAAA 2000 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 1933 ACTTCAGAAACTGATCTTTCACAAACTCATTTAGTTTTTAAAAGTCAAAA 1982 my_vip3Ca2 2001 TGGTTATGAGGCTTGGGGAGACAATTTTATTATTTTAGAAGCTAAGCTAT 2050 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 1983 TGGTTATGAGGCTTGGGGAGACAATTTTATTATTTTAGAAGCTAAGCTAT 2032 my_vip3Ca2 2051 TTGAAACTCCTGAAAGTCCAGAATTGATAAAATTTAATGATTGGGAAAGA 2100 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 2033 TTGAAACTCCTGAAAGTCCAGAATTGATAAAATTTAATGATTGGGAAAGA 2082 my_vip3Ca2 2101 TTTGGTACTACTTACATTACAGGAAATGAGCTTAGAATCGATCATAGTCG 2150 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 2083 TTTGGTACTACTTACATTACAGGAAATGAGCTTAGAATCGATCATAGTCG 2132 my_vip3Ca2 2151 TGGAGGTTATTTTAGACAATCTCTTAATATAGACAGTTATTCAACTTATG 2200 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 2133 TGGAGGTTATTTTAGACAATCTCTTAATATAGACAGTTATTCAACTTATG 2182 my_vip3Ca2 2201 ATTTGAGCTTTTCTTTTAGTGGATTATGGGCTAAGGTTATTGTGAAAAAT 2250 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 2183 ATTTGAGCTTTTCTTTTAGTGGATTATGGGCTAAGGTTATTGTGAAAAAT 2232

138

my_vip3Ca2 2251 TCCCGAGGGGTAGTATTGTTTGAAAAAGTGAAAAACAACGGTTCATCATA 2300 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 2233 TCCCGAGGGGTAGTATTGTTTGAAAAAGTGAAAAACAACGGTTCATCATA 2282 my_vip3Ca2 2301 TGAAGATATTAGTGAAAGTTTTACTACCGCATCAAATAAGGATGGATTTT 2350 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 2283 TGAAGATATTAGTGAAAGTTTTACTACCGCATCAAATAAGGATGGATTTT 2332 my_vip3Ca2 2351 TTATAGAACTAACAGCCGAAAGAACGAGCTCAACTTTCCATAGCTTTCGT 2400 |||||||||||||||||||||||||||||||||||||||||||||||||| JF916462.1 2333 TTATAGAACTAACAGCCGAAAGAACGAGCTCAACTTTCCATAGCTTTCGT 2382 my_vip3Ca2 2401 GATATTTCTATTAAGGAAAAGATTGAATAA 2430 |||||||||||||||||||||||||||||| JF916462.1 2383 GATATTTCTATTAAGGAAAAGATTGAATAA 2412

The derived amino acid sequence from vip3Ca2 >vip3Ca2_amino acid (809 amino acids) MHHHHHHNMNNTKLNARALPSFIDYFNGIYGFATGIKDIMNMIFKTDTGGDLTLDEILKNQQLLNEISGKLDGVNGSLNDLIAQGNLNTELSKEILKIANEQNQVLNDVNNKLDAINTMLNIYLPKITSMLSDVMKQNYALSLQIEYLSRQLQEISDKLDVINLNVLINSTLTEITPSYQRIKYVNEKFDKLTFATESTLRAKQGIFNEDSFDNNTLENLTDLAELAKSITKNDVDSFEFYLHTFHDVLIGNNLFGRSALKTASELITKDEIKTSGSEIGKVYSFLIVLTSLQAKAFLTLTTCRKLLGLSDIDYTSIMNEHLNNEKNEFRDNILPALSNKFSNPSYAKTIGSDNYAKVILESEPGYALVGFEIINDPIPVLKAYKAKLKQNYQVDNQSLSEIVYLDIDKLFCPENSEQKYYTKNLTFPDGYVITKITFEKKLNNLIYEATANFYDPSTGDIDLNKKQVESTFPQTDYITMDIGDDDGIYMPLGVISETFLTPINSFGLEVDAKSKTLTLKCKSYLREYLLESDLKNKETGLIAPPNVFISNVVKNWDIEEDSLEPWVANNKNAYVDNTGGIERSKALFTQGDGEFSQFIGDKLKPNTDYIIQYTVKGKPAIYLKNKSTGYITYEDTNGNSEEFQTIAVKFTSETDLSQTHLVFKSQNGYEAWGDNFIILEAKLFETPESPELIKFNDWERFGTTYITGNELRIDHSRGGYFRQSLNIDSYSTYDLSFSFSGLWAKVIVKNSRGVVLFEKVKNNGSSYEDISESFTTASNKDGFFIELTAERTSSTFHSFRDISIKEKIE*

Group 5: vip3Ba1

>vip3Ba1_nucleotide ATGCATCATCACCACCATCACAACATGAATAATACTAAATTAAACGCAAGGGCCTTACCAAGTTTTATTGATTATTTTAATGGCATTTATGGATTTGCCACTGGTATCAAAGACATTATGAACATGATTTTTAAAACGGATACAGGTGGTGGTAATTTAACACTAGATGAAATTTTAAAGAATCAAGATTTATTAAATCAAATCTCAGATAAACTCGATGGAATTAATGGAGATTTAGGTGATCTTATTGCACAAGGGAATTTGAATTCAGAACTAACTAAGGAATTATTAAAAATCGCCAATGAGCAAAATCTGATGTTAAATAATGTTAATGCTCAACTTAATTCAATAAATGCAACACTTAATACCTATCTTCCAAAAATTACATCTATGCTAAATGAGGTAATGAAACAAAACTATGTTTTAAGTCTACAAATAGAATTTCTTAGTAAACAATTACAAGAGATTTCAGATAAACTTGATATTATCAATTTAAATGTATTAATTAACTCTACATTGACAGAGATTACGCCTGCTTATCAACGTATTAAATATGTAAACGATAAATTTGATGAATTGACTTCTACTGTAGAAAAAAATCCAAAAATTAATCAAGATAATTTTACTGAAGATGTTATTGATAATTTAACTGATCTAACTGAACTAGCAAGAAGTGTAACGAGAAATGATATGGATAGTTTTGAATTTTATATTAAAACTTTCCATGATGTGATGATAGGAAATAATTTATTCAGTCGTTCTGCATTAAAAACTGCTTCAGAATTAATTGCTAAGGAAAATATACACACTAGGGGAAGTGAAATTGGTAATGTCTACACTTTTATGATTGTTTTGACTTCCTTACAAGCAAAAGCGTTCCTAACTTTAACTACATGTCGTAAATTATTAGGATTAGCAGATATCGATTATACACAAATTATGAATGAAAATTTAGATAGAGAAAAAGAGGAATTTCGCTTAAATATTCTTCCTACACTTTCTAATGATTTTTCTAATCCTAATTATACAGAAACTTTAGGAAGTGATCTTGTAGATCCAATTGTTACGTTAGAAGCTGAGCCTGGTTATGCTTTAATAGGTTTTGAAATTCTCAATGATCCACTTCCAGTATTAAAAGTATATCAGGCAAAGCTAAAACCAAATTATCAAGTCGACAAAGAGTCGATTATGGAAAATATTTATGGGAACATCCATAAACTACTTTGTCCAAAACAACGTGAACAAAAATATTATATAAAAGACATGACATTCCCTGAAGGTTATGTAATCACCAAAATTGTTTTTGAAAAAAAATTGAATCTATTAGGATATGAAGTAACAGCAAATCTTTATGATCCTTTTACAGGAAGTATCGATTTGAATAAGACTATTCTAGAATCATGGAAAGAAGACTGCTGTGAAGAAGACTGCTGTGAAGAAGACTGCTGTGAAGAAGATTGCTGTGAGGAATTATATAAAATTATAGAGGCGGATACTAACGGTGTTTATATGCCTTTGGGTGTAATTAGTGAAACATTTTTAACACCAATCTATAGTTTTAAACTAATTATTGACGAAAAAACAAAGAAAATATCTTTAGCGGGTAAATCTTATTTACGTGAATCTTTACTAGCCACAGATTTAGTTAATAAAGAAACAAATTTAATTCCTTCACCTAATGGTTTCATTAGCAGTATTGTGCAAAATTGGCATATAACATCGGATAATATAGAGCCATGGAAAGCGAATAATAAAAATGCATATGTCGATAAGACGGATGCCATGGTGGGATTTAGCTCTTTATATACTCATAAGGATGGGGAATTCTTGCAATTTATTGGAGCTAAGTTAAAGGCTAAAACTGAGTATATCATT

139

CAATATACTGTAAAAGGGAATCCGGAAGTTTATTTAAAAAACAATAAAGATATCTGTTATGAGGATAAAACAAATAATTTTGACACGTTTCAAACTATAACTAAAAAATTCAATTCAGGAGTAGATCCATCCGAAATATATCTAGTTTTTAAAAATCAAATTGGATATGAAGCATGGGGAAATAACTTTATTATACTTGAAATTAAGTCGCTTGAAACCCTACCACAAATATTAAAACCTGAAAATTGGATTCCTTTGGGTAATGCTGAGATTAAAGAAGATGGAAAAATTGAGATTTCAGGTAATGGAAGCATGAATCAATATATTCAATTAGAACAGAATTCCAAATATCATCTAAGATTCTCTGTAAAAGGAAAAGGTAGAGTAACGATGCAAGCTCAAACGTCCCATATAAATGTACCAGCTACAAACGAAGAGGTTTCTATAATGATTGAAACTACACGCTTATACGGCGAAGGTATAATTAGCCTATTAAATGATGAAGTGGAGAATTCCGGGGTTATTTTTTCGGATGTATCTATAGTTAAAGAATAA Alignment of vip3Ba1 with reference AY823271.1

my_vip3Ba1 1 ATGCATCATCACCACCATCACAACATGAATAATACTAAATTAAACGCAAG 50 ||| ||||||||||||||||||||||||||||| AY823271.1 1 ATG------------------AACATGAATAATACTAAATTAAACGCAAG 32 my_vip3Ba1 51 GGCCTTACCAAGTTTTATTGATTATTTTAATGGCATTTATGGATTTGCCA 100 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 33 GGCCTTACCAAGTTTTATTGATTATTTTAATGGCATTTATGGATTTGCCA 82 my_vip3Ba1 101 CTGGTATCAAAGACATTATGAACATGATTTTTAAAACGGATACAGGTGGT 150 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 83 CTGGTATCAAAGACATTATGAACATGATTTTTAAAACGGATACAGGTGGT 132 my_vip3Ba1 151 GGTAATTTAACACTAGATGAAATTTTAAAGAATCAAGATTTATTAAATCA 200 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 133 GGTAATTTAACACTAGATGAAATTTTAAAGAATCAAGATTTATTAAATCA 182 my_vip3Ba1 201 AATCTCAGATAAACTCGATGGAATTAATGGAGATTTAGGTGATCTTATTG 250 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 183 AATCTCAGATAAACTCGATGGAATTAATGGAGATTTAGGTGATCTTATTG 232 my_vip3Ba1 251 CACAAGGGAATTTGAATTCAGAACTAACTAAGGAATTATTAAAAATCGCC 300 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 233 CACAAGGGAATTTGAATTCAGAACTAACTAAGGAATTATTAAAAATCGCC 282 my_vip3Ba1 301 AATGAGCAAAATCTGATGTTAAATAATGTTAATGCTCAACTTAATTCAAT 350 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 283 AATGAGCAAAATCTGATGTTAAATAATGTTAATGCTCAACTTAATTCAAT 332 my_vip3Ba1 351 AAATGCAACACTTAATACCTATCTTCCAAAAATTACATCTATGCTAAATG 400 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 333 AAATGCAACACTTAATACCTATCTTCCAAAAATTACATCTATGCTAAATG 382 my_vip3Ba1 401 AGGTAATGAAACAAAACTATGTTTTAAGTCTACAAATAGAATTTCTTAGT 450 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 383 AGGTAATGAAACAAAACTATGTTTTAAGTCTACAAATAGAATTTCTTAGT 432 my_vip3Ba1 451 AAACAATTACAAGAGATTTCAGATAAACTTGATATTATCAATTTAAATGT 500 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 433 AAACAATTACAAGAGATTTCAGATAAACTTGATATTATCAATTTAAATGT 482 my_vip3Ba1 501 ATTAATTAACTCTACATTGACAGAGATTACGCCTGCTTATCAACGTATTA 550 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 483 ATTAATTAACTCTACATTGACAGAGATTACGCCTGCTTATCAACGTATTA 532 my_vip3Ba1 551 AATATGTAAACGATAAATTTGATGAATTGACTTCTACTGTAGAAAAAAAT 600 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 533 AATATGTAAACGATAAATTTGATGAATTGACTTCTACTGTAGAAAAAAAT 582 my_vip3Ba1 601 CCAAAAATTAATCAAGATAATTTTACTGAAGATGTTATTGATAATTTAAC 650 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 583 CCAAAAATTAATCAAGATAATTTTACTGAAGATGTTATTGATAATTTAAC 632 my_vip3Ba1 651 TGATCTAACTGAACTAGCAAGAAGTGTAACGAGAAATGATATGGATAGTT 700

140

|||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 633 TGATCTAACTGAACTAGCAAGAAGTGTAACGAGAAATGATATGGATAGTT 682 my_vip3Ba1 701 TTGAATTTTATATTAAAACTTTCCATGATGTGATGATAGGAAATAATTTA 750 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 683 TTGAATTTTATATTAAAACTTTCCATGATGTGATGATAGGAAATAATTTA 732 my_vip3Ba1 751 TTCAGTCGTTCTGCATTAAAAACTGCTTCAGAATTAATTGCTAAGGAAAA 800 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 733 TTCAGTCGTTCTGCATTAAAAACTGCTTCAGAATTAATTGCTAAGGAAAA 782 my_vip3Ba1 801 TATACACACTAGGGGAAGTGAAATTGGTAATGTCTACACTTTTATGATTG 850 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 783 TATACACACTAGGGGAAGTGAAATTGGTAATGTCTACACTTTTATGATTG 832 my_vip3Ba1 851 TTTTGACTTCCTTACAAGCAAAAGCGTTCCTAACTTTAACTACATGTCGT 900 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 833 TTTTGACTTCCTTACAAGCAAAAGCGTTCCTAACTTTAACTACATGTCGT 882 my_vip3Ba1 901 AAATTATTAGGATTAGCAGATATCGATTATACACAAATTATGAATGAAAA 950 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 883 AAATTATTAGGATTAGCAGATATCGATTATACACAAATTATGAATGAAAA 932 my_vip3Ba1 951 TTTAGATAGAGAAAAAGAGGAATTTCGCTTAAATATTCTTCCTACACTTT 1000 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 933 TTTAGATAGAGAAAAAGAGGAATTTCGCTTAAATATTCTTCCTACACTTT 982 my_vip3Ba1 1001 CTAATGATTTTTCTAATCCTAATTATACAGAAACTTTAGGAAGTGATCTT 1050 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 983 CTAATGATTTTTCTAATCCTAATTATACAGAAACTTTAGGAAGTGATCTT 1032 my_vip3Ba1 1051 GTAGATCCAATTGTTACGTTAGAAGCTGAGCCTGGTTATGCTTTAATAGG 1100 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 1033 GTAGATCCAATTGTTACGTTAGAAGCTGAGCCTGGTTATGCTTTAATAGG 1082 my_vip3Ba1 1101 TTTTGAAATTCTCAATGATCCACTTCCAGTATTAAAAGTATATCAGGCAA 1150 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 1083 TTTTGAAATTCTCAATGATCCACTTCCAGTATTAAAAGTATATCAGGCAA 1132 my_vip3Ba1 1151 AGCTAAAACCAAATTATCAAGTCGACAAAGAGTCGATTATGGAAAATATT 1200 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 1133 AGCTAAAACCAAATTATCAAGTCGACAAAGAGTCGATTATGGAAAATATT 1182 my_vip3Ba1 1201 TATGGGAACATCCATAAACTACTTTGTCCAAAACAACGTGAACAAAAATA 1250 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 1183 TATGGGAACATCCATAAACTACTTTGTCCAAAACAACGTGAACAAAAATA 1232 my_vip3Ba1 1251 TTATATAAAAGACATGACATTCCCTGAAGGTTATGTAATCACCAAAATTG 1300 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 1233 TTATATAAAAGACATGACATTCCCTGAAGGTTATGTAATCACCAAAATTG 1282 my_vip3Ba1 1301 TTTTTGAAAAAAAATTGAATCTATTAGGATATGAAGTAACAGCAAATCTT 1350 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 1283 TTTTTGAAAAAAAATTGAATCTATTAGGATATGAAGTAACAGCAAATCTT 1332 my_vip3Ba1 1351 TATGATCCTTTTACAGGAAGTATCGATTTGAATAAGACTATTCTAGAATC 1400 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 1333 TATGATCCTTTTACAGGAAGTATCGATTTGAATAAGACTATTCTAGAATC 1382 my_vip3Ba1 1401 ATGGAAAGAAGACTGCTGTGAAGAAGACTGCTGTGAAGAAGACTGCTGTG 1450 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 1383 ATGGAAAGAAGACTGCTGTGAAGAAGACTGCTGTGAAGAAGACTGCTGTG 1432 my_vip3Ba1 1451 AAGAAGATTGCTGTGAGGAATTATATAAAATTATAGAGGCGGATACTAAC 1500 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 1433 AAGAAGATTGCTGTGAGGAATTATATAAAATTATAGAGGCGGATACTAAC 1482 my_vip3Ba1 1501 GGTGTTTATATGCCTTTGGGTGTAATTAGTGAAACATTTTTAACACCAAT 1550

141

|||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 1483 GGTGTTTATATGCCTTTGGGTGTAATTAGTGAAACATTTTTAACACCAAT 1532 my_vip3Ba1 1551 CTATAGTTTTAAACTAATTATTGACGAAAAAACAAAGAAAATATCTTTAG 1600 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 1533 CTATAGTTTTAAACTAATTATTGACGAAAAAACAAAGAAAATATCTTTAG 1582 my_vip3Ba1 1601 CGGGTAAATCTTATTTACGTGAATCTTTACTAGCCACAGATTTAGTTAAT 1650 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 1583 CGGGTAAATCTTATTTACGTGAATCTTTACTAGCCACAGATTTAGTTAAT 1632 my_vip3Ba1 1651 AAAGAAACAAATTTAATTCCTTCACCTAATGGTTTCATTAGCAGTATTGT 1700 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 1633 AAAGAAACAAATTTAATTCCTTCACCTAATGGTTTCATTAGCAGTATTGT 1682 my_vip3Ba1 1701 GCAAAATTGGCATATAACATCGGATAATATAGAGCCATGGAAAGCGAATA 1750 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 1683 GCAAAATTGGCATATAACATCGGATAATATAGAGCCATGGAAAGCGAATA 1732 my_vip3Ba1 1751 ATAAAAATGCATATGTCGATAAGACGGATGCCATGGTGGGATTTAGCTCT 1800 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 1733 ATAAAAATGCATATGTCGATAAGACGGATGCCATGGTGGGATTTAGCTCT 1782 my_vip3Ba1 1801 TTATATACTCATAAGGATGGGGAATTCTTGCAATTTATTGGAGCTAAGTT 1850 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 1783 TTATATACTCATAAGGATGGGGAATTCTTGCAATTTATTGGAGCTAAGTT 1832 my_vip3Ba1 1851 AAAGGCTAAAACTGAGTATATCATTCAATATACTGTAAAAGGGAATCCGG 1900 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 1833 AAAGGCTAAAACTGAGTATATCATTCAATATACTGTAAAAGGGAATCCGG 1882 my_vip3Ba1 1901 AAGTTTATTTAAAAAACAATAAAGATATCTGTTATGAGGATAAAACAAAT 1950 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 1883 AAGTTTATTTAAAAAACAATAAAGATATCTGTTATGAGGATAAAACAAAT 1932 my_vip3Ba1 1951 AATTTTGACACGTTTCAAACTATAACTAAAAAATTCAATTCAGGAGTAGA 2000 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 1933 AATTTTGACACGTTTCAAACTATAACTAAAAAATTCAATTCAGGAGTAGA 1982 my_vip3Ba1 2001 TCCATCCGAAATATATCTAGTTTTTAAAAATCAAATTGGATATGAAGCAT 2050 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 1983 TCCATCCGAAATATATCTAGTTTTTAAAAATCAAATTGGATATGAAGCAT 2032 my_vip3Ba1 2051 GGGGAAATAACTTTATTATACTTGAAATTAAGTCGCTTGAAACCCTACCA 2100 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 2033 GGGGAAATAACTTTATTATACTTGAAATTAAGTCGCTTGAAACCCTACCA 2082 my_vip3Ba1 2101 CAAATATTAAAACCTGAAAATTGGATTCCTTTGGGTAATGCTGAGATTAA 2150 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 2083 CAAATATTAAAACCTGAAAATTGGATTCCTTTGGGTAATGCTGAGATTAA 2132 my_vip3Ba1 2151 AGAAGATGGAAAAATTGAGATTTCAGGTAATGGAAGCATGAATCAATATA 2200 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 2133 AGAAGATGGAAAAATTGAGATTTCAGGTAATGGAAGCATGAATCAATATA 2182 my_vip3Ba1 2201 TTCAATTAGAACAGAATTCCAAATATCATCTAAGATTCTCTGTAAAAGGA 2250 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 2183 TTCAATTAGAACAGAATTCCAAATATCATCTAAGATTCTCTGTAAAAGGA 2232 my_vip3Ba1 2251 AAAGGTAGAGTAACGATGCAAGCTCAAACGTCCCATATAAATGTACCAGC 2300 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 2233 AAAGGTAGAGTAACGATGCAAGCTCAAACGTCCCATATAAATGTACCAGC 2282 my_vip3Ba1 2301 TACAAACGAAGAGGTTTCTATAATGATTGAAACTACACGCTTATACGGCG 2350 |||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 2283 TACAAACGAAGAGGTTTCTATAATGATTGAAACTACACGCTTATACGGCG 2332 my_vip3Ba1 2351 AAGGTATAATTAGCCTATTAAATGATGAAGTGGAGAATTCCGGGGTTATT 2400

142

|||||||||||||||||||||||||||||||||||||||||||||||||| AY823271.1 2333 AAGGTATAATTAGCCTATTAAATGATGAAGTGGAGAATTCCGGGGTTATT 2382 my_vip3Ba1 2401 TTTTCGGATGTATCTATAGTTAAAGAATAA 2430 |||||||||||||||||||||||||||||. AY823271.1 2383 TTTTCGGATGTATCTATAGTTAAAGAATAG 2412

The derived amino acid sequence from vip3Ba1 >vip3Ba1_amino acid (809 amino acids) MHHHHHHNMNNTKLNARALPSFIDYFNGIYGFATGIKDIMNMIFKTDTGGGNLTLDEILKNQDLLNQISDKLDGINGDLGDLIAQGNLNSELTKELLKIANEQNLMLNNVNAQLNSINATLNTYLPKITSMLNEVMKQNYVLSLQIEFLSKQLQEISDKLDIINLNVLINSTLTEITPAYQRIKYVNDKFDELTSTVEKNPKINQDNFTEDVIDNLTDLTELARSVTRNDMDSFEFYIKTFHDVMIGNNLFSRSALKTASELIAKENIHTRGSEIGNVYTFMIVLTSLQAKAFLTLTTCRKLLGLADIDYTQIMNENLDREKEEFRLNILPTLSNDFSNPNYTETLGSDLVDPIVTLEAEPGYALIGFEILNDPLPVLKVYQAKLKPNYQVDKESIMENIYGNIHKLLCPKQREQKYYIKDMTFPEGYVITKIVFEKKLNLLGYEVTANLYDPFTGSIDLNKTILESWKEDCCEEDCCEEDCCEEDCCEELYKIIEADTNGVYMPLGVISETFLTPIYSFKLIIDEKTKKISLAGKSYLRESLLATDLVNKETNLIPSPNGFISSIVQNWHITSDNIEPWKANNKNAYVDKTDAMVGFSSLYTHKDGEFLQFIGAKLKAKTEYIIQYTVKGNPEVYLKNNKDICYEDKTNNFDTFQTITKKFNSGVDPSEIYLVFKNQIGYEAWGNNFIILEIKSLETLPQILKPENWIPLGNAEIKEDGKIEISGNGSMNQYIQLEQNSKYHLRFSVKGKGRVTMQAQTSHINVPATNEEVSIMIETTRLYGEGIISLLNDEVENSGVIFSDVSIVKE*

143

APPENDIX II: The DNA sequence of the vip3Ba gene modified for expression in plants

>Vip3BaORF_cowpea

ATGAACATGAACAACACCAAGCTCAACGCTAGGGCCCTCCCATCCTTCATCGATTACTTCAACGGAATCTACGGCTTCGCCACCGGCATCAAGGATATCATGAATATGATCTTCAAGACCGACACCGGCGGAGGTAACCTTACCCTTGATGAGATTssCTCAAGAACCAGGACCTCCTCAACCAGATCTCCGATAAGCTCGATGGAATCAACGGCGATCTCGGTGATCTTATCGCTCAGGGAAACCTCAACTCCGAGCTGACCAAAGAGCTTCTCAAGATCGCTAACGAGCAGAACCTCATGCTCAACAACGTGAACGCTCAGCTCAACTCCATTAACGCTACCCTCAACACCTACCTCCCCAAGATTACCTCCATGCTGAACGAGGTGATGAAGCAGAACTACGTGCTCAGCCTCCAGATCGAGTTCTTGTCCAAGCAGCTCCAAGAGATCAGCGACAAGCTCGACATCATCAACCTCAACGTGCTCATCAACTCCACCCTTACCGAGATTACTCCAGCCTACCAGAGGATCAAGTACGTGAACGATAAGTTCGACGAGCTTACCTCCACCGTTGAGAAGAACCCAAAGATCAACCAGGACAACTTCACCGAGGACGTGATCGATAACCTCACCGATCTTACCGAGCTTGCCAGATCTGTGACTAGGAACGACATGGACAGCTTCGAGTTCTACATCAAGACCTTCCACGACGTGATGATCGGCAACAACCTGTTCTCTAGGTCCGCTCTTAAGACCGCCTCTGAGCTTATTGCCAAAGAGAACATTCACACCAGGGGCTCCGAGATCGGAAACGTTTACACCTTCATGATCGTGCTCACCAGCCTTCAGGCTAAGGCTTTCCTTACTCTTACTACCTGCAGAAAGCTCCTCGGCCTCGCTGATATTGATTACACCCAGATCATGAACGAGAACCTCGACCGTGAGAAAGAAGAGTTCAGGCTCAACATCCTCCCCACCCTTTCCAACGATTTCTCCAACCCAAACTACACCGAGACTCTCGGATCTGATCTCGTGGATCCAATTGTGACTCTTGAGGCAGAGCCAGGATACGCTCTTATCGGATTCGAGATCCTCAACGATCCACTCCCAGTGCTTAAGGTGTACCAGGCTAAGCTCAAGCCCAACTACCAGGTGGACAAAGAATCCATCATGGAAAACATCTACGGCAACATCCACAAGCTCCTCTGCCCAAAGCAACGTGAGCAGAAGTACTACATTAAGGACATGACCTTCCCCGAGGGCTACGTGATCACTAAGATTGTGTTCGAGAAGAAGCTCAACCTCCTCGGATATGAGGTGACCGCTAACCTCTACGATCCATTCACCGGATCCATCGACCTCAACAAGACCATTCTCGAGTCCTGGAAAGAGGACTGCTGCGAGGAAGATTGCTGCGAAGAGGACTGTTGTGAAGAGGATTGCTGTGAGGAACTCTACAAGATCATCGAGGCTGACACCAACGGTGTGTACATGCCACTTGGAGTGATCTCCGAGACTTTCCTGACCCCCATCTACAGCTTCAAGCTCATCATCGACGAAAAGACCAAGAAGATCTCCCTCGCCGGAAAGTCCTACCTTAGAGAGTCTCTTCTTGCTACCGACCTCGTGAACAAAGAGACTAACCTCATCCCATCCCCCAACGGCTTCATCTCTTCTATTGTGCAGAACTGGCACATCACCTCCGACAACATTGAACCATGGAAGGCCAACAACAAGAACGCCTACGTTGACAAGACCGATGCTATGGTGGGATTCAGCTCTCTCTACACCCATAAGGATGGTGAGTTCCTCCAGTTCATCGGCGCTAAGCTTAAGGCTAAGACCGAGTACATCATCCAGTACACCGTGAAGGGAAACCCAGAGGTGTACCTCAAGAACAACAAGGACATCTGCTACGAGGACAAGACCAACAACTTCGACACCTTCCAGACCATCACCAAGAAATTCAACTCCGGCGTTGACCCCTCTGAGATCTACCTCGTTTTTAAGAACCAGATCGGCTACGAGGCTTGGGGGAACAACTTCATTATCCTCGAGATCAAGTCCCTCGAGACACTCCCACAGATTCTTAAGCCAGAGAACTGGATCCCACTCGGCAACGCTGAAATCAAAGAGGATGGCAAGATCGAGATCTCCGGCAACGGATCTATGAACCAGTACATTCAGCTTGAGCAGAACTCCAAGTACCACCTCAGGTTCTCTGTTAAGGGAAAGGGAAGGGTTACCATGCAGGCTCAGACCTCTCATATTAACGTGCCAGCTACCAACGAAGAGGTGTCCATCATGATTGAGACTACCAGGCTTTACGGGGAGGGGATTATTTCCTTGCTCAACGACGAGGTTGAGAACAGCGGTGTGATCTTCTCCGATGTGTCCATCGTGAAAGAGTGA

144

APPENDIX III: Sequence alignment of vip3Ba gene (optimized cowpea version vs the original bacterial version) vip3Ba_cowpea 1 ATGAACATGAACAACACCAAGCTCAACGCTAGGGCCCTCCCATCCTTCAT 50 |||||||||||.||.||.||..|.|||||.||||||.|.|||...||.|| vip3Ba_Bt 1 ATGAACATGAATAATACTAAATTAAACGCAAGGGCCTTACCAAGTTTTAT 50 vip3Ba_cowpea 51 CGATTACTTCAACGGAATCTACGGCTTCGCCACCGGCATCAAGGATATCA 100 .|||||.||.||.||.||.||.||.||.|||||.||.|||||.||.||.| vip3Ba_Bt 51 TGATTATTTTAATGGCATTTATGGATTTGCCACTGGTATCAAAGACATTA 100 vip3Ba_cowpea 101 TGAATATGATCTTCAAGACCGACACCGGCGGAGGTAACCTTACCCTTGAT 150 ||||.|||||.||.||.||.||.||.||.||.|||||..|.||.||.||| vip3Ba_Bt 101 TGAACATGATTTTTAAAACGGATACAGGTGGTGGTAATTTAACACTAGAT 150 vip3Ba_cowpea 151 GAGATTCTCAAGAACCAGGACCTCCTCAACCAGATCTCCGATAAGCTCGA 200 ||.|||.|.|||||.||.||..|..|.||.||.|||||.|||||.||||| vip3Ba_Bt 151 GAAATTTTAAAGAATCAAGATTTATTAAATCAAATCTCAGATAAACTCGA 200 vip3Ba_cowpea 201 TGGAATCAACGGCGATCTCGGTGATCTTATCGCTCAGGGAAACCTCAACT 250 ||||||.||.||.|||.|.|||||||||||.||.||.||.||..|.||.| vip3Ba_Bt 201 TGGAATTAATGGAGATTTAGGTGATCTTATTGCACAAGGGAATTTGAATT 250 vip3Ba_cowpea 251 CCGAGCTGACCAAAGAGCTTCTCAAGATCGCTAACGAGCAGAACCTCATG 300 |.||.||.||.||.||..|..|.||.|||||.||.|||||.||.||.||| vip3Ba_Bt 251 CAGAACTAACTAAGGAATTATTAAAAATCGCCAATGAGCAAAATCTGATG 300 vip3Ba_cowpea 301 CTCAACAACGTGAACGCTCAGCTCAACTCCATTAACGCTACCCTCAACAC 350 .|.||.||.||.||.|||||.||.||.||.||.||.||.||.||.||.|| vip3Ba_Bt 301 TTAAATAATGTTAATGCTCAACTTAATTCAATAAATGCAACACTTAATAC 350 vip3Ba_cowpea 351 CTACCTCCCCAAGATTACCTCCATGCTGAACGAGGTGATGAAGCAGAACT 400 |||.||.||.||.|||||.||.|||||.||.|||||.|||||.||.|||| vip3Ba_Bt 351 CTATCTTCCAAAAATTACATCTATGCTAAATGAGGTAATGAAACAAAACT 400 vip3Ba_cowpea 401 ACGTGCTCAGCCTCCAGATCG-AGTTCTT-GTCCAAGCAGCTCCAAGAGA 448 |.||..|.||.||.||.||.| |.||||| || ||.||..|.||||||| vip3Ba_Bt 401 ATGTTTTAAGTCTACAAATAGAATTTCTTAGT--AAACAATTACAAGAGA 448 vip3Ba_cowpea 449 --TCAGCGACAAGCTCGACATCATCAACCTCAACGTGCTCATCAACTCCA 496 ||| ||.||.||.||.||.|||||..|.||.||..|.||.|||||.| vip3Ba_Bt 449 TTTCA--GATAAACTTGATATTATCAATTTAAATGTATTAATTAACTCTA 496 vip3Ba_cowpea 497 CCCTTACCGAGATTACTCCAGCCTACCAGAGGATCAAGTACGTGAACGAT 546 |..|.||.||||||||.||.||.||.||..|.||.||.||.||.|||||| vip3Ba_Bt 497 CATTGACAGAGATTACGCCTGCTTATCAACGTATTAAATATGTAAACGAT 546 vip3Ba_cowpea 547 AAGTTCGACGAGCTTACCTCCACCGTTGAGAAGAACCCAAAGATCAACCA 596 ||.||.||.||..|.||.||.||.||.||.||.||.|||||.||.||.|| vip3Ba_Bt 547 AAATTTGATGAATTGACTTCTACTGTAGAAAAAAATCCAAAAATTAATCA 596 vip3Ba_cowpea 597 GGACAACTTCACCGAGGACGTGATCGATAACCTCACCGATCTTACCGAGC 646 .||.||.||.||.||.||.||.||.|||||..|.||.|||||.||.||.| vip3Ba_Bt 597 AGATAATTTTACTGAAGATGTTATTGATAATTTAACTGATCTAACTGAAC 646 vip3Ba_cowpea 647 TTGCCAGATCTGTGACTAGGAACGACATGGACAGCTTCGAGTTCTACATC 696 |.||.|||..|||.||.||.||.||.|||||.||.||.||.||.||.||. vip3Ba_Bt 647 TAGCAAGAAGTGTAACGAGAAATGATATGGATAGTTTTGAATTTTATATT 696 vip3Ba_cowpea 697 AAGACCTTCCACGACGTGATGATCGGCAACAACCTGTTCTCTAGGTCCGC 746 ||.||.|||||.||.||||||||.||.||.||..|.|||..|.|.||.|| vip3Ba_Bt 697 AAAACTTTCCATGATGTGATGATAGGAAATAATTTATTCAGTCGTTCTGC 746 vip3Ba_cowpea 747 TCTTAAGACCGCCTCTGAGCTTATTGCCAAAGAGAACATTCACACCAGGG 796 ..|.||.||.||.||.||..|.|||||.||.||.||.||.|||||.|||| vip3Ba_Bt 747 ATTAAAAACTGCTTCAGAATTAATTGCTAAGGAAAATATACACACTAGGG 796

145

vip3Ba_cowpea 797 GCTCCGAGATCGGAAACGTTTACACCTTCATGATCGTGCTCACCAGCCTT 846 |....||.||.||.||.||.|||||.||.|||||.||..|.| |..|||| vip3Ba_Bt 797 GAAGTGAAATTGGTAATGTCTACACTTTTATGATTGTTTTGA-CTTCCTT 845 vip3Ba_cowpea 847 -CAGGCTAAGGCTTTCCTTACTCTTACTACCTGCAGAAAGCTCCTCGGCC 895 ||.||.||.||.|||||.|||.|.|||||.||..|.||..|..|.||.. vip3Ba_Bt 846 ACAAGCAAAAGCGTTCCTAACTTTAACTACATGTCGTAAATTATTAGGAT 895 vip3Ba_cowpea 896 TCGCTGATATTGATTACACCCAGATCATGAACGAGAACCTCGACCGTGAG 945 |.||.|||||.|||||.||.||.||.|||||.||.||..|.||..|.||. vip3Ba_Bt 896 TAGCAGATATCGATTATACACAAATTATGAATGAAAATTTAGATAGAGAA 945 vip3Ba_cowpea 946 AAAGAAGAGTTCAGGCTCAACATCCTCCCCACCCTTTCCAACGATTTCTC 995 |||||.||.||..|..|.||.||.||.||.||.|||||.||.|||||.|| vip3Ba_Bt 946 AAAGAGGAATTTCGCTTAAATATTCTTCCTACACTTTCTAATGATTTTTC 995 vip3Ba_cowpea 996 CAACCCAAACTACACCGAGACTCTCGGATCTGATCTCGTGGATCCAATTG 1045 .||.||.||.||.||.||.|||.|.|||..||||||.||.|||||||||| vip3Ba_Bt 996 TAATCCTAATTATACAGAAACTTTAGGAAGTGATCTTGTAGATCCAATTG 1045 vip3Ba_cowpea 1046 TGACTCTTGAGGCAGAGCCAGGATACGCTCTTATCGGATTCGAGATCCTC 1095 |.||..|.||.||.|||||.||.||.|||.|.||.||.||.||.||.||| vip3Ba_Bt 1046 TTACGTTAGAAGCTGAGCCTGGTTATGCTTTAATAGGTTTTGAAATTCTC 1095 vip3Ba_cowpea 1096 AACGATCCACTCCCAGTGCTTAAGGTGTACCAGGCTAAGCTCAAGCCCAA 1145 ||.||||||||.|||||..|.||.||.||.|||||.|||||.||.||.|| vip3Ba_Bt 1096 AATGATCCACTTCCAGTATTAAAAGTATATCAGGCAAAGCTAAAACCAAA 1145 vip3Ba_cowpea 1146 CTACCAGGTGGACAAAGAATCCATCATGGAAAACATCTACGGCAACATCC 1195 .||.||.||.||||||||.||.||.||||||||.||.||.||.||||||| vip3Ba_Bt 1146 TTATCAAGTCGACAAAGAGTCGATTATGGAAAATATTTATGGGAACATCC 1195 vip3Ba_cowpea 1196 ACAAGCTCCTCTGCCCAAAGCAACGTGAGCAGAAGTACTACATTAAGGAC 1245 |.||.||.||.||.|||||.||||||||.||.||.||.||.||.||.||| vip3Ba_Bt 1196 ATAAACTACTTTGTCCAAAACAACGTGAACAAAAATATTATATAAAAGAC 1245 vip3Ba_cowpea 1246 ATGACCTTCCCCGAGGGCTACGTGATCACTAAGATTGTGTTCGAGAAGAA 1295 |||||.|||||.||.||.||.||.|||||.||.|||||.||.||.||.|| vip3Ba_Bt 1246 ATGACATTCCCTGAAGGTTATGTAATCACCAAAATTGTTTTTGAAAAAAA 1295 vip3Ba_cowpea 1296 GCTCAACCTCCTCGGATATGAGGTGACCGCTAACCTCTACGATCCATTCA 1345 ..|.||.||..|.||||||||.||.||.||.||.||.||.|||||.||.| vip3Ba_Bt 1296 ATTGAATCTATTAGGATATGAAGTAACAGCAAATCTTTATGATCCTTTTA 1345 vip3Ba_cowpea 1346 CCGGATCCATCGACCTCAACAAGACCATTCTCGAGTCCTGGAAAGAGGAC 1395 |.|||...|||||..|.||.|||||.|||||.||.||.||||||||.||| vip3Ba_Bt 1346 CAGGAAGTATCGATTTGAATAAGACTATTCTAGAATCATGGAAAGAAGAC 1395 vip3Ba_cowpea 1396 TGCTGCGAGGAAGATTGCTGCGAAGAGGACTGTTGTGAAGAGGATTGCTG 1445 |||||.||.|||||.|||||.|||||.|||||.||||||||.|||||||| vip3Ba_Bt 1396 TGCTGTGAAGAAGACTGCTGTGAAGAAGACTGCTGTGAAGAAGATTGCTG 1445 vip3Ba_cowpea 1446 TGAGGAACTCTACAAGATCATCGAGGCTGACACCAACGGTGTGTACATGC 1495 |||||||.|.||.||.||.||.|||||.||.||.||||||||.||.|||| vip3Ba_Bt 1446 TGAGGAATTATATAAAATTATAGAGGCGGATACTAACGGTGTTTATATGC 1495 vip3Ba_cowpea 1496 CACTTGGAGTGATCTCCGAGACTTTCCTGACCCCCATCTACAGCTTCAAG 1545 |..|.||.||.||....||.||.||..|.||.||.|||||.||.||.||. vip3Ba_Bt 1496 CTTTGGGTGTAATTAGTGAAACATTTTTAACACCAATCTATAGTTTTAAA 1545 vip3Ba_cowpea 1546 CTCATCATCGACGAAAAGACCAAGAAGATCTCCCTCGCCGGAAAGTCCTA 1595 ||.||.||.||||||||.||.|||||.||.||..|.||.||.||.||.|| vip3Ba_Bt 1546 CTAATTATTGACGAAAAAACAAAGAAAATATCTTTAGCGGGTAAATCTTA 1595 vip3Ba_cowpea 1596 CCTTA-GAGAGTCTCTTCTTGCTACCGACCTCGTGAACAAAGAGACTAAC 1644 .||| |.||.|||.|.||.||.||.||..|.||.||.|||||.||.||. vip3Ba_Bt 1596 -TTTACGTGAATCTTTACTAGCCACAGATTTAGTTAATAAAGAAACAAAT 1644

146

vip3Ba_cowpea 1645 CTCATCCCATCCCCCAACGGCTTCATCTCTT----CTATTGTGCAGAACT 1690 .|.||.||.||.||.||.||.|||| || .|||||||||.||.| vip3Ba_Bt 1645 TTAATTCCTTCACCTAATGGTTTCA----TTAGCAGTATTGTGCAAAATT 1690 vip3Ba_cowpea 1691 GGCACATCACCTCCGACAACATTGAACCATGGAAGGCCAACAACAAGAAC 1740 ||||.||.||.||.||.||.||.||.||||||||.||.||.||.||.||. vip3Ba_Bt 1691 GGCATATAACATCGGATAATATAGAGCCATGGAAAGCGAATAATAAAAAT 1740 vip3Ba_cowpea 1741 GCCTACGTTGACAAGACCGATGCTATGGTGGGATTCAGCTCTCTCTACAC 1790 ||.||.||.||.|||||.|||||.|||||||||||.||||||.|.||.|| vip3Ba_Bt 1741 GCATATGTCGATAAGACGGATGCCATGGTGGGATTTAGCTCTTTATATAC 1790 vip3Ba_cowpea 1791 CCATAAGGATGGTGAGTTCCTCCAGTTCATCGGCGCTAAGCTTAAGGCTA 1840 .|||||||||||.||.|||.|.||.||.||.||.||||||.|.||||||| vip3Ba_Bt 1791 TCATAAGGATGGGGAATTCTTGCAATTTATTGGAGCTAAGTTAAAGGCTA 1840 vip3Ba_cowpea 1841 AGACCGAGTACATCATCCAGTACACCGTGAAGGGAAACCCAGAGGTGTAC 1890 |.||.|||||.|||||.||.||.||.||.||.||.||.||.||.||.||. vip3Ba_Bt 1841 AAACTGAGTATATCATTCAATATACTGTAAAAGGGAATCCGGAAGTTTAT 1890 vip3Ba_cowpea 1891 CTCAAGAACAACAAGGACATCTGCTACGAGGACAAGACCAACAACTTCGA 1940 .|.||.|||||.||.||.|||||.||.|||||.||.||.||.||.||.|| vip3Ba_Bt 1891 TTAAAAAACAATAAAGATATCTGTTATGAGGATAAAACAAATAATTTTGA 1940 vip3Ba_cowpea 1941 CACCTTCCAGACCATCACCAAGAAATTCAACTCCGGCGTTGACCCCTCTG 1990 |||.||.||.||.||.||.||.||||||||.||.||.||.||.||.||.| vip3Ba_Bt 1941 CACGTTTCAAACTATAACTAAAAAATTCAATTCAGGAGTAGATCCATCCG 1990 vip3Ba_cowpea 1991 AGATCTACCTCGTTTTTAAGAACCAGATCGGCTACGAGGCTTGGGGGAAC 2040 |.||.||.||.||||||||.||.||.||.||.||.||.||.|||||.||. vip3Ba_Bt 1991 AAATATATCTAGTTTTTAAAAATCAAATTGGATATGAAGCATGGGGAAAT 2040 vip3Ba_cowpea 2041 AACTTCATTATCCTCGAGATCAAGTCCCTCGAGACACTCCCACAGATTCT 2090 |||||.|||||.||.||.||.|||||.||.||.||.||.|||||.||..| vip3Ba_Bt 2041 AACTTTATTATACTTGAAATTAAGTCGCTTGAAACCCTACCACAAATATT 2090 vip3Ba_cowpea 2091 TAAGCCAGAGAACTGGATCCCACTCGGCAACGCTGAAATCAAAGAGGATG 2140 .||.||.||.||.|||||.||..|.||.||.|||||.||.|||||.|||| vip3Ba_Bt 2091 AAAACCTGAAAATTGGATTCCTTTGGGTAATGCTGAGATTAAAGAAGATG 2140 vip3Ba_cowpea 2141 GCAAGATCGAGATCTCCGGCAACGGATCTATGAACCAGTACATTCAGCTT 2190 |.||.||.|||||.||.||.||.|||...|||||.||.||.|||||..|. vip3Ba_Bt 2141 GAAAAATTGAGATTTCAGGTAATGGAAGCATGAATCAATATATTCAATTA 2190 vip3Ba_cowpea 2191 GAGCAGAACTCCAAGTACCACCTCAGGTTCTCTGTTAAGGGAAAGGGAAG 2240 ||.|||||.|||||.||.||.||.||.||||||||.||.|||||.||.|| vip3Ba_Bt 2191 GAACAGAATTCCAAATATCATCTAAGATTCTCTGTAAAAGGAAAAGGTAG 2240 vip3Ba_cowpea 2241 GGTTACCATGCAGGCTCAGACCTCTCATATTAACGTGCCAGCTACCAACG 2290 .||.||.|||||.|||||.||.||.|||||.||.||.||||||||.|||| vip3Ba_Bt 2241 AGTAACGATGCAAGCTCAAACGTCCCATATAAATGTACCAGCTACAAACG 2290 vip3Ba_cowpea 2291 AAGAGGTGTCCATCATGATTGAGACTACCAGGCTT-TACGGGGAGGGGAT 2339 |||||||.||.||.||||||||.|||| ||.|||| |||||.||.||.|| vip3Ba_Bt 2291 AAGAGGTTTCTATAATGATTGAAACTA-CACGCTTATACGGCGAAGGTAT 2339 vip3Ba_cowpea 2340 TATTTCCTTGCTCAACGACGAGGTTGAGAACAGCGGTGTGATCTTCTCCG 2389 .|||..|.|..|.||.||.||.||.|||||...|||.||.||.||.||.| vip3Ba_Bt 2340 AATTAGCCTATTAAATGATGAAGTGGAGAATTCCGGGGTTATTTTTTCGG 2389 vip3Ba_cowpea 2390 ATGTGTCCATCGTGAAAGAGT 2410 ||||.||.||.||.|||||.| vip3Ba_Bt 2390 ATGTATCTATAGTTAAAGAAT 2410

enhancing the resilience of bt cowpea vigna …eprints.qut.edu.au/96968/4/bosibori bett...

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