studies on p-ine: ammonia-lyase peroxidases...
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STUDIES ON P-INE: AMMONIA-LYASE AND PEROXIDASES IN
CASSAVA (Manihot esculenta Crantz) DURING ROOT POST-HARVEST
DETERIORATION AND INTERACTION WITH XANTHOMONAS
A Thesis
Presented to
The Faculty of Graduate Studies
of
The University of Guelph
by
LUIZ FILIPE PROTASIO PEREIRA
In partial fulfilment of requirements
for the degree of
Doctor of Philosophy
February, 1998
@ Luiz Filipe P. Pereira, 1998
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ABSTRACT
STUDIES ON PHENYLALANINE AMMONIA-LYASE I L I PEROXIDASES IN
CASSAVA (Manihot esculenta Crantz) DURING ROOT POST-HARVEST
DETERIORATION AND INTEWCTION WITH XANTNOMONAS
L. Filipe P. Pereira
University of Guelph, 1998
Advisor:
Dr, L. ~rickson
The main objective of this research was to initiate a
molecular characterization of genes involved in the process
of resistance of cassava to Xanthomonas and in the
physiological post-harvest deterioration of the roots, which
are two major problems for this crop. The studies reported
in this thesis focused on characterization of genes for
phenylalanine ammonia-lyase (PAL) and peroxidase which are
involved in the initial steps of the phêryl-propa~oid
pathway and in the oxidation of the phenolic compocnds
produced by this pathway, respectively .
PCR clones of cassava genes for PAL ('IEP-AL) c ~ d
peroxidase (MEPX1) were isolated. MEPAL transcripcs were
detected in leaves, stems and petioles, and w e r e especially
prominently expressed in young tissues-
The plant-pathogen portion of the studies ch~racterized
the interaction between two cultivars of cassava (BICol 22
and CM 523-7) and two pathogens (X. axonopodis pv. manihotis
and X. cassavae). A resistant interaction was observed only
when cultivar MCol 22 was inoculated with X. cassavae,
wherein no disease symptoms were observed in the leaf and
there was a reduction of bacterial growth and induction of
both PAL and peroxidase enzymes. MEPAL mRNA was also
detected during the resistance interaction between cultivar
Mc01 22 and X. cassavae. Both clones showed potential as
molecular markers relevant to breeding for resistance to
bacterial blight.
During post-harvest deterioration of the roots, it was
demonstrated that there were differences in the enzyme
activity of both PAL and peroxidase enzymes, concomitant
with the level of deterioration of the roots, and probably
related to the rate of oxidation which is higher in the air-
exposed parts of the injured root, than in the inner layers.
In both layers, however, there was an increase of PAL and
peroxidase enzyme activity during deterioration, as well of
the PAL rnRNA transcription.
I am very grateful to Dr. Larry Erickson for his
academic support and encouragement during this study. 1 also
appreciate the participation and advise of Drs. Paul
Goodwyn, Ken Kasha and Peter Pauls for their critical review
and comments of this thesis.
Special thanks for Akwasi Agyare-Tabbi and Sean Rogers
for their friendship and support during the course of this
work. 1 also thank the technicians, fellow graduate
students, staff and faculty of Crop Science Department for
their assistance when it most needed.
1 gratefully acknowledge the Brazilian Ministry of
Education - CAPES, for financial support, as well the
Agronomy Institute of Parana State - IAPAR for granting me a
leave of absence during this study.
Finally 1 would like to express my deep appreciation to
Marcela, Alexandre and Marina, for their encouragement,.
support and patience throughout the course of this graduate
program.
Appendix 1.1 Methods of inoculation and bacterial growth in selective media . . . . . . . . . . . . . . . . . . . 150
Appendix 1.2 Alignment of peroxidase genes for . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . primer design 151
Appendix 1.3 Protocols for cassava DNA and RNA extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
Appendix 1.4 Partial sequence of two clones of amplified cassava DNA using peroxidase
. . . . . . . . . . . . . . . . . . . . . . . . . . . . desigmed primers 156
Appendix 1.5 Southern blot of cassava DNA probed with MEPX1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Appendix 2.1 Alignment of PAL oenes for primer design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
Appendix 2.2 Construction of a genomic library . . . . . . . . . . . . and efforts to isolate PAL genes 159
Appendix 2 - 3 Southern blot of lamda clones probed with MEPAL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
Appendix 3.1 Effect of enzymatic and transcriptional PAL inhibitors in the deterioration of inner
. . . . . . . . . . . . . layers of cassava root sections 173
. . . . . . . . . . . . . . . . . . . . . . . . Appendix 3.2 Anova Tables 174
List of F i g u r e s
Literature Review
1 World cassava production during 1962-1995 - - - . . . . . . . . 4
2 General phenylpropanoid pathway . . . . . . . . . . . . . . . . . - ..25
Chagter 1
1.1 Cassava leaves from cultivar MC0122 after inoculation with Xanthomonas . . . . . . . . . . . . . , , . . . . . . . . 5 0
1-2 Cassava leaves £rom cultivar CMS23-7 after inoculation with Xanthomonas . . . . . . . , . . . . . . . . . . . . . . . 51
1.3 Multiplication of Xanthomonas in cassava leaves . . . . 53
1.4 Electrolyte leakage £rom cassava leaf disks inoculated with Xanthomonas . . . . . . . . . . . . . . . . . . . . . . . . 55
1.5 Peroxidase activity in cassava leaves inoculated with Xanthomonas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
1.6 Gel of PCR products using peroxidase-specific primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
1-7 Sequence of cassava peroxidase clone (MEPX1) . . . . . . . 60
1.8 Alignment of the predicted amino acid sequence of MEPXl and plant peroxidases sequences . . , . . . . - . . . . . . 6 2
1.9 Southern Blot of cassava DNA probed with MEPX1 . . . . . 64
1.10 Northern blot analysis of total RNA in cassava leaves inoculated with Xanthomonas and probed with MEPXl , . . . . . . . . . . . . . . . . . . . . . . - . . . . . . . . . . . . . . . . . 66
Chagter 2
2.1 PAL enzyme activity in cassava leaves after inoculation with Xanthomonas . . . . . . . . . . - . . . . . . . . . . . . 7 9
2.2 Gel of PCR products using PAL-specific primers . . . . . 81
2.3 Sequence and alignment of a cassava amplified
transcription rates of PAL during root deterioration in cassava .................................
3 . 1 1 ~eroxidase activity i n outer layers of cassava root . . . . . . sections after treatment with PAL inhibitors 1 1 6
. . . 1 .1A Apparatus used to wound leaf before inoculation 1 5 0
1 . 1 B Syringe inoculation method . . . . . . . . . . . . . . . . . . . . . . . . 1 5 0
1.E Cassava leaf inoculated with X. axonopodis pv. manihotis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - . . - . . . . . . l 5 O
. . . . . . . . . . 1 . l D Cassava leaf inoculated with X. cassavae 1 5 0
1. I E X. axonopodis pv. manihotis and X. cassavae . . . . . . . . . growing in specific media for Xanthomonas 1 5 0
1 . 5 Southern blot of cassava DNA probed with m P X 1 ............-..--..-......--.-----....-.l57
2 . 2 . 1 Membrane plaque lifts screened with heterologous PAL probe . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 6 3
2 . 2 . 2 Southern blots of lambda clones with cassava DNA inserts .....................-................l64
. . . . . . . . 2 . 2 . 3 Membrane plaque lifts screened with MEPAL 165
2 . 2 . 4 Southern blots of lambda clones with cassava DNA inserts . . . . . . . . . . . . . . . . . . . . . - . . . . - . . . 1 6 6
2 . 2 . 6 Sequence and GenBank homology search of clone 3 . 4 3 ...........----.-..........-...........l67
2 . 2 . 7 Sequence and Genbank homology search of clone 3 . 4 1 . . . . . . . . . . . . . . . . . . . . . . . . . . - . . . . . . . - . . - - 1 6 8
2 . 2 . 8 Sequence and Genbank homology search of clone 5 - 1 1 ..-............................-.........l69
2 . 2 . 9 Partial Sequence and Genbank homology search of clone 5.16 (5' end) . . . . . . . . . . . . . . . . . . . . . l 7 O
2 . 2 . 1 0 Partial Sequence and Genbank homology . . . . . . . . . . . . . . . . . . . . search of clone 5.16 ( 3 end) L71
2.2.11 Partial Sequence and Genbank homology search of clone 4.27 . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 7 2
Southern blot of cassava DNA probed with MEPU . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
3.1 Effect of enzymatic and transcriptional PAL inhibitors in the deterioration of i m e r layers of cassava root sections . . . . . . . . . . . . . . . . . . 1 7 4
viii
List of Abbreviations
4CL
AVR
C4H
CAD
CBB
CBN
C IAT
HCN
HPRP
HR
HRP
MEPAL
MEPOX
PAL
PPP
PR
R
- coumarate:CoA ligase
- avirulent genes
- cimarnate 4-hydroxylase
- cinnamyl alcohol dehydrogenase
- cassava bacterial blight
- cassava bacterial necrosis
- ~nternational Center for Tropical ~griculture
- Hydrogen Cyanide
- hydroxy-proline r i c h proteins
- hypersensitive response
- hypersensitive response genes
- Manihot esculenta C. PAL gene
- Manihot esculenta C. peroxidase gene
- phenylalanine ammonia-lyase
- phenylpropanoid pathway
- pathogenesis related proteins
- resistance gene
General introduction
Cassava is one of the most important tropical crops,
being the main source of calories for more than 400 million
people in Africa and South America (Cock 1985) . Two major problems with the crop are post-harvest root deterioration
that occurs a few days after harvest, and bacterial blight
disease that can cause significant yield losses. One of the
biochemical mechanisms involved in both problems is the
production of several secondary plant products originating
from or passing through the phenylpropanoid pathway (PPP) .
Phenylalanine ammonia-lyase (PAL EC 4 . 3 . 1 . 5 ) is a critical
enzyme in this pathway, working as a trigger for the
production of these secondary compounds.
The PAL enzyme is part of plants defence against many
different stresses and an increase O £ PAL activity in plants
under different stresses has been extensively reported
(Hahlbrock and Scheel 1989; Dixon 1986; Jones et al, 1984).
In the last 5 years, PAL genes have been isolated £rom
several specieç including parsley, french-beans, potato,
alfalfa, tomato (reviewed by Douglas et al. 1991, van de
Meer et al. 1993). Since the PPP is one of the biosynthetic
pathways for diverse plant natural products, studies on PAL
have not only a practical importance in understanding the
defence and the wound response mechanisms in plants, but can
also be used as a mode1 to study the basic mechanisms of
gene regulation and expression.
Peroxidases are also intrinsically related to plant
response to stresses, and play a key role in the protective
mechanism of the plant. An increase in peroxidase activity
occurs during both incompatible plant pathogen interactions
and wounding of plant tissues. The peroxidase enzymes
participate in the oxidation of phenolic products
originating £rom the PPP that will be cross-linked in the
ce11 wall to form lignin and suberin.
The objective of this work was to identify and
characterize the expression of PAL and peroxidase genes in
cassava involved in root post-harvest deterioration and in
the interaction with Xanthomonas axonopodis pv. manihotis
and Xanthomonas cassavae. Isolation and characterization of
genes that are involved in these processes will allow a
better understanding of the molecular defense mechanisms of
this important crop, providing information that can be used
in both conventional breeding programs as well as genetic
improvement via biotechnology methods.
Literature review
Cassava
Cassava (Man iho t esculenta Crantz) is a woody shrub
that grows mainly in tropical lowland areas, although it is
also cultivated in tropical highlands and subtropical areas.
It originated in north-east Brazil with the likelihood of an
additional center of origin in Central America (Rogers
1963). Cassava belongs to the ~uphorbiaceae family. It is an
allotetraploid with 2n=36 chromosomes (Umanah and Hartmann
1973) . The plants can reach maturity and produce seeds, although the conventional method for propagation is through
the use of stem cuttings. The cassava roots are an important
source of calories in developing countries, having a major
role in the nutrition O£ poor people in South America and
Africa. They are one of the most important sources of
calories for more than 400 million people throughout al1
tropical regions (Cock 1985). Cassava is planted in several
regions as a security crop to avoid famine after a drought
season due to the loss of other crops (IITA 1995). It is
also a very important cash crop in South-East Asian
countries, where most of the production is exported to
developed countries as pellets for animal feed.
The world production of cassava is estimated at 163
million tonnes of fresh roots with an average yield of 10
tonnes/ha. Nigeria, Brazil, Thailand, Zaire and Indonesia
are the major producers (FA0 1968-1996). Figure 1 shows the
trend in production of cassava between 1962 and 1995. It is
interesting to observe the fast growth of cassava production
in both Asia during 1972-1980 and in Africa during the last
10 years.
The plant can be grown on marginal land with
soils, low pH and a high concentration of soluble
3
sandy
aluminum,
1980 Year
World Africa S America Asia -+- C
Figure 1 World cassava production during 1962-1995 (FA0 1 9 6 8 - 1 9 9 6 ) .
which are al1 characteristics of depleted tropical soils
that are unsuitable for the majority of other crops.
Although the optimum conditions for growth are in the
subhumid and humid tropics with annual rainfall of 1500 mm,
cassava is drought tolerant and can be grown in locations
with less than 600 mm of precipitation. with erratic
rainfall and where the dry season lasts as long as eight
months (El-Sharkawy 1993) .
Cassava cultivars have been differentiated on the basis
of morphology of the plant, tuber shape, earliness to
maturity. yield and the content of cyanogenic glucosides in
the roots. This cyanogenic content has been used to
distinguish two major groups of cassava: the bitter
varieties. in which the roots contain a high and well-
distributed amount of cyanogenic glucoside and the sweeter
varieties in which low levels of the glucoside is confined
to the peel (Conceicao 1980). Linamarin and lotaustralin are
the major cyanogenic glucosides, found in a 9 3 : 7 ratio.
Linamarase is the main enzyme responsible for break-dom of
iinamarin, and further release of Hydrogen Cyanide (HCN) .
Although the cyanogenic glucoside content is considered a
drawback for the crop, especially for fresh consumption of
roots. the industrial processes for producing different
cassava flours have easily eliminated these compounds from
cassava products due to the action of heat. Approximately 5
70% of the linamarin is removed by enzymatic hydrolysis
during processing, and almost al1 cyanide generated is
eliminated by volatization or solubilization (Nambisan and
Sundaresan 1985).
frica an mosaic virus is another limitation to
production of the crop, although the disease is concentrated
in frica an countries. Due to the heavy losses caused by
planting cuttings infected with virus, research is currently
underway at the Center for International Tropical
Agriculture (CIAT, Colombia), Scripps Institute (San Diego,
CA) and the International Institute for Tropical Agriculture
(IITA, Nigeria), aimed at developing sources of resistance
to the virus through the use of both conventional and
biotechnological breeding and rnethods (Asiedu et al. 1992,
Thro 1993). In addition, the same institutes have devoted
considerable effort to the production and distribution of
virus-free cuttings, originating £rom meristem-tip tissue
culture .
Two other important constraints on the crop, bacterial
blight and root post-harvest deterioration, are reviewed
below in more detail.
Xanthanonas
X a n thomonads are gram negative, obiigate aerobic, plant
associated bacteria. Infections caused by this genus have
been reported in almost 400 species of mono- and
dicotyledonous plants (Hayward 1993). Symptoms of the
disease caused by Xanthomonas bacteria include necrosis,
blight, vascular wilt, pustules and leaf spots in different
tissues such as leaves, stems and fruits. The proposed
reclassification of the Xanthomonas genus by Vauterim e t al.
(1995) will be used throughout this report.
Cassava and Xanthmonas
Two bacterial diseases in cassava are caused by
Xarithomonas: cassava bacterial blight (CBB) and cassava
bacterial necrosis (CBN) . Xanthomonas axonopod i s pv.
m a n i h o t i s is the causal agent of Sacterial blight, while
cassava bacterial necrosis occurs results £rom infections by
Xanthomonas cas savae (Onyango and Mukunya 1982) . The initial
symptoms of disease caused by both pathovars are similar and
both produce blight symptoms and leaf defoliation.
Differences in symptoms between the diseases occur only when
x. a x o n o p o d i s pv. m a n i h o t i s systemically invades the plant
causing vascular wilt and tip dieback.
Cassava bacterial necrosis (CBN) is not as widespread
as CBB, having been reported mainly in Eastern Africa and
Zaire. The disease symptoms are very similar to CBB, with
water-soaked and yellow necrotic regions around the
infection points. Defoliation may occur depending on the
severity of the infestation, but there is no systemic
invasion of the plant as in CBB.
Cassava bacterial blight
Cassava bacterial blight (CBB), caused by X. axonopodis
pv. manihotis is considered the rnost important bacterial
cassava disease. It was reported initially in Brazil, but
today occurs world-wide. It can result in significant yield
reductions in the crop. Umemura and Kawano (1983) reported a
yield reduction of up to 92% in susceptible cultivars, when
compared with resistant ones.
The bacteria, dispersed by rain splash or insects,
penetrate hosts via stomata and epidermal wounds, destroying
the mesophyll and penetrate the vascular tissues (Lozano
1986, Boher et al. 1995). Infected leaves exhibit water-
soaked deposits due to production of bacterial
exopolyssacharides followed by necrosis of the areas
surrounding the initial points of infection. The necrotic
spots, characteristically yellow, increase and cause
defoliation. This is followed by spread of the bacteria into
the stem and petioles through the xylem vessels and phloem.
The infection occurs frequently in young plant tissues
causing extensive breakdown of parenchymatous tissues with
subsequent wilting and die-back of the plants (Lozano 1986) .
The epidemiology of the disease is dependent on
environmental conditions. Tropical regions. where maximum
and minimum temperatures between 20 and 30°C are observed,
are ideal for survival of the bacteria. However, the disease
occurs mainly when the temperature fïuctuates to more than
15O Celsius. The bacteria have a low survival rate in moist
soil, and the disease is more prominent in savannah areas
than in forest zones in Africa (Persley 1980). The bacteria
can survive the dry season in symptomless plants or
epiphytically on leaves and debris, building up an inoculum
for the next rainy season (Daniel and Boher 1985).
The disease can be controlled with specific cultural
practices. the use of resistant vârieties, biological
control and phytosanitary methods. Since a six-month
interval is sufficient to avoid infestation £rom the field,
the use of crop rotation. where the debris of cassava is
either incorporated into the soil or removed, has been
successfully used to control the disease when disease-free
cuttings are also used in the next cassava planting (Cock
1985). Utilization of such phytosanitary practices. combined
with the planting of resistant clones. increased the
production £ r o m 7-8 to 20 tonnes/ha in regions with previous
high incidence of the disease (Lozano 1986. Umemura and 9
Kawano 19 8 3 ) .
Characterization of resistant and tolerant cultivars
was initiated by Lozario and Laberry (1982). Experiments
indicated the existence of genotypes with resistance to CBB
and these genetic lines have been incorporated into the
cassava breeding program at CIAT (Lozano 1986). ~esistance
is controlled by more that one gene with additive effects
(Umemura and Kawano 1983, Hahn 1989). Although the resistant
cultivars cari be easily propagated vegetatively, breeding
and testing of this material is a lengthy process. ~ccording
to Lozano (1986) four cycles of evaluation are necessary to
identify truly resistant cultivars. For breeding purposes
the challenge is increased due to the long life cycle of
some cultivars where seed production can take up to three
years .
Kpemoua et al. (1996) characterized the defense
response to CBB in cassava stems at the cytochemical level.
The production of phenolic compounds in the phloem and xylern
cells of the resistant cultivars was signif icantly higher
than that in susceptible ones. There was also a higher
accumulation of lignin and a greater formation of calloses
and tyloses in resistant cultivars.
Boher et al. (1995) reported the capacity of X.
axonopodis pv. manihotis to degrade the ce11 walls and
middle lamella of susceptible cultivars of cassava. They
observed the release of p-glucans and pectin oligomers in
the infected tissue- Their results also indicated the
secretion of active lytic enzymes by the bacteria which
could degrade the plant ce11 wall.
Flood e t al, (1995) reported that there were
differences in ion leakage of cassava leaves inoculated with
different pathogens. There was a different pattern of
conductivity in compatible and incompatible reactions
between X a n t h o m o n a s and cassava. However, there was no
difference in the multiplication of the bacteria in the
susceptible and resistant plants during the 18-day period
af ter inoculation.
PAL genes in cassava are certainly involved in disease
response to either CBB and CBN. Although a characterization
of the interaction between cassava and CBB has been reported
at the biochemical and histochernical levels (Kpernoua et al.
1 9 9 6 , Boher et al. 1995) , there are no studies at the
molecular level for either X . axonopodis pv. manihotis or X.
cassavae. Characterization of PAL genes during interaction
with those X a n t h o m o n a d s would allow us to gain valuable
information regarding the defense mechanism of this plant
with possible applications in improvement in the resistance
against both diseases,
Plant x gathogen interactions
Plants employ a variety of defense mechanisms during a
resistance response to pathogens. These mechanisms can work
as a cascade of events or simultaneously depending on the
stage of the interaction and the defensive strategy used.
Generally the defense mechanisms include the use of
mechanical barriers, defensive proteins and defensive
enzymes.
Fritig et al. (1987) and Bent (1996) described the
general mechanism of a plant's defensive response to
pathogens. There is a level of recognition involving the
avirulence (Avr) gene from the pathogen and the resistance
( R ) gene in the plant host. This model, proposed by Flor
( 1 9 4 7 ) , postulated that resistance is achieved whenever the
Avr gene product is recognized by the R gene product.
Several Avr genes £ r o m different pathogens have been cloned
as well as a few R genes, and molecular characterization of
this interaction is under investigation (Table 1).
Interestingly, most of the R genes have no strong
similarity, other than leucine-rich repeat (LRR) regions.
LRRs have been reported to be involved in protein-protein
interactions in different organisms (Kobe and Deisenhofer
1994). Anderson et al. (1997) reported a mutant allele of
the M rust resistance gene in flax that resulted in the loss
Table 1. Cloned Plant Disease Resistance Genea
R Gene/Plant Pa thogen Avr Gene structureb Ref erence
H m l maize
Pto Tomato
Xd21 Rice
RPS2 Arabidopsis
RPMl Arabidopsis
Prf Tomato
N Tobacco
r W
L6 Flax
M Flax
CE-9 Tomato
C f - 2 Tomato
12C Toinato
Cochl iobolus carbonum
P. syringae pv. tomato
X. campestris pv. oryzae
P. syringae pv. comato
P. syringae pv. macul icola
P. S. pv. tomato
Tobacco mosaic virus
Malampsora lini
Malampsora lini
Cladospori um tu1 vum
C l adospor i um f 11 l vum
P.oxvsporum t.sp.lycopersici
None
avrpto
Unknown
avrRp t2
a v f fpml ,
avr-pt : O
Unknown
Unknowri
Unknown
Avrg
Avr2
Uriknown
Toxin reductase
Protein kinase
LRR, protein kinase
LRR, NBS, LZ
LRR, N B S , LZ avrb
LRR, NBS, LZ
LRR, NES
LRR, NBS
LRR
LRR
LRR
LRR, NBS
Johal and Briggs ( 1 9 9 2 )
Martin et al. ( 1 9 9 3 )
Song et al. ( 1 9 9 5 )
Bent et al. ( 1 9 9 4 ) ; Mindrinos et al. 1 1 9 9 4 )
Grant et al. ( 1 9 9 5 )
Salmeron et al. ( 1 9 9 6 )
Whitham et al. ( 1 9 9 4 )
Lawrence et al. (1995)
Anderson et al. ( 1 9 9 7 )
Jories et al. ( 1 9 9 4 )
Dixon et al. 11996)
Qri et dl. ( 1 9 9 7 1
"Adapted from Bent (1996). b~trrcture refers to protein structure motifs recognizable in the derived amino acid sequences of the listed genes: leucine-rich repeat (LRR; Kobe and Deisenhofer, 1994), nucleotide binding site (NBS; Saraste et al., 19901, and leucine zipper (LZ; Albert, 1992).
of its LRR site which in turn led to an inability to confer
resistance. However, other R genes encode for totally
different proteins, some with nucleotide binding sites, that
may be related to regulatory functions.
Tang et aL(1996) reported an interaction between the
products of the AvrPto gene from Pseudomonas syringae pv.
tomato and the Pto R gene in tomato. Modifications in either
gene can lead to non-interaction of the gene products
resulting in infection of the plant by the pathogen. The Pto
gene encodes for a protein kinase that is targeted to the
cytoplasm. It is also involved in phosphorylation of P t i l , a
serine/threonine kinase, that is related to the
hypersensi t ive response (HR) agains t P. syr ingae ( Zhou e t
al. 1995). The Fen gene, with high homology to the Pto gene
and also located in the same cluster, cannot either interact
with the avr product or phosphorylate P t i l .
The resulting recognition of the gene-for-gene
interaction or other incompatible interaction can activate
hydrolases in both pathogen and plant that can degrade the
ce11 wall of their opponents. There is also the production
of chitinases and polygalacturonidases that will attack the
pathogen and the plant ce11 wall, respectively. Elicitors
£ r o m the pathogen or
be produced via ce11
endogenous elicitors £rom the plant can
wall degradation or also directly from
the initial gene-for-gene interaction. Chitinases and
glucanases, in addition to their antimicrobial lytic
activities, may be involved in the amplification of
elicitors signalling the initial stages of the microbial
attack. These elicitors will woxk on regulation of the
expression of several genes that rnight produce an HR. At
this level of interaction more hydrolases will be expressed,
as well as proteases, polygalacturonidases, pathogen-related
(PR) proteins, antimicrobial enzymes, hydroxy-proline rich
proteins (HPRP), and enzymes involved in the production of
lignin, suberin, phenolic polymers and polysaccharides
(McNeil et al. 1984, Corbin et al. 1987, Bowles 1990) .
One of the first events during the HR and several other
mechanism of resistance is the induction of the PPP and the
production of several phenolic compounds related to plant
defence. During the first step of the pathway, phenylalanine
or tyrosine is deaminated by phenylalanine ammonia-lyase or
tyrosine ammonia-lyase, producing respectively cinnamic acid
or p-coumarate. This is the initial step for the production
of several secondary metabolites that will lead to the
formation of lignin, suberin, phytoalexins, coumarins and
others. It is interesting to note that the production of PAL
and other enzymes involved in the pathway is very rapid and
specific in resistant plants with PAL rnRNA being extensively
produced in the cells surrounding the infection site (Lawton 15
and Lamb 1987).
From the PPP, a variety of defensive compounds are
produced. There is the formation of lignin, reinforcing the
ce11 wall, and making it more resistant to degrading enzymes
as well as more resistant to diffusion of toxins from the
pathogen. Isolation of the pathogen behind "barricades" of
lignin is mediated by peroxidases, another group of
defensive enzymes, which promote cross-linking of phenolic
compounds and proteins in the matrix of the ce11 wall. The
phenylpropanoid pathway is also the source of precursors for
the synthesis of sorne phytoalexins such as coumarins and
pterocarpans. These latter products are synthesized by
living cells around the infection site and are exported to
the dying cells. The accumulation of phytoalexins and
pathogenesis related (PR) proteins occurs both in the
apoplast and in the vacuole creating two lines of defense
for the plant against a pathogen (Ohashi and Ohshime 1992).
Mechanical barriers include aromatic macromolecules
that are produced in large amounts and incorporated into the
host ce11 wall. There is also the increase in hydroxy-
proline rich proteins (HPRP) which becomes insoluble after
secretion into the ceIl wall. These proteins may function in
the defense response by forming a physical barrier or
allowing the deposition of lignin reinforcing the barrier.
It is also possible that these proteins contribute to the
16
immobilization of a pathogen in the cell wall (McNeil et al.
1984) .
Synergistic interactions can also occur between
different compounds during the defense response. It has been
demonstrated that two products involved in signalling,
jasmonate and ethylene, can increase the production of the
PR protein, osmotin (Xu et al. 1994). The same work suggests
that the induction of different PR proteins is controlled
via different pathways. Alternative pathways for induction
of PR proteins rnay be beneficial for the plant since it
would allow a more specific and coordinated production of
the PR proteins.
Jasmonate is a signal rnolecule that is related to the
induction of defense proteins such as: proteinase
inhibitors, thionins, proline-rich proteins, enzymes in the
PPP and ribosome inactivation proteins. mile proteinase
inhibitors are more related to defense against insects and
herbivores, thionins accumulate in the vacuole and the ce11
wall and have antimicrobial properties (Bohlmann 1994).
Interestingly, jasmonate also induces ribosome inactivating
proteins. that will lead to decay of cytoplasrnic polysomes,
dom-regulation of protein synthesis and ce11 death
(Reinbothe et al, 1994). The jasmonates rnay play two major
functions in the defense mechanisrn, producing defensive
proteins at initial stages of infection or promoting ce11
suicide if necessary at later stages.
Plants can also develop systemic acquired resistance
(SAR) where resistance to a fungus, bacterium or virus is
induced by prior inoculation with an avirulent pathogen.
Plants with SAR showed an accumulation of PR proteins and HR
to the pathogen (Alexander et al. 1993). It is not clear in
SAR what kind of signaling is involved to transport the
irnmuno-like response in the plant. Possible candidates
include ethylene. systemin. ce11 wall fragments, salicylic
acid or a combination of signals. Although these signals
show a good correlation with induction of PR proteins, they
do not appear to move systematically in the plant (Eneydi et
al. 1 9 9 2 , Delaney 1997) .
Post-harvest deterioration in cassava
In contrast to cassava's robust qualities in the field,
cassava roots undergo a rapid process of deterioration after
harvesting. Roots start to perish 2-5 days after harvest,
which constitutes a major constraint on utilization of the
crop. Losses in cassava due to post-harvest deterioration
are hard to quantify and Vary £rom 3 to 15% of the roots.
three days after harvest. There is also a loss in market
value, where remaining deteriorated roots are commercialized
for industrial purposes at lower values than fresh roots
(Wenham 1995). This rapid process of deterioration has also
implications in production systems, processing and
consumption of the roots or its products.
A survey in Ghana reported that 30% of the cassava
farmers mention perishibility of the roots as a major risk
in cassava production (NRI 1992 in Wenham 1995). Although it
is possible to delay the harvest of the roots even for one
full season, this practice entails an economic loss due to
the occupation of the land. There is also a decrease in the
quality of the roots by reduction of their s t a rch content,
and an increase in fiber and cooking time (Rickard and
Coursey 1981, Wheatley and Gomez 1985) -
According to Booth (1975) there are two types of post-
harvest deterioration in cassava. The primary or
physiological deterioration is the initial cause of loss of
acceptability of roots for fresh consumption and is
identified by fine blue-black streaks in the root vascular
tissue. The secondary, or microbial deterioration of the
roots occurs when the roots have already become unacceptable
due to the primary deterioration. It is caused by pathogenic
rots, fermentation and/or softening of the roots. Several
microorganisms have been associated with microbiological
deterioration, including Aspergillus f l a v u s , Fusarium
solani, ~otryodiplodia theobromae, Trichodema har iz ianwn, 19
among others. These microorganisms can generally be isolated
at 7 days post-harvest after signs of physiological
deterioration have become apparent. Application of
fungicides and bactericides can reduce microbiological
deterioration, and decrease perishability when used with
techniques to inhibit physiological deterioration (Wenham
1995)-
Early studies in cassava physiological post-hanrest
deterioration have postulated that it was due to an
oxidative process (Drummond 1953, Averre 1967). However the
first detailed studies of Plumbey et al. (1981) reported an
increase in total peroxidase activity in the initial stages
of deterioration. In electrophoretic studies, they showed
not only the intensification of peroxidase bands during the
deterioration process, but also the appearance of a new
peroxidase band.
Rickard (1981) reported that the major seconda-
product in roots following injury was the coumarin
scopoletin, although many changes in other phenolic
compounds also occurred. Tanaka et al. (1983) also reported
scopoletin, scopolin and esculin as the major products
formed during primary deterioration. In addition,
significant amounts of catechin and chlorogenic acid were
observed. The topical application of different phenolic
compounds to fresh root tissue showed that only scopoletin 20
produced a significant amount of deterioration (wheatley and
Schwabe 1985) . In the same work, pruning and/or curing the
root before scopoletin application significantly reduced the
deterioration process by the inhibition of scopoletin
degradation.
Coumarins are synthesized £rom p-coumaric acid, one of
the products of the general PPP. Initially, there are two
successive hydroxylations from caffeic acid. The subsequent
steps leading to the formation of scopoletin have not been
totally clarified, althougb there is evidence for the
formation of ferulic acid by methylation as an intermediate
step before the cyclization of the scopoletin molecule
(Fritig et al. 1970). However, results from recent attempts
to correlate visible symptoms of deterioration with
scopoletin were non-significant (CIAT Annual Report 1995).
PAL in root post-hanmst deterioration
The presence of PAL enzyme during the prirnary stage of
cassava deterioration has been reported (Tanaka et al. 1983,
Rickard 1985). Both reports demonstrated a significant
increase in PAL activity with a peak at 30-40 hours after
harvest or injury of roots. The production of scopoletin and
other secondary compounds seems to be correlated with the
increase in PAL activity (Tanaka et al- 1983).
An increase in total phenolic compounds, especially
proanthocyanidins, in cassava roots after wounding was
reported by Rickard (1982). The production of phenolics, due
to de novo PAL activity was greater than the accumulation of
coumarins. Proanthocyanidins are probably involved in the
formation of tannin deposits in the wounded surface of the
roots.
The induction of PAL in wounded plant tissue has been
reported for several other species such as potato (Ishizuka
et al. 1991; Smith and Rubery 1979). lettuce (Ke and
Saltveit 1989) , french beans (Bolwell and Rogers 1991) . and cucumber (Hyodo and Fuj inami 1989) . Wounding of the roots
during harvest is probably the main reason for the
physiological deterioration of cassava roots, and production
of PAL is directly related to wounding. Suppression of PAL
activity in the roots could decrease the production of
phenolic compounds, coumarins and subsequent formation of
oxidative products. It is possible that reduction of these
compounds would decrease the post-harvest deterioration of
the root.
Although there are studies of PAL enzymatic activity in
cassava roots under deterioration, there are no reports at
the molecular level assessing PAL gene expression and its
correlation with enzyme production. Identification and
characterization of PAL genes and other genes in the PPP in
cassava would give us the opportunity to study their
importance in the post-harvest deterioration process, The
recent advances in cassava transformation (Schopke et al-
1997, Li et al. 1997) might allow studies in suppression or
overexpression of genes in the PPP in cassava in a search
for genetic approaches to decrease root post-harvest
deterioration.
Phenylgroganoid gathway
Following wounding, plants increase the production of
cornpounds involved in the repair of the damaged tissue and
in the defense against microorganisms. The wound response in
plants is closely associated with the de£ense response since
many pathogens create or enter the plant through wounded
tissue (Dyer et al. 1989)- Different signal responses are
activated in plants after wounding (Lamb et al. 1989, Graham
and Graham 1991), including production of several secondary
metabolites with origin in the PPP. The phenylpropanoid
derivatives include flavonoids and isoflavonoids, coumarins,
soluble esters, suberin, lignin, and tannins. The function
of these compounds is also diverse. They are involved in the
repair of wounded tissue through production of suberin,
lignin, pigments such as anthocyanins, and antibiotic-like
compounds such as the isoflavonoid phytoalexins and
coumarins .
Smit and Dubery (1997) demonstrated induction of
several enzymes of the PPP in cotton hypocotyls after
inoculation with Verticillium dahlia. There was a rapid
increase of PAL follawed by increases in cinnamyl alcohol
dehydrogenase (CAD) and peroxidases. Reinforcement of the
ce11 wall was also observed with the formation of lignin-
like polymers.
Today the general PPP is well understood (Dixon and
Paiva 1995, Meer et al. 1993, Hahlbrock and Schell 1989,
Fig. 2 ) . and comprises three enzymatic steps where L-
phenylalanine is converted into 4-coumaryl CoA. In the first
s t e p , phenylalanine ammonia-lyase (PAL) acts on L-
phenylalanine producing cinnamic acid and ammonia. The
second step is t h e hydroxylation of cinnamic acid by the
enzyme cinnamate 4-hydroxylase ( C 4 H ) producing coumaric
acid. The third step is the activation of coumaric acid by
coenzyme A producing 4-coumaryl CoA catalyzed by t h e enzyme
coumarate:CoA ligase (4CL).
Molecular characterization of t he several enzymes in
the PPP has been reported (Whetten and Sederoff 1995). The
4CL gene has been cloned in parsley and potato, and it is
encoded by two highly homologous genes (Douglas et al. 1987,
Dangl et al. 1987, Dangl 1990). Identification of
General phenylpropanoid metabolism
COOH COOH COOH COSCoA
- 6:iL / - 6 / - a,, 6 "-c 1 /
R y R '
OH OH Phenylalanine Cinnamic acid 4-Coumaric acid 4-Coumaroyl CoA
Fig 2 . General phenylpropanoid metabolism and secondary products . Dashed arrows indicated products formed £rom branch pathways. PAL. phenylalanine ammonia-lyase; C4H. cimamate 4-hydroxylase; 4CL, 4-coumarate:CoA ligase. Adapted £rom Douglas et al. 1991
cis-controlling elements on these genes has also been
reported (Lois et al. 1989, Douglas et al. 1987, 1991) .
~henylalanine nmmonia-lyase
Phenylalanine ammonia-lyase (PAL) was first described
in the early 1960's by Koukol and Corn (1961). PAL is one of
the most important enzymes involved in a plants defensive
response to environmental stress. It has been demonstrated
respond that PAL activity increases rapidly after wounding,
pathogen infection, and U.V. illumination (Hahlbrock and
Griesebach 1979). The PAL enzyme is the first enzyme in the
PPP and catalyzes the elimination of ammonia from L-
phenylalanine to form trans-cinnamate. This is the first
step for several phenylpropanoid derivative products
including tannins, coumarins, flavonoids, isoflavonoids and
others .
The molecular weight (MW) of the enzyme is in the range
of 280-330,000, depending on species, with subunits of
approximately 83,000 PlIW and two active sites per tetramer
(Hanson and Havir 1981). Although knom for its activity on
L-phenylalanine, the enzyme also acts on L-tyrosine in
grasses and fungi to yield tram-coumarate, showing tyrosine
ammonia-lyase activity (Roder et al. 1997).
PAL activity is inhibited by the concentration of
cinnamic acid produced, Bolwell et al. (1986) demonstrated
that cinnamic acid inhibits the synthesis of the one subunit
of the enzyme due to the appearance of an active factor
which may bring a specific and irreversible PAL activity
without an appreciable increase in the synthesis of intact
enzyme subuni ts .
The induction of PAL activity is strongly related to
the physiological state of the plant tissue. Changes in
environmental conditions such as light, wounding, plant
hormone application or pathogen infection stimulate a rapid
increase in PAL activity (reviewed by Dixon and Paiva 1995).
Howles et al. (1996) demonstrated that overexpressing
PAL genes in leaves of transgenic tobacco plants produced an
accurnulation of chlorogenic acid, indicating that PAL is the
key enzyme controlling the flux of this product. However, no
increase in PAL activity, flavonoids or lignin was observed.
Furthermore there was an accumulation of 4-cournaric acid,
suggesting that the enzyme 4-coumarate coenzyme-A might be
limiting the pathway after PAL. Nevertheless, the
accumulation of chlorogenic acid has the potential to
increase plant resistance to pathogens due to its
antimicrobial activity (Maher et al. 1994) .
Tobacco plants containing transgenic PAL genes, which
reduce PAL expression, failed to develop SAR resistance. The
transgenic plants contained reduced levels of PAL
transcripts and salicylic acid (Pallas et al. 1996). In
another example of suppressed expression of PAL genes,
tobacco plants showed higher susceptibility to disease than
non-tracsgenic plants (Maher et al. 1994). It was observed
that as a consequence of PAL suppression, there was a
reduction in the formation of chlorogenic acid and the
flavonoid rutin, and an increase in susceptibility to the
f ungus Cercospora nicotianae .
Disturbance of PAL activity in tapetum tissue induced
partial male sterility in transgenic tobacco (Matsuda et al.
1996). The plants contained a sweet potato PAL gene under
the control of a tapetum-specific promoter and produced
rnicros~oros lacking flavonoids and starch. The fertility of
pollen grains was reduced to 8% and 10% with sense and
antisense orientation constructs respectively.
Molecular characterization of PAL genes
PAL genes have been recently isolated and characterized
for several species, as will be described below. The reports
indicate that there is a family of PAL genes, and that cis-
elements in the promoter region can control the spatial and
temporal expression of the genes under different
environmental conditions.
Edwards et al. (1985) identified 5 PAL cDNA clones from
elicitor-treated suspension cells of bean, using hybrid-
selected translation and cross hybridization. One of these 5
clones, PALS, was used to screen for the complete gene in
two independent genomic libraries of the bean cultivars
Tendergreen and Canadian Wonder. Fourteen clones were
identified and grouped into classes 1, 11 and III. Classes 1
and II were found in libraries of cv. Tendergreen and cv.
Canadian Wonder respectively, while class III was found in
both libraries. The genes of class 1 (PAU) comprised a set
of clones with a truncated version of the gene, very similar
to the cDNA probe, Genes of class II (PAL2) and III (PAL31
showed the presence of two conserved regions in the exons,
but they were separated by an intron that is divergent with
respect to both size and nucleotide seqence. Furthermore
PAL3 showed differential regulation compared with PALl and
PALS; the last two were induced by elicitor treatment of
suspension cells, while PAL3 is not responsive. The spatial
expression of these genes during plant development was also
differentiated. Transcripts of al1 three genes were present
at relatively high levels in roots, but only PALl and PAL2
were expressed in shoots, and only PAL1 was expressed in
leaves. With mechanical wounding al1 three genes were
expressed, but illumination of etiolated hypocotyls
activated only PALl and PAL2 (Liang et al. 1989a). It was 29
also reported that the PAL gene family exhibits a complex
pattern of regulation during plant development, and it is
possible that within the same organ and at the same
developmental stage, individual genes within the gene family
might be activated selectively by different stimuli (Liang
et al. 1989a) .
Leyva et al. ( 1 9 9 2 ) demonstrated that the bean PAL2
promoter has a modular organization and that a negative
combinatorial interaction between cis elernents defined
tissue specificity for the gene in the vascular system.
Transgenic tobacco plants carrying the chimeric construct
PAL2-GUS, but with internal deletions of the promoter
region, produced different patterns of expression. Although
a deletion from -119 to -74 nucleotides did not show any
difference from the complete promoter, a deletion from
-134 to -74 produced weak GUS staining in the xylem but
strong expression in internal and external phloem and
surrounding perivascular parenchyma tissue, which was not
observed with the complete promoter. A deletion from -289 to
-74 abolished the expression in the xylem, but the
expression of the phloem was maintained. There was no
expression in any vascular tissue when the -480 to -74
internal deletion was used. The results indicated that the
-289 to -74 promoter region ccntains essential sequences for
xylem expression. Furthermore, the expression in the phloem 3 O
is probably inhibited by a downstream negative regulatory
cis elernent that might be located between -135 and -119
nucleotides.
Lois et al. (1989) described a family of 4 genes
encoding PAL in parsley. Expression of three genes were
tested with U.V. light, fungal elicitor and wounding of
leaves and roots. An increase in mRNA content was observed
in al1 cases studied. Further studies of PAL1 revealed that
the promoter region contained motifs involved in the
response to both U.V. irradiation and elicitor application.
Working with PAL antibodies, Gowri et al. (1991)
immuno-screened a cDNA library of alfalfa suspension cells
induced with a fungal elicitor. One clone, pPAL1, showed a
high degree of similarity with the deduced amino acid
sequence of PAL sequences £rom bean, parsley, sweet potato
and rice. Rescreening of the cDNA library with the pPALl
cDNA resulted in hybridization to sixty putative PAL clones.
Five clones were sequenced and three were identical to
pPAL1, but the two others were different but highly
homologous. Southern blot analysis of alfalfa genomic DNA
using the entire pPALl cDNA as a probe resulted in more than
six distinct bands indicating the presence of a rnultigene
family for PAL in alfalfa similar to other species. The
pattern of expression of the genes in a Northern blot of
mRNA from alfaifa suspension cells exposed to a fungal
elicitor correlated with the PAL activity measured in
spectrophotometric assays.
Kawamata et al. (1992) cloned a PAL gene £rom a cDNA
library of pea ( P i s r u n çativum) epicotyls after elicitor
treatment. Six clones were obtained and were divided into
four groups according to restriction rnapping analysis. The
deduced amino-acid sequence was between 65% to 88%
homologous to PAL genes frorn othex species.
PAL genes £rom rice were isolated by ina ami et al.
(1989) using a sweet potato cDNA as a probe. The cDNA clone
obtained was then used to screen a genornic library and where
ten clones were obtained. One genornic clone had very similar
restriction enzyme patterns to the cDNA probe. Genomic
Southern hybridizations of rice DNA with the cDNA probe
indicated a srnall multi-gene family encoding PAL, In
subsequent work Minami and Tanaka (1993) reported another
PAL gene in rice, but with a different restriction map £rom
the one previously described.
Also working with rice, Zhu et al. (1995) cloned a PAL
gene (ZB8) using a PCR-amplified rice fragment as a probe.
The authors designed the primers according to two conserved
regions of PAL genes in beans (GIRFEILEA and QIAAAIME) . The
ZB8 gene was found to be highly expressed after wounding or
pathogen attack. Spatial and temporal studies demonstrated
that the gene was expressed in meristematic regions. and
vascular and epidermal parts of roots and stems.
Tanaka et al. (1989) isolated a PAL cDNA clone £rom
mechanically-wounded roots of sweet potato. Using an
antibody against PAL. a cDNA library was screened and the
clone obtained was also analyzed using immunoreaction tests.
The cDNA clone had a nucleotide sequence with 75.9% homology
to PAL clones in beans for the coding region and an amino
acid deduced sequence with homology of 78.9%.
PAL genes in Arabidopsis were isolated £rom a genomic
library using a bean cDNA probe (Oh1 et al. 1990). The
Southern blot of Arabidopsis genomic DNA showed four to five
distinct bands which indicates a small family of genes.
Using a partial bean cDNA clone. Lee (1992) was able to
identify 5 classes of PAL genes £rom a genomic library of
tomato. He demonstrated that differences in the protein size
might be the result of the truncation of the genes
identified producing truncated polypeptides. Tt was also
reported that one PAL gene. PALS, might utilize alternate
transcription initiation sites to produce two different
proteins according to different environmental stresses. When
the tomato plants were stimulated by light. wounding or
Verticillium infection, the site of initiation was different
depending on the stimulus. Light illumination stiinulated one
site, while both sites were stimulated by wounding leaves
and roots. When petioles were exposed to Verticillium, there
was no induction of either site, but in roots only one site
was stimulated by the elicitor.
Shufflebottom et al. (1993) reported more evidence for
a specific spatial and temporal control of PAL genes during
plant developrnent. Tobacco, Arabidopsis and potato were
transformed with PAL2- and PAL3-GUS constructs and it was
observed that the latter was expressed in the xylern, in
etiolated, light-induced seedlings, and pollen grains, while
PAL3-GUS expression was absent in these tissues. The
wounding response with PALS-GUS was slower and more limited
than PAL3-GUS, although in potato tubers the expression of
those constructs showed identical patterns. Leaves treated
with elicitor in the three species studied showed that the
PALS-GUS foms a narrow ring of expression around the
bleached tissue, which remains unchanged in size after
several hours. However, the PAL3-GUS construct produced
large halos of expression around the lesions, which
increased and covered the whole leaf disc after 24 hours.
To date there are no reports of molecular work related
to PAL genes in cassava. ~solation and characterization of
the expression of PAL genes in cassava might provide
relevant information for understanding both the post-harvest
deterioration process of the roots and the interaction of
cassava with X. axonopodis pv. manihotis and X.cassavae .
Plant gexoxidases
Plant peroxidases ( E L . 1.11.1.7) are enzymes involved
in the oxidation of compounds at the expense of H202 . They
play a key role in several aspects of plant physiology and
development, such as lignification and suberization of ce11
walls, IAA oxidation and post-harvest deterioration of
fruits and vegetables. An increase in peroxidase activity
generally occurs after wounding, incompatible pathogenic
interactions and physiological stress such salinity,
radiation, and pollution (see review by Campa 1991) .
Peroxidases can be classified on the basis of their
isoelectric points. Basic or cationic peroxidases have
isoelectric points in the pH range 8.0-10.0, while the
acidic or anionic have isoelectric points between 3.5-6.0.
The latter are also divided intc highly (pH 3 . 5 - 4 . 5 ) and
moderately (pH 4.5-6.0) anionic forms (Robinson 1990 ) .
Peroxidases can also be classified according to
solubility during extraction (Robinson 1990, 1991). After
low speed centrifugation of an extract (buffer plus a tissue
sample), the supernatant contains the soluble fraction with
both anionic and cationic cytoplasmic peroxidases. The
pellet is washed and extracted with a salt solution to
obtain ionically ce11 wall-bound peroxidases, which are
mainly cationic in nature. Ce11 wall- and ionically-bound
peroxidases have a high affinity for phenolic compounds.
They promote the oxidation of hydroxy cimamyl, coniferyl
and sinapyl alcohol producing phenoxy free radicals that can
be cross-linked in the ce11 wall for the formation of lignin
and suberin.
Plant peroxidases are grouped in families and
subfamilies according to their homology and function
(Welinder 1986). Peroxidase gene families have been
identified in several plants including horseradish (Fujiyama
et al. 1988), tomato (Robberts and Kolattukudy 1989),
tobacco (Lagrimini et al. 1987) and rice (Ito et al. 1994).
Chittoor et al. (1997) cloned three peroxidase genes
£rom rice that were induced according to different plant
stresses, including pathogen interaction and tissue
wounding. Wound inducible peroxidases were also reported in
horseradish (Kawaoka et al. 1994) and tomato (Mohan et al.
1993)
Reimer and Leach (1991) were able to identify two
anionic and one cationic rice peroxidase after an
inoculation and incompatible interaction with X. campestris
pv. oryzae. Total peroxidase activity from the apoplast of
inoculated leaves showed an increase of two to four fold over
the compatible reaction aï~d the control, respectively. In
immunocytochemistry experiments , an accumulation of the
cationic peroxidase was observed in both xylem vessels and the
apoplast of rnesophyll tissue during an incompatible
interaction (Young et al. 1995). Induction of both cationic
and anionic peroxidases was also observed when Arabidopsis
thaliana plants were inoculated with X. awonopodis pv.
campestris (Lummerzheim et al. 1995) . Furthemore, an increase
was reported in both callose deposition and activity of B-
glucanases and chitinases. Cationic peroxidases are also
involved in the wounding response and pathogen interactions
(McDougall 1993, Gotthardt and Grambow 1992) .
mer-expression of an anionic peroxidase in transgenic
tomato lead to an increase in lignin compared with non-
transformed plants (Lagrimini et al. 1993). The increase in
lignin production ranged from 20% in leaves to 106% in fruit.
W h e n transgenic fruits were wounded, soluble phenolics
increased more than 300% while the lignin content increased
20 fold relative to non-transgenic, wounded fruits. However,
initial tests screening for resistance against pathogens did
not show increases in resistance against Fusarium, Verticilium
or tobacco mosaic virus.
In cassava, during a resistance reaction to X. axonopodis
pv. manihotis, the presence of lignin, callose and tyloses in
the vessels was reported to obstruct the passage of the
bacteria (Kpemoua et al. 1996) . An increase in peroxidase
activity is thought to occur prior to the formation of those
structural barriers
Tanaka e t aL(1983) reported that an increase in
peroxidase activity in wounded cassava roots correlated with
the formation of a lignin layer on the wound surface. Pfumbey
et al. (1981) showed that soluble and ionically bound
peroxidases increased in wounded cassava roots with de novo
activity of one soluble peroxidase.
In the case of both post-harvest root deterioration in
cassava and the resistance interaction with Xanthomonads an
increase in the phenylpropanoid pathway is expected beginning
with an increase in the level of PAL and leading to an
accumulation of phenolic compounds. In turn, peroxidases will
catalyse the oxidation of those phenolics, producing
structural barriers for the pathogens.
The analysis of peroxidases in both the post-harvest
deterioration of cassava and the interaction with Xanthomonas
will provide a better understanding of the protective
mechani sms
developing
of the plant, with
strategies for the
potential applications in
molecular breeding of cassava.
C h a p t e r 1
The role of peroxidase during the interaction between
cassava and Xanthomonas axonopodis QV. manihotis and
Xanth-nas cassavae
Introduction
Cassava bacterial blight (CBB) and cassava bacterial
necrosis (CBN) are the two main bacterial diseases of
cassava caused by Xanthomonads . Xan thomonas axonopodis pv.
manihotis is the etiological agent of CBB, while CBN is
caused by Xanthomonas cassavae. Both bac teria produce the
characteristic blight lesion in leaves, but only X-
axonopodis pv. manihotis systernically invades the plant
through the xylem vessels (Maraite 1993) .
Plants utilize different mechanisms of defence response
to avoid infection, including production of phytoalexins, PR
proteins. and structural changes of the ce11 through
deposition of lignin and suberin (Borchert 1978. Vance et
al. 1980). Plant peroxidases cari be directly involved in
defence mechanisms working as catalysts for polymerization
of phenolic compounds to form lignin and suberin in the ce11
wall. Those structures act as physical barriers to block or
isolate the pathogen and its spread into the plant (Fritig
1987) .
39
The importance of peroxidases during plant resistance
against pathogens has been demonstrated in interactions
between rice and X. o r y z a e pv. o q z a e (Chittor 1997, Young
et a l . 1995, Reirner and Leach 1991) and between cotton and
X . c a m p e s t r i s pv. malvacerum (Dai et al. 1996). Flood et al.
(1995) suggested that peroxidases might also play an
important role during the interaction between X. axonopodis
pv. m a n i h o t i s and cassava.
This chapter describes the interaction between two
cultivars of cassava (KCol 22 and CM 523-7) and Xanthomonas
axonopodis pv. maniho tis and Xanthomonas cassavae . The
growth of bacteria in inoculated leaves was analysed as well
as ion leakage using conductivity experiments. Peroxidase
activity was also measured in resistant and susceptible
interactions. Finally, characterization and sequence
analysis of a PCR clone of a peroxidase gene in cassava are
also described.
Materials and methods
Bacteria and plant material
An isolate of Xanthomonas axonopodis pv. m a n i h o t i s
( 9 6 4 6 ) and one isolate of X. cassavae (9018) were used.
These bacteria were obtained £rom infected cassava plants
from the northwest region of Parana State, Brazii. The
bacteria were grown in YPGA media (Daniel and Boher 198%
with the pH adjusted to 7.2 (before autoclaving) and kept at
-70"~ until used for experiments.
For inoculation the bacteria were grown for 24 hours in
YPGA liquid medium at 28 to 30°C. centrifuged and
resuspended to 0.D .,,,, of 0.11-0.12 in water or in a 0.5 rnM
CaCl,, 0.5 mM MES buffer pH 6.2 (Flood et al. 1995). This
O.D.,,, , is equivalent to 4x107 colony forming units (cfu)
per ml. A one ml syringe without the needle was appressed to
the abaxial leaf surface and the bacteria were infiltrated
into the leaf. Inoculation by spraying the bacteria on a
wounded plant surface was used initially and is described in
Appendix 1.1.
Inoculations were performed on the third, fourth and
fifth fully-expanded leaves £rom the apex of Manihot
esculenta cultivars MCol 22 and CM 523-7 growing in a growth
room with day/night temperatures of 27 /22 "C and 12 hours of
daylight (250 pmol m-2s -1 ) . Plants from cultivar MCol 22 were
propagated from cuttings of plants from the same growth room
and were inoculated six to seven weeks, after the cuttings
were taken. Plants £rom cultivar CM 523-7 originated
directly from in vitro micropropagation and were inoculated
10 to 12 weeks after transplanting to the growth room. After
inoculation, plants were transferred to a clear plastic
chamber in the same growth room with constant mist for five
days .
Cultivars CM 523-7 and MCol 22 are considered resistant
and susceptible to CBB. They are ranked respectively as a 2
and a 4 on a 1-to-5 scale, based on the lesions observed in
the leaf, with 1 being resistant and 5 being susceptible to
X. axonopodis pv. manihotis (Table 1.1). No information was
available regarding the interaction of those cultivars with
X. cassavae . Experiments were performed to test the
following hypothetical interactions:
X. axonopodis pv. manihotis vs. cv. MCol 22 - susceptible;
X. cassavae vs. cv. MCol 22 - resistant;
X. axonopodis pv. manihotis vs. cv. CM 523-7 - susceptible;
X. cassavae vs. cv. CM 523-7 - resistant.
Measurement of bacterial growth in the leaves
Three plants from cultivar MCol 22 and CM 523-7 w e r e
inoculated by the syringe method as described above.
lnoculated leaves were detached from each plant and surface
sterilized with a 2.1% sodium hypochlorite solution for 5
minutes, followed by washing with distilled sterilized
water. Leaf disks were cut from the inoculated area using a
8 mm diameter cork borer. One leaf disk from each plant was
collected, and grouped with disks £rom the other plants.
Table 1.1 Classification of different cassava cultivars in relation to resistance and susceptibility to bacterial blight disease (CBB)
Cultivar Resistance to CBB' rig gin'
BRA 7 0 CG 1 - 5 6 CG 403-18 CM 523- 7 ESPETO I A C 24 MCHN MCOL 22 MIND 8 MIND 27 MMAL 2 MMAL, 4 MNGA 1 MNGA 2 MTAI 1 MTAI 8 SG 107-5
Brazil,
Colombia Colombia Brazil, Brazil,
Colombia, India, India, ~alaysia, Malaysia, Nigeria, ~igeria, Thailand, Thailand, Colombia,
VEN 7 0 ND Venezuela, SA
'classification from CIAT ûatabank. CBB susceptibility was ranked on a 1-to-5 scale where 1 was very low and 5 very high. Cultivars with no information about CBB are rnarked ND.
2 ' 3 Personal communication by Claudia Guevara, Curator CIAT Databank (CIAT).
Resistant cultivar reported by Flood et a1.(1995) but not ranked on 1-5 scale.
' Country and/or continent of origin: South Arnerica (SA), Asia (AS) ~frica (AF)
Each sample with three disks was ground in 0.7 ml of 0 . 5 mM
CaClJMES buffer (Flood et al. 1995) in a mortar and pestle
at room temperature. The volume of the extract was adjusted
to 1 ml with bu£ fer, serially diluted (10-2, IO-' and 10-4
fold) and plated on YPGA medium. The bacteria were incubated
at 28°C for three days before counting the colonies. The
experiment was repeated once for the cultivar Mc01 22, where
a lower inoculum of bacteria, 2.3x103 cfu/ml, was also used.
Leaf samples were collected 0, 6, 12, 24, 48, 72, 96 and 120
hours after bacterial inoculation.
Conductivity exgeriments
Electrolyte leakage experirnents were conducted
according to Brisset and Paulin (1991). Al1 glassware was
washed in IN HC1, rinsed with distilled water and autoclaved
at 120"~ for 20 minutes. Cassava leaves were surface
sterilized in 2.1% sodium hypochlorite for 10 minutes and
rinsed three times with sterile distilled water. Leaf disks
were cut with a 8 mm diameter cork borer, and transferred to
50 ml glass tubes containing either 15 ml of bacterial
suspension (10' cfu/ml in 0.5 mM MES/CaCl, buffer, pH 6.2)
or buffer only for control. ~noculation was performed by
placing the tubes under vacuum for 15 minutes. Leaf disks
were air-dried on paper towels for 30 minutes and
transferred to glass tubes with 15 ml of the same buffer
(four leaf disks /15 ml tube) . The tubes were incubated
under light 150 pmol N 2 s - ' at 26°C in a shaker at 1000 r p m .
Conductivity was measured in a conductance meter (YS1 32@,
Yellow Spring Inc-, Yellow Spring, OH, USA) every 24 hours
for at least eight days after inoculation. Each cultivar
(MCol 22 and CM 5 2 3 - 7 ) x pathogen (X. axonopodis manihotis
or X. cassavae) interaction had four repetitions plus four
controf samples.
Peroxidase assay
Soluble and ionically-bound peroxidases were extracted
as described by Lee and Lin (1995). Inoculated leaves were
homogenized in liquid nitrogen and 10 mM phosphate buffer
(pH 6.4) was added at 1:5 w/v ratio. Smples were
centrifuged at 1000 X g at 4OC for 10 minutes, The
supernatant was used to measure soluble peroxidase, while
the pellet was resuspended and centrifuged three times in
the same buffer to remove the remaining traces of soluble
peroxidase. The pellet was incubated in 1 M NaCl for 2 hours
at 30°C in a shaker and centrifuged at 1000 X g, for 10
minutes at 4OC . The supernatant was used to assay the
ionically-bound peroxidase.
Guaiacol peroxidase activity was measured according to
Chance and Maehly (19551, where 5 0 pl of plant extract was
added to a 3 ml reaction mixture containing 20 mM phosphate
buffer pH 6.0, 20 mM guaiacol (Sigma, North York, ON,
Canada), and 0-03% H,O,. Tetraguaicol formation was measured
at 470 nm, two and three minutes after adding H202.
Peroxidase activi ty was expressed as AOD ,,, ,/minute/pg
protein. Total protein was determined using the coomassie
blue assay (Bradford 1976). Samples were collected at 0, 4,
8, 12, 24, 48, and 72 hours after inoculation.
Amplification and cloning of a peroxidase gene in cassava
Sequence comparison of peroxidase genes £rom different
plants was performed using the ALIGN program £rom PCGENE
(Appendix 1.2). The regions that showed high homology were
submitted for a homology search in GenBank using the BlastN
(Altschul et al. 1990) program to check the homology with
other species. Two primers were designed £rom those regions
as follows:
PX1 5' - CGT CTC CAC TTT CAT GAC TGC - 3 '
PX2 5' - GAA ACC TAC CGT GTG TGC ACC - 3'
with sense and antisense orientation respectively. PCR
reactions were performed with 200 pM of each dNTP, 10mM
Tris-HC1, 50mM K C 1 and 1.5 U of Taq polymerase, and 200-400
ng of genomic DNA £rom cultivar MCol 22 at three
concentrations of MgCl,: 1.5, 2.25 and 3.0 mM. PCR
conditions were 4 minutes at 94"C, followed by 30 cycles of
1 minute at 94"C, 1 minute at 46°C and 2 minutes at 72"C, in
46
a GTC-2 thermocycler (~recision Scientific, Chicago, Il,
USA). After the last cycle the samples were left for 5
minutes at 72"C, and cooled to S O C . PCR products w e r e
visualised in 1% agarose gels stained with ethidium bromide
and cloned in the pGEM-T vector £rom Promega (Madison, WI,
USA) .
The cloned PCR products were sequenced using the Taq
Dye Deoxy Terminator Cycle sequencing kit ( A B I ) on an AB1
PRIS^ mode1 377 DNA sequencer. A sequence homology search
of GenBank w a s performed using BlastN. The predicted
translation product was obtained using TRANSL, CODING and
SIGNAL programs £rom PCGENE and GENSCAN 1 - 0
(http://gnomic.stanford.edu/genscanw.html) . A search for
specific motifs in the translated sequence was performed
using PROSITE from PCGENE and MotifFinder
(http: //www.genome .al. jp/htbin/) .
Southern and Northern blot analysis
DNA and RNA extraction methods are described in detail
in appendix 1.3. For Southern blot analysis, 10 pg of DNA
£rom different cassava cultivars was double-digested with
Eco RI/Bam HI respectively. Digestions were conducted
overnight at 37OC with 2-3 units of enzyme. Transfer of DNA
to GeneScreen Plus@ nylon membranes (DUPONT, Boston, MASS,
USA) and hybridization conditions were according to the
membrane manufacturer's manual. The peroxidase clone was
labelled with ~cTP~*-" by the random primer method using a
commercial kit (Boehringer Mannheim, Laval, QB, Canada) .
Hybridization was performed overnight at 4 2 ' ~ with 50%
formamide, 2X SSC, 1% SDS, 10% dextran sulfate, 5X
Denhardt's and 100 pg/ml of denatured salmon sperm DNA. The
membranes were washed at low stringency as follows: 2X SSC
at room temperature for 5 minutes; 2X SSC and 1% SDS at 4 2 " ~
for 30 minutes; and 0.2X SSC and 1% SDS for 5 minutes at
room temperature. Membranes w e r e exposed to X-ray film
overnight for low stringency blots. After developing the
film, membranes were washed with 0.2X SSC and 1% SDS at 6 3 ' C
for 30 minutes and re-exposed for the high stringency blot.
RNA was extracted £rom 9-12 leaf disks (three to four
£rom each plant) , cut by a 8 mm diameter cork borer, and
irnmediately frozen in liquid nitrogen. Samples were
collected at 0, 4, 8, 12, 24 and 48 hours after inoculation.
For Northern blots, 10 pg of total RNA samples were heat-
denatured ( 6S0C/ 15 minutes ) according to Sambrook et
aL(1989). RNA was separated by electrophoresis in 1.2 %
agarose gels with 2.2 M formaldehyde, and transferred to
GeneScreen Plus@(DUPONT) nylon membranes by the capillary
method (sambrook et al. 1989). The peroxidase clone was
labelled as described above. Blots were also hybridized with
a fragment corresponding to exon regions of the clone,
digested with Eco RV and Nde 1. Blots were hybridized at
4 2 " ~ overnight in 5X SSPE, 50% formamide, l%SDS, 10% dextran
sulfate, 5X Denhardt's and 100 pg/ml of denatured salmon
sperm DNA. Washing conditions and exposure of membranes were
as described for Southerns blots above but using SSPE
instead of SSC. To reuse the blots, the probe was stripped
by heating the membranes at 90-95"~ for at least 30 minutes
in a solution with 1% SDS and 0,lX SSC.
Resuïts
Plant inoculation and disease symptoms
Xanthomonas axonopodis pv. manihotis was virulent on
both cultivars, MC01 22 and CM 523-7 , resulting initially in
dark water-soaked regions at seven days after inoculation
(Fig. 1.1, 1.2, Appendix L I ) , and subsequently extensive
yellow necrosis around the inoculation areas. The leaves
after two weeks started to wilt and were chlorotic around
the area inoculated. The leaves eventually desiccated and
fell off. The regrowth of the plant showed stem die-back,
browning and wilting of tips, typical symptoms of CBB.
The two cultivars tested showed a different response
when inoculated with X. cassavae. Cultivar MCol 22 did not
show any symptom of CBN, and small necrotic regions were
visible at the inoculation points one to two weeks following
inoculation (Fig. 1.1 El, D). The leaves did not desiccate or
F i g u r e 1.1. Cassava leaves £rom c u l t i v a r MCol 22 i n o c u l a t e d w i t h e i t h e r X. axonopodis pv. manihotis ( A , C and E ) o r X. cassavae (B and D ) . P i c t u r e s were t a k e n two weeks p o s t i n o c u l a t i o n excep t for E, where p i c t u r e was taken a f t e r s i x weeks.
Figure 1.2. Cassava leaves from cultivar CM 523-7 inoculated with either X. axonopodis pv. manihotis (A and C) or X. cassavae (B and D) . Pictures were t aken two weeks after inocula t ion .
fa11 off even two months after inoculation. In contrast,
cultivar CM 523-7 showed symptoms of CBN after inoculation
with X I cassavae (Fig. 1.2 A, B) A large necrotic region
around the inoculation point developed in 14 days, but the
advance of the disease was very slow in comparison with CBB.
The leaves did not fa11 off as quickly as was observed with
X, axonopodis pv, manihotis and remained on the plant for
more than four weeks.
acter rial growth in the leaves
Bacterial populations in inoculated leaves of cultivar
MCol 22 reached 7.8~10' cfu/ml in 120 hours following
inoculation with X. axonopodis pv. manihotis (Fig. 1.3 A) .
However, with X I cassavae the growth in MCol 22 was
inhibited and the number of bacteria only reached 3.7x105
cfu/rnl in 120 hours. Inoculation with a lower concentration
of bacteria ( 2 . 3 ~ 1 0 ~ cfu/ml instead of 4.0x107 cfu/ml) in
cultivar Mc01 22 produced a similar trend in bacterial
growth, and a reduction of growth was observed only with X.
cassavae (Fig. 1 . 3 B).
The growth of either Xanthomonads in inoculatiocs with
cultivar CM 523-7 (Fig. 1.3 C), occurred in similar pattern.
X. axonopodis pv. manihotis reached 9x106 cfu/ml and X.
cassavae reached concentrations of 2.8~10' cfu/ml at 120
hours. These results were consistent with the observations
52
a. pv. manihalis
cassa va e
X. a. pv. manihotis
cassa va e
Figure 1.3
1 523-7 - -*-- +-- -
7- - 2 7 -
I '/ I I --y- -
6 J
! / !
W d t i p l i c a t i o n of Xan tbomonads in cassava leaves . A)
X. a. pv. rnaninotis
p X. cassavae
-
cultivar MC0122 inoculated with X. axonopodis pv. manihotis and X. cassavae. B) Same as A but inoculation was performed with a low concentration inoculum. C ) cultivar CM 523-7, inoculated with X. axonopodis pv. manihotis and X. cassavae. Each data point is the mean of six (A) or three (B and C ) replicates for the number of bacterial colony-forming units (cfu/ml) £rom three leaf disks. Bars indicate the standar error (mean I SE)
5 " m, ,'i' 1
1 '" 1 I
1 4 -i 7 q
O 20 ‘KI 60 80 lm 120 140
Hours after inoculation
of symptoms in the plant described above. Whereas cultivar
CM 523-7 showed susceptibility to both bacteria, cultivar
MCol 22 was resistant and susceptible to X. cassavae and X.
axonopodis pv. manihotis, respectively.
Conductivity exgeriments
Conductivity experirnents showed variation in ion
leakage related ta the bacteria used, but independent of the
cultivar (Fig. 1.4 A, B). When X. axonopodis pv. manihotis
was used, a steady increase in ion leakage occurred until
day 8 in both cultivars tested. The increase started at 2 or
3 days after inoculation in cultivars CM 523-7 and MCol 22
respectively. During the resistant interaction between X.
cassavae and MCol 22, there was a small increase in ion
leakage relative to the control samples starting two days
after inoculation (Fig 1.4 A). In contrast, during the
susceptible interaction between X. cassavae and CM 523-7,
the ion leakage differed from the control only £rom at 6 and
increased thereafter (Fig. 1.4 B).
Peroxidase activity
Soluble and ionically-bound fractions were analysed for
peroxidase activity in Mc01 22 plants inoculated with either
X. axonopodis pv. manihotis or X. cassavae and bu£ f er as
control (Fig. 1.5 A, B) . There were no signif icant
54
Days after inoculation
A X. a. pv. manihotis X. cassavae
Figure 1.4 Electrolyte leakage from cassava leaf disks inoculated with Xanthomonas. A)cultivar MCol 2 2 , inoculated with either X. axenopodis pv. manihotis or X. cassavae . B) Cultivar C M 523-7, inoculated with either X. axenopodis pv. manihot is or X . cassavae . Bars indicate the standard error (rnean r SE, n=4) .
IO-
Hours after inoculation
e control A X. a. pv. manihotis X. cassavae
Figure 1.5 Guaiacol peroxidase activity in cassava leaves of cultivar M C o l 22 after inoculation with X. axonopodis pv. manihotis, X. cassavae and MESKaC12 bu£ fer. Soluble (A) and ionically-bound peroxidases (B) are expressed as A O.D.470nm/min/pg of protein. Bars indicate the standard error (mean 2 SE, n=3).
56
differences in soluble peroxidase activity between the
different inoculations, although there was a small but not
statistically significant increase in the resistant
interaction with X I cassavae. The cell-wall-bound peroxidase
activity showed a similar trend during the first 24 hours,
but increased rapidly thereafter in the resistant
interaction at 48 and 72 hours (Fig. 1.5 B ) . At 72 hours the
cell-wall-bound peroxidase activity of MCol 22 in the
resistant interaction was two-fold higher than that of the
control and the susceptible one.
Amplification and sequence analysis of a cassava peroxidase
gene
Several products were amplified from MCol 22 DNA using
the peroxidase primers PX1 and PX2 (Fig. 1.6). The primers
were £rom a highly conserved region and PCR products were
also obtained when tomato DNA was used as template. A cloned
PCR product of 1501 bp (MEPX1) £rom MCol 22 showed sequence
homology with other peroxidase genes and was analyzed in
more detail. Two other amplified products, of approximately
700 and 900 bp, were partially sequenced, but did not have
any homology with other peroxidases or any other sequences
in GenBank (Appendix 1.4) .
The predicted transcription product of MEPXl indicated
the presence of two introns of 160 and 923 bp starting at
Figure 1.6 Gel of PCR products using peroxidase-specif ic primers and different sources of template DNA. Lane 1: 100 bp DNA ladder marker; lane 2 - 4: amplification of MCOL 22 DNA with 5, 7.5 and 10 rnM of MgCl2 added on the PCR reaction; lane 5 : amplification of tomato DNA. Arrow indicates peroxidase band (1501 bp) .
nucleotides 31 and 392 respectively (Fig. 1-71. A SlastX
search showed the highest hornology to peroxidases frorn
Arabidopsis thaliana, Lycopersicon esculen tum and Spinacia
oleracea. Alignment of those sequences with the predicted
translation product of MEPXl showed homology of 73% with two
A, thaliana peroxidase genes, 58% with a L. esculentum
peroxidase and 53% with a S. oleracea peroxidase (Fig. 1.8) .
The calculated pI (CHARGPRO, PCGENE) for those peroxidases
indicated that al1 but one, the ATP20 peroxidase, (Welinder
et al. 1996) were cationic.
A search for specific protein motifs in the predicted
translation product revealed the presence of the heme-ligand
on the 3' end of the gene, similar to other peroxidases
(Fig. 1.8). Potential phosphorylation, myristoylation and
glycosylation sites were also detected (Fig. 1.8).
The introns were A + T rich (71 % ) and were located in
similar positions as the introns in two horseradish
(Arrnoracia rus ticana) peroxidase genes (Fuj iyama et al.
1990) and a tomato (L, esculentum) peroxidase gene (Roberts
and Kolattukudy 1989) , but the length of the introns were
different. No significant homology was found when only the
introns were submitted to a B l a s t N search at GenBank.
Southern and Noxthern blot analysis
Southern blots of different cassava cultivars probed with
CGTCTCCACTïTCATGACTGCTFETAGAAgtaa tc taaat tc t a c t 5 1 R L H F H D C F V E - - - - - - -
ttctccatgcatgcaaattttagccaaaatttcctcctcacatttcaagcaat 102 - - - - - - _ _ _ _ - - - - - - -
cattctctgtcatcttaattagcagatgctaattagcttttggtttgcat 153 - - - _ - _ _ _ _ _ - - - - - - -
ataattctttattttctttttggttggattattgaa- 204 - - _ _ _ - _ - _ - - - C C D A S E
T A T C ~ T C A A C G A A G C C T G G A A G C A A A G A G ~ ~ M ~ ~ 2 5 5 I L I S T K P G S K E L A E K D A
AGAFGATAArnGGATITGAGGGTGGAGGGATGTGAGAGCArnAGAATGGC 3 0 6 E D N K D L R V E G C E S I R M A
TAAGGCAmTGGAGAGCAAGTGTCCTGGTGTTGTATCmTGCAGATAT 3 5 7 K A L V E S K C P G V V S C A D I
TCTTGCAA~n;CCAGAGATTATGTCCACCT~ta tgcc tc t g t tc 4 08 L A I A A R D Y V H L - - - - - -
E aattcttgatatcccctactcaatccttaattaactatttcaaactctaga 459
tcttatcccactcaatcaaaacttattaacaatttggaatatattgatggt 510 - - _ - - - - - _ _ - - - - - - -
aacaaagtcctataaataatccaaagcatagggctggtttgttgatataag 561 _ - - - _ - - _ _ - - - - - - _ -
ggaaatcaaatttctcgactgtaggtgaaaatatatgttggggtgctcata 612 - - - _ - _ - _ _ - - - - - - - -
ctcataatgcttccaaagtagaaritgtggaaaaaggaagatgttttgtc 663 - - - - - - - - _ - - - - - - - -
atttttgacnaagtatttagaacaaaacaaactcttctaaaagggcaagaa 714 - - - _ - - - _ _ - - - - - - - -
agtataaaaaatcattaagtccatgtgatttgaacagctacgttatttgtc 765 _ _ _ _ _ - _ - _ - _ - - - - _ - E
ctttgctacaancnatatcctctntgaaaagtcaagaantatnaatcaatt 816 - _ _ _ - - _ _ _ - - - - - - - -
aatcctzccaaaaataggaccaatgctgtgaaaaaccaaatgcctcatca 867 - _ _ - - - - - - _ - - - - - - -
ctggtaacatgatgagagaactaatagacaataariactggcatttgcttg 918 _ _ _ _ - - _ _ _ _ - - - - - _ -
tattggttttctaaatgtctcattcattggtaactggatgtggtcaatgat 969 - - - _ - - _ _ _ - - - - - - - -
t t tna t t t tc tcaaaactg tac tc t t t ta t ta t t tnc tg tgataacaa 1020 _ _ _ - - - - - _ _ _ _ _ _ - - -
tttgtattattattaaattagraatttatattcaaatttctatataaattt 1122 - _ _ - _ - - - _ - _ _ - _ - _ -
tagaaaaattaactatttaaattatgggtaaattttgaaaaatatattaaa 1173 _ _ _ - - - _ - _ _ _ - - - - _ -
atatttttgaaatattaaaaattttttcataaatttccctaaattttaatt 1224 - - _ - _ - - - _ - - _ - - - _ -
tatagaagtcctttttataggcgtctattttcttctaaataatcctataat 1275 - - - - - - - - _ _ - - - - - - -
N g g a g t g c t c t a a t t c c a t a t g c t a c a t t t t c a t g a c g c a ~ C 1326 - - - - - - - - - - _ - - A G G P
TTATI:ACCAPGTGAAGAiMGGGAGATGGGATGGCAAAATATCAATGGCATC 1 3 7 7 Y Y Q V K K G R W D G K I S M A S
GAAGCTTITCAA?TCCAAAGGATTAACACCACAAGATCTAGTGGTTC:TCTC 14 2 9 K L F N S K G L T P Q D L V V L S
AGGTGCACACACGGTAGG~ 1501 G A H T V G F
Figure 1.7 Sequence of a cassava peroxidase clone (MEPX1) with predicted translation products. Introns are represented i n lower case. Primers used for amplification are underlined. Restriction enzyme sites for Eco RV (E) and Nde 1 (N) are marked i n bold.
Myr/Pho Pho Pho Cassava RLHFHDCFVEGCDASILISTKPGSKELAEKDAEDNKDLRVEGCESI~ 50
.. Arabidopsis F..........G....E..K...K...RE.YE..E..E..FD..IK.. 127 ArabidopsisS .MF............VF.---ASEN.D.....D...S.AGD.FDTVIK.. Il2 Spinach .. F......S......I.--QSTGTNT....HPPPLSSAGDFD1K 113 T O K E L ~ O .M.......R...G.V.LNFTSsT.NQT..V.W.QT- .--.FSF.DGV 111 R i c e . . . . . . . . . Q . . . . . V.L.--- .QNAGPNVGSLRGFSVIDN------- . . 102
N y r / Pho Cassava ALVESK--CP~SWILAIAARDYVHLAGGPriQVKKGRWDGKISMAS 98
. . . . . . . Arabidopsis H-- SL . . . S.........FI-...-.-......-.--.R.T.K 175 Arabidopsis2 TA ...Q--............ L . . . . V - V . V V V . E F K . E L . . R L V K . 160 Spinach .A.DAVPG.T.NN........L.T..V-N.S..~FWE.EL..F..LV.K.. 163 Tomato KA.. m-- . . . . . . . . . . V-LV . . . S-VVT . . . . WK.PT..R..E..N.. 159 R i c e .R..AI--.NQT........V....S-VALALALALSWT.LLttRRSTTASEA 150
G ~ Y Perox Cassava RVPYNLPQANSTIDQLLKLFNSKGLTPQDLVVLS~F 139 Arabidopsis N..P.I.RS...V...I...A.....VEE......S..I.. 216
. . . . . . . . .. . . ArabdopsisS TGK EPGLDVRG.VQ1.A.N SLT.MIA 1.- 202 Spinach S.NGR . . . PTDELNR.NS..A.N. ..QAEM.A. . A . . . . . . 204 Toma t O EALh.I.PPT.NFSS.QTS.A....DLK...L......I.- 199 Rice LANTD . . APS.SLAE.1GN.SRKGLDAT.M.A . . . . . . 1 . Q 200
Figure 1.8. Alignment of the predicted amino acid sequence of MEPXl peroxidase of cassava and peroxidases of di£ f erent plant species. putative protein motifs are marked in bold as myristoylation (Myr), phosphorylation (Pho), glycosylation (Gly) and peroxidase signature (Perox). Individual homologies - similarities and identities - between MEPX1 and each peroxidase amino acid sequence are: 84 and 73% for Arabidopsis 1 (Rounsley and Lin 1997) ; 71 and 56% for Arabidopsis 2 (ATP20- Welinder et al. 1996); 65 and 52% for spinach (Simon 1993); 63 and 52% for tornato (Botella et al. 1993)-; and 57 and 48% for rice (Chittoor et al. 1997) . Dots ( ) represent perfect homology with MEPX1.
the MEPX1 clone showed different banding patterns, depending
on the cultivar or enzyme used (F ig . 1.9). When cassava DNA
was double-digested with Eco RI/Bazn HI, different
hybridization patterns were observed between some of the
cultivars. Most cultivars showed a unique band of 5 Kb, but
cultivars CM 523-7, MNGA-2 and MIND-8 also produced a higher-
weight band of approximately 8.8 K b . The presence of two
bands, as well as the dif ference in the intensity of the
hybridization signal, suggests a heterozygous condition
for MEPXl in those three cultivars. Interestingly, cultivar CG
107-3 was distinct £rom other cultivars analysed in a number
of features. First, only partial digestion was observed when
the DNA was double digested with Eco RI/Bam HI. No digestion
was observed when either Hind III or Xho 1 enzymes were used
(Appendix 1.5). Also the bands hybridizing in the double
digest were of higher rnolecular weight than the other
cultivars (between 16-21 K b ) .
Cultivars MNGA 2 and CMS23-7 that had a distinct 8.8 Kb
band are classified as tolerant or resistant to CBB, and the
same pattern was also observed for MIND8, whose resistance
to CBB has not yet been reported (Table 1.1). Cultivars
Espeto, MChn, BRA 70, VEN 70, CG 403, IAC 24 and CG 107-5
showed bands of much higher molecular weight only. Among
these cultivars, resistance to CBB has been reported only
for CG 403, IAC 24 and CG 107-5, which are ranked 3, 2 and
1, respectively, which is a range of moderate to high CBB
F i g u r e 1 . 9 . Southern blots of cassava DNA probed with MEPXl. Genomic DNA of dif f e r e n t cassava cultivars double digested with Eco RI/ Bam HI.
resistance. These cultivars, plus cultivar Espeto,
originated £rom South America and have a banding pattern
distinct from most of the cultivars coming from Asia or
Africa. There was no difference in the number of the bands
on the blots exposed after low or high stringency washes-
Northern blot assays did not detect the presence of any
homologous mRNA following inoculation of cultivars MCol 22
(Fig. 1.10 A) or CM 523-7 (data not shown) with X.
axonopodis pv. rnanihotis or X. cassavae. Low stringency
washes of the blots lead to hybridization with the ribosomal
RNA band (285 rRNA). As the presence of the two introns in
the MEPXl could have possibly interfered with hybridization,
attempts were made to probe the Northern blots with part of
the exons from the clone, but again only ribosomal bands
were detected following low stringency washes (data not
shown). The use of a heterologous peroxidase probe £rom
tomato (TPX1) was also not able to detect any peroxidase
transcripts (Fig. 1.10 B ) -
Discussion
In the inoculation experiments two dif f erent responses
occurred in the interactions between cultivars Mc01 22 and CM 523-
7 and X. cassavae. A resistant response was obsenred only when
cultivar MCol 22 was inoculated with X. cassavae. In this case,
there were no symptorns of the disease, and there was suppression
of the growth of bacteria in cornparison with cultivar CM 523-7,
where a susceptible interaction occurred. In contras t , a
65
Hours after inoculation
MEPX1
28s rRNA
MEPXI
28s rRNA
TPX2
28s rRNA Figure 1.10. Northern blot analysis of total RNA in cassava leaves £rom cultivar MCOL22, after inoculation with either X. cassavae or X. axonopodis pv. m a n i h o t i s . Blots were hybridized with MEPXl (A and B) and TPX2 (C) and wheat ribosomal gene for loading control .
susceptible response was observed when X. axonopodis pv.
m a n i h o t i s was inoculated, as there was no inhibition of
bacterial growth. These results were surprising since
cultivar CM 523-7 was expected to be more tolerant to X,
axonopodis pv. manihotis than cultivar MCol 22: according to
information about the cultivar from the CIAT (C. Guevara,
personal communication). However, experirnental conditions,
as well as the different bacterial isolates £rom Brazil,
might be reasons for the apparent discrepancy.
ït is important to note that in the resistant
interaction between cultivar MCol 22 and X. cassavae, a
hypersensitive response (HR) was not observed. The necrotic
tissue around the inoculation areas was apparent only after
one to two weeks. If an HR had occurred, one would have
expected rapid ce11 death within 2 days of inoculation .
During the susceptible interaction between cultivar
Mcol 22 and X. cassavae, there was an increase in ion
leakage, whereas, during the resistant interaction ion
leakage was lower with values close to the control. These
results were expected since an interaction was occurring,
and there was gradua1 damage to the cells due to the action
of the bacteria- Flood et a l . (1995) reported sirnilar
results during resistant and susceptible interactions of
cassava cultivars MNGA 1 and MCol 22 with X. axonopodis pv.
manihotis. However, the ion leakage during the susceptible
interaction between X. cassavae and cultivar C M 523-7 was
lower than expected, being more similar to the ion leakage
from the resistant interaction between X. cassavae and MCol
2 2 . However, a trend of increasing ion leakage was observed
in the susceptible interaction and it is possible that had
measurements continued for a few more days, the conductivity
would have reached higher levels, such as those found in the
susceptible interaction between X. axonopodis pv. manihotis
and MCol 22 . The conductivity pattern that w a s observed may
be related to the type of bacteria. It was observed that CBN
shows a delay in necrosis formation in cornparison to CBB,
even in cultivars highly susceptible to both diseases. It
was also observed that the in v i t r o growth of X . axonopodis
pv. m a n i h o t i s was generally £aster than X. cassavae .
In the resistant interaction between X. cassavae and
MCol 2 2 , an induction of the plant defence system against
the pathogen was expected, and peroxidases can be an
important part of the defence response of plants.
Phenylalanine ammonia-lyase (PAL) activity would lead t o an
increase in phenolic compounds which can be oxidized by
peroxidases to form lignin and suberin-containing
structures. The results reported in this chapter showed an
increase in ionically bound peroxidase levels in cultivar
MCol 22 only in t h e resistant reaction. Biochemical and
cytochemical studies during the resistant interaction of
cassava and X. axonopodis pv. m a n i h o t i s showed that lignin
was incorporated into pectin polymers in the phloem and
xylem vessels, thereby inhibiting the action of pectinases
exuded by the bacteria (Kpemoua et al. 1996, Boher e t al.
1995). Boher et al. (1995) also described the deposition of
suberin, callose and tyloses in cassava tissue resistant to
X. axonopodis pv. manihotis .
It has been reported that ionically-bound peroxidases
are predominantly cationic. although a small fraction of
anionic peroxidases are also present (Scholls 1987, Robinson
1991). Cationic plant peroxidases can be involved in the
lignification process, to produce a physical barrier against
the pathogen. Reimers et al. (1992) reported increases in
cationic and anionic peroxidase enzyme activity 24 hours
after inoculation, during a resistant interaction between
rice and X. oryzae pv. oryzae. In subsequent work with the
same host and pathogen, Young et al. (1995) showed that a
rice cationic peroxidases gene (PO-Cl) was induced during
the resistance reaction. PO-Cl accumulation coincides with
deposition of lignin, and imrnuno-localization studies showed
the presence of the enzyme in the apoplast of the mesophyl
cells, as well as in the ce11 walls and the vessels of the
xylem during the resistant interaction.
Increases in ionically-bound peroxidase activity during
the resistant interaction has also been observed in several
interactions with pathogens other than bacteria. For
example, Ikegawa et al. (1996) observed an increase in
ionically-bound peroxidases during a resistant interaction
between oat and Puccinia coronata F. sp. avenae. They noted
that the increase in peroxidase started at 24 hours and
reached the maximum point at 48 hours after inoculation of
the oat leaves. Tomato fruits inoculated with Botrytis
cinerea also contained increased levels of ionically-bound
peroxidase at 48 hours after inoculation (Lurie et al.
1997), this timing being comparable to the results presented
on this thesis.
Plant peroxidases are encoded by a rnulti-gene family
with closely-related genes that can be differentially
regulated by different stress signals or during different
phases of plant developrnent (Gaspar et al. 1982). Chittoor
et al. (1997), for example, showed that specific peroxidase
genes were induced during the resistant interaction between
rice and X. ozyzae pv. oryzae. They observed that for three
highly homologous genes. FOX 5.1, POX 8.1 and POX 22 .3 , only
the latter two showed an increase in expression during the
resistant reaction. The three peroxidase genes are also
differentially regulated during wounding, and only FOX 5.1
and POX 8.1 showed an increase in expression of wounded rice
leaves. Furthemore, only POX 22.3 was highly expressed in
fully-expanded leaves and in roots, while POX 5.1 and POX
8.1 were detected in young leaves. Other multigene families
in plants, such as those encoding PAL and chalcone synthase,
have also been reported to be specifically regulated during
plant development or according to different environmental
stimuli (Liang et al. 1989a, 1989b, Harker et al. 1990) .
MEPX1 was obtained £rom amplification of genomic
cassava DNA, but peroxidase transcripts were not observed
either during the resistant or during the susceptible
interactions. M E P X l was also not able to detect transcripts
£rom roots, storage roots, petioles, stems, leaves at
different growth stages or wounded tissue (data not shown),
in similar Northern blots as described in Chapters 2 and 3 .
It was hypothesized that the presence of two introns which
constitute more than 70% of the clone, might be one of the
reasons for the lack of hybridization with peroxidase rnRNA.
Since the exons and the predicted translated product of
MEPXl exons showed high homology to other peroxidase genes,
attempts were made to detect peroxidase mRNA with the MEPXl
exons and also with a highly homologous peroxidase clone
from tomato (Botella et al. 1993). However, rnRNA homologous
to either probed was not detected. Attempts to characterize
temporal and spatial peroxidase transcripts with MEPX1 exons
were also unsuccessful, and only hybridization with
ribosomal bands were again observed (data not shown).
The occurrence of large introns in peroxidase genomic
clones, such as that observed in MEPX1, have also been
reported in horseradish (Fujiyama et al. 1988, 1990) and
tomato (Roberts and Kolattukudy 1989). As well, the
predicted translation product of MEPXl has features similar
to other peroxidases in the literature, such as the heme
signature, and glycosylation and phosphorylation sites.
Glycosylation is an important feature for the functioning of
the enzyme. Inhibition of glycosylation in peroxidases is
correlated with both a decrease in activity of the enzyme
and an increase in enzyme susceptibility to proteolysis (Hu
and Huystee 1989). The phosphorylation motifs are probably
involved with cellular localization. Kuan and Tien (1989)
showed that the presence of mannose 6-phosphate moieties in
a lignin peroxidase was involved as a signal for liposomal
targeting in the f ungus Phanerochaete chrysospori um . Southern blot analysis of several cultivars of cassava
revealed significant polymorphisms at the MEPXl locus.
Interestingly, two of three cultivars with a unique Eco
RI/Bam HI 8.8 Kb hybridizing band (CG 107-5, MNGA 2, and CM
523-7) are classified as resistant or tolerant to CBB,
indicating a potential use of the clone as a molecular
marker for disease resistance. Although the peroxidase clone
MEPXl did not show any expression on Northen blots with MCol
22, the Southern blot analysis of cassava cultivars with
different levels of resistance for CBB, indicated that this
gene might be involved in a resistance mechanism. Future
work might include PCR amplification of peroxidase genes
from those cultivars with hybridizing bands other than the
frequently observed 5 Kb band and then examining the
expression of those genes. Such genes could be valuable
tools for breeding programs against bacterial diseases in
cassava, where they could be used either as a markers for
assisted selection or in the production of transgenic
plants.
Chapter 2
The role of a PAL gene during cassava bacterial blight and
cassava bacterial necrosis.
Introduction
The production of several secondary metabolites which
originate from the phenylpropanoid pathway (PPP) is one of the
important plant defense mechanism against pathogens or
environmental stresses. Those compounds c m be involved in the
repair of wounded tissue through production of suberin and
lignin, or have antibiotic-like activity such as the
isoflavonoid phytoalexins and coumarins . The function of these
cornpounds is not exclusive to defense mechanisms, and they are
also involved in the overall development of the plant, having
roles such as the production of anthocyanins for ce11
pigmentation. Phenylalanine ammonia lyase (PAL) is the first
enzyme in the PPP, and its transcription is regulated by
different environmental stimuli, including mechanical wounding,
interaction with pathogens, and stages of plant development
(Dixon and Paiva 1995) . PAL activity has been associated with an
increase in both lignin deposition (Whetten and Sederoff 1995)
and production of phytoalexins (Graham 1995) , which cari
participate in plant defences against pathogens.
Increased PAL activity has been demonstrated in many
plant-pathogen systems. Lummerzheim et al. (1993) showed
that there was an increase in PAL mRNA during a
hypersensitive response of Arabidopsis thaliana to X.
axonopodis pv. campestris, and PAL activity in soybean
cultivars resistant to Phytophtora megaspema f . sp .
qlycinea increased 7 to 10 fold in six-hour period following
inoculation. An increase in PAL transcription was also
observed in potato tubers in£ ected wi th Phytophthora
infestans (Yoshioka et al. 1996) . Campbell and Ellis ( 1 9 9 2 )
reported an increase in PAL activity in cultured cells of
Pinus banksiana treated with different elicitors. Elicitors
f rom Colletotrichum lindmuthianum also increased PAL mRNA
levels in ce11 suspension cultures of french beans (Mavandad
et al. 1990).
In chapter one, it was demonstrated that there was an
increase in peroxidase levels during the resistant
interaction between cultivar Mc01 22 and X I cassavae, but
not in the susceptible interaction with X. axonopodis pv.
manihotis. The same mechanism of resistance could also
involve increases in PAL activity during the resistant
interaction.
In this chapter studies of the enzyme activity of PAL
during resistant and susceptible interactions of cassava
with X. axonopodis pv. nianihotis and X. cassavae are
reported. The amplification and characterization of a PAL
gene is described, along with the spatial and temporal
7 4
transcription of the cloned gene and transcription after
mechanical wounding of leaves. Finally, to verify the
importance of PAL during cassava bacterial blight (CBB) and
cassava bacterial necrosis (CBN), the transcription af PAL
during susceptible and resistant interactions was also
analysed -
Material and methods
Primer design and amplification of a PAL gene
Similar to the isolation of the cassava peroxidase
gene, MEPX1, described in Chapter 1, a sequence comparison
of PAL genes from different plant species was performed
using the ALIGN program from PCGENE. The regions that showed
high homology were subrnitted for a second homology search
using the BlastN program at GenBank to check the homology
with other genes (Appendix 2.1). Two prirners were designed
£rom those regions as follows:
PAL81 5' - GCT GCT GCT ATT ATG GAA CAC - 3 '
PAL82 5' - CAT CTT GGT TGT G T / = ~ GCT CA/cG - 3 '
with sense and antisense orientation respectively. PCR
reactions were performed with 200 p.M of each dNTP, 10 mM
Tris-HC1, 50 mM KC1, 1.5 MgC12, 1.5 U of Taq polymerase, and
200-400 ng of template DNA £rom: cassava cultivars MCol 22
and 1-56, alfalfa and tomato. Amplification was also
performed on plasmids containing PAL genes £rom tomato
(pPALtom, Lee 1992) and alfalfa (pPALalf, ~owri et al.
1991). PCR conditions were 4 minutes at 9 4 " ~ ~ followed by 30
cycles of 1 minute at 94°C 1 minute at 5 2 " ~ and 2 minutes
at 7 2 ° C After the last cycle, the samples were left for 5
minutes at 7 2 ° C and cooled to 5°C. PCR products were
visualised in 1% agarose gels stained with ethidium bromide
and cloned in the pGEM-T vector from Promega (Madison, WI,
USA) .
Sequencing and sequence cornparison
PCR products were sequenced using the Taq Dye Deoxy
Terminator Cycle sequencing kit (ABI) on an AB1 PRIS^
mode1 377 DNA sequencer. A sequence hornology search of
GenBank was performed using BlastN. The predicted
translation product w a s obtained using the TRANSL program
from PCGENE.
Southern and Northern blot zmalysis
Southern and Northern blot analyses were carried out as
described in Chapter one, using as a probe a 520 bp cassava
PAL clone isolated by PCR as described above. The sizes of
mRNA products were estimated according to RNA molecular
markers in the gel. For spatial expression studies, total
RNA was extracted from leaf, root, storage roots, root
cortex, stems and nodal sections from cultivar MCol 22. To
characterize temporal expression of PAL in cassava leaves,
RNA was extracted £rom leaves 1-3.0 cm, 5-6.0 cm, 10.0 cm
and 15.0 cm in length.
To determine if PAL was induced after wounding stress,
leaves from cultivar MCol 22 were perforated with a circular
path of 12 needles attached to a piece of cork. The wounded
sections were collected at 6-hour intervals during a 48-hour
period for RNA extraction,
PAL enzyme activity and mRNA levels during interaction with
X . cassavae and X . axonopodis gv. manihotis
Enzyme assays for PAL activity were performed in
cultivar MCol 22 with either X. axonopodis pv. manihotis
(CBB) or X. cassavae (CBN) . Leaf disks were collected at O,
4, 8, 12, 24, 48 hours after inoculation £rom three
different plants and frozen in liquid nitrogen. Tissue was
homogenized in l i q u i d nitrogen and mixed in 1:5 w/vol ratio
with 0.1 M sodium borate, pH 8.8 and 54 mM of B-
mercaptoethanol. The samples were centrifuged at 8800 X g
for ten minutes and the supernatant was transferred to a new
tube prior to enzyme assays. Total protein was determined
using the coomassie blue assay (Bradford 1976). The PAL
assay was performed by incubating 10 to 50 pg of total
protein in a 0.1 M sodium borate buffer, ph 8.8 and 20 mM of
L-phenylalanine. The mixture was incubated at 40°C for two
hours and the absorbante was measured in a spectrophotometer
at O. D. against a control without L-phenylalanine. PAL
activity was calculated on the basis of 3.09 moles of
trans-cimamic acid for each 0.01 O - D - ,,,, increase (Zucker
1968) .
For mRNA assays, three plants from cultivars Mc01 22
and CM 523-7 were inoculated as described in chapter one.
RNA was extracted £rom 9 to 12 leaf disks (three/ four disks
£rom each plant), using a brass cork borer, and imrnediately
frozen in liquid nitrogen. RNA extraction £rom cassava
leaves is described in appendix 1.3. Samples were collected
at 0, 4, 8, 12, 24 and 48 hours after inoculation-
Resuïts
PAL enzyme activity in resistant and susceptible
interactions between cassava and Xanthomonads
PAL enzyme activity in the resistant interaction of
MCol 22 plants inoculated with X. cassavae was higher than
when inoculated with X. axonopodis pv. maniho t i s or the
control (Fig. 2.1). PAL activity increased dramatically in
the initial hours after inoculation. There was a drop in PAL
activity after four hours, but it remained higher in the
resistant reaction than in both the control and susceptible
interaction. The PAL activity in the susceptible interaction
Hours after inoculation
= X. cassavae A X. a. pv. manihotis
Figure 2.1 PAL enzyme activity i n cassava leaves of cultivar MCol 22 af t e r inoculation with X. axonopodis pv. manihotis, Xanthomonas cassavae and MES/CaC12 buf f er (control). PAL activity is expressed as m o l of cinnamic acid produced /min/pg protein. Bars indicate the standard error (mean I SE, n=3 ) .
was higher than the control, with a small peak at 4 hours after
inoculation, as in the resistant interaction, but the
levels of activity were rnuch lower .
Amplification and ssguence analysis of PAL gene
A product of 520 bp was obtained using primers PAL81 and
PAL82 (Fig . 2.2 ) . The primers were £rom a highly conserved
region and amplified PAL homologues not only £rom cassava DNA
but also from tomato and alfalfa DNA. The PCR product £rom
cultivar MCol 22 was cloned and called MEPAL for Manihot
esculenta PAL. Attempts to clone the genornic clone using MEPAL
and heterologous PAL genes as probe are described in appendix
2.2.
A BlastN search of GenBank with MEPAL demonstrated high
homology with other PAL genes at the nucleotide level such as
soybean (83% Frank and Vodkin 1991), alfalfa (83% Gowri et al.
1991) and pea (82% Y a m a d a et al. 1992) (Fig. 2.3). BlastP
searches and Clustal analysis (Thompson et al. 1994) of the
predicted translation product resulted in more than 90% homology
with several PAL genes including grape (Sparvoli et al. 1994 ) ,
soybean (Estabrook and Sengupta-Gopalan 1991), parsley (Appert
et al. 1994) and sorghum (Cui and Maguill 1996) (Fig. 2 . 4 ) .
Southern blot analysis
Southern blots of di£ ferent cassava cultivars (see Table 1,
80
Figure 2.2 Gel of PCR products using PAL-specific primers and different sources of DNA as template. Lane 1: 100 bp DNA marker; lanes 2 & 3: amplification £rom MCOL 22 and 1-56 cassava DNA respectively; lanes 4 & 5 : amplification £rom alfalfa and tomato DNA; lanes 6 & 7 : amplification f rom pPALtom and pPALalf plasmid DNA containing PAL genes; lane 8: pBluescript plasmid DNA (negative control) .
Cassava GCTGCTGCTATTATGGAACACATTTTC-GATGG-r-2? 50 Soybean GCTGCTGCTATTP,TGGAGC$-TATCTTGGATGGPPh-GTTCCTACATG.GC 2 6 9 7 A l f a l f a GCTGCTGCGATTATGGAACACATTTTGGACGGCAGCTCTTATGTCAAAGC 1122 Pea GCTGCTGCTATTATGGAACACAYM'TGGATGGAAGTGCTTATGTCAAAGC 3197
* * * * * * * * * * * * * * * * - * * t * t t f f * * * * * * ** * * * *
Cas s zva PqGC-Ana-4G,9AGTTGCATGAGPPTGGFFTCCTTTGCAGL4GCCTA.GCATC 1 0 0 Soybean TGCTMGAAGTTGCATGAGATTGATCCCTTGCFAAAGCCAAAACAAGATA 2 7 4 7 A l fa 1 fa AC-CTPAGG4AGTTGCATGAGATAGATCCTTTGCAGAAGCCAA,4,9CFAGATA 11 7 2 P e a TGCTUGAAGTTGCATGAGATGGATCCTTTGCAGGAhAGCC~4CCX4GATA 3 2 4 7
e t t t f t * t t f f * t f f t f t f - t t r t t * * t * * - * r * * * t * t * * * * . . . - -
Cas sava GGTATGCTCTTCGTACATCP.CCPPCPATGGTTACGCCCACAAATTGFF9GTG 1 5 0 Soybean C-ATATGCCCTTAG.zL9CTTCACCPPCPPGTGCCTTGC-TCCTCTTArIY:-PP~.GTG 2 7 9 7 A l f a l f a G A T A T G C A C T T A G N U Y T C A C C A C A A T G G C T T G G C T T T T G 1222 Pea GATATGCACTTAGA-2\CTTCACCACMTGGCTTGGTCCTCTTATTGP4GTC 3 2 9 7
* * * * * * * * * * - * * . * * * * * * * * - * * * * - * * f*. * * * * * * * * * *
Cas sava ATTCGATTCTCMCCAAGTCCATTGPPP4GAGPPGATTTTCTGTGTG 2 0 0 Soybean ATTCGTTTCTCPUCCMGTC~TTGAGPPGAGAGATCP-4CTCTGTGF-9TGA 2 8 4 7 A l fa l fa ATTAG3~TTCTCTACCAAGTCAATTGAGAGXGAGXTC3-4CTCTGTCM-TGA 12 7 2 P e a ATTAGATTCTCTACTAAGTCA-&TTGAGAGGGAGATC;WCTCTGTTPATGA 3 3 4 7
* r t * e t * * t t t**f*.*f f * t t f f t t f t r t * e x * *
Ca s s ava T.~-~TCCXCTGATTGATGTCTCMC-G.~ACR9.OGCCTTGCATGGAGGPPE~nCT 2 5 0 S O y De an C-~J-CCCTTTGATTGATGTTTCCuGGF-9CFF\C-GCCATTGCATGGT~CPP4TT 2 8 9 7 A l f al f a C-~-~-CCCTTTGATTGATGTTTCCUGU;CL1.AGCTTTGCACGGCCG~CT 1 3 2 2 Pea TAdaPCCCTTTGATTGATGTTTCM.GM-~-CA9GGCTTTGCATGGTC-CZD-CT 3 3 9 7
t r t e - f t * t * r f f t * * t f f t - * t t + t . f i t r t * * t t r * * * *
Cas sava T C C A G G G G A C C C C , n , 9 T T G G T G T C T C F P P T G G P T A 3 0 0 So ybean TCC-~-C,GG.~ACCCCTATTGGAGTCTCT-'TCGAC-L\CACACGTTTC~ACTT 2 9 4 7 A l fa l fa TTCPPAGGAiICACCTATTGGAGTATCCATGG-CC 13 7 2 P e a TTC,-;?GG.~PPCPA-CCTATTGGTC-TATCCC~TGGG=?T-~-ATT=.CC4CGTTTGCTCTT 3 4 4 7
f r t t r * f t f t X t t t t t f i t f t t * X w T t * r X r I r * . . . .
Ca s s âva GC?TCP~-~..TTGC-GP~-~;14CTCATGTTTC-CTC?-ZTTC~CTGAGCTTGTT>-~TGA 3 5 0 Soybean GCATCTATTGGCLVKTCATGTTTGCTCAAT'TCTCTGTGA 2 9 9 7 Al fa 1 fa GC,kTCP~-~TTGGC-~nV~CTTATGTTTTGCTCC4~TTCTCTGGCTTGTT~TG.4 14 2 2 P e a GCGTCPPPTTGGTAa-ACTCTTGTTTGCTCC~-qTTCTCTGLL4CTCGTC>-a-TGG\ 3 4 9 7
t t - t + . t t t C t e t * * * * f f C t * f r t r t t f t t * r t t t t X r t t t t f
Cas s ava TTTTTAC,9P,TA.9TGGGTTGCCTTCAA.9TCTCACAC-G-GAffiUC-G-G~-~--CCTA 3 9 9 Soybean CTTTTPPCF-4CMTGGATTGCCTTCMATCTCALTGCTAGCAC--&~-9TCCAA 3 0 4 7 A l fa l fa CTTTTACPUCAATGGATTGCCTTCA9~-TCTTTCTGCTAGTAGAE4TCCTA 1 4 7 2 P e a TTTTT?-CS-~CA~CGGGTTGCCTTGCCTTCTPP9TCTCTCAGCTAGTAGO-L9TCCCA 3 5 4 7
r f * f f t C * t * * t . t ~ f * t f * * - * t f f C = * . . - . * * t t * * *
Cassava GCTTGGATTATGGTTTCA9GGGTGCTGAGATTGCCATGGCAGCCTATTGC 449 Soybean GCTTÛGACTATGGTTTCAAGGGAGCTGAAATTGCCATGGCTTCTTACTGC 3097 Alfalfa GCTTGGATTATGGTTTCAAGGGAGCTGAAATTGCCATGGCTTCCTATTGT 1522 Pea GCTTGGATTATGGATTCAAGGGATCCGAAATTGCCATGGCTTCTTATTGT 3597
* * * * * * * * * * * * ~ * * * * * * * * - - * * * * * * * * * * * * * * - - * * * t *
Cassava TCTGAGCTTCAATACCTTGCAAATCCTGTCACAAACCATGTTCACAGTGC 499 Soybean TCTGAACTCCAATATCTTGCAAATCCAGTAACTACCCATGTCCAAAGTGC 3146 Alfalfa TCTGAGTTGCAATATCTTGCAAATCCGGTTACAACCCACGTCCAAAGTGC 1571 Pea TCGGAGTTACAATATCTTGCGAACCCAGTTACAACTCATGTTCAAAGTGC 3646
* * . * * * * * * * * * * * * * * * * * * * ** * * . * * * * * * * * * * *
Cassava TGAGCAGCACAACCAAGATG- - 520 Soybean TGAGCAACACAACCAGGATGTC 3179 Alfalfa TGAGCAGCACAACCAAGATGTG 1594 Pea TGAGCAACACAACCAAGATGTG 3679
* + * * * * * * * * * * * * * ~ * * * *
Figure 2.3. Sequence and alignrnent of a cassava amplified fragment with other PAL genes. Sequences were identical ( * ) at 75% of the nucleotides. Individual homologies of sequences with MEPAL are: 83% with soybean (Frank and Vodkin 1 9 9 1 ) ; 83% with alfalfa (Gowri et al. 1991) ; 82% with pea (Yamada et al. 1992) . Primer binding regions are markeii in bold. Alignment was performed using t h e Clustal program £rom PCGENE.
Cassava AAAIMEHILDGSSYVQEAKKLHEMDPLQKPKQDRYALRTSPQGPQI 50 Grape AÀAIMEHILDGSSY7ICEAKKLHEMDPLQKPKQDRYALRTSPQWLGPHIEV 67 Parsley A A A I M E H I L D G S A Y V K A A Q K L H E M D P L Q K P K Q D R Y A L R T 240 Soybean ~ I M E H I L E G S S Y V K A A K K L H E I D P L Q K P K Q D R Y A L R T S Q G 373 Sorghum AAAIMEHILEGSSYMKLAKKLGELDP~PKQDRYALRTSPQWLGPQIEV 5 4
* * * * * * * * * ~ * * ~ * ~ ~ * - * * * * * * * * * * * * * * * * * * * * * * * * * * *
Cassava IRFSTKSIEREINSVNDNPLIDVSRNKALHGGNFQGTPIG 100 G r a p e IRASTKSIEREINÇVNDNPLIDVSRNKALHGGNFQGTPIGVSMDNTRLAI 117 Parsley IRSSTKMIERE;IINSVMiNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAI 290 Soybean IRFSTKSIEREINÇVNDNPLIDVSRNKALHGGNFQGTPIWSMDNTRLAL 423 Sorghum IRFATKSIEREINSVNDNPLIDVSRGKALHGGNFQGTPIGVSMDNTRLP 104
* * - * * . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Cassava ASIGKLMFAQFSELVNDFYNNGLPSNLTGGRNLSLDYGFKGAEIAMAAYC 150 Grape AAIGKLMFAQFSELVNDFYNNGLPSNLSGSRNPSLDYGFKGAEIAMASYC 167 Parsley AAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGIYC 340 Soybean ASIGKLMFAQFSELVNDYYNNGLPSNLTASRNPRLDYGFKGAEIAMASYC 473 Sorghum AAIGKLMFAQFSELVNDYYNNGLPSNLSGGRNPSLDYGFKGISYC 154
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * * * * * * * * * * * * * - * *
Cassava SELQYLANPVTNHVHSAEQHNQD- 17 3 G r a p e SELQFLANPVTNHVESAEQHNQDV 1 9 1 Parsley SELQFLANPVTNHVQSAEQHNQDV 354 Soybean SELQYLANPVTSHVQSAEQHNQDV 487 Sorghum SELQFLGNPVTNHVQSAEQHNQDV 178
* * * * * ~ * * * * * * * ~ * * * * * * * *
Figure 2.4. Alignment between predicted amino acid sequence of cassava (MEPAL) and PAL protein sequences. Characters show that a position in the alignment is perfectly rnatched ( * ) o r well consenred ( . ) . Perfect homology between sequences was at 82%. Homology between MEPAL and individual sequences are: 93.6% with grape (Sparvoli et al. 1994) ; 92% with parsley (Appert et al. 1994) and soybean (Estabrook and Sengupta- Gopalan 1991) and 90% with sorghum (Cui and Maguill 1996).
Chapter 1) probed with MEPAL showed di£ ferent banding
patterns depending on the cultivar used (Fig 2.5). From 17
cultivars double-digested with BAM HI/Eco RI, a strong band
of -6.6 Kb hybridized for most of the cultivars except for
cultivars MNGAI, where a 8.6 Kb band occurred (Fig. 2.5).
Along with the 6.6 Kb fragment, the 8.6 K b hybridizing band
was also present in cultivar MNGAS and CM 523-7, suggesting
an heterozygous condition for M E P U in those cultivars. All
three cultivars, MNGAI, MNGA2 and 523-7, with the 8.6 K b
band are also reported as resistant to CBB (Table 1.1,
Chapter 1). Weak hybridization with several other bands was
also observed, especially following low stringency washing.
Two weak bands of 4.6 and 1.8 K b were present in most
cultivars double digested with Eco RI/Bam HI. A weak band of
9.6 Kb also occurred in cultivars M I n d 8, M I n d 27, MMai 2 ,
MMal 48, MTai 8 and IAC-24. The presence of polymorphisrns
was also observed in cultivars CM 523-7 and CG 107-5
diges ted with H i n d III (Appendix 2.3 ) .
Clustal analysis to measure the genetic distance
between the cultivars was performed using the polymorphism
observed on Southern blots probed either with MEPAL or MEPXl
£rom chapter 1, The analysis was able to separate the
cassava cultivars based in their origin ( F i g . 2.6). One
cluster contained most of South American cultivars, while
Figure 2.5 Southern blots of cassava DNA probed with MEPAL. Genomic DNA of d i f f e r e n t cassava cultivars double digested wi th Eco RI/Bam H f .
lAC24 CG107 CG403 VEN70 BRA70 MCHN
MMAL48 MIND27 MMAL2 MIND8 CM523
MNGA2 MNGAI
MTA18 MTAI 1
MCOL22
1 South American Cluster
AfricadAsian Cluster
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
Distances
Figure 2.6 Dendogram of cassava cclcivars baseci oi, the banding patterns of Southern blocs of cassava DXFs hy~ridized with MEPAL and E P X l . Genetic distance wcs czlculated using c lu s t a l anztlysis in SYSTAT Software.
another was composed mainly of Asian and African cultivars.
Analysis of PAL activity using Northera blots
Northern blot analysis was performed on different
cassava tissues £rom cultivar MCol 22 using MEPAL as a
probe. MEPAL hybridized to 2.3 tCb rnRNA (Fig. 2.7 A, 8 , C ) , and
a few blots showed hybridization with another transcript of
4.8 K b (Fig 2.7 B), Transcripts accumulated mainly in
leaves, young stems and young petioles (Fig. 2.7 A),
suggesting that there is a temporal and spatial regulation
of the gene. When leaves at different growth stages were
analysed, there was a progressive decrease in MEPAL mRNA
levels as leaves rnatured (Fig. 2.7 B) .
In wounded leaves the highest level of PAL mRNA was
observed between 12 to 18 hours after the wounding treatment
(Fig. 2.7 C) - No significant increase in PAL activity w a s
found after 18 hours either in the wounding treatment or in
undamaged leaves (data not shown) .
PAL RNA levels during plant-pathogen interaction
PAL mRNA was detected during the resistant reaction
when cultivar Mc01 22 was inoculated with X, cassavae (~ig.
2 . 8 ) . An increase in mRNA levels began a few hours after
28s r RNA
Hours after wounding O 6 12 18 24 30
28s r RNA
Figure 2 . 7 Northern b l o t analysis of t o t a l cassava RNA , c u l t i v a r MC01 22, probed with MEPAL. A) RNA from d i f f e r e n t casssava t i s s u e s . B) RNA from leaves a t d i f f e r e n t growth s t a g e s . Size of t h e leaves i n lanes 1 t o 4 a r e : 1-3 cm; 5-6 cm; lOcm and 15 c m . C ) RNA from cassava leaves after wounding. Blots were probed wi th rRNA f o r loading c o n t r o l .
Hours after inoculation
CM 523-7 + X. a. pv. manihotis
CM 523-7 + X. cassavae
28s rRNA
28s rRNA
MEPAL
285 rRNA
$&..gpg;: . ? . .-
!<y+. :-, . - ..- 28s rRNA
Figure 2.8 Levels of MEPAL rnRNA in cassava leaves of two cassava cultivars after inoculation with either Xanthonzonas cassavae or X. axonopodis pv. manihotis. A) Cultivar MCol 22; B) Cultivar CM 523-7. Total RNA was hybridized with MEPAL and wheat ribosomal DNA for control.
inoculation and was observed up until 12 hours- In the
susceptible reaction, when MCol 22 plants were inoculated
with X. axonopodis pv. manihotis, PAL rnRNA was observed only
at 48 hours, and this was at a much lower level than in the
resistant interaction. No transcription of PAL was observed
in the susceptible interaction of CM 523-7 plants inoculated
with either bacteria. It is interesting to observe that the
transcriptional activity detected in the resistant
interaction was at a relatively lower level than that
observed in young stems, petiole and young leaf tissue
(Fig.2.7 A) as well as that observed after injury of the
roots (Chapter 3) .
Discussion
The activation of enzymes in the PPP during resistant
interaction can provide several lines of defence for plants
against pathogens. In chapter one, an incompatible
interaction between cassava cultivar MCol 22 and X. cassavae
lead to an increase in peroxidase activity. In this chapter,
it was observed that there was an increase in PAL activity
in the same interaction but with a sharp peak at 4 hours
after inoculation; this was not observed with the increase
in ionically-bound peroxidase, which was not significantly
greater than the control until 24 hours following
inoculation (chapter 1). PAL enzyme activity in resistant
soybean hypocotyls inoculated with P. megaspenna f. sp.
glycinea was also reported to be approxirnately seven times
higher than the control and susceptible cultivar at 4 hours
after inoculation (Bhattacharyya and Ward 1986). A peak in
the increase of PAL enzyme activity was also found in
resistant pearl millet seedlings (Pennisetum glaucum (L,)
R-Br,) following inoculation with downy mildew disease
(Sclerospora graminicola (Sacc,) Schroter), but the
accumulation was not detected until 12 hours after
inoculation (Nagarathna et al. 1993).
One important effect of the increase in PAL activity
and the activation of PPP, is the production of cinnamoyl
alcohols. Cinnamoyl alcohols are used in the formation of
structural barriers, such as lignin and suberin, against the
pathogen via oxidation catalysed by peroxidases (~olattukudy
et al. 1992). It is not surprising that one would observe an
increase in PAL activity of cultivar MCol 22 with X.
cassavae prior to increases in peroxidase activity which is
not observed until at least 24 hours after inoculation
(chapter 1). Boher et al. (1995) reported the production of
lignin, tyloses and calloses during resistant interactions
be tween cassava and X. axonopodis pv . maniho tis .
Another main function of the PPP in plant defence is
92
the production of phytoalexins, which might have
antimicrobial activity. An increase in phenolic compounds
and phytoalexins during a resistant interaction between X.
axonopodis pv. manihotis and cassava has been demonstrated
by Kpemoua et al. (1996). Similarly, soybearis inoculated
with X. campestris pv. glycines showed an accumulation of
the flavonoids glyceollin and coumesterol, which reduced the
growth of bacteria (Fett and Osman 1982). Essenberg et al.
(1990) also identified sesquiterpenoids produced in cotton
leaves inoculated with X. campestris pv. vesica toria. The
accumulation of phytoalexins was around the infection site,
providing resistance toward the pathogen (Pierce and
Essenberg 1987). An increase in PAL activity followed by an
increase in phytoalexins was also reported in wounded
cassava roots (Tanaka et al. 1983).
A Southern blot of cassava DNA digested with different
restriction enzymes and probed with MEPAL exhibited a
multiple hybridization pattern suggesting that there is a
srnall family of PAL genes in cassava, similar to r ice
(Minami et al. 1989). alfalfa (Gowri et al. 1991) and
arabidopsis (Wamer et al. 1995). It was possible to
differentiate the cassava cultivars tested on the basis of
the polymorphism present in the blots. ~nterestingly, the
few cultivars that are classified as resistant to CBB
93
demonstrated that such regulation is related to cis-elements
in the prornoter region. Liang et al. (1989b) showed strong
evidence that the pattern of expression of a bean PAL 2 gene
is es tablished at the transcriptional level . They
transformed tobacco plants with a chimeric construct (PAL&
GUS), that showed localized activity in different organs.
After stress treatments, such as wounding and light, there
were tissue-specific modifications in the pattern of GUS
expression, indicating that the PAL2 promoter was able to
decode developmental and environmental stimulation into an
integrated spatial and temporal program of gene expression
to control the following cascade of events in the PPP.
~nterestingly, some blots showed not one but two RNA
hybridization bands. One possible explanation is that there
is a small family of PAL genes in cassava and the MEPAL
probe is not able to distinguish among those genes when they
are expressed. Hybridization to multiple PAL transcripts has
been reported in bean and alfalfa (Elkind et al. 1990, Gowri
et al. 1991). The presence of more than one band could also
be the result of either mRNA splicing or an alternative
transcriptional initiation site as previously suggested
(Gowri et al. 1991) .
The results during this resistant plant-pathogen
interaction showed an increase in the level of MEPAL mRNA-
Similar results were obtained with PAL RNA transcripts
during the resistant interaction between white bean and P.
syringae pv. tabaci (Jakobek and Lindgren 1993a) . In this
latter study, PAL transcripts reached a maximum at 4 hours
after inoculation, followed by increases in chalcone
synthase and chalcone isomerase transcripts. Meanwhile,
during a susceptible interaction between white bean and P.
S. pv. phaseolicola, the defence via PPP was cornpletely
inhibited and no PAL transcripts were detected (Jakobek and
Lindgren 1993b). Another example of PAL expression after
infection is in potato leaves inoculated with P. infestans
which showed higher accumulation of PAL transcripts in
resistant cultivars 8 hours following inoculation than in
susceptible cultivars. Increases in PAL transcripts after
inoculation or exposure to microbial elicitors have been
reported in several species. including alfalfa (Ni et al.
1996), sorghum (Cui et al. 1996), potato (Joos and Halbrock
1992, Yoshioka et al. 1996), French beans (Bolwell et al.
1986) , arabidopsis (Lummerzhei rn et al. 1993) .
The transcription of MEPAL during the resistant
interaction was apparently at a lower level than that when
transcripts of PAL were detected in wounded roots (chapter
3 ) . Northern blot analysis following shorter periods after
inoculation might show higher levels of PAL expression than
96
those observed-
Although MEPAL is not a full length clone, it could
potentially be used in antisense research and as a molecular
marker- Furthemore it could be used in expression studies
to verify the activation of the PPP during different stress
situations. Further studies might also include the
characterization of the 8.6 Kb band that hybridized in
cultivars resistant to CBB in order to explore its potential
as a molecular marker in breeding programs. The promoter
regions of MEPAL and other cassava PAL genes also merit
investigation to provide information about the mechanism
regulating the expression of different PAL genes in
resistant and susceptible cultivars,
Chagter 3
Phenylalanine amnonia-lyase and peroxidases activity during
root gost-harvest deterioration of cassava
Introduction
Following harvest cassava roots undergo a rapid process
of deterioration that is generally divided into
physiological and microbiological components (Booth 1 9 7 5 ) .
The primary or physiological deterioration is the initial
cause of loss of acceptability of roots for fresh
consumption and is identified by fine blue-black streaks in
the root vascular tissue. This physiological process is
quite variable, wherein cultivar and environmental
conditions play important roles in the speed and degree of
deterioration that occurs. The secondary or rnicrobial
deterioration occurs when the roots have already become
unacceptable due to t h e primary deterioration. It is caused
by pathogenic rots, fermentation and/or softening of the
roots (Noon and Booth 1977).
Wounding of the roots during harvesting is probably the
initiation signal for the physiological deterioration. After
injury of the roots, there is water loss £rom tissues
provoking an oxidative stress (Aracena 1993). The wounding
response involves the production of several plant defence
metabolites frorn the phenyl-propanoid pathway ( P P P ) , which
eventually leads to the production of coumarins, tannins and
lignin. These polymers subsequently form a peridem layer to
seal the exposed tissue (Beeching et al. 1994). Furthemore.
there is the oxidation of flavonoids, proanthocyanidins,
cathechins and other phenolic cornpounds not only in the
injury site of the roots, but also in inner layers producing
blue and dark brown streaks in the storage parenchyma
(Tanaka 1983).
Rickard (1982) suggested that studies involving changes
in phenolic content. PAL, peroxidase and polyphenol oxidases
would be important to elucidate the basis of post-harvest
deterioration. Data regarding PAL and peroxidase activity in
cassava have been reported (Plumbey et al. 1981. Tanaka
1983. Rickard 1985). However. there has been no detailed
work at the biochemical or molecular level on those enzymes,
especially comparing different layers of the root during
deterioration.
In this chapter the expression of MEPAL (frorn chapter
2 ) , PAL enzyme ac tivi ty and peroxidase ac tivi ty in di£ f erent
layers of the root during deterioration are described. To
evaluate the importance of PAL in root post harvest
deterioration, an inhibitor of PAL transcription and one
inhibitor of PAL enzyme activity were applied to root
sections. PAL transcription, PAL and peroxidase enzyme
activity and root post harvest deterioration with and
without the inhibitors were analysed.
Material and methods
Cassava roots were obtained £rom 12-14 month-old plants
(cultivar MCol 22) grown under growth room conditions, as
described in chapter one. To simulate post-harvest
deterioration, roots £rom two plants were transversely cut
into slices 3.5-4-0 cm thick, and incubated in the dark on
the top of moistened paper towels in covered plastic
containers (4Ox25x9cm), at 20-2S°C, which were similar to
conditions used in previous studies on cassava post-harvest
deterioration (Rickard 1982 and 1986, Tanaka 1983). From the
c u t root slices, two sub-sections were collected for
analysis, an outermost layer of 0.5 cm, and an inside layer
of 0.5-1.0 cm thick ( F i g . 3.1). Samples (outer and inner
layers) were randomly collected in three replicates at O , 6,
12, 24, 36, 48, 72 and 96 hours after cutting f o r PAL enzyme
analysis. One sample was also used for mRNA extraction. For
peroxidase analysis samples were collected at 0, 6, 24, 48
and 72 hours, also in three replicates. The tissue was
imrnediately frozen in liquid nitrogen, ground and stored at
-80°C prior to RNA or protein extraction.
Both inner and outer layers were stained f o r lignin,
using phloroglucinol-RC1 (Speer 1987) at 0, 24, 48 and 72
hours. The samples were immersed in a 1:I solution of 92%
outer layer -+ Cassava root section
Figure 3.1 Diagram with casava roots (A) and cassava root sections used in root deterioration experiments. To simulate post-harvest deterioration, roots were transversely cut into slices 3 . 5 - 4 . 0 cm thick, and incubated in the dark, on the top of moistened paper towels in covered plastic containers From the cut root slices, two sub-sections were collected for analysis, an outermost layer of 0.5 cm. and an inside layer of 0.5-1-0 cm thick. Dotted lines in A represent points where wounding during harvest might occur.
ethanol and glacial acetic acid for five minutes, followed
by three minutes in a solution of 1% phloroglucinol (Sigma,
North York, Canada) in 92% ethanol, The sections were
transferred to 25% HC1 for 30 seconds and washed in
distilled water.
PAL inhibitors assay
For these experiments, cassava roots were obtained £rom
a local market. The roots originated from the Caribbean
region, and were coated with a paraffin to reduce the rate
of deterioration. The roots were transversely cut into
slices 2 . 0 - 3 . 0 cm thick and inoculated in a solution of PAL
inhibitor under vacuum for ten minutes. Two inhibitors were
tested: 250 and 500 p M of trans-cinnamic acid (Sigma, North
York, Canada) , and 100 pM 2-aminoindan-2-phosphonic acid
(AIP) . AIP was kindly supplied by Dr. Jerzy Zon, University
of Wraclaw, Poland. Trans-cinnamic acid has been reported to
decrease PAL transcription and enzyme activity (Mavandad et
al. 1994), while AIP has been reported to be a PAL enzyme
inhibitor (Zon and Amrhein 1992). Samples were also
inoculated with sterile distilled water for control- The
root slices were incubated in the dark on top of moistened
paper towels in closed plastic containers (4Ox25x9cm) at 2 0 -
2Z0c. Samples were taken from the outer layer of the cassava
root sections between O and 72 hours following inoculation
for different assays. Samples were frozen in liquid nicrogen
and ground with a mortar and pestle. RNA extraction,
Northern blot analysis, PAL and peroxidase enzyme assays
were performed as described previously in chapters one and
two. For each data point three samples were measured and
analysed for PAL and peroxidase activity. One sample was
also used for RNA extraction.
Resuïts
PAC enzyme assays and MgPAL expression during root post-
harvest deterioration
As was expectrd, the degree of deterioration was
visibly less in the inner layer than in the outer layer of
the cassava root sections (Fig. 3 .2 , 3 . 3 ) . Sections stained
with phloroglucinol-HC1 revealed an increase in lignin
(purple-red colour) during the time course of the study in
both layers (Fig. 3 . 4 , 3 . 5 ) . The yellow colouration in the
roots were probably due to the presence of condensed
tannins. The increase in lignin and tannins was also higher
in the outer layers than in inner layers.
The PAL enzyme assay showed distinctly different
activity patterns between the two layers analysed (Fig. 3 . 6
A, B). The outer layer produced high enzyme levels at 12
hours after injury and decreased subsequently, but still
Figure 3.2 Outer layers of cassava root sections during deterioration. Cassava roots were transversally cut in slices 3 . 5 - 4 . 0 cm thick (Fig. 3.1), and incubated in the dark, on top of mois tened paper towels in covered plas tic containers (4Ox25x9cm), at 20-22OC. Pictures, £rom the top and left to right, were taken at 0 , 24, 48,72 and 96 hours after injury.
Figure 3.3 Inner layers of cassava root sectians during deterioration. Cassava roots were transversally cut in slices 3 . 5 - 4 . 0 cm thick (Fig. 3 . 1 , and incubated in the dark, on top of moistened paper towels in covered plastic containers (4Ox25x9cm) , at 20-22OC. Pictures from the top and left to right were taken at 24, 48, 7 2 and 96 hours after injury.
Figure 3 - 4 O u t e r Iayers of cassava root sections during deterioration, after lignin staining wi th phloroglucinol-HC1. Cassava roots were transversally cut in slices 3.5-4.0 cm thick, and incubated i n the dark, on top of moistened paper towels i n covered plastic containers (4Ox25x9crn), at 20-2Z0C. Pictures £rom the top and left to right were taken at 24, 48 and 72 hours after injury.
Figure 3.5 Inner layers of cassava root section during deterioration, after lignin staining with phloroglucinol-HC1. Cassava roots were transversally cut in slices 3 - 5 - 4 . 0 cm thick F i g . 3 -1.. and incubated in the dark, on top of moistened paper towels in covered plastic containers (4Ox25x9cm), at 20-2Z°C . Pictures £ r o m the top and left to right were taken at 24, 48 and 72 hours, after injury.
outer layers
H o m after injury
inner layers
Hours after injury
Figure 3.6 PAL enzymatic activity in ou te r (A) and inne r (B) layers of cassava roots sections after injury. Bars indicate the standard error (mean k SD, n=3).
continued at higher levels than observed before injury (O
hour) . In the inner layers of the root section, PAL enzyme
activity increased gradually until 48 hours after injury,
decreasing also gradually after that point.
The level of MEPAL transcripts in both layers increased
rapidly after injury reaching a maximum at the 12-hour point
and followed by a sharp decline at the 24-hour point (Fig.
3.7 A, B). Surprisingly, PAL mRNA increased thereafter up to
96 hours in inner layers, and to a lesser extent in the
outer layer. Furthemore, outer layer samples at 72 and 96
hours showed degradation of the MEPAL mRNA.
Peroxidase activity during root post harvest deterioration
Soluble and ionically-bound peroxidase activity was
measured in both outer and imer layers of the cassava root
sections. Ionically-bound peroxidase activity showed an
increase at 24 hours and was significantly higher (P<0.05)
in outer layers than in the inner layers at 48 and 72 hours
after injury (Fig. 3.8 A). Soluble peroxidasa activity also
showed an increase at 24 hours after injury in both layers
(Fig. 3.8 B).
Effects of different PAL inhibitors
The effects of PAL inhibitors on root sections were
analysed at the enzymatic and transcriptional levels. Enzyme
Hours after injury
O 6 12 24 48 72 96
inner layers
28s rRNA
Hours after injury
6 12 24 48 72 96
MEPAL
28s rRNA
Fig 3 . 7 Expression of PAL gene i n o u t e r ( A ) and inner (B) l a y e r s of cassava roo t s e c t i o n s . RNA was i s o l a t e d from l a y e r s of cassava roots a t various t i m e a f t e r i n j u r y . Tota l RNA was probed with MEPAL and a wheat ribosomal gene f o r con t ro l .
lonically bound peroxidase
lnner layers
i Outer layers
O 10 20 30 40 50 60 70 80
Hours after iniurv
Soluble peroxidase
lnner layers
i Outer layers
L - -- - - I --- 7'
O 10 20 30 40 50 60 70 80
Hours after injury
Figure 3.8 Guaiacol peroxidase activity in inner and outer layers of cassava root sections. ïonically-bound (A) and soluble (B) peroxidase fractions were measured at various times after injury. Bars indicate the standard error (mean .t SE, n=3).
inhibition relative to control samples was observed only
when 100 pM AAIP was used at 12 and 24 hours, but none was
observed at 48 hours after injury (Fig. 3.9) . No enzyme
inhibition was observed when trans-cimamic acid was used
(data not show). The data from the latter experirnent were
extremely variable, probably due to the fact that the PAL
assay was based on the formation of trans-cinnamic acid,
which was also applied in the treatments, and residual acid
£rom the application might have contributed considerably to
the variability in the results.
The level of PAL transcripts was reduced when root
sections were incubated with a trans-cimamic acid solution
(Fig. 3.10 A). Both treatments of 250 and 500 pM trans-
cinnamic acid decreased transcript levels measured at the 6-
hour point and at the 24-hour point, compared with the
water-treated roots. No inhibition of MEPAL transcription
was observed when AIP was used (Fig . 3 -10 B) .
Although a reduction of PAL was observed at the
enzymatic and transcriptional levels, there waç no
difference between control and treated samples regarding
root deterioration (Appendix 3.1) .
Peroxidase activity
If there is a reduction in PAL enzyme activity, Zhen a
decrease in the pool of phenolic compounds followed by a
Hours after injury
m control i 100uM AIP
Figure 3 . 9 PAL enzymatic activiïy in cassava roooc sections after inoculation with 100 uM A I P . Bars indicate the standard error (rnezn 2 SE, n=3).
A Trans-cinnamic acid
AIP
MEPAL
MEPAL
18s rRNA
18s rRNA
Figure 3.10 Effect of trans-cimamic acid (A) and AIP ( B I on transcription rates of PAL during root deterioration in cassava. Roots sections were incubated 15 minutes with the inhibitor and RNA was extracted at different time interval (hours) after inoculation.
reduction in the peroxidasa level might occur. Peroxidase
activity was measured in samples treated with 500 pl trans-
cinnamic acid or 100 pM AIP, as well as in control samples.
Soluble peroxidase activity was significantly higher
(P<0.05) in samples treated with AIP ( F i g . 3.11 A). The
increase was observed as early as six hours after injury and
was three-fold higher than both control and trans-cinnamic
acid-treated samples at 48 and 72 hours. There was no
difference in soluble peroxidase activity between control
and trans-cinnamic acid-treated samples.
Ionically-bound peroxidase showed an extremely high
level of activity for samples treated with trans-cinnamic
acid (Fig. 3.11 B ) . The activity was 3.5 and 12 fold higher
(P~0.05) than control and AIP-treated samples respectively.
Discussion
The characterization of MEPAL expression was performed
on inner and outer layers of cassava root sections. The
objective was to obtain information about the root
deterioration mechanism at both the wounding site (outer
layer) and inside the roots (inner layer), which is similar
to the in jury occurring when cassava is harvested (Rickard
1982). As expected, deterioration of the exposed wounded
surface layer was more rapid than deterioration of the inner
layers with a greater and more rapid deposition of lignin;
Soluble peroxidase A O D ~ ~ ~ J r n i n l g F.W. root
lonically-Bound peroxidase A O D ~ ~ d r n i n I g F.W. root
likewise, there was more rapid deposition of lignin in the
outer layers, as determined by the red staining with
phloroglucinol-HC1. The deposition of lignin on the injured
surface of cassava roots has been previously reported
(Tanaka 1983). The yellow colouration throughout the root
tissue is probably due to the presence of proanthocyanidins
and catechins (Rickard and Gahan 1983, Tanaka 1983). These
compounds are involved in the formation of condensed
tannins, and can produce a similar lignin staining pattern
with phloroglucinol-HC1 (Vance et al. 1980).
The analysis of PAL enzyme activity in sections of
cassava roots reported in this chapter demonstrated two
different patterns according to the layer analysed (Fig.
3 . 3 ) . The inner layers showed a gradua1 increase in PAL
activity, which is very similar to the activity previously
reported for root sections in similar experiments but
without the two layers subdivision (Tanaka et al. 1983,
Rickard 1985). In contrast, in the outer layers, the
increase in PAL activity was more rapid with a high peak in
the initial hours after injury. It is possible that this
different pattern of PAL activity reflects two mechanisms
regulating its activity in different layers of the root
section, and the level of PAL enzyme activity might be
related to the level of deterioration in roots. Thus, the
pattern of PAL activity in the outer layers appears to be
related to the accelerated process of oxidation and
lignification of the cells. This process would lead to ce11
death in the exposed outer region, followed by a decrease in
PAZ, accivity of this layer, which would be composed of a
mix of dead and live cells. Meanwhile, in the inner layers,
deterioration as well as the PAL activity had a gradua1
increase. A gradient of activation of PAL enzymes in tissue
at different distances from the wounding site was also
observed in lettuce (Lactuca sativa L.), where layers
closest to the wounding also showed a rapid increase in PAL
activity (Ke and Saltveit 1989).
MEPAL transcription levels in the outer layers
correlated with PAL enzyme activity. The mRNA transcripts
accumulated more rapidly during the first 12 hours,
decreasing thereafter. In the imer layers, the pattern of
transcription was sirnilar to that in the outer layers in the
first 24 hours, but increased to much higher levels than in
the outer layers in the 48 to 96 hours period. It is
possible that in the inner layers, the first MEPAL peak is
associated with a wounding signal. A . increase in PAL,
transcription would increase the amount of trans-cinnamic
acid, but since the oxidation process inside the roots is
slower than in the outside layers, an accumulation of the
level of trans-cinnamic acid might occur to produce a
negative feedback on the PAL transcription. As the root
deterioration accelerates in the inner layers, after 24
hours, the trans-cimamic acid concentration would decrease
and MEPAL transcription would increase again. The inhibition
of MEPAL transcription by trans-cinnamic acid observed in
this thesis partially supports this hypothesis. Mavandad et
al. (1994) also showed that trans-cinnamic acid inhibited
PAL activity in suspension ce11 cultures of French bean.
The induction of PAL in wounded plant tissue has been
reported for several other species such as potato (Ishizuka
et al. 1991; Smith and Rubery 1979), lettuce (Ke and
Saltveit 1989), French beans (Bolwell and Rogers 19911, and
cucumber (Hyodo et al. 1989). The wounding stress increases
the levels of phenolic cornpounds in different branches of
the PPP, such as coumarins, chlorogenic acid, and coumaryl
alcohols, which are used to produce lignin and suberin. In
addition, flavonoids, cathechins and proanthocyanidins might
also be produced for the formation of tannins.
An increase in the level of peroxidases during cassava
root post-harvest deterioration is expected since there is a
preliminary increase in PAL-produced phenolic compounds. The
peroxidases catalyse the oxidation of such phenolics to form
a lignin layer on the injury surface (Tanaka 1983, Plumbey
et al. 1981). Furthermore, the peroxidases might also be
involved in the oxidation of proanthocyanidins and
cathechins in the formation of condensed tannins (Haslam
1989) . The results reported in this thesis show that total
peroxidase activity started to increase 24 hours after
wounding in both layers. However, the inner layers showed an
increase in both soluble and ionically bound-peroxidase,
whereas the outer layers showed a higher level of the
ionically-bound form relative to the soluble form. As
discussed in chapter one. ionically-bound peroxidases are
mainly cationic and increased levels of this form have been
associated with deposition of lignin-like polymers on
wounded plant surfaces. These results are different from
those previously reported on cassava root deterioration.
where the increase in soluble peroxidase activity was
greater than that of the ionically-bound form (Plumbey et
al. 1981). However, the above study was performed only
during a 22-hour period after root injury, whereas the
difference between the two peroxidase forms reported in this
thesis were observed at 48 and 72 hours after injury.
McDouglas (1993) also reported an increase in ionically-
bound peroxidases after wounding of flax stems, most of
which were cationic in nature- It is interesting that the
increase of total peroxidase activity in the outer layers at
the site of injury was far greater than in the inside
layers, where there was a delay in the onset of PAL activity
and less deposition of lignin or tannins and probably less
oxidation,
When AIP, an inhibitor of the PAL enzyme, was applied
to the roots, there was a decrease in PAL activity. AIP
inhibits PAL enzyme activity and the accumulation of
products related to the PPP (Zon and Amrhein 1992) . Schmutz
et a1.(1993) reported the inhibition of PAL and lignin
formation with AIP in fiber walls of cotton. PAL inhibition
by AIP also decreased the production of salycilic acid and
inhibited SAR in tobacco (Pallas et al. 1996) and cucumber
(Meuwly et al. 1995); in addition, a decrease in ionically-
bound peroxidase and an increase in soluble peroxidase were
observed, There were no visible differences in the pattern
of root deterioration when AIP was applied to cassava roots
in research conducted for this thesis. However, there were
differences in the balance of peroxidases produced. Roots
treated with AIP showed a reduced amount of ionically-bound
and an increase in the soluble peroxidases. The decrease in
the ionically-bound peroxidase fraction was expected with
the decrease in PAL activity and a potential decrease of
substrate for those peroxidases. However, the increase in
the soluble fraction is intriguing- It is possible that
there is a switch in the phenolic pool, due the PAL
inhibition or the presence of AIP, with the production of
phenolics that are not involved in the reaction with
ionically-bound peroxidase but which are substrates for the
soluble form. It is also possible that AIP is interfering in
the synthesis of peroxidase isoenzymes (Sato et al. 1993).
This change in the balance of peroxidases has been
previously reported, but in the reverse order, L e . a
surprising increase and decrease in ionically-bound and
soluble forms respectively in suspension cells cultures of
Zinnia elegans L, treated with AIP (Sato 1993).
A decrease in root deterioration was expected as a
consequence of the decrease in both PAL activity and
ionically-bound peroxidases. However, there were no visible
differences between roots treated with AIP and control
samples 72 hours after in jury . It is possible that
oxidation of previously formed tannins by other enzymes such
as polyphenol oxidases and laccases also contribute to
oxidation of phenolics on the injured surface and the
visible symptoms of deterioration.
It was observed that PAL enzyme activity and MEPAL
transcription were related to the root deterioration
process. There is a possible regulation of transcription of
PAL by the level of the phenolics in the cell, which control
both PAL transcription and enzyme activity differentially in
imer and outer layers, according to the degree of oxidation
of the tissue. Further work to characterize the role of
MEPAL could include in s i t u hybridizations to observe its
expression not only in different parts of the roots but also
in cassava cultivars with longer shelf-life.
General discussion
The main objective of this research was to initiate a
molecular characterization of genes involved in the process
of resistance to xanthomonads in cassava and in the
physiological post-harvest deterioration of the roots, which
are two major problerns for this crop. Although these
problems have been studied at the biochemical level (Rickard
1985, Boher et al. 1995), there are no reports of such
characterizations at the molecular genetic level, including
studies of genes related to the phenylpropanoid pathway
(PPP). The studies reported in this thesis were focused on
characterization of genes for phenylalanine ammonia-lyase
(PAL) and peroxidases which are involved respectively in the
initial steps of the PPP and in the oxidation of the
phenolic compounds produced by this pathway.
The plant-pathogen portion of the studies characterized
the interaction between two cultivars of cassava (MCol 22
and CM 523-7) and two pathogens (X. axonopodis pv. m a n i h o t i s
and X. cassavae). A resistant interaction was observed only
when cultivar MCol 22 was inoculated with X . cas savae , where
no disease symptoms were observed in the leaf and there was
a reduction of bacterial growth and induction of both PAL
and peroxidase enzymes. In addition, increased levels of
MEPAL transcripts were detected during the onset of
resistance but not in the susceptible interaction. These are
the first reports showing the direct involvement of both PAL
genes and enzyme activity during a plant-pathogen
interaction in cassava- The activation of peroxidase enzymes
during the resistance interaction is also described although
other reports indirectly indicate peroxidase activity during
resistance between cassava and X. axonopodis pv. manihotis
(Kpemoua et al. 1996, Boher et al. 1995) .
One of the major differences between X. axonopodis pv.
manihot is and X . cassavae is that the former promotes
defoliation and systemically invades the plant, while X.
cassavae is restricted to the infected leaf causing
defoliation. The results described in chapter one showed
that there is resistance on the leaf during the interaction
between MCol 22 and X, cassavae. Since susceptibility of
this cultivar to X. axonopodis pv. manihotis was also
observed, comparison of these two interactions could be used
to examine the mechanism of resistance by the plant.
It is also possible to use the differences between the
susceptible and resistant interaction to compare and
characterize the genes involved not only on the plant side
but also £rom the pathogen side of the equation. For
example, it would be interesting to observe the differences
in both avr and hrp genes occurring in both interactions.
During post-harvest deterioration of the roots, it was
demonstrated that there were differences in the activities
of both PAL and peroxidase enzymes, concomitant with the
level of deterioration of the roots, and probably related to
the rate of oxidation that is higher in the air-exposed
parts of the injured root, than in the inner layers. In both
layers, however, there was an accumulation of PAL and
peroxidase enzyme activity during deterioration, as well as
an increase in PAL rnRNA. These are the first studies to
report molecular data pertinent to PAL activity during
deterioration of cassava roots. The analysis of peroxidase
in the roots also showed interesting results that deserve
further research. Ionically-bound peroxidases appear to be
associated with the oxidation of phenolic compounds produced
through the PPP, compounds which are used in the formation
of lignin or suberin (Kolattukudy et al. 1992). Roots with
lower levels of deterioration or treated with a PAL enzyme
inhibitor showed a decrease in ionically-bound peroxidases
relative to the soluble form. In addition, when trans-
cinnamic acid, which is one of the main initial products of
the PPP, was applied to the roots, ionically-bound
peroxidase activity increased. Further studies of root
deterioration should include characterization of these
enzymes, to determine possible effects on lignin deposition-
It would also be interesting to observe changes in the
phenolics pool after the use of the PAL inhibitors to assess
if there is any correlation between such changes and
peroxidase activity, or if there is any effect on the
defense mechanism through the formation of lignin or
oxidation of quinones.
During research for this thesis, PCR clones of two
cassava genes of significance were isolated. MEPAL was able
to successfully detect PAL transcripts in leaves, stems and
petioles, and was preferentially expressed in young tissues.
MEPAL could also detect transcripts during the two stress
situations studied: plant-pathogen interaction and post-
harvest deterioration of the roots. In contrast transcripts
of M E P X l were not detected either in the post-harvest
deterioration studies or during plant-pathogen interactions,
even when only exons of the clone were used as a probe. In
addition, both clones showed potential as molecular markers
relevant to breeding for resistance to bacterial blight. It
will be also interesting to check these as markers for
another important disease in cassava, such as cassava mosaic
virus (CMV), since there are indications that the genes for
resistance for both diseases are linked (Hahn et al. 1989).
Finally, cloning the promoter region of these genes would
provide the opportunity to study the cis and trans-acting
factors necessary for their regulation.
A comparison of both kinds of studies in this thesis,
plant pathogen-interaction and post-harvest deterioration,
reveals several similarities during the production of both
PAL and peroxidase enzymes and the transcription of MEPAL.
The plant responses to those stresses together with those
observed in symbiotic interactions are probably "...a
variation on a common theme" (Baron and Zambrynski 1995) . In
other words, plants utilize the same defense mechanism in
response to a pathogen attack, a symbiotic interaction or an
injury due to either a biotic or abiotic source. The
responses are basically the same, with increases in ion
flux, hydroxy proline-rich glycoproteins . enzymes of the PPP, peroxidases and chitinases.
Given the similarity in such responses, it is important
to keep in mind that attempts to modify the phenylpropanoid
pathway via traditional methods or via biotechnology might
solve one set of problerns but not necessarily another. For
example, a decrease in the PPP in the roots might decrease
the deterioration of the roots. but rnight also produce
plants more susceptible to diseases. The inverse, an
increase in the PPP to obtain more disease resistant plants,
could produce roots that will oxidize Vary rapidly due to
the high level of phenolic compounds or peroxidase activity.
However, the latter option might be a valid approach if the
wounding response is sufficiently rapid to produce a sealing
peridem on the injured surface of the root, thus preventing
the spread of oxidation to inner parts of the root.
One question for researchers in cassava post harvest
deterioration is how to stop the process of deterioration in
regions of the root distal to the wounded surface. In other
words: How do you prevent transmitting the wound signal to
the storage parenchyma of the roots? One possible approach
would be breeding znd selection for cassava roots with
higher contents of antioxidant compounds, such as B-carotene
and vitamin C, which can work as sequester phenofic
compounds or act as inhibitors of the enzymatic mechanism of
deterioration. Recent work reporting improvements in
breeding cassava for B-carotene content might give some
insight into this possibility (Iglesias et al. 1997). The
reduction of ethylene content in the roots would also be
another approach to consider. It has been dernonstrated that
roots with lower levels of ethylene content deteriorated
more slowly than roots with higher levels (Hirose et al.
1984). The reduction of ethylene might be obtained through
production of transgenic cassava plants carrying antisense
genes for ACC-synthase or genes for ACC-deaminase.
In conclusion the information presented in this thesis
can be applied in programs to irnprove the resistance of
cassava to Xanthomonas, either in molecular studies of the
plant-pathogen interaction or in applied research using the
MEPAL and MEPXl clones as molecular markers. M ' P A L was also
directly involved in the physiological deterioration of the
roots. Further investigation of PAL and peroxidases would
help to clarify this process and provide a direction for
research with the objective of increasing the shelf-life of
the roots of this important crop-
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Appendix 1.1 Methods of inoculation: A) Apparatus used to injure the leaf before spraying of bacterial- solution; B) ~yringe method, in which the bacteria suspension is inoculated by pressure in the abaxial part of the leaves; C,D) Cassava leaves from cultivar MCol 22 inoculated with either X. axonopodis pv. manihotis (C) or X . cassavae ( D ) using the wounding/spray method. Pictures were taken two weeks post inoculation. E) X. axonopodis pv. manihotis and X. caçsavae growing i n media with powdered m i l k and bromocresol-blue, specific for Xan thomonas.
PX1 Tomato TAPI Petunia Potato Tornato TAP2 Arabidopsis Tobacco Alf aif a
GGAGCTTCTCTCATTCGTCTCCACTTTCATGACTGCTTCGTTGATGTA
A T C
Tomato TAPl gene for anionic peroxidase Petunia hybrida anionic peroxidase Potato anionic peroxidase mRNA Tomato TAP2 gene for anionic peroxdase A-thaliana mRNA for peroxidase N i c o t i a n a sylves tris anionic Medicago truncatula peroxidase
PX2 ATGGTTGCACTAGCTGGTGCACACACGGTAGGTTTCGCCAGG Tomato ATPi Potato Tomato ATP2 Petunia C. cilaris Alfalfa
Tomato TAPl gene for anionic peroxidase Potato anionic peroxidase mRNA Tomato TAP2 gene for anionic peroxidase Petunia hybrida anionic peroxidase Cenchrus ciliaris clone PX18 peroxidase M. sativa rnRNA for peroxidase
Appendix 1.2 Alignment of conserved regions £rom different peroxidases genes for primer design. Primers PX1 and PX2 are marked bold. Peroxidases genes aligned are listed according to database, accession number and name.
Protocols for cassava DNA extraction
DNA extraction from in vitro tissue
For tissue originating from in vitro plants, DNA was
extracted using a modified method from Dellaporta et al.
(1983) as follows: leaf tissue (1-5 g ) was frozen in liquid
nitrogen. ground using a mortar and pestle and transferred
to a 50 ml tube. Twenty-five ml extraction buffer (100 mM
Tris pH 8, 5 0 rnM EDTA pH 8, 5OOmM NaC1. 1 . 5 % SDS) was added
and mixed vigorously. followed by an incubation at 65" C for
10 minutes. For precipitation of proteins, 8.5 ml of 5 M KAc
was added, the tube mixed again and incubated in ice for 20
minutes. The tubes were centrifuged for 20 minutes at 5000 X
q. and the supernatant was filtered through Miradoth@
(Calbiochem, California) or four layers of cheesecloth into
a clean tube with 15 ml isopropanol, gently mixed and
incubated at -20° C for 30 minutes. The DNA was spooled
using a pasteur pipette and rinsed in 75% cold ethanol.
After air drying for 10-15 minutes the DNA was dissolved in
2-3 ml of TE buffer. Ten pg of RNAse was added and the
sample was incubated 30 minutes at 37O C. Proteinase K (100
pg/ml, final concentration) was added and the mixture
incubated for 1 h at 37°C. For purification from
polysaccharides 500 pl of 5M NaCl was added. followed by the
addition of 400 pl of pre-warmed 10XCTAB solution. The
sample was incubated at 6 5 ° C for 20 minutes. One volume of
a 1/1 phenol/chlorofom was added for the separation of
organic and aqueous phases, with precipitation of most
polysaccharides with the CTAB, after centrifugation (20,000
g, 5 minutes). The aqueous phase was extracted twice with
chloroform for removal of phenol residues. The DNA was
spooled again after adding 2.5 vol cold 95% EtOH. The DNA
was rinsed in 75% ethanol, air dried and dissolved in TE
buffer.
DNA extraction from growth room plants
Extraction of DNA £rom leaves in plants from growth
room required a modified protocol from Doyle and Doyle
(1990), where repeated CTAB extraction removed most of the
starch from the samples and resulted in higher DNA yields.
This protocol involves the following steps: pre-heated (60"
C ) of extraction buffer: 1.4M NaCl, lOOmM Tris- pH 8.0, 2OmM
EDTA, 3% CTA8. To 25 ml of buffer, 5g of leaf tissue, ground
with liquid nitrogen, was added and mixed by inversion.
Fifty pl of B-mercapto-ethanol (0.2% v/v) was added and the
tube inverted several times, followed by incubation at 60" C
for 30 minutes, with occasional mixing. The same volume of
chloroform 24:l isoamyl-alcohol, was added and the tube was
inverted several times. The sample was centrifuged at 5000 g
for 10 minutes and the aqueous phase was transferred to a
new tube. CTAB was added to a final concentration of 3% and
the sample was incubated at 60' C for 20 minutes, with
occasional mixing. Extractions with chloroform:isoamyl-
alcohol ( 2 4 : 1) were repeated one or two tintes. until the
interface between aqueous and organic phase was clear.
Isopropanol (2/3 of the sample volume) was added and the
sample was incubated in ice for 30 minutes. The DNA was
spooled and washed in 75% ethanol with 10 rnM ammonium-
acetate during a minimum of 20 minutes. The DNA was dried at
room temperature and dissolved in TE buffer. RNAse and
proteinase K was added as described above. The sample was
extracted with phenol/chlorofom, mixed and centrifuged for
10 minutes at 10.000 X g to separate the layers. The aqueous
layer was transferred to a new microtube and extracted twice
with chloroform. DNA was precipitated with 2.5 volumes of
cold 95% EtOH and spooled again. After air drying. the DNA
was redissolved in TE buffer.
RNA extraction
RNA extraction was performed according to Chang et al.
(1993) where 5 grams of tissue was ground in liquid nitrogen
and mixed with a 65" C pre-warmed extraction buffer (2%
CTAB, 2% PVP K30, 100 mM Tris-HC1 pH 8 , 25 mM EDTA, 2 M NaCl
and 0.5 g / L spermidine and 2% p-mercaptoethanol. An equal
volume of chloroform:isoamyl alcohol (IAA) (24:l) was added
and the sample was centrifuged at 6-8000 g for 10 minutes t o
separate the phases, The aqueous phase was extracted again
with chloroform:IAA, centrifuged and transferred to a new
tube to which 1/4 volume of 10 M LiCl was added. The sample
was incubated overnight at 0-5" C and centrifuged at 5,000 g
for 30 minutes. The precipitated RNA was resuspended in 500
pl SSTE (1 M NaCl, 0.5% SDS, 10 mM Tris-HCI pH 8 , 1 rnM EDTA
pH 8). The sample was extracted with an equal volume of
chloxoform:IAA, and centrifuged 10 minutes at 10,000 X g.
In a new microcentrifuge tube, 2 volumes of ethanol were
added to the aqueous phase. The sample was incubated at -20°
C for at least 2 hours, followed by centrifugation at 10,000
X g for 20 minutes. The precipitated RNA was dried and
resuspended in DEPC treated water and stored at -70° C - The
protocol was scaled dom, according to the initial weight of
the tissue.
10 20 3 O 40 50 I I I I I
l TCATTTCTCGTGGCTGCCTTAAGATATTGGCNTNNAGAGGGAGACAATTT 51 TCAGAGAACTGCATCAACNTTGGCATTCAGCAAGACTTTTTTTTTATCAT
101 TGCAAAACCTNATTTTCAGAAAACTTCACGAGTGTCTGACTACTCGATCA 151 CTTGATTTTCCTTCAANAATCCAAAACATCAGTTGAANGTTTTTTTTACA 201 AAAAAAAGTGTCAATTTTCGCTTTTTGCTAATATAAATTAAACACGCATC 251 CTAGCATCTCAGAGAATTATTAACGCAATCAAGTGTGAGAGCATAAAAAA 301 TGCNGATACTTGTACAATTTTATTRTTCTATACAAAGAAAAATGTACGGC 351 AACTGCGGATGAATTGCCAACATTCTAAGCTTCACCACTATCTACATATT 4 0 1 CN4AAAAAGAAG"GATCCAGGATCTGCAATTCAACCGACTGACC 451 GACTGATTTCAAAATCCACACATTCNAAATCTCGAACAAGCTGCAGTCTT 501 CCATTTCATTAGCAAAAATCTTTCATTGCATCTATCTTACAGGACAAGCT 551 ACAGTCTCTTTCATCCATCCAACTGGGACTTGAACTTGAACTCGCACTGG 601 GCAAA
10 20 30 40 50 l I I I I
1 TCATTTCTCGTGGCATGTGATGTTTACCATGCTAGAGTTTTGATGAGGAC 51 TCTAGTGGAGGTTTTATGTTTTATGTTTTGGATGCATGCACAGGTTAGGT
101 TTTTGGTTCATCAGATGTGACCCGTGCTTGATGTATGATATGTTTACCCA 151 GTTAGAGCATTTGATAGGGCTCTAGCAGGGGGTTCTTTATGTTTTTAGTT 201 ATGTAGCATACAGGTCAAGCTCGGTATATACATCAGATGTTTAAGTTTTT 251 GTGTTAATGTTTTGATCATGTATGGGATTTGACCAGGTGATAGGAGGTAT 301 GTCAGGCTTGCTACGGGTCCCGGCGACCTTAAGCCGATCTGGATCCTAGC 351 GCCGGTAGCGGTCCGGTTTCCGGGTGGTTACAGAGTGGTATCAGMCCCT 401 AGGTTCATATGGTCGGACCTANANTGTGTTGGGCTCATAAGGGTCATANA 451 AGGGCAAGCATAATANGAAAAACATGTCCACTAGGATAGGATGTA
Appendix 1.4 Partial sequence of two clones of amplified cassava DNA using peroxidase designed prirners, Clone A corresponds to the 900 bp band in Figure 1.5. Clone B corresponds to the 700 bp band in the same picture. A BlastN search in GenBank did not show significant homology with any peroxidase or other relevant sequence.
Appendix 1.5. Southern blot of cassava DNA probed with M E P X l . Genomic DNA of different cassava cultivars digested with H i n d III (H) or double digested with Eco RI/ Bam HI ( B / E ) .
PAL 81 Pea Potato Tobacco Toma t O Alf alf a Soybean P. crispum White Beans Ipomoea
5' ATTGAGGCTGCTGCTATTATGGAACACATTTTGAT 3 '
P . sa t i vum gene for phenylalanine .. . S . t u b e r o s m PAL-1 gene for phenyla . . . N. tabacum (Samsun NN) mRNA for phe. . . Lycopersicon e s c u l e n t u m phenylalan . . . M . s a t i v a P A L mRNA for phenylalanin . . . phenylalanine ammonia-lyase [soybe . . . P. cr i spum mRNA for phenylalanine a. . . Phase01 us vulgaris L. phenylalanin . . . Ipomoea batatas mRNA for phenylala. . .
PAL 82 5 ' CAAAGTGCTGA-CIAWCCAACATGTGAATTC 3 '
PEAPALS STPALl NTPHEAL
dbj emb emb
Potato Tomato White Beans C i t r u s Grapes Aifalf a Soybeans
~ ~ I M ~ O ~ ~ ~ ~ T O M P A L ~ A embIx58180 IMSPAL gb1~469881~46988 e m b 1 ~ 8 1 1 5 9 1 ~ ~ ~ ~ ~ 3 gb1~11939 1 PHVPAL ~ ~ ~ I D ~ ~ ~ ~ O I I P B P A L A
Dl0003 X63103 X78269
emb1~63lO4 ~ S T P F L ~ gb M83314 TOMPHEAMLY gb 1 Ml1939 1 PHVPAZI dbj I~10002 1 PEAPAL~ gbl~43338 1 ~ ~ ~ 4 3 3 3 8
gbl~469881~46988
S.tuberosum P A L 2 gene for phenyla . . . Tomato phenylalanine ammonia lyase,. . Phaseolus v u l g a r i s L. phenylalanin... P.sativum gene for phenylalanine a..- Citrus limon phenylalanine ammonia . . . V - v i n i f e r a PAL mRNA for phenylalan... M. s a t i v a PAL mRNA for phenylalanin. . . phenylalanine ammonia-lyase [soybe. . .
Appendix 2.1 Alignment of conserved regions from di£ f erent PAL genes for primer design. Primers PAL 81 and PAL 82 are marked bold. PAL genes aligned are listed according to database, accession number and name.
Aggendix 2 - 2
Construction of a g e n d c library and efforts to isolate PAI;
genes . Attempts to isolate genomic clones of PAL are described
herein. Initially, two heterologous probes were used to
screen the original library. Subsequently, an amplified
library was screened using a PCR cloned PAL gene from
cassava (MEPAL, chapter 2) as a probe.
Material and methods
Construction and amplification of a genomic library
A cassava library was constructed using genomic DNA
partially digested with X h o 1 £rom MC0122 plants grown under
growth room conditions as described in chapter 1. DNA
inserts of 16-19 K b were isolated by sucrose gradient
centrifugation, purified in dialysis tubes and ligated in a
Lambda D A S H ~ II/Bam HI vector from Stratagene (La Jolla,
CA, USA). For packaging the GigapackmII Gold kit £rom
Stratagene was used. The library was plated in NZY media
using top agar with a low concentration of agar or agarose,
according to the Stratagene manual. Library efficiency was
calculated according to the formula in manufacturer's
manual. The library was plated in petri dishes at a density
of 10-20,000 plaques/dish. After a first round of screening,
the library was amplified according to Sambrook (1990) and
159
stored with 7% DMSO at -70°C-
Screening of original library with heterologous probes
Two heterologous probes were used to screen the
original library. Those cDNA PAL genes from affal£a and
tornato were kindly provided by Dr. Richard Dixon £rom the
Nobel Foundation, Oklahoma, and Dr. Ross Nazar from the
Department of Molecular Genetics, University of Guelph,
respectively. Screening of the library was performed also
according to the Stratagene protocol from the GigapackBII
Gold kit. Forty-three original plates were screened using
nylon membranes. Membrane plaque lifts were performed in
duplicate and hybridized with PAL cDNA probes from alfalfa
and tomato. Plaques showing hybridization on both membranes
were selected £rom a second screening with a low
concentration of plaques. Individual clones £rom the
secondary screening were selected for a plaque grid assay.
Eight individual lambda clones were selected for DNA
extraction. The clones were digested with Barn HI and a
Southern blot was performed to check hybridization of the
inserts with the heterologous probe.
Screening of the amglified library with an amplified eassava
PAL gene
An amplified cassava PAL fragment (MEPAL, described in
chapter 2) was used as probe to screen the amplified
library. The screening protocol was the same as described
above, but the density of plaques on the petri-dish were
between 20-25000 plaques/dish. Thirty-£ive plates were
originally screened. followed by secondary screening and
Southern blot of seven isolated lambda clones digested with
Barn HI. On the basis of data from the Southern blots,
inserts were subcloned in pBluescript0 II vector from
Stratagene. Clones were totally or partially sequenced using
the Taq Dye Deoxy Terminator Cycle sequencing kit (ABI) on
an A B 1 PRISP mode1 377 DNA sequencer. Sequences were
submitted to GenBank for both BlastN and BlastX homology
searches .
Resuïts
Screening of the original library w i t h heterologous probe
After the first screening of 38 plates, 67 clones were
selected for a second screening ( F i g . A 2.2.1A). After a
second round of hybridization, 53 clones were selected and a
plaque grid assay was performed (Fig. A 2.2.1B). The results
of the plaque grid assay were not encouraging, with no
differences between selected clones and a negative control.
Meanwhile, lambda DNA was extracted £rom 8 clones that
showed a strong signal in either the f i r s t or second
screening. The Lambda DNA was digested and inserts of
different sizes were obtained. Southern blots of these
digested clones under high stringency conditions did not
show any hybridization with the probe, and under lower
stringency conditions only the lambda DNA am,s hybridized
with the probe (Fig A 2.2.2) .
Screening of the amplified library with MEPAL probe
From the screening of the amplified library 15 clones
showing hybridization with MEPAL were selected (Fig.
A.2.2.3). After secondary screening (Fig. A.2.2.4), seven
clones were selected for Southern blot analysis of the
inserts ( F i g . A.2.2.5). Those inserts were cloned in
pBluescript and sequenced. A search in GenBank did not show
any homology with PAL genes. Figures A 2.2.6-11 shows the
sequence and homology search results for those clones.
Figure A 2.2.1 Membrane plaque l i f ts showing hybridization with a heterologous PAL probe during first (A) and second round of screening (B) .
Figure A 2.2.2 Southern blots of lambda clones with cassava DNA inserts, digested withBam HI and hybridized with a heterologous PAL gene under low (A) and high stringency conditions (B) .
Figure A 2.2.3 Membrane plaque l i f t s showing hybridization with MEPAL during f i r s t (A) and second round of screening (B) .
Figure A 2 . 2 . 4 Southern blots of lambda clones with cassava DNA i n s e r t s , digested with Bam HI and hybridized with MEPAL under high stringency conditions.
10 20 3 O 40 5 0
I I I I I ATCCCTTTGTTGTCAACTCTGAACTTTGCACTGTTGCCTGACTGAACAGT CCTGACAATTTTCATCAACTCTGGGTCCTCGTGCTGTTCCTGAGCTATCT GCTCCAGAAACATAGGTGTCACTCTCATCTGTGCTATCAACGCACCTGTA CCAGACAACTCTAGCTGTAACCCCTCTTCTAAGAGCTTGTAAAGCTCCAT CACGACTAGTCTCCGCTCTGCTGCTATATGGGATAFACTGCCTAGTAACT TCCGGCTTAGGGCGTCTGCCACAACATTCGCCTTACCCGGATGATACTGG ATCTTACAATCATAATCACTGAGCAATTCTACCCATCTTCTCTGCCTCAA ATTCAGCTCTCTCTGGCTCAAGGTATACTGTAAACTCTTGTGATTAGTGA AGATTTCGCATTTAACCCCGTAGANGTAATGCCGCCACATCTTAAGTGCA AAGATAACTGCTGCCATCTCTANGTCATGGGTGGGGTAATTCAACTCGTG CTTCTTCAACTGTCTAGAANCATAAGCTATCACCCTATCACTCTGCATCA AAACACAGCCCAATCCCACTCGAGATGCATCACAGAATACTGTGAAATCC TCATTACTGACAGGCAGANCTAACACTGGGTGCTGATGTCAATCTCCTCT TGANCTCCCNUIGCTCTCTGCACACTGGTCTGACAAACAAACCTCTGGGT TCTCTGANTCMTTTGGTCATAGGAACCGCTATCTTT
Tota l number of bases is: 737. DNA sequence composition: 185 A; 207 C; OTHER ;
Smal lest sum
Reading High Probability Sequences producing High-scoring Segment Pairs: Frame Score P(N) N
prf1 11510387~ retrotransposon dell-46 [Lilium h... -1 286 gi11402848 (U60529) pol-like protein [Cerati. . . -1 156 sp[PO4323 1~0~3-RETROVIRUS-REUTED POL POLYPROTEI . . . -1 259 pirl 1~34639 pol protein - fruit fly (Drosophi . . . -1 252 spIP20825 1~0~2-RETROVIRUS-RELATED POL POLYPROTEI . . . -1 243 pirl 1~36329 hypothetical protein 2 - cabbage . . . -1 219 gil1226168 (M32662) ORF B (bases 1850-5560) . . . -1 219 gil1905852 (U89994) reverse transcriptase po . . . -1 145 gi 1929567 (X03734) reverse transcriptase-li . . . -1 133 s~~P~O~O~~POLY-RETROVIRUS-RELATED POL POLYPROTEI . . . -1 132
Figure A.2.2.6 Sequence of cassava DNA clone 3.43 and results of homology search in GenBank (BlastX) .
10 20 3 0 40 50
I I I I I AGTGGATCCCCCCCTGAAATCTACTTGNCTTCTGCGGATGTCTGCATAAC TCTACTGTCTGCTTGCAGCTGTCTTGATTCTCTCTCTGATTAAGGGTACC ACCCTGCTGGTGATCTCTACTAGCTCAGGCCCTGNCANGGCCTTTTCTCC AACTTCTTCCCAGCAAACAGGTGATCTGCACTTCCTCCCATAT?UUIGCTT CATATGGAGCCATCCCGATGCTAGCATGATGACTGCTTATTGTAGGCAAA CTCCACCAAAGGTAGGATGNTGTCTCCAAGNAACCGCAAAGTCCAGCACA CACATNCTTAGCATATCTNCTATGGTCTGGATGGNCCTCTCTGACTGTCC GNCTGTCTGTNGATGGGAAGGGGGNCCCNCTGAAATCCAACCTGGTACCC ATGGNATTCTGCAGACTCTGCCAAAACCTGGAGGNAWXTGGGGCCCTCT ATCAGACACTATAGGAAACAGGAACCCANACAGTCTGACNATCTCATCTA CGNACAACCTGCNCCAACNTGNCCACAGAATANCCACNCCTGACAGGGAT GAAGTGAGCAGATTTGGTGAGTCTGNCCACAATCACCCATATGGAGNCCA ATCTGTTGGACNTCGCCGGTAACCCCACNACGAAGNCCATANCTATGTTC CTCCCATTTCCACTCTGGAATAGGTAGGTAGCGGGNTAANCATTCCANCCGNCT TCTGATGTTCCAGCATCACCCNCTGACANACNTCACAGACTGACACAAAC TGTGCCACNTCTCTCTTCATAACTGACCACCAGTAAACTTTCTTCAGATT TTGATACATCTTGGTGGTTCCGGGGTGAATACTGTATCTTGCATTATGAG CCTCTCTCATAATGTCTCCTTTTAGCCCTATGTCATCTGGTACACACAAT CGACTCCCATAGCGGAGGAT
Total number of bases is: 920. DNA sequence composition: 317 A; 3 6 OTHER;
Srnallest Sum
Reading High Probability Sequences producing High-scoring Segment Pairs: Frame
prf( 11510387~ retrotransposon dell-46 [Lilium h... -3 gi 1522302 (L35053) endonuclease [Magnaporth . . . -1 pirl 1~69842 TyB protein - yeast (Saccharomyce . . . -1 gnll~1~le243484272894) TY3B [Saccharomyces cere. .. -1 pir1 1S53577 TyB protein - yeast (Sacchaxomyce . . . -1 gi 1536873 (M34549) POL3 gene product [Sacch.. . -1 gi 1763265 (247047) unknown [Saccharomyces c . . . -1 gi 1173088 (M23367) has hornology to retrovir.. . -1 gi11326016 (246728) Y19910.15, incomplete TY ... -1 gi12367675 (AF017040) Pol [Dictyostelium dis. . . -1 pirl 1~60179 pol polyprotein homolog - fungus ... -3 pirl 1~23570 pol polyprotein homolog - fungus ... -3 gi 1538067 (M77661) putative pol polyprotein ... -1 pirI 1~36373 hypothetical protein Tfl - fissio ... -1 gi 1 805078 (U09586) integrase [Tribolium cas ... -1 S~~QO~~~~[RDPORETROTRANSPOSABLE ELEMENT TF2 155 ... -1 pirl lJN0791 Tf2 protein, Retrotransposon - fi ... -1
Score P ( N )
Figure A.2.2.7 Sequence of cassava DNA clone 3.41 and results of homology search in GenBank ( B l a s t X) .
CTGTCÛGATGAAGCCCTTTTCTACCAGTTCTTGCJULCTGTCCTTTC-CT CCTTCAACTCGGCTGGAGCCATCCTGTAGGGAGGGATAGAGATCGGTCTA GTTCCAGGCACCAACTCTATCTCGAACTCTATCTCCCTAGCAGGTGGTAA ACCTGGAAGCTCATCCGGAAAAACATCCTGAAACTCTCTGACAACTGGCA CCGAGGNCGGNTCCCTGANCTGACTGTCTAGCTCTCTCACGTGAGCTAAG TACCCCTGACATCCCTTCCTAAGCAACTTACGAGCCTGAAGAGCTGATAT CAGACCTCTAGGTGTGCCCCTCATGTCTXTTTGAAGACGANCTCTGACC CGNTCTGATCTCTGAACCTGACTACCTTGTCCCTGCAGTCCAAGGTAGCA NCATGGGTAGATAGGCAATCCATCCCTAGAATGACGTCIUAGTCTGTCAA ATCTAGAACCACAAGGTCGGGGGAGAGGGATCTTCCCTCATG GACTGTACTGGGAGACTGACANTGNCACTGACGGGTCACANTTGGGTNCA CTGACCCATAGGGGGAAATCTNACCCAGAGANTATCAGACCCAACTCTCA ACGGGTCTCGG
Total number of bases is: 661. DNA seqgence composition: 166 A; 12 OTHER;
Smallest sum
Reading High Probability Sequences producinq High-scoring Segment Pairs: Frame Score P ( N ) N
prf~~1510387Aretrotransposondell-46 [Liliumh ... -3 274 1.3e-38 2 pir[1~23570 pol polyprotein homolog - fungus . . . -3 90 3 .se-07 2 gi 1522302 (L35053) endonuclease [Magnaporth ... -3 96 2.le-05 2 pir11~02021 micropia polyprotein - fruit fly . . . -3 85 0.0059 1 gi11030731 (X14037) polyprotein [Drosophila . . . -3 85 0.0059 1 grill ~1~Ie304159 (6099) UL5 [human herpesvirus 21 -3 79 0.039 1 pir( ~WMBEUS gene UL5 protein - human herpesvi . . . -3 79 0.039 1 S~~PIO~~~~HELIPROBABLE HELICASE /gi/330231 (Ml9 . . . -3 79 0.039 1 pirl [SI8211 hypothetical pxotein 2 - fruit fl . . . -3 78 0.053 1 pirl 1~34639 pol protein - fruit fly (Drosophi . . . -3 78 0.053 1 pirl 1~36373 hypothetical protein T i 1 - Eissio . . . -3 72 0.12 2 gi12723362 (AF023459) lustrin A [Baliotis ru. . . +l 74 0.16 1 s ~ ~ Q O ~ ~ ~ ~ ~ R D P O R E T R O T R A N S P O S A B L E ELEMENT TF2 1 5 5 . .. -3 72 0.27 2 pirl lJN0791 T i 2 protein, Retrotransposon - fi . . . -3 72 0.27 2
Figure A.2.2.8 Sequence of cassava DNA clone 5.11 and results of homology search in GenBank ( B l a s t X) .
10 20 3 O 40 50
I I I I I l CTGAGCTACCTGCTCTAGRAACACGGGTGCCACTCTCATCTGGGCCACCA
5 1 AGGCACCTGTACCAGACAACTCCATCTGTAGACCTTCCTCAATGAGCTTG 101 TAGAACTCCTTCACCACTGGNCTCCTCTCTGCCGATATGTGGGATMCT 151 ACCGAGTGATTTCCGGNTTAAGGNGTCTGCCACAACATTCGCCTTACCCG 201 GATGGTACTGAATCTTGCAATCATAGTCACTCAGNAGGTCCACCCATCTC 251 CTCTGTCTCAAATTCAAATCTCTTTGACTCAGGATGTACTGCAGGCTTTT 301 ATGATCTGTAAAGATCTCACATTTANCCCCATAGAGGTNGTGNCTCCACA 351 TCTTGAGTGCAAAGATTACTGNTGNCATCTCAAGATCATGTGTGGGGTNA 401 TTCAACTCATGNTTCTTCAACTGNCTAGAAGCATAAGNAATCACCCTCTC 451 ATTCTGNATCAGTACACAACCCAGTCCCACATGGGGACGGATCACWG
T o t a l number of bases is: 500. DNA sequence composition: 126 A ; 137 C; 93 G; 130T; 14 OTHER ;
Smallest Sum
Reading High Probability Sequences producing High-scoring Segment Pairs: Frame Score P ( N ) N
prf((l510387A retrotransposon dell-46 [Lilium h... -3 249 2.8e-37 2 sp 1~04323 1 PoL~RETRovIRUS-RELATED POL POLYPROTEI. . . - 3 197 1.5e-18 1 gi11402848 (U60529) pol-like protein [Cerati . . . -3 145 4.3e-18 2 pir1 1~34639 pol protein - fruit fly (Drosophi ... -3 189 1.9e-17 I pir/ 1~36329 hypothetical protein 2 - cabbage . . . -3 188 2.6e-17 1 gi 11226168 (M32662) O R F B (bases 1850-5560) . . . -3 188 2.6e-17 1 gi11905852 (U89994) reverse transcriptase po . . . -3 126 2.8e-17 2 ~ ~ ( P ~ O ~ ~ ~ ~ P O L ~ R E T R O V I R U S - R E L A T E D POL POLYPROTEI . . . - 3 186 4.8e-17 1 s~~P~O~O~~POLYRETROVIRUS-RELATED POL POLYPROTEI ... -3 133 1.3e-16 2 gi 1495770 (M12927) unknown protein [Drosoph. . . -3 133 1.3e-16 2 gi 1929567 (X03734) reverse transcriptase-li. . . -3 133 1 -3e-16 2 pirl 1~26840 retrovirus-related pol polyprocei ... -3 129 2.5e-16 2
Figure A.2 .S. 9 Partial sequence of cassava DNA clone 5.16 (5' end) and results of homology search in GenBank (Blast X ) .
10 20 3 O 40 50 I I I I I
GGATCCTTTAGACTTTGTATCGACTACAGACAGTTGGAACAAAGTCACTA CCAAGNAATAAGTACCCATTGNCmGGATCGATGATCTATTCGACCAGCT AGCCGGAGCAGGTTGTTTCTCCAAAATAGATCTGAGATCGGGGTACCATC AGTTAAGGATAAGGGAGGAGGATGTGCCGAAGACAGCTTTCAGGACCAGA TATGGGCATTTTGAGTTCCTTGTAATGTCGTTCGGGTTAACMLCGCCCCT GCAGGATTCATGGATCTCATGAACAGAATATTTAGCCAATACCTGGATCA CTTTGTTATTGTCTTCATAGATGATATCTNAGTGTATTCTAGGAACGCAG AGGAGCATGCCCATCATCTGAGGTTGGTTCTGCAGACTTTGAGGGMCAT NGNTTGTATGCCAAGTNCTCTAAGTATGAGTTCrTGGNTMGGAGCATTTC GNTCTTGGGGGATGTNGTGTCAGAGAATGGGATTGAGGTGGACCCCANGA GGACAGAAATGTGG
Total number O£ bases is: 514. DNA sequence composition: 10 OTHER;
Smalles t sum
Reading High Probability Sequences producing High-scoring Segment Pairs: Frame Score P ( N ) N
prf111510387~ retrotransposondell-46 [Liliumh . . . +2 269 1.2e-51 5 gi11658455 (U75247) reverse transcriptase [G.-. +2 275 3.8e-42 2 S ~ ~ P ~ ~ ~ ~ ~ I R R P O R N A - D I R E C T E D DNA POLYMERASE HOMOL ... +3 213 5.7e-33 3 gi 1522302 (L35053) endonuclease [Magnaporth . . . +2 184 6.2e-33 3 prf111312271~ nuclear 18s rRNA [Oenothera sp.] +3 205 6.9e-32 3 pirl 1~60179 pol polyprotein homolog - fungus . . . +2 159 3.le-30 3 spl~043231~0~3~ETROVIRuS-RELATED POL POLYPROTEI . . . +3 146 l.le-27 3 pirl 1~23570 pol polyprotein homolog - fungus . . . + 3 167 l.6e-27 4 sp 1 ~20825 1 POL~RETROVIRUS-RELATED POL FOLYPROTEI . . . +3 150 2.1e-27 3 pir 1 1~34639 pol protein - fruit fly (Drosophi. . . +2 154 3.8e-27 3 pirl 1~36329 hypothetical protein 2 - cabbage . . . +3 150 7.le-27 3 gi11226168 (M32662) ORF B (bases 1850-5560) . . . +3 150 7.le-27 3 gi11402848 (U60529) pol-like protein [Cerati . . . +2 158 3.4e-26 3 S ~ ~ Q O ~ ~ ~ ~ ~ R D P O R E T R O T R A N S P O S A B L E ELEMENT TF2 155 . . . +3 151 8.4e-26 3 pirl 1 ~ ~ 0 7 9 1 Tf2 protein, Retrotransposon - fi . . . +3 151 8.4e-26 3
Figure A.2.2.10 Partial sequence of cassava DNA clone 5.16 (3' end) and results O £ homology search in GenBank (Blast XI-
4.27 ( 5 ' end) 10 20 3 0 40 5 0
I I I I I GAGATGGAAGAGCTTCTCACCATCATGTCTCTCCTTATGATTAAACCMT TAGGGAACATCCTCTAATTAATTACTAATTAACAAATTGCCAAGGAACGT CCTTGGGCCTTAGGCATCAAACAATTGTTANTTGTNTGTNTG~GAAAGAGAGA GATCAAATCCTAACAACTCAAACGCATGAGATGTTGCTAGATCATACAAT TTCCTTGGTTTTTACACCAAGTGTTCTTTATGTTTGANTAATCCAAGCAA TTACGGACTTAAATTACCCAAACTAACATATTATTACCTTGCAATCAAGA ATCAATTGGNCATATTGATCAAAACAACAAAGCAACANTAGAATTANGCA T G A G A T T G T A T G A A T A T T G A A T A A T A A A A G A T A A T G TCTCCC-9ATCCATAAAACAACTAAAGCATCACCTAATCTTCAACTAGATA AAAAGGTTTCAGCCACTCATGGNTGAACCANANCAAANNTNAAAGANAAG ANGAAAGGTAGAGGANGAGATGTGMTCCCGTAGGTGTCTCCAAGGTGGT GTGTNAAAGTTCTGTGGNGGGTCCTTATGTGTCTTTNATGGTGGAAGNCT GNCCTAGGTCGGNCTGGTTTTTAATTAGTGAGGAGATGTCCANAGTGCTG AAAAGGGAAGATCGCGTGGNTTATCCAGA
Total number of bases is: 681. DNA sequence composition: 233 A; 113 C; 129 G; OTHER ;
4.27 ( 3 ' end) 10 20 30 40 5 0
I I I I I CACTCCAGCGNTGGGATTGATAATTGACACTTAGTTTCTCTTGATTGATT CAATAGATCACTCATGGAGGATGGGAAGACGCTTCTCACCATCATGTCTC TCCTTATGATT~CCAATTAGGGGAACATCCCCTTNTTAATTACTAANN AACAAAATTGGNCAANGGACNGCCCTGGGNCNTAAGGANCNAACAAATGG TAATTGGNTTGAGGAAGGGGGGGGTCNAATCCCTACAACTNNAACGGANT NAGNNNTGGTNGGTCNANCNAATTCCCGGGGNTTNACAACCANGGGTCNT TTNGTTGGGTTATCCCAGNANTTTTTTGTTTTTTTTTCCCCAACTTACAA ATTNTTTACCNGGGANWCCANNTNCATGGGNCCATTTGGCCANAC ANAANAGGGANCAATCGGGNTTTACATATGATTNGGTNTATTTTNGGTTT TTTCAAGATTTTNTNNTNTGGGCACAANTCNCCCNCCCNATACAAATTTT NGGNTCNCCCCCNNTTCCCCCCNGGGTAAAGGNTCCCNCCCCCCAGGNGT TGCCCCCCCCCTTTTTNATNAGANGAAGGGNGCNGAAGC
Total number of bases is: 589. DNA sequence composition: 134 A; 122 C; 113 G; OTHER ;
Figure A.2.2.11 Partial sequence of cassava DNA clone 4.27 ( 5 ' end, and 3' end!. No significant homology was found in BlastX or B l a s t N search in GenBank.
Appendix 2.3 Southern blot of cassava DNA probed with MEPAL. Genomic DNA of different cassava cultivars digested with H i n d III (H) or double-digested with Eco RI/Bam HI (B/E) .
AIP 100 FM
24 hours
trans-cinnamic 500 FM
72 hours
Appendix 3.1 Effect of enzymatic and transcriptional PAL inhibi tors in the deterioration of imer layers of cassava root sections. Cassava roots were transversally cut in 3 cm slices and vacuum in£ iltrated in solutions with H,O, 100 pM AIP and 500 pl of trans-cimamic acid. Pictures were taken at 24 and 72 hours from inner layers, after inoculation.
Appendix 3.2 ANOVA tables
ANOVA for the multiplication of bacteria in cassava leaves, cultivar MCol 22 ( F i g . 1 . 3 A )
Source DF SS MS F P Bact 1 99. O 98 .98 4 5 . 8 1 <O. 0 0 0 1 Hours 5 3 4 7 . 7 6 9 . 5 5 3 2 . 1 9 < O . O001 Bact x Hours 5 6 8 . 1 1 3 . 6 2 6 . 3 1 < o . O001 ~esidual 59 1 2 7 . 5 2 . 1 6 Total 7 0 8 0 2 . 9 1 1 . 4 7
ANOVA f o r the multiplication of bacteria in cassava leaves, cultivar 523-7 (Fig. 1 . 3 C )
Source DF SS MS F P Bact 1 2 6 . 7 9 2 6 . 7 8 5 1 4 2 . 8 0 <O. 0 0 0 1 Hours 7 370 .05 5 2 . 8 6 5 281 .83 <O. O001 Bact x Hours 7 3.46 O . 495 2 . 6 4 0 . 0 2 8 5 Residual 3 2 6 . 0 0 O. 188 Total 47 406 .30 8 . 6 4 5
ANOVA for the ionically-bound peroxidase activity in cassava leaves after inoculation with Xanthomonas ( ~ i g . 1.5B)
Source DF SS MS F P Bact 2 7327667 .2 3 6 6 3 8 3 3 . 6 5 . 1 5 O . O090 Hours 6 49444728 .1 8240788 .0 1 1 . 5 8 <O. O001 Residual 54 3 8 4 1 3 7 2 5 . 1 711365 .3 Total 62 9 5 7 9 3 1 4 9 . 1 1 5 0 5 0 5 0 . 8
ANOVA for the ionically-bound peroxidase data after treated with inhibitors of PAL (Fig. 3 . 1 1 A )
Source DF SS MS F P Treat 2 565873.9 2 8 2 9 3 6 . 9 1 9 9 . 5 <O. O001 Hours 4 1905954 .7 476488 .7 335.9 < O . 0 0 0 1 Treat x Hours 8 1613400. 3 201675 . O 1 4 2 . 2 < O . 0 0 0 1 Residual 3 O 42553.7 1 4 1 8 . 5 Total 44 4127782 .5 9 3 8 1 3 . 2
ANOVA for the soluble peroxidase data after treated with inhibitors of PAL (Fig. 3.11 B
Source DF SS MS F P Treat 2 1 8 . 8 9 9 . 4 4 6 7 3 . 3 6 C O . O001 Hours 4 1 0 3 . 7 4 2 5 . 9 3 4 2 0 1 . 4 1 C O . O001 Treat x Hours 8 1 0 . 2 0 1.275 9 . 9 0 <O. 0 0 0 1 Residual 30 3 . 8 6 O . 129 Total 44 1 3 6 . 6 9 3 . 1 0 7
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