Overview of Efforts to DevelopHLB-Resistant Transgenic

Citrus

Kim Bowman, Jude Grosser, Marcos A.Machado, Greg McCollum, Erik

Mirkov, Gloria Moore, RandyNiedz, and Ed Stover

Focus on Developing HLB-and ACP Resistant Citrus

• HLB likely the single greatest threat to citrus

• No HLB resistance has been identified in cultivated Citrus scion varieties

• Transgenics appear to be the only medium term solution for HLB resistance

• Goal: incorporate genes to reduce survival, growth, and/or virulence of causal pathogen, as well as genes to deter psyllid vector

• With little known about host /pathogen interaction, antimicrobial peptides have been a major focus- may also confer resistance to canker and CVC

• Other types of genes also being explored………..

Image: www.plantsci.cam.ac.uk/.../GFP/plantrans.html

Harnessing nature’s genetic engineer: Agrobacterium tumefaciens(being used in most but not all citrus transgenic projects described)

A. tumefaciens causes crown gall disease in many plant species

By removing At genes for growth regulators and replacing with: 1)promoter 2)gene of interest etc.3)gene for selectable marker (antibiotic)can express genes when and where you want, without gall formation.

AgrobacteriumAgrobacterium ––Mediated Genetic Transformation Mediated Genetic Transformation of Citrus Epicotyl Segmentsof Citrus Epicotyl Segments

Transgenic scion 

Non‐TransgenicRootstock

Etiolatedseedlings

Epicotyl sections inAgrobacterium carrying gene of interest

Epicotyl sections inCo-cultivation medium

Epicotyl sections after1 month in selection medium

PCR confirmation of presence of transgenePCR positive and acclimatized transgenicplants

Transgenic plants in the greenhouse

M    1    2    3   4    5    6    7    8    9  10  11  12  13  14  NC PC  M

Transgenic plants grafted onto rootstock

Slide: Jude Grosser, CREC, UF

Antimicrobial Peptides• Broadly active against groups of micro-organisms

• Widespread in multicellular organisms

• First line of active defense to combat infection

• Most are very small molecules

• Numerous distinct AMPs produced in each organism, with somewhat different activities

• Most function by inserting into microbial membranes causing leakage etc.

• Results in microbial death or prevents growth

Genetic Transformation of Citrus with Spinach Defensins for BroaGenetic Transformation of Citrus with Spinach Defensins for Broadd--Spectrum Spectrum Resistance to Bacteria and FungiResistance to Bacteria and Fungi

T. Erik MirkovT. Erik MirkovDepartment of Plant Pathology and MicrobiologyDepartment of Plant Pathology and Microbiology

Texas A&M AgriLife Research Center for Crop Improvement and BiotTexas A&M AgriLife Research Center for Crop Improvement and Biotechnologyechnology

Spinach Defensins: Mirkov

•Plant defensins are small (c.a. 5 kDa), basic, cysteine-rich proteins with broad-spectrum antimicrobial activities

•None have been reported to be toxic or allergenic

•Trees in FL greenhouse and field --8 events--Red Grapefruits (2 varieties) and Sweet Orange (3 varieties) Furthest along in deregulation

•Trees in TX greenhouse ~71 events in 3 Sweet Orange varieties

•Some sweet orange with both spinach defensins in same tree

•Need to produce fruit on mature transgenic trees for composition analysis-slow going since started with juvenile tissue -2-

LB

Pnos-nptII-Tnos P35S-SoD2-T35S Tnos-GUS intron -P34SRB

SoD2 and SoD7 Constructs:

•pBinPlus Binary Vector Backbone•Synthetic Genes--Codons Optimized for Citrus• PR-1 Signal Peptide (Secretion to Apoplast)•Dual Enhanced CaMV 35S Promoter with TEV Translational Enhancer• Intron Containing GUS Gene-- FMV 34S Promoter•nptII selection

Agrobacterium Transformation Constructs

1-GIFSSRKCKTPSKTFKGICTRDSNCDTSCRYEGYPAGDCKGIRRRCMCSKPC-52

LB

Pnos-nptII-Tnos P35S-SoD7-T35S Tnos-GUS intron -P34SRB

1-GIFSSRKCKTPSKTFKGYCTRDSNCDTSCRYEGYPAGD-38

p35S::SoD2

p35S::SoD7

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Attempted antibody

23 419

201

205

141

108

302

316

NT

-H

NT

-Ru

968

905

933

877

881

861*

882

420kb

11.5

5.1

SoD2/7 Copy Number in Transgenic Citrus

-5-

•Most events are single copy insertions

•Multi-copy insertions lead to gene silencing*

Southern Blot of Independent Events

*

2 421

824

867 88

0R

u-N

T

877

881

861

882

420

53 Ri -N

T

5 6

SoD2

SoD2/7 mRNA Accumulation in Transgenic Citrus

-6-

*

Northern Blot of Independent Events

•Varying expression levels in different events

•Multi-copy insertions lead to gene silencing*

Grapefruit Cultivar ‘Rio Red’ Inoculated with the Citrus Bacterial Canker Pathogen, Xac: Mirkov

Non-transgenic

Transgenic for Spinach Defensin 2

-11-

Greening inoculation test on Greening inoculation test on ‘‘Rio RedRio Red’’ GrapefruitGrapefruit

NonNon--Transgenic buds grafted on Transgenic buds grafted on infected rootstockinfected rootstock

SoD2 Transgenic buds grafted on SoD2 Transgenic buds grafted on infected rootstockinfected rootstock Mirkov

J.W. GrosserM. Dutt, A. Omar and G.A. Barthe

University of Florida, Citrus Research and Education Center,

Lake Alfred, FL USA

Multiple approaches for Multiple approaches for genetic engineering of genetic engineering of

citrus for disease and pest citrus for disease and pest resistanceresistance

Antimicrobial gene(s) currently under Antimicrobial gene(s) currently under evaluation: Grosser et al. evaluation: Grosser et al.

• AttacinE - Lytic peptide gene from Hyalophora cecropia.

• CEAD - Codon optimized cecropin A-cecropin D lytic peptide gene.

• CEMA - Codon optimized cecropin A-melittin lytic peptide gene.

• CEME - Codon optimized cecropin A-melittin lytic peptide gene (differs at the C terminus from CEMA).

• LIMA - Lytic peptide gene obtained from Dr. Dennis Gray, MREC, UF/IFAS. Shown effective vs. Xylella

• PTA - Codon optimized N terminally modified Temporin A gene.

Systemic acquired resistance Systemic acquired resistance (SAR)(SAR)Pathogen

attackPathogen

attack

SA synthesized Induction of PR proteins

Increased resistance to further attack

Increased resistance to further attack

SA dispersedsystematically

SAR genes currently under SAR genes currently under evaluation: Grosser et al.evaluation: Grosser et al.

• SABP2 (tobacco Salicylic Acid-Binding Protein 2 gene)– High Affinity for SA.– It may be required to activate systemic acquired

resistance.

• NPR1 (Nonexpresser of PR Genes1 gene from Arabidopsis)– NPR1 is a key regulator in the signal transduction

pathway that leads to SAR.– Mediates the salicylic acid induced expression of

pathogenesis-related (PR) genes and systemic acquired resistance.

Strategies for Psyllid control: : Grosser et al. Grosser et al.

• HLB pathogen is transmitted by the Asian citrus psyllid [Diaphorina citri ].

• Snowdrop lectin (Galanthus nivalisagglutinin; GNA) is a potential insecticidal protein which is knownto act on sucking insects.• Transgenic plants have been produced to

express this gene and several are being multiplied for insect resistance studies –transgenic Carrizo plants killed aphids (psyllid testing next! )

Genetic transformation Genetic transformation of juvenile citrusof juvenile citrus

• Three methods now available– Agrobacterium mediated transformation of

nucellar seedling stem sections– Agrobacterium mediated transformation of

embryogenic callus– PEG mediated Protoplast transformation

AgrobacteriumAgrobacterium Mediated Genetic Transformation Mediated Genetic Transformation of Citrus Callusof Citrus Callus

Citrus Callus Citrus Callus with Agrobacterium Transgenic callus/ embryosregenerating on selection medium

GFP expressing transgenic citrus callus

Closeup of GFP expressing transgenic citrus somatic embryos

Transgenic citrus plants

protoplastsprotoplasts 24 hours after transformation24 hours after transformation

4-6 weeks4-6 weeks6-8 weeks6-8 weeks

3-4 months

3-4 months

5-6 months Transgenic5-6 months Transgenic

5-6 months non-transgenic5-6 months non-transgenic

8-9 months

8-9 months

One yearOne year8-9

months8-9

months

3-4 months

3-4 months

Use of phloem specific promoters to Use of phloem specific promoters to restrict transrestrict trans--protein in phloem tissues: protein in phloem tissues:

Grosser et al.Grosser et al.

• HLB resides in the phloem.•Targeting the trans‐protein in the phloem resolves issues of the     presence of the protein in the fruit and juice.• Two phloem specific promoters are currently under evaluation1) Arabidopsis Sucrose synthase promoter and2) Rice Sucrose synthase promoter.

GUS expression in citrus leaf phloem tissue using the Rice Sucrose Synthase promoter

Transgenic plant regeneration Transgenic plant regeneration –– lytic lytic peptide: Grosser et al. peptide: Grosser et al.

Misc grapefruit includes Duncan, Marsh and Flame cultivars

Results with SAR genes: Results with SAR genes: Grosser et al.Grosser et al.

PCR and Southern analysis of plants

1830bp

NPR1

831bp

SABP2

120 bp

LIMA

Copy number varied from 1 to 5 copies.

Rapid propagation of Rapid propagation of transgenicstransgenics

Transgenic trees ready for testingTransgenic trees ready for testing

Disease resistance studiesDisease resistance studies

• Canker resistance– Detached leaf assay– Greenhouse inoculation

• HLB resistance—Grafting challenge in greenhouse—Field challenge in high pressure area

Canker (Xcc) assay on transgenic Duncan leaves Canker (Xcc) assay on transgenic Duncan leaves containing the lytic peptide gene(s): Grosser et al. containing the lytic peptide gene(s): Grosser et al.

A B C

A – Mild tissue hypertrophy in transgenic LIMA leafB – Moderate tissue hypertrophy in transgenic Attacin leafC – Severe water‐soaking and tissue hypertrophy in non‐transgenic control leaf

qRTqRT--PCR analysis of selected transgenic PCR analysis of selected transgenic lines infiltrated with Xcc by the detached leaf lines infiltrated with Xcc by the detached leaf

assay methodassay method: Grosser et al.: Grosser et al.Transgen

ic lineAttacinE LIMA

Mean cT Bacterial cells / mg tissue*

Mean cT Bacterial cells / mg tissue

MP1 14.775 194975 18.285 18623MP2 16.565 61453 18.362 17619MP3 14.180 318000 26.625 70MP4 15.935 90065 23.365 629MP5 18.670 14369 14.040 324000

Control 15.560 115031 15.560 115031

Transgenic plant challenge with Transgenic plant challenge with HLB : Grosser et al.HLB : Grosser et al.

• Carried out in an approved secure facility.• Transgenic plants graft challenged with

HLB infected sweet orange budwood. • Several Transgenic lines are currently

under evaluation. • Transgenic plants evaluated for HLB

symptoms and infection verified by qRT-PCR.

HLB inoculation results: Grosser et al.: Grosser et al.Plant ID Gene Line # qPCR  Visual Symptoms 

566 LIMA MP13 + +567 LIMA MP5 + +568 LIMA MP1 + +569 ATTE MP11 + -570 LIMA MP3-C7 + +571 LIMA MP14 + -572 LIMA MP18 + +573 LIMA MP16 UNDET -577 LIMA UNDET -578 LIMA MP4-C7 UNDET -579 ATTE MP2 + +580 LIMA MP3-C6 + +585 ATTE MP4 + +586 ATTE MP29-C8 + -587 LIMA MP7 + +588 ATTE MP16 + +589 LIMA MP21 + +590 ATTE MP1 + +591 ATTE MP8 + +592 ATTE MP14 UNDET -600 LIMA MP2-C5 UNDET -601 LIMA MP2-C6 UNDET -602 ATTE MP19 UNDET -603 LIMA MP5 UNDET -604 LIMA MP9 + +605 LIMA MP20 UNDET +606 ATTE MP3 UNDET -706 Control + -

Future approaches for genetic Future approaches for genetic resistance studies: Grosser et al.resistance studies: Grosser et al.• Screen a large number of transgenic trees

to select individuals with the highest level of resistance.

• Challenge putative tolerant plants with “hot” psyllids.

• Field testing in a high disease pressure environment. APHIS permit issued, 900 trees ready to go.

Genetically transformed sweet orange for HLB resistance in Brazil

Marcos A. MachadoCentro de Citricultura Sylvio Moreira

Some approaches for GM citrus to disease resistance

• Antimicrobial peptides (AMP) of insects•Attacin A •Cecropin

•AMPs from citrus •from genome screening •use of transgenes from same species likely to facilitate acceptance

Machado et al.

•Resistance genes• R genes (Xa21 and NPR1 of citrus and At)• PR gens (PR4, PR5, PR10)

Methodology: Machado et al.

100 10-1 10-2 10-3 10-4 10-5

Varieties (juvenile tissue)

•Sweet orange Hamlin, Pera, Natal and Valência

Constructs

•35S promoter and kanamycin resistance

Checking HLB symptoms and bacteria population

Symptoms in leaves• Leaf mottle• Every three months after grafting during 17 months

Bacterial titer testing•qPCR of Ca. Liberibacter asiaticus•Taq-Man

Methodology: Machado et al.

Vectors

•Attacin A and Xa21 (CENA/USP)

RB35S-TAtNPR1NPTII 35S-PLB nos-T nos-P

35S-P 35S-PXa21cLB RB35S-Pnos-T nos-TNPTII

35S-P 35S-PAtaccin ALB RB35S-Pnos-T nos-TNPTII

• Npr1 (CCSM)

Results: Machado et al.

Number of Transformed Plants_____________________________________________________Variety Gene Nr of Events Nr of Tested Plants_____________________________________________________Natal Attacin A 5 30Pera Attacin A 3 18Valencia Attacin A 8 48Hamlin Attacin A 12 60Valencia Xa21 9 54Hamlin npr1 4 24Natal npr1 1 6_____________________________________________________

Npr1Northern blot

H1 H4 H6 H10 N2 C-

Southern blot

Results: Machado et al.

Symptoms progress of HLB in leaves

Symptom progress of HLBVariety Gene Nr of Events 3 Months 6 Months 9 Months 12 Months

Natal Attacin A 5 0 5+ 5+ 5+

Pêra Attacin A 3 0 3+ 2+ 3+

Valência Attacin A 8 0 6+ 7+ 8+

Hamlin Attacin A 12 0 6+ 8+ 12+

Valência Xa21 9 0 3+ 8+ 9+

Hamlin npr1 4 0 0 0 0

Natal npr1 1 0 0 1+ 1+

Results: Machado et al.

Symptoms progress of HLB in leaves

3 Months12 Months

6 Months17 Months

• All plants transformed with attacin A show symptoms of HLB and high titer of the bacteria

• AtNpr1 reached high expression level in some GM plants, was able to induce the expression of PR-protein (R1), and provided apparent HLB resistance

• There is great variation on bacteria titer during the year

• The plants were evaluated only in screen house conditions

• The GM plants are originated from seedlings (juvenile) and need more evaluation.

General considerations: Machado et al.

•have several candidate Citrus AMPs and synthetic peptides for screening against Xylella fastidiosa and Xanthomonas axonopodis pv citri in the lab.

•In collaboration Embrapa ,screening the database of citrus looking for new sequences for possible AMPs.

•An AMP from sweet orange has been tested and shown effictive against citrus canker, and used for transformation.

•All our plants are in screen-house. We do not have permission to bring them to the field.

•I would like to point out that all our work on genetic transformation will be focused on AMPs, R genes or iRNA from citrus to citrus.

•We will not continue working with insect AMPs.

General considerations: Machado et al.

Production of Transgenic Plants Expressing the NPR1

Defense Gene to Develop Resistance to Citrus Canker

and HLBVicente Febres

Gloria A. Moore

Plant-derived defense genes and transformation

• They can induce wide-spectrum disease resistance

Advantages:

Biosafety issues:

• The use of endogenous genes may be more acceptable to consumers concerned with GMOs

NPR1

• Required for SAR and ISR

• Overexpression induces wide-spectrum disease resistance

• This resistance does not activate constitutive expression of PR proteins

• Resistance against herbivore insects (Spodoptera) has been observed in transgenic tobacco

Agrobacterium-mediated transformation: Febres and Moore

• The NPR1 gene from Arabidopsis was transformed into ‘Carrizo’ citrange and grapefruit plants.

• Some lines have been evaluated for PR-1 gene expression and resistance to canker

Results: Febres and Moore• We have obtained stably transformed Carrizo and grapefruit

lines with the Arabidopsis NPR1 defense gene.

• The transgenic lines show normal growth and phenotype.

• Some transgenic lines express the AtNPR1 and higher levels of endogenous PR1 than WT.

• Some transgenic lines show delay and attenuation in canker lesion formation. This is a promising results that needs to be corroborated. Also the role of AtNPR1 in the observed response needs to be established

• The transgenic plants are in the process of being tested for HLBresistance/tolerance

• Evaluation of the most promising lines under field conditions isunderway

Field evaluation transgenic plants (AtNPR1 and others)

Horticulture and Plant Breeding Unit

Update on transgenic programKim Bowman

Greg McCollum

Randy Niedz

Ed Stover

U.S. Horticultural Research LaboratoryFt. Pierce, Florida

YongPing DuanDavid HallWilliam BelknapWilliam Dawson

Ute AlbrechtRic StangeMizuri HertLesley Benyon

Transgenic Project: Parallel Tracks

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• Fastest track- possible “home run” using best available technology on rootstocks, sweet orange and grapefruit-high throughput.

• Goal is earliest possible resistant variety in field

• Emphasizing components which are deregulated in crop plants

• Experiments to overcome transformation bottlenecks

• Identifying new targets for transgenes

• Using other promoters etc.

Selection of AMPs 1

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• Plant-derived or synthetic for greater consumer acceptance

• Low potential for adverse health effects

• Reports of effectiveness against related bacteria

• Screening in-vitro (in the lab), model systems (tomato), and citrus rootstocks

Selection of AMP Transgenes

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• D4E1: a synthetic AMP of 17 amino acids• active against Agrobacterium in poplar• undergoing extensive tests for use in human

medicine- should help fast-track deregulation • May be desirable to pyramid several AMPs with very

different modes of action• New cooperative agreement to identify new synthetic

AMPs

In –Vitro AMP Screening

•Agrobacterium and Sinorhizobium are related to Liberibacter

•Also using Xanthomonas citri

•Best AMPs, including D4E1 are effective in 1 μM range

•Getting Dr. Mirkov’s spinach defensins to serve as benchmarks

MIC (μM)Hemolytic Activity (%)

AMP Source At Sm XccTachyplesin I crustac 0.3 0.3 0.3 3.0SMAP-29 sheep 1 0.3 1 3.2D4E1 synth 1 0.3 1 3.6D2A21 synth 1 0.3 1 8.4LL-37 human 1 1 1 5.1Melittin bee 1 1 1 100.8Cecropin A insect 3 3 10 1.1Cecropin B insect 10 3 10 1.2Indolicidin cow 10 3 3 2.0Apidaecin IA insect >30 1 >30 1.6Drosocin insect >30 3 >30 1.6α-Purothionin plant 30 10 1 22.5Pyrrhocoricin insect >30 10 >30 1.9Magainin I frog >30 >30 >30 1.3Magainin II frog >30 >30 >30 1.5Histatin-5 human >30 >30 >30 1.8Ib-4 plant >100 100 >100Cn-1 plant >100 >100 >100P4c plant >100 >100 >100

In-vitro assays: broad group

•Quite repeatable across multiple runs

•Most were comparable in effectiveness across species

•“Best” AMPs in trial were animal or synthetic , but broader sampling of plant AMPs may be valuable

Other Transgenes for HLB Resistance

• Working with Duan group at USHRL have identified a target from the newly sequenced Liberibacter asiaticus genome

• A phloem-specific protein is induced during infection (Bowman data), appears be an attempt seal sieve tubes as a barrier against an increasing bacterial population.

• MAY be primary cause of symptoms?

• Working with ARS scientists in California to develop constructs so that ALL inserted genes are from Citrus!

Promoters being tested• D35S

• 2 Ubiquitin promoter variants: from Bill Belknap who indicates that they are extremely phloem active and have less IP entanglement than D35S

•Arabidopsis and Citrus Sucrose Synthase, Wheat Dwarf Geminivirus promoters: all phloem specific

• Citrus PP-2 promoter: lectin-like gene promoter strongly up-regulated by Las infection in Albrecht & Bowman microarray

•PR-1: an inducible promoter (acibenzolar-S- methyl) reported to have strong phloem expression- also many plants transformed with FT driven by this promoter

330 independent scion (Hamlin, Midsweet, Valencia, and Ray Ruby) and >3000 independent rootstock regenerants established as micrografts

Testing for HLB Resistance• Still slow going from transformant to multiple

plants suitable for testing• Sometimes get >90% infection with graft

inoculation- 2 buds and 2 leaf midribs per plants. PCR detection and symptoms within 6+ weeks. Sometimes very fast and uniform but VARIABLE!

• Directly compared graft vs. caged psyllid vs. “natural” psyllid – appeared that natural infection was almost as fast and much more efficient in this experiment, but in heavily HLB-infected area

• Numerous plants from UF and ARS will soon be in field

Transgenic Project: Challenging with HLB etc.

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• Lots of plants of US-812 and US-802 transformed with D35S/D4E1 have been challenged

•No major differences, so far, in HLB symptoms or Las

•However, D4E1plants have better growth

•Encourages selection of more active promoters

•Initiated psyllid challenge of US-812 transformed with garlic lectin gene

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Transgenic Project Plan-Major Bottlenecks

• Juvenility- standard protocols use seeds.• Transformation is VERY poor with mature tissues, and

will absolutely need for monoembryonic and seedless types, earlier fruiting for all

• Trifoliate types have 10X transformation rate unifoliates

• Transformation of mandarins is much more difficult than sweet orange or grapefruit

• Rapid throughput- need higher transformation % and quicker passage from Agro to grafted plant

Mirkov- Regeneration from Mature Wood

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Genetic transformation Genetic transformation of mature citrusof mature citrus‐‐ Grosser et al.Grosser et al.

• 6 transgenic ‘Hamlin’ shoots recovered from 2nd experiment– testing rootstock effect on explant quality

– testing improved slow‐release fertilizer products as alternative to fertigation 

– efforts to minimize contamination

•published reports of HLB resistance in different species in the Aurantiodeae•some clearly reflect different strain host range•however, broad search may yield true resistance•experiments in progress include more than 50 genotypes.- collaboration R. Lee, Riverside

Evaluating distant citrus and citrus relatives for HLB-resistance

•May identify genes which can then be used to transform commercial citrus•Some possibility of using hybrids directly as rootstocks…. •Transforming citrus with FT gene to permit flowering within year of seed germination

>Will permit rapid introgression of genes from distant relatives into commercial citrus rootstocks and scions

Evaluating distant citrus and citrus relatives for HLB-resistance

Carrizo transformed with D35S:: Citrus FT Gloria Moore- UF Horticulture

What does this all mean?•Many groups working on numerous strategies to develop HLB/ACP resistant citrus, with little duplication

•Seriousness of the HLB threat and availability of grant funds has made this a high-priority, highly active area of research

•Ultimately only a few HLB-resistance solutions will undergo expensive deregulation for commercial use

•My crystal ball suggests these will likely be resistant but not immune to HLB….

•Absolutely best to keep HLB away from your industry and your farm

•When HLB IS present, there will be no better solution than durable host-plant resistance

The Future?•The US now produces GM crops (primarily corn and soybeans) on X million acres of cropland, with substantially increases annually

•New products such as soybeans modified to produce omega-3 rich oil, will likely quiet many opponents to GM crops, opening the door for many more GM solutions

•More genomic data and increased understanding of gene expression will permit ever greater control over our crops

•Likely that HLB-resistance may just be the first in a non-stop series of GM traits that will enhance opportunities for citrus growers and consumers

THANKS!THANKS!• FCPRAC• NVDMC/FDOC• Industry Collaborators• USDA-CSREES• UF/CREC Core Citrus Transformation

Facility • CREC Faculty and Staff• USDA/ARS USHRL Staff• A cast of hundreds (OK dozens at least)!!!