Breeding for HLB Resistance

Transcription

1 Breeding for HLB Resistance

2 Overview of Efforts to Develop HLB-Resistant Transgenic Citrus Kim Bowman, Jude Grosser, Marcos A. Machado, Greg McCollum, Erik Mirkov, Gloria Moore, Randy Niedz, and Ed Stover

3 Focus on Developing HLB-and ACP Resistant Citrus HLB likely the single greatest threat to citrus No strong 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 focusmay also confer resistance to canker and CVC Other types of genes also being explored..

4 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. Image:

5 Agrobacterium Mediated Genetic Transformation of Citrus Epicotyl Segments Transgenic scion Etiolated seedlings Epicotyl sections in Agrobacterium carrying gene of interest Epicotyl sections in Co-cultivation medium Epicotyl sections after 1 month in selection medium Transgenic plants grafted onto rootstock Non-Transgenic Rootstock M NC PC M Transgenic plants in the greenhouse PCR positive and acclimatized transgenic plants PCR confirmation of presence of transgene Slide: Jude Grosser, CREC, UF

6 Antimicrobial Peptides Broadly active against groups of micro-organisms First line of active defense to combat infection in multicellular organisms Most are very small molecules, MAY move systemically Results in microbial death or prevents growth

7 Genetic Transformation of Citrus with Spinach Defensins for Broad-Spectrum Resistance to Bacteria and Fungi T. Erik Mirkov Department of Plant Pathology and Microbiology Texas A&M AgriLife Research Center for Crop Improvement and Biotechnology

8 Spinach Defensins: Mirkov Furthest along in deregulation : Trees in FL greenhouse and field --8 events--red Grapefruits (2 varieties) and Sweet Orange (3 varieties) Trees in TX greenhouse ~71 events in 3 Sweet Orange varieties Some sweet orange with both spinach defensins in same tree -2-

9 Grapefruit Cultivar Rio Red Inoculated with the Citrus Bacterial Canker Pathogen, Xac: Mirkov Non-transgenic Transgenic for Spinach Defensin 2-11-

10 Greening inoculation test on Rio Red Grapefruit Non-Transgenic buds grafted on infected rootstock SoD2 Transgenic buds grafted on infected rootstock Mirkov

11 Multiple approaches for genetic engineering of citrus for disease and pest resistance J.W. Grosser M. Dutt, A. Omar and G.A. Barthe University of Florida, Citrus Research and Education Center, Lake Alfred, FL USA

12 Antimicrobial gene(s) currently under 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.

13 Systemic acquired resistance Pathogen attack (SAR) SA synthesized Induction of PR proteins SA dispersed systematically Increased resistance to further attack

14 Transgenic plant regeneration lytic peptide: Grosser et al. Cultivar Gene No. of plants in soil Duncan AttacinE 27 Hamlin AttacinE 15 Misc Grapefruit LIMA 45 Valencia, Hamlin,OLL-8 LIMA 56 Carrizo LIMA 8 Flame LIMA-SN 10 Misc Grapefruit PTA 12 Valencia, Hamlin, OLL8 CEMA 21 Carrizo CEMA 20 Key Lime CEMA 6 Misc Grapefruit CEME 18 Hamlin CEME 6 Valencia CEAD 14 Carrizo CEAD 12 Carrizo LIMA under AtSuc2 promoter 25 Misc grapefruit includes Duncan, Marsh and Flame cultivars Valencia, Hamlin, OLL-8 LIMA under AtSuc2 promoter 23 Key Lime LIMA under AtSuc2 promoter 17 Misc Grapefruit LIMA under AtSuc2 promoter 12

15 Canker (Xcc) assay on transgenic Duncan leaves containing the lytic peptide gene(s): Grosser et al. A B C A Mild tissue hypertrophy in transgenic LIMA leaf B Moderate tissue hypertrophy in transgenic Attacin leaf C Severe water-soaking and tissue hypertrophy in non-transgenic control leaf

16 HLB inoculation results: Grosser et al. Plant ID Gene Line # qpcr Visual Symptoms 566 LIMA MP LIMA MP LIMA MP ATTE MP LIMA MP3-C LIMA MP LIMA MP LIMA MP16 UNDET LIMA UNDET LIMA MP4-C7 UNDET ATTE MP LIMA MP3-C ATTE MP ATTE MP29-C LIMA MP ATTE MP LIMA MP ATTE MP ATTE MP ATTE MP14 UNDET LIMA MP2-C5 UNDET LIMA MP2-C6 UNDET ATTE MP19 UNDET LIMA MP5 UNDET LIMA MP LIMA MP20 UNDET ATTE MP3 UNDET Control + -

17 Future approaches for genetic 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 already in the field

18 Genetically transformed sweet orange for HLB resistance in Brazil Marcos A. Machado Centro de Citricultura Sylvio Moreira

19 Machado et al. Some approaches for GM citrus disease resistance Antimicrobial peptides (AMP) of insects AMPs from citrus use of transgenes from same species likely to facilitate acceptance Systemic Acquired Resistance genes

20 Results: Machado et al. Symptoms progress of HLB in leaves Symptom progress of HLB Variety Gene Nr of Events 3 Months 6 Months 9 Months 12 Months Natal Attacin A Pêra Attacin A Valência Attacin A Hamlin Attacin A Valência Xa Hamlin npr Natal npr

21 General considerations: Machado et al. An AMP from sweet orange has been tested and shown effective against citrus canker, and used for transformation. All Machado et al. work on genetic transformation will be focused on AMPs, R genes or irna from citrus to citrus.

22 Production of Transgenic Plants Expressing the NPR1 Defense Gene to Develop Resistance to Citrus Canker and HLB Vicente Febres Gloria A. Moore

23 Results: Febres and Moore Transformed Carrizo and grapefruit lines with the Arabidopsis NPR1 defense gene. The transgenic lines show normal growth and phenotype. Some transgenic lines show slowing and reduction of canker lesion formation. The transgenic plants are in the process of being tested for HLB resistance/tolerance Evaluation of the most promising lines under field conditions is underway

24 Field evaluation transgenic plants (AtNPR1 and others)

25 U.S. Horticultural Research Laboratory Ft. Pierce, Florida Horticulture and Plant Breeding Unit Update on transgenic program Kim Bowman Greg McCollum Randy Niedz Ed Stover Ute Albrecht Lesley Benyon Randy Driggers Mizuri Hert Ric Stange YongPing Duan David Hall Bob Shatters William Belknap William Dawson Ute Albrecht Ric Stange Mizuri Hert Lesley Benyon

26 Transgenic Project: Parallel Tracks 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 Exploring other promoters, constructs, etc. 1a 2a 3a a 1 mm Shoot-tip 9 10

27 Other Transgenes for HLB Resistance Target from the newly sequenced Liberibacter asiaticus genome (working with Duan group at USHRL) Phloem-specific protein induced during HLB infection (Bowman data USHRL) Working with ARS scientists in California to develop constructs so that ALL inserted genes are from Citrus!

28 400 independent scion (Hamlin, Midsweet, Valencia, and Ray Ruby) and >3500 independent rootstock regenerants established as micrografts

29 Transgenic Project: Challenging with HLB etc. 2a 3a a 1 mm 4a Shoot-tip Lots of plants of trifoliate hybrids transformed with D35S/D4E1 have been challenged No major differences, so far, in HLB symptoms or Liberibacter levels However, D4E1-GE plants have better growth Now using more active promoters Psyllid challenge of plants transformed with garlic lectin gene

30 All the labs discussed under transgenics (and others) are working to develop system for mature citrus transformation, many approaches and most showing progress Mirkov- Regeneration from Mature Wood -9-

31 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 Solutions should emerge soon that will help sustain a commercially productive industry, likely in combination with improved psyllid control, HLB diagnostics/roguing etc.

32 U.S. Horticultural Research Laboratory Ft. Pierce, Florida Other breeding approaches to HLB resistance or tolerance Ed Stover Kim Bowman Greg McCollum Randy Niedz YongPing Duan David Hall Richard Lee

33 Evaluating distant citrus and citrus relatives for HLB-resistance published reports of HLB resistance in different species related to citrus broad search may yield true resistance experiments in progress include more than 50 genotypescollaboration R. Lee, Riverside

34 Evaluating distant citrus and citrus relatives for HLB-resistance May identify genes which can then be used to transform commercial citrus Have transformed citrus with gene that should permit flowering within year of seed germination >Will permit rapid use of genes from distant relatives in commercial citrus rootstocks and scions

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

36 Evaluating varieties for HLB-tolerance Apparent that citrus varieties vary widely in their rate of developing HLB- commercially valuable? Looks like Sunburst and Temple may be much slower to succumb to HLB than sweet orange? Triumph slower than grapefruit. All still get HLB.. What current varieties may show useful tolerance? Need to test value in replicated trial. In many reports, trifoliate hybrids seem to have substantial ACP/HLB resistance >will use citrange mapping population to identify critical genes-collaboration with Fred Gmitter of UF and David Hall and Bob Shatters of USHRL

37 THANKS! FCPRAC/DOC: 20 million funding/yr for HLB NVDMC/FDOC Industry Collaborators USDA-CSREES UF/CREC Core Citrus Transformation Facility Faculty and Staff of our research organizations A cast of hundreds (OK dozens at least)!!!