Outline

1. Outline • State of transgenic disease resistance in the market • Intro to Two Blades Foundation • Projects that have successfully breached taxonomic barriers: Bs2, EFR • Trying to do so: wheat stem rust resistance • Effectors and their importance for finding new R genes • Outlook/vision/dreams

2. Yield losses in agriculture • Abiotic stresses - drought, wind, frost, flood • Pests - insects, nematodes, etc • Weeds - compete with crop • Disease - fungi, bacteria, oomycetes, viruses, post-harvest losses

3. Disease Control Market • Large • Current global fungicide sales ~ $8billion • Disease resistance traits present or sought in all crops • Growing • Asian soybean rust • Increased consumption of fruits and vegetables in developing world • Unsolved problems • Bacterial diseases • Soilborne diseases

4. Crop Disease • Disease problems are increased with high intensity agricultural practices • Monocultures provide “buffet” for successful pathogen • Narrow genetics base • Pressure on evolutionary arms race • Tremendous specificity in plant-pathogen interaction

5. Traditional disease control measures • Chemicals: broad spectrum, but • Expensive to develop, and to buy • Hard to distribute • Safety concerns • Evolution of pathogen resistance, sometimes quickly

6. Traditional disease control measures • Genetics: narrow spectrum • Limited number of genes in intercrossing species • Desirable traits often linked to bad traits, making breeding difficult and slow • Evolution of pathogen resistance, sometimes quickly

7. Value shift from chemical to seed & trait US Agricultural Industry Revenue 2003 $8.4 Billion 2008 E $9.1 Billion 1996 $7.6 Billion $0.1 $1.3 $2.7 $2.6 $3.6 $4.5 $3.0 $3.5 $3.8 Biotech Traits Germplasm Crop Chemicals Sources: 1996-2003 Doane Agrotek and Seed Studies; Monsanto estimates

8. Difficulties with genetic approaches to disease problems • No single “killer app” like Bt toxin or glyphosate to control diseases • Disease control market is widely spread across many small crops • Regulatory costs considered too high to support development in nearly all non-hybrid or “minor” crops

9. Industry Consequences • Primarily academic institutions pursuing disease resistance • Significant value attributed only after proof-of-concept in the crop • Dramatic downturn in early stage venture investing 2001 – present • Despite major advances in the lab: • No real innovation in crop disease resistance

10. Two Blades Foundation Mission: 2Blades Foundation supports the development and deployment of new ways of producing durable disease resistance in crop plants.

11. Rationale for 2Blades’ Existence • Progress in plant-pathogen research has been extraordinarily good • None of it has reached commercialization • Next generation sequencing and bioinformatics make feasible advances in crops that were nearly impossible a few years ago • Unique opportunity to take advantage of a know-how base and network of contacts that would be difficult to duplicate

12. 2Blades Strategy Identify promising disease targets Drive technology development through grants Provide project management support Develop and manage IP portfolios by in-licensing and new filings Provide technology at no cost to least-developed countries Partner with seed companies or grower groups for commercial deployment Partner with other charities to increase reach Recycle any revenue into more research

13. Program Selection Criteria • An unmet agricultural need in a major crop • Dearth of breeding solutions • A scientifically credible strategy • Outstanding collaborators • [Minimum regulatory and consumer issues]

14. People Roger Freedman Chairman, CEO Diana Horvath COO Susan Nycum non-executive director Eric Ward President

15. Scientific Advisory Board Jeff Dangl Jeff Ellis Jonathan Jones Sophien Kamoun Paul Schulze-Lefert Brian Staskawicz

16. Bacterial Spot Disease of Tomato • Constant major disease losses (10% – 30%) in commercial production (FL) Two Blades Foundation

17. Bacterial Spot Disease of Tomato • 92% of FL acreage affected • 40% of U.S. tomato acreage affected • Chemical control inadequate • Effective genetic resistance not available • Major resistance genes identified in wild species of Capsicum chacoense and introgressed into Capsicum annuum. Two Blades Foundation

18. Bacterial Spot Disease of Tomato PNAS November 23, 1999 vol. 96: 14153–14158 • 16 tomato events (VF36) demonstrate BS2 “resistance” (HR) via leaf infiltration of pathogen (Xanthomonas) • Bacterial growth curves on T2 plants show growth suppression with BS2 • Field test in 2000 of one BS2 event vs untransformed VF36: BS2 line shows resistance to Xcv infection and greater fruit yield. • Continued resistance and yield testing in 2007 - 2009 Two Blades Foundation

19. VF36 2008 Field Trial, Balm, FL VF36-Bs2 Two Blades Foundation

20. UF Field Station (Balm), Fall 2007 and Spring 2008 1 Means in column with the same letter are not significantly different, P ≤ 0.05, (Duncan's multiple range test). 2 Disease severity based on the Horsfall-Baratt scale. Two Blades Foundation

21. Spring 2009 Field TrialDisease Ratings1 1 Horsfall-Barratt defoliation scale: 1=0%; 2=0-3%; 3=3-6%; 4=6-12%; 5=12-25%; 6=25-50%; 7=50-75%; 8=75-87%; 9=87-93%; 10=93-97%;11=97-100%; and 12=100%. 12 plants per plot Two Blades Foundation

22. EFR: PAMP Receptor that recognizes EF-Tu Zipfel et al (2006) Cell 125:749.

23. Can we transfer PAMP perception systems between plant families? All plants outside the family of Brassicaceae tested so far are non-responsive to EF-Tu. Kunze et al. (2004) Plant Cell Nicotiana benthamiana (Solanaceae) Arabidopsis thaliana (Brassicaceae) Transformation of AtEFRp::EFR Test elf18 responses Stevens, P. F. (2001 onwards). Angiosperm Phylogeny Website. Version 9, June 2008

24. Lacombe et al (2010) Nature Biotechnol. In press

25. EFR WT EFR WT P. syringe pv. syringe B728a P. syringe pv. tabaci 11528 Agrobacterium tumefaciens A281 WT EFR WT EFR EFR Transgenic EFR Nicotiana benthamiana WT Transgenic EFR tomatoes C. Zipfel group MoneyMaker WT MM 35S::EFR L16 MM 35S::EFR P1 Ralstonia solanacearum GMI1000

26. Lacombe et al (2010) Nature Biotechnol. In press

27. Banana Xanthomonas Wilt • Devastating disease in great lakes region of Africa Tripathi et al (2009) Plant Disease 93:440.

28. Wheat Stem Rust (Puccinia graminis f.sp. tritici Ug99) • Already pandemic in E. Africa; headed for Pakistan and India • Potential for devastating impact on global wheat supply USDA Cereal Disease Laboratory • Seek new R genes from wild wheat relatives • Find important effectors in pathogen

29. Variation in Pgt Isolates Data from Y. Jin and L. Szabo, USDA Cereal Disease Lab

30. Why Have Major R Genes BeenDe-Emphasized? • Individually, they always break down • Introgressing one major R gene from a wild relative of wheat is just as time-consuming and difficult as introgressing a partial resistance gene • Partial resistance genes last longer/indefinitely and provide useful resistance when deployed with other partial genes • But, technology will make identification and deployment of multiple major R genes ever easier

31. Wheat Stem Rust Resistance Strategy • Premise: The best resistance package will combine partial resistance genes with major, dominant R genes • Approach: • Identify and isolate multiple major R genes from wild species • Deploy at least 3 in combination as a single transgenic locus • Supply resistant transgenic event to wheat breeding programs for rapid introgression into locally-adapted varieties

32. Aegilops sharonensis

33. Aegilops sharonensis as a source of R genes effective against Ug99 • Collaboration with B. Steffenson/E. Millet • Screen Aegilops accessions for resistance to Ug99 (TTKSK,04KEN156/4), and its presumed derivatives, virulent on Sr24 (TTKST, 06KEN19-V-3) and Sr36 (TTTSK, 07KEN24-4) • Make segregating populations and score F2 and F3 for R and S • Find co-segregating R genes in pooled R and S individuals

34. Bulked Segregant Analysis • First published by Michelmore et al. 1991 • Allows high resolution mapping without needing to test each individual • Principle: • from a population segregating for a trait of interest, mix together individuals of identical genotype w.r.t. the trait • Screen the pooled samples for DNA markers that co-segregate with the trait of interest

35. Hypothetical Example F1 Parents X Susceptible (rr) Resistant (RR) X Susceptible (rr) Resistant (Rr) Resistant (RR)

36. Hypothetical Example F1 Parents X Susceptible (rr) Resistant (RR) X Susceptible (rr) Resistant (Rr) Resistant (RR)

37. Simple Case — 4 markers From Michelmoreet al. 1991. PNAS 88:9828

38. Whole Genome Sequencing – “Infinite” Number of Markers From Schneebergeret al. 2009. Nature Methods 6:550

39. Challenges to Our Approach • Ae. sharonensis has a big genome; no reference sequence available • How to functionally test candidate genes? • Will the genes work across species? • [Acceptance of GM traits in wheat]

40. Rust infection and host immunity Host Manipulation Courtesy of Peter Dodds, CSIRO

41. Rust infection and host immunity Immune Recognition Courtesy of Peter Dodds, CSIRO

42. Effectors and R Genes Can Work Across Species • Potato R gene and late blight effector functioning in Nicotiana benthamiana • Gives access to R genes in species that are not hosts for the pathogen Vleeshouwerset al. 2008. PLoS ONE 8:e2875

43. Why Pathogen Effectors? • Figure out what’s essential for pathogenicity—which ones show greatest conservation • Use as tools to find new R genes from non-hosts • Understand variation in pathogen population and anticipate breeding needs • Create breeding tools—proxy assays for critical pathogen components

44. Conclusions/Outlook • Next generation sequencing is generating a “data tsunami” • R genes are now accessible from any species, not just crops or model species • Transgenic technology lets us expand the limits of sexual crossing • Isolated, critical pathogen effectors allow searching for R genes in non-hosts