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THE HOST: RESISTANCE GENE ISOLATION AND REALISING THE POTENTIAL

THE HOST: RESISTANCE GENE ISOLATION AND REALISING THE POTENTIAL. Kim Hammond-Kosack Wheat Pathogenesis Programme Rothamsted Research. 19 th December 2006. kim.hammond-kosack@bbsrc.ac.uk. PLANT PATHOGENS ARE PICKY. Most plants are naturally resistant to most

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THE HOST: RESISTANCE GENE ISOLATION AND REALISING THE POTENTIAL

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  1. THE HOST: RESISTANCE GENE ISOLATION AND REALISING THE POTENTIAL Kim Hammond-Kosack Wheat Pathogenesis Programme Rothamsted Research 19th December 2006 kim.hammond-kosack@bbsrc.ac.uk

  2. PLANT PATHOGENS ARE PICKY Most plants are naturally resistant to most pathogenic microbial species Plant disease is rare

  3. R PROTEIN RACE - SPECIFIC RESISTANCE NON-HOST RESISTANCE PREFORMED STRUCTURAL BARRIERS and ANTIMICROBIAL COMPOUNDS ESCAPE BASAL RESISTANCE TOLERANCE

  4. RACE - SPECIFIC GENETIC INTERACTIONS FLOR’s ‘Gene-for-Gene’ Hypothesis Host plant genotype r1 R2 R1 r2 C I Avr1, avr2 Pathogen genotype I C avr1, Avr2 I - incompatible - no disease C - compatible - disease

  5. 3 COMPONENTS TO INDUCIBLE DEFENCE PATHOGEN RECOGNITION R PROTEINS ONLY FUNCTION HERE DEFENCE SIGNALLING A MULTI-COMPONENT RESISTANCE RESPONSE

  6. RESISTANCE GENE ISOLATION Started in late 1980s Several groups successful by 1994 Approach – molecular genetics

  7. MAP - BASED ISOLATION OF R GENES Genetic marker 2 Genetic marker 1 R gene M3 M4

  8. MAP - BASED ISOLATION OF R GENES 5. Transform a susceptible genotype with a single cosmid clone R S S R Disease reaction • Types of problems encountered • A ‘LONGER WALK’ than expected - lack of recombination • An unknown ‘HOLE IN THE BAC CLONE’ RPM1 • Which gene is it ?PTO

  9. Rare outcome or inactive R gene TRANSPOSON TAGGING OF R GENES The two-component system Ds transposase gene in a T-DNA R gene Ac transposon in a T-DNA

  10. 30 C 22 C TMV nn NN TRANSPOSON TAGGING OF R GENES with genetic selection Tomato Cf-9 : Avr9 Tobacco N gene Transposon inactivation of R gene

  11. Effective against Host species Fungi Oomycetes Bacteria Viruses Nematodes Aphids Whiteflies Model Crop Monocotyledonous Dicotyledonous RESISTANCE GENES ISOLATED

  12. What protein types do R genes code for ?

  13. RACE NON-SPECIFIC R PROTEINS RACE SPECIFIC R PROTEINS CC Pi-d2 Xa21 Ve1 Ve2 Cf-2, 4, 5, 9 5 LRR 6 cell wall plasma membrane cytoplasm 3 TIR 1 CC PEST CC 4 NBS NBS ECS 2 kinase kinase kinase LRR RPW8 kinase LRR kinase NLS RPG1 WRKY Pto PBS1 Xa27 RRS1 RPS2 RPM1 BS2 N, L6 RPP5

  14. A single pathogen species can be controlled by different types of R protein Hyaloperonospora parasitica – Downy mildew of Arabidopsis CC-NBS-LRR RPP8 gene TIR-NBS-LRR RRP4 gene eLRR RPP27 gene* RACE – SPECIFIC R PROTEIN (SUMMARY) Still a very limited number of R protein types and motifs A single R protein class can confer resistance to different pathogen types Intracellular CC-NBS-LRR – bacteria, fungi (Basidiomycetes and Ascomycetes), Oomycetes,virus,root knot and cystnematodes and an aphid * Retracted by authors Jan 2007 – Plant Physiology see original article

  15. R PROTEIN / R LOCI SPECIES SPECIFICITY Most R proteins / R loci confer resistance to just specific isolates within a single species A few exceptions Tomato Mi protein ( CC-NBS-LRR) - root knot nematode, aphid and whitefly Potato - neighbouring genes Rx1 and Gpa2 Both CC-NBS-LRR types (88% amino acid identity) Rx1 potato virus X Gpa2 cyst nematode

  16. R gene expression Very low and constitutive Occasionally low level induction following infection but only in the vicinity of the pathogen Rarely expressed only in resistant genotype - Rice Xa27 – Xanthomonas oryzae pv. oryzae Susceptible alleles 10 bp and a 25 bp insertion in the promoter (Gu et al, (2005) Science 435: 1122-1125)

  17. How do R proteins function ? Four examples Direct pathogen molecule recognition Indirect pathogen molecule recognition

  18. Direct recognition Tomato Pto - AvrPto Pseudomonas syringae AvrPto or AvrPtoB Resistant Pto host Susceptible host cell wall plasma membrane pto Pto Prf AvrPto and AvrPtoB effectors bind to unknown host target Enhanced pathogen virulence HR Less pathogen proliferation Adapted from Jones and Dangl (2006) Nature

  19. Indirect recognition Arabidopsis RPM1- AvrRpm1 Pseudomonas syringae AvrRpm1 or AvrB Resistant RPM1 host Susceptible host NDR1 NDR1 P P RIN4 RIN4 P P P P No RPM1 RPM1 AvrRpm1 effector bind to host target RIN4 and other targets HR Less pathogen proliferation Enhanced pathogen virulence Adapted for Jones and Dangl (2006) Nature

  20. Virulence Target Avr R PROTEINS GUARD THE VIRULENCE TARGET A. COMPATIBLE INTERACTION Susceptible response to favour pathogen growth and development Virulence Target Avr B. INCOMPATIBLE INTERACTION Resistance by guarding R3 Resistance response AvrB/ RIN4 RPM1 AvrRpm1 Avr2 Rcr3 Cf-2

  21. Tomato Cf-4 – Cladosporium fulvum Avr4 Cf-4 eLRR Cf-4 Chitinase Avr4 Avr4 Avr4 Resistant host Susceptible host Rcr3 Fungal chitin NRC1 CC-NBS-LRR Enhanced pathogen virulence Activation of Cf dependent defence response Suzan Gabriels, Matthieu Joosten (2007) Plant J (in press) Less pathogen proliferation

  22. Barley Mla10 and powdey mildew Avra10 spore appressorium Resistant host Susceptible host penetration peg haustorium fungal plasma membrane Modified plant plasma membrane WRKY1/2 Avra10 or EHM Mla10 Avra10 effector bind to host target ? ? Plant nucleus plant cell cytoplasm Enhanced pathogen virulence ECM – extracellular haustorial matrix HR Less pathogen proliferation Shen, Q-H …Schultz-Lefert (Science 21st Dec 2006) Chris Ridout (2006) Plant Cell 18, 2402-14

  23. R PROTEIN DEFENCE ACTIVATION Early cytoplasmic to nuclear shuttling of R proteins Cytoplasmic route CC-NBS-LRR TIR-NBS-LRR RPW8 Pto kinase early NDR1 EDS1 Plant nucleus RAR1 SGT1 HSP90 TIR-NBS-LRR-NLS-WRKY late CC-NBS-LRR SA WRKY HR OB TGAs W box MAP kinase cascades NPR1 NO Early defence responses PR and other defence proteins

  24. STUBBORNLY UNANSWERED QUESTIONS 1. How to extracellular LRR-TM class of R proteins work ? Cladosporium fulvum Tomato N-terminal LRR confer specificity Cf-2, Cf-4 Cf-5, Cf-9 Small secreted Avr peptides Avr2 LRR (24 aa) Avr5 Avr4 Avr9 cell wall plasma membrane cytoplasm

  25. STUBBORNLY UNANSWERED QUESTIONS 2. What is the crystal structure of a plant LRR domain ? Porcine ribonuclease inhibitor < 30% amino acid identity with plant LRR containing proteins

  26. STUBBORNLY UNANSWERED QUESTIONS (2) 3. What other types of host targets are guarded by R proteins? 4. What function(s) to these host targets provide to successful pathogens? 5. Why do only a few R genes confer durable resistance ? Tomato Tm22, Barley Rpg1

  27. NEW INFORMATION OBTAINED THROUGH SEQUENCING PLANT GENOMES Putative R gene numbers Rice R protein class Poplar Arabidopsis 104 0 95 TIR-NBS-LRR 46 400 141 CC-NBS-LRR 56 90 105 RLP (eLRR-TM and eLLR-TM-kinase) CC-NBS-LRR – common ancestral origin Some gene classes have other functions plant development

  28. THE POST - GENOMICS ERA – What are the possibilities ? 1. Proteomics – phospho-proteomics Use of epitope – tagged R proteins to identify the direct interactors FRET–Fluorescence resonance energy transfer to confirm interactions in vivo 2. VIGS – Virus induced gene silencing to explore gene function in a high throughput mode

  29. a  ßb ßd ß a ßc B A NotI PacI  a b plant seq 80-120nt Barley stripe mosaic virus Identification of wheatleaf rust resistance gene Lr21 VIGS vector R S Scofield et al. (2005) Plant Physiology 138: 2165–2173

  30. Barley rym4 soil-borne viruses R R R S S R S R R R K Kanyuka R GENE APPLICATIONS – REALISING THE POTENTIAL Non- transgenic approaches 1. Development of markers ‘within-the-R gene’ or closely linked to the R gene - Useful in R gene introgression by plant breeders - minimises the need for bioassays 2. Pyramiding of R genes each working in a different way is a real possibility

  31. EACH RACE SPECIFIC R GENES HAS ONLY A LIMITED LIFE SPAN BOOM YEARS BUST YEARS

  32. R GENE APPLICATIONS – REALISING THE POTENTIAL Transgenic approaches Sophisticated Simple

  33. THE CONSEQUENCES OF CONSTITUTIVELY ACTIVATING PLANT DEFENCES Tomato Cf-9 : Avr9 Plant death 30 C 22 C Reduced plant growth rates TMV nn Slower plant development Transposon inactivation of R gene Lower crop yield NN

  34. PATHOGEN - INDUCIBLE PROMOTERS

  35. 4 x W1 Synthetic promoters – pathogen inducible - 46 - 8 add motif tetramers CaMV 35S GUS from WRKY promoter Hyaloperonospora parasitica – Arabidopsis Incompatible Paul Rushton et al. (2002) The Plant Cell 14: 479-762

  36. Pathogen debilitation Variant virulence target Shuffled chitinase/glucanase Enzyme to block / degrade a pathogenicity factor Durable disease control achieved by using two contrasting transgenes in each T-DNA cassette Plant defence LB pPI Gene 1 pP2 Gene 2 RB Examples Self activated R* protein Broader recognition R protein Improved defence signallingprotein Major QTL enzyme P - borders

  37. R GENE APPLICATIONS – REALISING THE POTENTIAL Transgenic approaches Sophisticated Simple

  38. UK GM field trials for 2007 Announced Dec 2006 by BASF Control of late blight by two R genes from potato Rpi-blb1 and Rpi-blb2 CC-NBS-LRR proteins evolutionarily related to tomato Mi Cloning of Rpi-blb2 van der Voissen et al., (2005) Plant Journal 44, 208-222

  39. Transfer of resistance Tedious and time-consuming breeding schemes involving ploidy manipulations and a series of bridge crosses gave P. infestans resistant inter specific hybrids - ABPT lines ABPT lines in field trials at multiple sites in Netherlands over 20 years gave no or only a few sporulating lesions at the end of the season Organic and intensive farming conditions tested UK GM late blight field trials for 2007 Rpi-blb1 and Rpi-blb2both originally from the diploid potato species Solanum bulbocastanum Both confer high level resistance to P. infestans isolates with complex race structures – BROAD SPECTRUM

  40. Limited number of R protein classes with conserved structural domains NBS-LRRs - largest class no TIR-NBS-LRR in monocots SUMMARY (1) Isolation of the first R genes was difficult now far easier through homology searches and transient transformation techniques R proteins possess both recognition and signalling domains Pathogen effector (Avr) signals are far more variable

  41. Many unanswered questions on R protein function as well as R gene evolution Exploitation - Marker assisted selection and breeding - Transformation of broad spectrum and durable R genes into elite genotypes - Sophisticated R-Avr cassettes and R* genes using pathogen inducible promoters SUMMARY (2) Some R proteins may guard pathogen virulence targets Plant R proteins function only in closely related species Several different types of R locus organisation

  42. BioExploit Many thanks to … Rothamsted Research Elsewhere Kostya Kanyuka Hai-Chun Jing Jason Rudd John Lucas Ian Crute Jonathan Jones Matthieu Joosten Paul Schultz-Lefert Chris Ridout Greg Martin

  43. Extra slides

  44. TIR:NBS:LRR - RACE-SPECIFIC R PROTEINs Drosophila Mammals Plants Interleukin receptors Toll N - tobacco L6 - flax RPP5 - Arabidopsis LRR LRR extracellular cell wall plasma membrane cytoplasm CC TIR NBS TIR TIR NBS LRR LRR Immunity and defence Dorsal - ventral polarity and defence

  45. TIR :NBS:LRR - RACE-SPECIFIC R PROTEINs Plants Mammals TLRs N - tobacco L6 - flax RPP5 - Arabidopsis Signals - LPS, unmethylated DNA flagellin LRR extracellular cell wall plasma membrane LRR cytoplasm TIR CC TIR NBS NBS NBS LRR LRR Immunity and defence CARD NOD 1, NOD 2

  46. THE LEUCINE-RICH REPEAT DOMAIN Contains the reiterated sequence 23 aa (I) missing LxxLxxLxxLLxLxx(N)xLxGxIPxx 24 aa (E) b - strand / b - turn structural motif Within this motif, leucine residues project into the hydrophobic R protein core whilst x amino acids are solvent exposed and very variable Detection of pathogen - derived signals?

  47. TRANSCRIPTION PROFILING TO IDENTIFY PLANTPROMOTERS INDUCED BY MULTIPLE PATHOGENS RNA Probe sources Control healthy infected Different biotrophic pathogens Different necrotrophic pathogens Compatible interactions overlay images and normalise BIOINFORMATICs (eg. MEME) to identify common motifs in promoters (arabidopsis, rice) analysis

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