How has lr34 yr18 conferred effective rust resistance in wheat for so long

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Beat Keller, University of Zurich

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How has lr34 yr18 conferred effective rust resistance in wheat for so long

  1. 1. How has Lr34/Yr18 conferred effective rust resistance in wheat for so long? BGRI Technical Workshop 2012, Beijing, 2.9.2012 Beat KellerInstitute of Plant Biology, University of Zurich, Switzerland
  2. 2. The Holy Grail of resistance breeding: Durable resistanceResistance is defined as „durable“ when it remains effective in cultivarsthat are widely grown for long periods and in environments favorable tothe disease (Johnson 1983).There are single, major resistance genes which are durable, but these areexceptionsDurable resistance is mostly polygenic and caused by genes actingquantitatively
  3. 3. Lr34: a durable leaf rust resistance gene R S Thatcher + Lr34 Thatcher
  4. 4. Lr34 is a durable disease resistance gene•  Has been effective against leaft and stripe rust for more than100 years in the field, certainly 40 years at large scale•  Confers partial resistance•  Prolongs the latency period and reduces the production ofspores (slow-rusting gene)•  Is associated with the morphological trait Leaf Tip Necrosis(Ltn1)•  Is not race specific
  5. 5. Lr34 confers resistance against multiple pathogens Lr34 Pm38 Yr18 All these effects are caused by a single gene Bdv1
  6. 6. Durable leaf rust resistance in the Swiss cultivar ‘Forno’ The Lr34 gene is also an important determinant of durable resistance in winter wheat, i.e. it is active in a broad germplasm and highly differing climatic conditions cv.‘Arina‘: Swiss winter wheat, susceptible cv.‘Forno‘: Swiss winter wheat, durably resistant
  7. 7. Documented history of Lr34 begins at the end of the 19th century in Italy and is restricted to hexaploid wheat China Italy Marco Polo?Adapted from Kolmer et al. 2008. Crop Science. Pedigree of Lr34/Yr18 cultivar entries in different wheatbreeding programs.
  8. 8. Deployment of Lr34: the example of Canadian germplasm Lr34 is present in many cultivars released since the 1970ies, but not in older cultivars McCallum et al. Euphytica, 2012
  9. 9. Proportion of the total Canada Western Red Spring seeded area which wasseeded to Lr34 carrying cultivars. Data from Canadian Wheat Board varietalsurveys (McCallum and DePauw 2008). No prairie wide variety surveys wereconducted 1993–1997
  10. 10. Map-based cloning of the Lr34 gene Lr34Wheat chrom. 7D 7DS 7DL XSWSNP2 XSWDEL1 XSWDEL2 XSWDEL3 Xgwm1220 XSWSNP3 Lr34 XcsLVE17 XSWM10Genetic mapchromosome 7DS 0.75 0.12 0.03 0.03 SWDEL1 SWDEL2 SWSNP2 SWDEL3 Hexose Cytochrome Cytochrome Glycosyl carrier P450 P450 transferase (Ψ)Physical map‘Chinese Spring’(363 kb) ABC Cysteine transporter Lectin receptor proteinase (Ψ) 120,000 363,640 kinase Eight open reading frames within a 363 kb target interval
  11. 11. Eight Lr34 mutants: for example splice site mutations Hexose Cytochrome Cytochrome Glycosyl carrier P450 P450 transferase (Ψ) ABC Cysteine transporter Lectin receptor proteinase (Ψ) 120,000 kinase 363,640 ATG TAA 4E 3E m19 2B 2G 4C 2F m21 1 kbInfection experiments revealed that the mutants are more susceptible to leafrust, stripe rust, powdery mildew and stem rust and lost leaf tip necrosis
  12. 12. The molecular basis of the resistance effect of the Lr34 gene: Lr34 encodes a putative ABC transporter (ABCG or PDR) protein What about Lr34-type of gene in susceptible lines (orthologous region in susceptible lines)? àSequence analysis in reference wheat lines with and without Lr34 Findings: Lr34- lines also contain the ABC transporter coding gene (allele Lr34sus) There are only three sequence differences between Lr34res and Lr34sus lines. Because of the dominant nature of Lr34res, these are gain-of-function mutations
  13. 13. Sequence differences between susceptible and resistant Lr34 alleles ATG TAA Lr34res: 5’-CCGACTT-3’ Lr34res: 5’-TCC ATC ATG-3’ Lr34res: 5’-TCG CAG CAT CGA-3’ 1 kb Lr34sus: 5’-CCGTCTT-3’ Lr34sus: 5’-TCC ATC TTC ATG-3’ Lr34sus: 5’-TCG CAG TAT CGA-3’ Deletion of a phenylalanine Conversion of tyrosine to histidine residue in ‘Chinese Spring’ in ‘Chinese Spring’Resistant and susceptible allele differ by only two amino acid polymorphisms
  14. 14. Development and application of molecular markers for theLr34 gene:• Perfect markers derived from the gene sequence• Markers have been adopted in the last two years in mostwheat breeding programs worldwide where leaf and/orstripe rust is of relevance
  15. 15. Functional studies of the Lr34 gene in transgenic wheat: cold- treated seedlings Seedlings of transformed Bobwhite Cold treatment at 4oC Risk, Selter et al. 2012 Plant Biotech. J.
  16. 16. Functional studies of the Lr34 gene in transgenic wheat: flag leaf of adult plant Risk, Selter et al. 2012 Plant Biotech. J.
  17. 17. Microscopic analysis of leaf rust infection using WGA-alexa staining Risk, Selter et al. 2012 Plant Biotech. J.
  18. 18. Leaf tip necrosis is identical in Lr34 transgenic wheat Risk, Selter et al. 2012 Plant Biotech. J.
  19. 19. Genetic background of transgenic wheat plays an important role: seedling resistance in BW26SUI but not BW26AUS at 20oCà  Additive interaction(s) with other gene(s)   Risk, Selter et al. 2012 Plant Biotech. J.
  20. 20. Conclusions from transgenic wheat lines with Lr34•  The transgene is fully functional against leaf rust and results in LTN•  This complementation demonstrates that Lr34 is indeed pleiotropic•  The genetic background can result in improved resistance: one should broadly cross the most active transgene into a broad set of breeding material (…normal breeding procedure…)•  Combination with other durable resistance genes in a cassette or by crossing seems a good strategy
  21. 21. Why durable, how does the gene confer resistance? Some evolutionary findings and conclusions
  22. 22. PDRs in Arabidopsis and rice There are 15 PDRs in Arabidopsis and 23 PDRs in riceClosest homolog of Lr34 in rice: PDR23 (86% identity at AS level) second hit: OsPDR2 (55% identity) in Arabidopsis : PDR5 / PDR9 (56% identity at AS level)There is one clear Lr34 homolog in rice, but none in barley, Brachypodium andArabidopsis
  23. 23. Observations from the molecular nature of LR34:• It is not an NBS-LRR encoding protein (such as e.g. Lr1, Lr10,Lr21, the other cloned Lr resistance genes)• This suggests a completely different molecular mechanism ofresistance• For example by reducing the quality of the leaves as «food»for biotrophic pathogens• Transporting an antimicrobial substance (phytoalexins)• Priming of resistance response?
  24. 24. Lr34 encodes an ATP-binding cassette (ABC) transporterTransporters sharing a common basic structure: Nucleotide binding fold (NBF) Trans-membrane domain (TMB)Lr34 belongs to the subfamily of the pleiotropic drug resistance (PDR) transportersPDRs are only found in plants and fungi. ??? TMD TMD TMD TMD plasma membrane NBF NBF NBF NBF ATP substrate ADP + P Lr34 protein: 1402 amino acids
  25. 25. Structure of the ABC exporter Sav1866 from Staphylococcusaureus with bound nucleotide (Dawson and Locher 2006). Source:http://en.wikipedia.org/wiki/ATP-binding_cassette_transporter.Most exporters in prokaryotes, such as the multidrug exporterSav1866, are made up of a homodimer consisting of two half-sizetransporters.
  26. 26. What is Lr34 doing?• Lr34 confers resistance against multiple obligate biotrophic pathogens.• Lr34 is associated with reduced intercellular hyphal growth but not with ahypersensitive response or papilla formation.• The level of Lr34-mediated resistance for leaf rust infection correlated with the development of leaf tip necrosis. These observations suggest that Lr34 could be due to a general physiological effect. Microarray studies revealed that a similar set of genes was up-regulated in uninfected flag leaves of Lr34 containing near-isogenic lines and senescing flag leaves. A. Senescence-related resistance?
  27. 27. Leaf tip necrosis in Lr34 containing lines
  28. 28. HvS40: a barley gene that is up-regulated during leaf senescence
  29. 29. Nonfluorescent chlorophyll catabolites (NCC): hallmarks of leaf senescenceLr34 regulates senescence-like processes in the flag leaf of resistant plants.
  30. 30. Transcriptomics on Lr34 linesTwo transcriptomic studies on uninfected and infected wheat leaves with orwithout Lr34 were made (Hulbert et al. 2007, Phytopathology; Bolton et al.2008, MPMI)Main conclusions:1.  After infection, there is a high demand of cellular energy (increased carbonflux)2.  Expression of defense genes was often higher in resistant plants: is Lr34involved in priming of defense responses? B. Resistance priming BEFORE infection?
  31. 31. PEN3/PDR 8 contributes to non-host resistance to inappopriate pathogens (Stein et al, Plant Cell 2006) Pen3 mutants of Arabidopsis were more susceptible to infection with barley powdery mildew, pea powdery mildew and potato late blight.Lipka et al.,CurrentOpinon inPlant Biology,2008 C. Transport of an antimicrobial metabolite?
  32. 32. From the FIELDTo the FIELD!
  33. 33. AcknowledgmentsUniversity of Zürich CSIRO Canberra, AustraliaLiselotte Selter Joanna RiskSimon Krattinger Evans LagudahThomas WickerChauhan HarshIPK GaterslebenJochen KumlehnGötz HenselThis work was supported by the European Research Council, GRCD and theSwiss National Science Foundation

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