1. 1.Survey the entire panel of maize diversity lines (association mapping panel) for their ability to impact HR induced by Rp1-D21. 2.Clone and characterize Hrml1 by using a combination of linkage and association mapping tools, especially the nested association mapping (NAM) RIL populations. 3.Evaluate NAM RIL populations for additional Hrml genes/QTL and clone at least three of them. Mutant-assisted exploration of natural variation underlying R gene-mediated immunity in maize And the outreach effort

2. The idea is to find the missing pieces of the HR pathway puzzle by taking advantage of natural variation in maize MAGIC: Mutant-assisted gene identification and characterization

3. HR stands for: Hypersensitive Reaction (hypersensitivity, or HR cell death) Hypersensitive Response The term was first coined by Stakman (1915) He defined ‘hypersensitiveness’ to indicate the abnormally rapid host plant death when attacked by rust fungi’ (Stakman, 1915)

4. Compatible vs. incompatible plant- pathogen interaction

5. Biochemical mechanisms underlying the HR response

7. The HR response - updated definition

8. Genetics of the HR response HR is induced Specifically by the interaction of R genes and their corresponding Avr genes following a gene-for-gene interaction (Flor 1941)

9. The HR is controlled by R gene-encoded receptor proteins, a major proportion of which are ‘nibblers’ CC/

10. Molecular basis of the HR response In the absence of the pathogen AVR proteins (now called effector proteins), nibblers are quiescent. This seems to be because of their ability to undergo intermolecular interactions, as well as association with a number of other proteins, including chaperones such as Hsp90

11. Molecular basis of the HR response

12. R gene activation

13. Pathways linking R gene activation with the HR response In Arabidopsis, two major signaling pathways have emerged that link R genes with their downstream responses NDR1 - non race-specific disease resistance EDS - enhanced disease susceptibility CC type NBS-LRR genes TIR type NBS-LRR genes

15. R genes have two functions The first is to recognize the pathogen The second is to trigger the HR response following recognition Means mutations can in either function that can cause the R gene to fire HR constitutively, in the absence of the pathogen

16. In fact many R genes have been identified that are autoactive The first cases of which came from the maize Rp1 locus 32 5 4 7 196 8 Rp1 - a complex locus The Rp1-D allele – is made up of 9 NBS-LRR genes

17. 32 5 4 7 196 8 Undergoes unequal crossing over frequently 32 5 4 7 196 8 32 5 4 7 196 8 32 5 4 7 196 8 X X

18. 7 9 8 32 5 4 7 196 8 X 2/9 1 Rp1-D21 Generation of the Rp1-D21 recombinant A dominant lesion mimic mutant

19. The HR phenotype of Rp1-D21 in H95

20. Rp1-D21 plants exhibit all the hallmarks of the HR response 18S rRNA PR1 Rp1-D21 WTmut PR5 PRms WIP1

21. The HR phenotype of Rp1-D21 is cell autonomous

22. Rp1-D21 expression in different genetic backgrounds

23. Expression of Rp1-D21 in B73 vs. Mo17

24. IBM RIL population 302 lines strong recombinant inbred lines (RILs) population Derived from a cross between B73 with Mo17 F2 plants were allowed to intermate for 4 generations before inbreds was derived by the single seed descent method

25. MAGIC screen of the IBM population for modifiers of Rp1- D21 (HR response) - Rp1-D21::H95/+ was crossed with the entire IBM RIL population - The F1 progenies were evaluated for the severity of the Rp1-D21 population

26. Other RILs significantly suppressed Rp1-D21

27. MAGIC screen of the IBM population for modifiers of Rp1- D21 (HR response) - Rp1-D21::H95/+ was crossed with the entire IBM RIL population - The F1 progenies were evaluated for the severity of the Rp1-D21 population - The phenotypic data thus collected was correlated with the genotypic data (which was already available for all these IBM RILs) using a QTL software (cartographer)

28. QTL scan

29. Rp1 Chromosome 10 Identification of Hrml1 (HR modulating locus-1)

30. To clone Hrml1 and to identify additional modifiers of the HR response We have started a MAGIC screen of the NAM resource

31. Nested association mapping (NAM) resource

32. Rp1-D21 phenotype in hybrids of H95 with the NAM founders

35. In some cases HR becomes dependent on temperature

36. 1.Survey the entire panel of maize diversity lines (association mapping panel) for their ability to impact HR induced by Rp1-D21. Mutant-assisted exploration of natural variation underlying R gene-mediated immunity in maize Between Peter’s and our lab, we have crossed most diversity inbreds with Rp1-D21(H95). This includes almost all NAM founders. Peter also crossed some (~50) with Rp1-D21(B73) The resulting F1 progenies from all of these crosses were planted this past summer (2009) at ACRE as well as at NCSU These were screened at multiple times using various approaches and parameters

37. 1.Survey the entire panel of maize diversity lines (association mapping panel) for their ability to impact HR induced by Rp1-D21. Mutant-assisted exploration of natural variation underlying R gene-mediated immunity in maize Between Peter’s and our lab, we have crossed most diversity inbreds with Rp1-D21(H95). This includes almost all NAM founders. Peter also crossed some (~50) with Rp1-D21(B73) The resulting F1 progenies from all of these crosses were planted this past summer (2009) at ACRE as well as at NCSU These were screened at multiple times using various approaches and parameters This strategy identifies dominant modifiers only To unveil and map recessive modifiers, we have also made F2 populations for a number of diversity inbreds (especially the NAM founders) by making F1 x F1 crosses between WT plants with their mutant F1 siblings

38. 1.Survey the entire panel of maize diversity lines (association mapping panel) for their ability to impact HR induced by Rp1-D21. Mutant-assisted exploration of natural variation underlying R gene-mediated immunity in maize Between Peter’s and our lab, we have crossed most diversity inbreds with Rp1-D21(H95). This includes almost all NAM founders. Peter also crossed some (~50) with Rp1-D21(B73) The resulting F1 progenies from all of these crosses were planted this past summer (2009) at ACRE as well as at NCSU These were screened at multiple times using various approaches and parameters This strategy identifies dominant modifiers only To unveil and map recessive modifiers, we have also made F2 populations for a number of diversity inbreds (especially the NAM founders) by making F1 x F1 crosses between WT plants with their mutant F1 siblings In fact, nine such F2 populations were planted at ACRE, which involved Mo17, A632 and 7 NAM founders. These were screened multiple times for various Rp1-D21 severity parameters and sampled for DNA (mutant plants) for genotyping purposes.

39. 1.Survey the entire panel of maize diversity lines (association mapping panel) for their ability to impact HR induced by Rp1-D21. 2.Clone and characterize Hrml1 by using a combination of linkage and association mapping tools, especially the nested association mapping (NAM) RIL populations. 3.Evaluate NAM RIL populations for additional Hrml genes/QTL and clone at least three of them. Mutant-assisted exploration of natural variation underlying R gene-mediated immunity in maize

40. 1.Survey the entire panel of maize diversity lines (association mapping panel) for their ability to impact HR induced by Rp1-D21. 2.Clone and characterize Hrml1 by using a combination of linkage and association mapping tools, especially the nested association mapping (NAM) RIL populations. 3.Evaluate NAM RIL populations for additional Hrml loci and clone at least three of them. Mutant-assisted exploration of natural variation underlying R gene- mediated immunity in maize Strategy: Cross Rp1-D21(H95) mutants with 3,400 of the 5,000 NAM RILs, screen their F1 progenies at both Purdue and NCSU, and then do QTL analysis just as it was done in the experiment involving IBM RILs.

41. 1.Survey the entire panel of maize diversity lines (association mapping panel) for their ability to impact HR induced by Rp1-D21. 2.Clone and characterize Hrml1 by using a combination of linkage and association mapping tools, especially the nested association mapping (NAM) RIL populations. To confirm that we have cloned the right gene for Hrml1, we are following at least two strategies: Mutant-assisted exploration of natural variation underlying R gene- mediated immunity in maize Directed EMS mutagenesis of Hrml1 Transposon tagging of Hrml1

42. Mutant-assisted exploration of natural variation underlying R gene- mediated immunity in maize Directed EMS mutagenesis of Hrml1 Using A632 for this purpose because: it has been shown to have an effective Hrml1 gene capable of suppressing Rp1-D21 A632 plants homozygous for Rp1-D21 produce pollen H95 x Rp1-D21/Rp1-D21(A632) M1 - few thousand seed has been generated (Treated with EMS)

43. Mutant-assisted exploration of natural variation underlying R gene- mediated immunity in maize Transposon tagging of Hrml1 Using both B73 and A632 Crosses were made this summer (2009) to transfer Mutator (Mu) into B73 and A632. - The progenies will be selfed/sib-mated to generate Mu- active plants homozygous for Hrml1 - Such plants will be selected and propagated by either selfing or sib-mating. - Next summer (2010), these plants will be planted in the field and fertilized with Rp1-D21(H95) pollen to generate at least 50,000 F1 seed, which will be screened in the greenhouse/field for plants with enhanced severity of the Rp1-D21 phenotype

44. Mutant-assisted exploration of natural variation underlying R gene- mediated immunity in maize Transposon tagging of Hrml1 Next summer (2010), these plants will be planted in the field and fertilized with Rp1-D21(H95) pollen to generate at least 50,000 F1 seed, which will be screened in the greenhouse/field for plants with enhanced severity of the Rp1-D21 phenotype B73 (Mu) X Rp1-D21(H95) F1 (Screen for F1 plants that have a relatively severe Rp1-D21 phenotype)

45. Mutant-assisted exploration of natural variation underlying R gene- mediated immunity in maize Transposon tagging of Hrml1 The same procedure can be used to tag and clone additional Hrml loci that we find in other lines (e.g., Tzi8, Co255, CML228) We will start crossing these lines with Mu stocks soon

46. Mutant-assisted exploration of natural variation underlying R gene- mediated immunity in maize Transposon tagging of Hrml1 In fact, the same procedure can be used to tag and clone additional Hrml loci, and this includes enhancers. For example, TX303 and M37W have a very strong enhancing effect on Rp1-D21 in their hybrids with H95 TX303 (Mu) X Rp1-D21(H95) F1 (Screen for F1 plants that no longer have a severe Rp1-D21 phenotype) EMS mutagenesis of seed may work even better in knocking out the enhancer

47. Other intellectual considerations Why Rp1-D21 is constitutively active? Why the HR underlying Rp1-D21 does not express in very young leaves? Why the HR underlying Rp1-D21 becomes cold sensitive in certain genetic backgrounds? What about some other recombinant alleles of Rp1 that exhibit a relatively weaker HR response? These alleles include Rp1- Kr1N and Rp1-nc3. The latter normally exhibits a recessive inheritance in most genetic backgrounds but we have found one exception thus far. In the Mo20W background, the Rp1- nc3 phenotype can be observed in a heterozygous condition. Should we raise antibodies against the Rp1-D21 protein to address these questions?

48. Mutant-assisted detection and capture of useful variation By MAGIC Its underlying principle is: The phenotype of a mutant (in the trait of interest) can be used to unveil relevant variation present in any line, and also to consolidate this variation in individual plants - which can then be used for breeding purposes

49. F1 hybrid To perform MAGIC: X Diversity parent mutant F2 progeny

50. Second-site suppressor screen following intentional mutagenesis

51. MAGIC is even more effective with a dominant mutant