Dissecting the Mystery Behind the Rpg5 Mediated Puccinia graminis Resistance in Barley Using Genetics, Molecular and Bioinformatics Approaches
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Abstract
Barley rpg4/Rpg5 locus harbors three tightly linked genes, two NLRs Rpg5 and HvRga1, and HvAdf3, togather providing resistance against Puccinia graminis f. sp. tritici, causal agent of wheat stem rust including race TTKSK, considered a threat to global food security. The integrated decoy hypothesis proposes role for head-to-head genome architecture present in the dual plant NLR immunity receptors, where one NLR partner contains an integrated sensory domain (ISD). The ISDs represent mimics of virulence effector targets translocated to the immunity receptors and act as baits to recognize virulent effectors to initiate defense responses. Alleles of Rpg5 contain two diverse C-terminal, the Rpg5 resistance allele has a serine threonine protein kinase (STPK) ISD whereas the major class of rpg5 susceptible alleles contain a protein phosphatase 2C (PP2C) ISD. Genetic and functional analysis shows that in the heterozygous state rpg5-PP2C allele acts as a dominant susceptibility factor suppressing Rpg5-STPK mediated Pgt resistance. This is the first integrated decoy NLR gene identified that contains two distinct ISDs. Liabrary scale Y2H screeing using Rpg5-STPK as bait identified HvVoz1. HvVoz1 was also interacts with the HvRga1, Rpg5-LRR, and rpg5-PP2C domains suggesting that it may act as a scaffold to hold the R-protein complex together until effector manipulation. We identified Rpg5-STPK ISD progenitor HvGak1, ortholog of the Arabidopsis guard cell AT5G15080 and AtAPK1b, shown to function in stomatal aperture opening in response to light. We hypothesize that several forma specialis of P. graminis contain virulence effector/s, that manipulate HvGak1, mimicking the presence of light to open the stomates, allowing the pathogen to gain entry in to the plant during dark period that P. graminis spores evolved to germinate. We identified dark period pathogen penteration through stomata by deveoping a novel staining method and using confocal microscopy. To further characterize the Rpg5 immunity pathway fast-neutron irradiation was utilized to generate rpr9 mutant, compromised for rpg4/Rpg5-mediated resistance. Utilizing genetic mapping and exom capture we identified candidate genes for rpr9 mutants. Based on our understanding of this resistance mechanism it would be a good candidate system for generating synthetic resistances utilizing different ISD baits fused to the Rpg5 NLR.