Domain/domain interactions in RPS4/RRS1 immune complex activation by bacterial effectors

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Plant disease can cause big crop losses, which is costly to farmers. For example, control of potato late blight costs UK farmers ~ £60M/year. Resistance (R) genes enable plants to detect and resist pathogens, but how R proteins work is very poorly understood. Plant pathogens deliver molecules called effectors into host cells to interfere with host immune mechanisms. R genes enable plants to recognize such effectors and then activate immunity. To overcome R genes, pathogens must evade detection by mutations in recognized effectors. We aim to understand R protein molecular mechanisms in such detail that we can design new R genes to recognize effectors from any pathogen. A shorter-term goal is generate intelligently designed libraries of R protein variants that can be screened for recognition of previously unrecognized effectors. Before we can do either, we need to thoroughly understand how R proteins convert recognition of effectors into activation of defense.We study an R gene locus in the model plant Arabidopsis that confers resistance to two different bacteria, and to a fungus. The locus comprises two R genes, RPS4 and RRS1, which encode two proteins that associate to form a receptor complex that recognizes AvrRps4 and PopP2 bacterial effectors. A similar, linked R gene pair, RPS4B and RRS1B, also recognizes AvrRps4, but not PopP2. Although RPS4 and RPS4B, and RRS1 and RRS1B, are closely related (each pair ~ 60% identical), RPS4 and RRS1B associate with each other but do not function; this also true of RPS4B and RRS1. We aim to define the specific interactions between protein domains of RPS4 and RRS1, or of RPS4B and RRS1B, that enable formation of a functional complex that (i) perceives effectors and (ii) then activates signaling. We also aim to understand why non-authentic combinations fail to function.RPS4 & RPS4B comprise 4 domains (AAAA or BBBB), and RRS1 & RRS1B comprise 5 domains (AAAAA or BBBBB). We will exchange domains between homologs, and test, for example, whether RPS4/RPS4B domain swaps BAAA, ABAA, AABA or AAAB function in combination with RRS1, and ABBB, BABB, BBAB and BBBA function in combination with RRS1B. We will also test RRS1/RRS1B swaps such as BAAAA, ABAAA etc in combination with RPS4. We can test function of RPS4 and RRS1 domain swap variants, within 3 days after transient expression of these genes in tobacco leaf tissue using Agrobacterium ("agroinfiltration"). We will also investigate domain/domain interactions by expressing each of the domains of these 4 proteins and looking for domain/domain interactions, both in yeast and after co-expression in plants and coimmunoprecipitation.By removing a domain from the end of RRS1, or by introducing mutations into this domain, we create an RPS4/RRS1 complex that constitutively activates defense. We will investigate the domain requirements of this constitutive activity. Wild type RRS1 suppresses this constitutive defence activation triggered by RPS4 and constitutive forms of RRS1; we will investigate the mechanisms of this suppression.RRS1 can form higher order associations with itself, so we will investigate RRS1 domain/domain interactions that might explain this multimerisation. Furthermore, the two ends of RRS1 appear to be able to interact with each other; we will investigate this in more detail.We can monitor protein/protein or domain/domain interactions in living cells using split fluorescent proteins. These do not fluoresce separately. When attached to proteins of interest, they only become active when brought together by protein/protein interactions of the domains to which they are attached. Using this method, we have found that the N terminal domains of RPS4 and RRS1 become closely associated only upon perception of AvrRps4 or PopP2 effectors. We will investigate the temporal and genetic requirements for this association, which we believe to be key to the reconfiguration of the protein complex that leads to activation