RESUMO
Detection of pathogens by plants is mediated by intracellular nucleotide-binding site leucine-rich repeat (NLR) receptor proteins. NLR proteins are defined by their stereotypical multidomain structure: an N-terminal Toll-interleukin receptor (TIR) or coiled-coil (CC) domain, a central nucleotide-binding (NB) domain, and a C-terminal leucine-rich repeat (LRR). The plant innate immune system contains a limited NLR repertoire that functions to recognize all potential pathogens. We isolated Response to the bacterial type III effector protein HopBA1 (RBA1), a gene that encodes a TIR-only protein lacking all other canonical NLR domains. RBA1 is sufficient to trigger cell death in response to HopBA1. We generated a crystal structure for HopBA1 and found that it has similarity to a class of proteins that includes esterases, the heme-binding protein ChaN, and an uncharacterized domain of Pasteurella multocida toxin. Self-association, coimmunoprecipitation with HopBA1, and function of RBA1 require two previously identified TIR-TIR dimerization interfaces. Although previously described as distinct in other TIR proteins, in RBA1 neither of these interfaces is sufficient when the other is disrupted. These data suggest that oligomerization of RBA1 is required for function. Our identification of RBA1 demonstrates that "truncated" NLRs can function as pathogen sensors, expanding our understanding of both receptor architecture and the mechanism of activation in the plant immune system.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/química , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Doenças das Plantas/genética , Proteínas de Plantas/química , Arabidopsis/imunologia , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/imunologia , Sítios de Ligação , Morte Celular/genética , Morte Celular/imunologia , Cristalografia por Raios X , Erwinia/patogenicidade , Erwinia/fisiologia , Interações Hospedeiro-Patógeno , Modelos Moleculares , Mutação , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Imunidade Vegetal/genética , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Pseudomonas syringae/patogenicidade , Pseudomonas syringae/fisiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Transdução de Sinais , Nicotiana/genética , Nicotiana/imunologia , Nicotiana/microbiologia , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/metabolismoRESUMO
Pseudomonas syringae strains deliver variable numbers of type III effector proteins into plant cells during infection. These proteins are required for virulence, because strains incapable of delivering them are nonpathogenic. We implemented a whole-genome, high-throughput screen for identifying P. syringae type III effector genes. The screen relied on FACS and an arabinose-inducible hrpL sigma factor to automate the identification and cloning of HrpL-regulated genes. We determined whether candidate genes encode type III effector proteins by creating and testing full-length protein fusions to a reporter called Delta79AvrRpt2 that, when fused to known type III effector proteins, is translocated and elicits a hypersensitive response in leaves of Arabidopsis thaliana expressing the RPS2 plant disease resistance protein. Delta79AvrRpt2 is thus a marker for type III secretion system-dependent translocation, the most critical criterion for defining type III effector proteins. We describe our screen and the collection of type III effector proteins from two pathovars of P. syringae. This stringent functional criteria defined 29 type III proteins from P. syringae pv. tomato, and 19 from P. syringae pv. phaseolicola race 6. Our data provide full functional annotation of the hrpL-dependent type III effector suites from two sequenced P. syringae pathovars and show that type III effector protein suites are highly variable in this pathogen, presumably reflecting the evolutionary selection imposed by the various host plants.