RESUMO
Resistance (R) proteins recognize pathogen avirulence (Avr) proteins by direct or indirect binding and are multidomain proteins generally carrying a nucleotide binding (NB) and a leucine-rich repeat (LRR) domain. Two NB-LRR protein-coding genes from rice (Oryza sativa), RGA4 and RGA5, were found to be required for the recognition of the Magnaporthe oryzae effector AVR1-CO39. RGA4 and RGA5 also mediate recognition of the unrelated M. oryzae effector AVR-Pia, indicating that the corresponding R proteins possess dual recognition specificity. For RGA5, two alternative transcripts, RGA5-A and RGA5-B, were identified. Genetic analysis showed that only RGA5-A confers resistance, while RGA5-B is inactive. Yeast two-hybrid, coimmunoprecipitation, and fluorescence resonance energy transfer-fluorescence lifetime imaging experiments revealed direct binding of AVR-Pia and AVR1-CO39 to RGA5-A, providing evidence for the recognition of multiple Avr proteins by direct binding to a single R protein. Direct binding seems to be required for resistance as an inactive AVR-Pia allele did not bind RGA5-A. A small Avr interaction domain with homology to the Avr recognition domain in the rice R protein Pik-1 was identified in the C terminus of RGA5-A. This reveals a mode of Avr protein recognition through direct binding to a novel, non-LRR interaction domain.
Assuntos
Proteínas Fúngicas/genética , Magnaporthe/genética , Oryza/genética , Proteínas de Plantas/genética , Processamento Alternativo , Sequência de Aminoácidos , Sítios de Ligação/genética , Resistência à Doença/genética , Transferência Ressonante de Energia de Fluorescência , Proteínas Fúngicas/metabolismo , Interações Hospedeiro-Patógeno , Immunoblotting , Magnaporthe/metabolismo , Magnaporthe/fisiologia , Microscopia Confocal , Dados de Sequência Molecular , Mutação , Oryza/metabolismo , Oryza/microbiologia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Attack and counter-attack impose strong reciprocal selection on pathogens and hosts, leading to development of arms race evolutionary dynamics. Here we show that Magnaporthe oryzae avirulence gene AVR-Pik and the cognate rice resistance (R) gene Pik are highly variable, with multiple alleles in which DNA replacements cause amino acid changes. There is tight recognition specificity of the AVR-Pik alleles by the various Pik alleles. We found that AVR-Pik physically binds the N-terminal coiled-coil domain of Pik in a yeast two-hybrid assay as well as in an in planta co-immunoprecipitation assay. This binding specificity correlates with the recognition specificity between AVR and R genes. We propose that AVR-Pik and Pik are locked into arms race co-evolution driven by their direct physical interactions.