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.
抗性（R）蛋白通過(guò)直接或間接結(jié)合方式，能夠識(shí)別病原菌的無(wú)毒（AVR）蛋白，R蛋白含有多個(gè)結(jié)構(gòu)域，通常含有NB（核苷酸結(jié)合位點(diǎn)）和LRR（富含亮氨酸重復(fù)結(jié)構(gòu)域）。水稻中2個(gè)編碼NB-LRR蛋白基因RGA4和RGA5，是識(shí)別稻瘟病效應(yīng)子AVR-CO39所必需的。RGA4和RGA5也能介導(dǎo)非相關(guān)的稻瘟病原菌效應(yīng)子AVR-Pia的識(shí)別，表明R蛋白具有雙重識(shí)別特異性。RGA5存在2個(gè)剪切轉(zhuǎn)錄本，RGA5-A和RAG5-B。遺傳分析表明只有RGA5-A具有抗性，而RGA5-B處于失活狀態(tài)。酵母雙雜、免疫共沉淀以及熒光共振能量轉(zhuǎn)移壽命成像表明AVR-Pia和AVR1-CO39能夠直接結(jié)合RGA5-A，這為直接結(jié)合單個(gè)抗性蛋白的多個(gè)Avr蛋白的識(shí)別提供了分子證據(jù)。直接結(jié)合好像是產(chǎn)生抗性所必需的，因?yàn)榉腔钚訟VR-Pia的等位基因不能結(jié)合RGA5-A。我們發(fā)現(xiàn)RGA5-A的C末端有一個(gè)小Avr互作域，與水稻抗性蛋白Pik-1的Avr識(shí)別結(jié)構(gòu)域同源。以上通過(guò)直接與新的非LRR互作結(jié)構(gòu)域結(jié)合方式揭示了一個(gè)Avr無(wú)毒蛋白識(shí)別模型。