Rice expressing the Pi-ta gene is resistant to strains of the rice blast fungus, Magnaporthe grisea, expressing AVR-Pita in a gene-for-gene relationship. Pi-ta encodes a putative cytoplasmic receptor with a centrally localized nucleotide-binding site and leucine-rich domain (LRD) at the C-terminus. AVR-Pita is predicted to encode a metalloprotease with an N-terminal secretory signal and pro-protein sequences. AVR-Pita176 lacks the secretory and pro-protein sequences. We report here that transient expression of AVR-Pita176 inside plant cells results in a Pi-ta-dependent resistance response. AVR-Pita176 protein is shown to bind specifically to the LRD of the Pi-ta protein, both in the yeast two-hybrid system and in an in vitro binding assay. Single amino acid substitutions in the Pi-ta LRD or in the AVR-Pita176 protease motif that result in loss of resistance in the plant also disrupt the physical interaction, both in yeast and in vitro. These data suggest that the AVR-Pita176 protein binds directly to the Pi-ta LRD region inside the plant cell to initiate a Pi-ta-mediated defense response.
水稻表達(dá)Pi-ta基因?qū)Φ疚敛”磉_(dá)AVR-Pita的生理小種以基因?qū)蚣僬f(shuō)的方式產(chǎn)生抗性。Pi-ta具有位于序列中部的核苷酸結(jié)合位點(diǎn)并在其C端具有亮氨酸富集區(qū)（LRD），它編碼一個(gè)可能的細(xì)胞質(zhì)受體。AVR-Pita在其N端具有分泌信號(hào)區(qū)和蛋白前體序列，它編碼可能編碼一種非金屬蛋白酶。AVR-Pita176缺失分泌信號(hào)區(qū)和蛋白前體序列。我們的實(shí)驗(yàn)結(jié)果表明在植物細(xì)胞中短暫表達(dá)AVR-Pita176會(huì)產(chǎn)生Pi-ta依賴型抗病反應(yīng)。AVR-Pita176蛋白在酵母雙雜系統(tǒng)和體外結(jié)合實(shí)驗(yàn)中都特異性的結(jié)合到Pi-ta的LRD區(qū)。在Pi-ta的LRD區(qū)或AVR-Pita176蛋白酶基序的單核苷酸替換都會(huì)導(dǎo)致植物抗性的喪失，也會(huì)在酵母或體外破壞兩者的結(jié)合。這些結(jié)果表明AVR-Pita176蛋白在植物細(xì)胞中直接結(jié)合到Pi-ta的LRD區(qū)來(lái)啟動(dòng)Pi-ta介導(dǎo)的防衛(wèi)反應(yīng)。
結(jié)果：使用瞬時(shí)表達(dá)系統(tǒng)驗(yàn)證AVR-Pita176與Pita176互作；酵母雙雜實(shí)驗(yàn)證明AVR-Pita176特異性的結(jié)合到Pita的LRD區(qū)；通過(guò)體外遠(yuǎn)外雜交（far-western）蛋白與蛋白互作實(shí)驗(yàn)也證明AVR-Pita176與Pita及它的LRD區(qū)結(jié)合。
討論：AVR-Pita176與Pita的互作及抗病基因與無(wú)毒基因的配體-受體模型。