Kong Guanghui, Zhao Yao, Jing Maofeng, Huang Jie, Yang Jin, Xia Yeqiang, Kong Liang, Ye Wenwu, Xiong Qin, Qiao Yongli, Dong Suomeng, Ma Wenbo, Wang Yuanchao
College of Plant Protection, Nanjing Agricultural University, Nanjing, China.
Department of Plant Pathology and Microbiology, University of California, Riverside, Riverside, California, United States of America; National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing, China.
PLoS Pathog. 2015 Aug 28;11(8):e1005139. doi: 10.1371/journal.ppat.1005139. eCollection 2015 Aug.
Plant pathogens secrete an arsenal of effector proteins to impair host immunity. Some effectors possess enzymatic activities that can modify their host targets. Previously, we demonstrated that a Phytophthora sojae RXLR effector Avr3b acts as a Nudix hydrolase when expressed in planta; and this enzymatic activity is required for full virulence of P. sojae strain P6497 in soybean (Glycine max). Interestingly, recombinant Avr3b produced by E. coli does not have the hydrolase activity unless it was incubated with plant protein extracts. Here, we report the activation of Avr3b by a prolyl-peptidyl isomerase (PPIase), cyclophilin, in plant cells. Avr3b directly interacts with soybean cyclophilin GmCYP1, which activates the hydrolase activity of Avr3b in a PPIase activity-dependent manner. Avr3b contains a putative Glycine-Proline (GP) motif; which is known to confer cyclophilin-binding in other protein substrates. Substitution of the Proline (P132) in the putative GP motif impaired the interaction of Avr3b with GmCYP1; as a result, the mutant Avr3bP132A can no longer be activated by GmCYP1, and is also unable to promote Phytophthora infection. Avr3b elicits hypersensitive response (HR) in soybean cultivars producing the resistance protein Rps3b, but Avr3bP132A lost its ability to trigger HR. Furthermore, silencing of GmCYP1 rendered reduced cell death triggered by Avr3b, suggesting that GmCYP1-mediated Avr3b maturation is also required for Rps3b recognition. Finally, cyclophilins of Nicotiana benthamiana can also interact with Avr3b and activate its enzymatic activity. Overall, our results demonstrate that cyclophilin is a "helper" that activates the enzymatic activity of Avr3b after it is delivered into plant cells; as such, cyclophilin is required for the avirulence and virulence functions of Avr3b.
植物病原体分泌一系列效应蛋白以削弱宿主免疫力。一些效应蛋白具有能够修饰其宿主靶标的酶活性。此前，我们证明大豆疫霉RXLR效应蛋白Avr3b在植物中表达时作为一种Nudix水解酶发挥作用；并且这种酶活性是大豆疫霉菌株P6497在大豆（Glycine max）中完全致病所必需的。有趣的是，大肠杆菌产生的重组Avr3b没有水解酶活性，除非它与植物蛋白提取物一起孵育。在这里，我们报道了植物细胞中脯氨酰 - 肽基异构酶（PPIase）亲环蛋白对Avr3b的激活作用。Avr3b直接与大豆亲环蛋白GmCYP1相互作用，后者以PPIase活性依赖的方式激活Avr3b的水解酶活性。Avr3b含有一个假定的甘氨酸 - 脯氨酸（GP）基序；已知该基序可赋予其他蛋白质底物与亲环蛋白结合的能力。假定的GP基序中的脯氨酸（P132）被取代会损害Avr3b与GmCYP1的相互作用；结果，突变体Avr3bP132A不再能被GmCYP1激活，也无法促进疫霉感染。Avr3b在产生抗性蛋白Rps3b的大豆品种中引发超敏反应（HR），但Avr3bP132A失去了触发HR的能力。此外，GmCYP1的沉默导致由Avr3b引发的细胞死亡减少，这表明GmCYP1介导的Avr3b成熟对于Rps3b识别也是必需的。最后，本氏烟草的亲环蛋白也可以与Avr3b相互作用并激活其酶活性。总体而言，我们的结果表明亲环蛋白是一种“辅助蛋白”，在Avr3b被递送到植物细胞后激活其酶活性；因此，亲环蛋白是Avr3b无毒和致病功能所必需的。
