Liu Yunxiao, Lan Xia, Song Shiren, Yin Ling, Dry Ian B, Qu Junjie, Xiang Jiang, Lu Jiang
College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China.
Center for Viticulture and Enology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China.
Front Plant Sci. 2018 Apr 13;9:286. doi: 10.3389/fpls.2018.00286. eCollection 2018.
Downy mildew is one of the most destructive diseases of grapevine, causing tremendous economic loss in the grape and wine industry. The disease agent is an obligate biotrophic oomycete, from which over 100 candidate RXLR effectors have been identified. In this study, 83 candidate RXLR effector genes () were cloned from the isolate "JL-7-2" genome. The results of the yeast signal sequence trap assay indicated that most of the candidate effectors are secretory proteins. The biological activities and subcellular localizations of all the 83 effectors were analyzed via a heterologous -mediated expression system. Results showed that 52 effectors could completely suppress cell death triggered by elicitin, 10 effectors could partially suppress cell death, 11 effectors were unable to suppress cell death, and 10 effectors themselves triggered cell death. Live-cell imaging showed that the majority of the effectors (76 of 83) could be observed with informative fluorescence signals in plant cells, among which 34 effectors were found to be targeted to both the nucleus and cytosol, 29 effectors were specifically localized in the nucleus, and 9 effectors were targeted to plant membrane system. Interestingly, three effectors PvRXLR61, 86 and 161 were targeted to chloroplasts, and one effector PvRXLR54 was dually targeted to chloroplasts and mitochondria. However, western blot analysis suggested that only PvRXLR86 carried a cleavable -terminal transit peptide and underwent processing . Many effectors have previously been predicted to target organelles, however, to the best of our knowledge, this is the first study to provide experimental evidence of oomycete effectors targeted to chloroplasts and mitochondria.
霜霉病是葡萄最具毁灭性的病害之一，给葡萄和葡萄酒产业造成了巨大的经济损失。病原菌是一种专性活体营养卵菌，已从中鉴定出100多个候选RXLR效应子。在本研究中，从“JL-7-2”分离株基因组中克隆了83个候选RXLR效应子基因。酵母信号序列捕获试验结果表明，大多数候选效应子是分泌蛋白。通过异源介导的表达系统分析了所有83个效应子的生物学活性和亚细胞定位。结果表明，52个效应子能完全抑制激发素引发的细胞死亡，10个效应子能部分抑制细胞死亡，11个效应子不能抑制细胞死亡，10个效应子自身能引发细胞死亡。活细胞成像显示，大多数效应子（83个中的76个）在植物细胞中可观察到有意义的荧光信号，其中34个效应子定位于细胞核和细胞质，29个效应子特异性定位于细胞核，9个效应子定位于植物膜系统。有趣的是，三个效应子PvRXLR61、86和161定位于叶绿体，一个效应子PvRXLR54双定位于叶绿体和线粒体。然而，蛋白质免疫印迹分析表明，只有PvRXLR86携带可裂解的C末端转运肽并经历加工过程。此前许多效应子被预测定位于细胞器，然而，据我们所知，这是第一项提供卵菌效应子定位于叶绿体和线粒体的实验证据的研究。
