Berkey Robert, Zhang Yi, Ma Xianfeng, King Harlan, Zhang Qiong, Wang Wenming, Xiao Shunyuan
Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, Maryland (R.B., Y.Z., X.M., H.K., Q.Z., S.X.).
The Rice Research Institute, Sichuan Agricultural University, Chengdu 611130, China (W.W.); and.
Plant Physiol. 2017 Jan;173(1):600-613. doi: 10.1104/pp.16.01539. Epub 2016 Nov 17.
Upon penetration of the host cell wall, the powdery mildew fungus develops a feeding structure named the haustorium in the invaded host cell. Concomitant with haustorial biogenesis, the extrahaustorial membrane (EHM) is formed to separate the haustorium from the host cell cytoplasm. The Arabidopsis resistance protein RPW8.2 is specifically targeted to the EHM where it activates haustorium-targeted resistance against powdery mildew. RPW8.2 belongs to a small family with six members in Arabidopsis (Arabidopsis thaliana). Whether Homologs of RPW8 (HR) 1 to HR4 are also localized to the EHM and contribute to resistance has not been determined. Here, we report that overexpression of HR1, HR2, or HR3 led to enhanced resistance to powdery mildew, while genetic depletion of HR2 or HR3 resulted in enhanced susceptibility, indicating that these RPW8 homologs contribute to basal resistance. Interestingly, we found that N-terminally YFP-tagged HR1 to HR3 are also EHM-localized. This suggests that EHM-targeting is an ancestral feature of the RPW8 family. Indeed, two RPW8 homologs from Brassica oleracea tested also exhibit EHM-localization. Domain swapping analysis between HR3 and RPW8.2 suggests that sequence diversification in the N-terminal 146 amino acids of RPW8.2 probably functionally distinguishes it from other family members. Moreover, we found that N-terminally YFP-tagged HR3 is also localized to the plasma membrane and the fungal penetration site (the papilla) in addition to the EHM. Using this unique feature of YFP-HR3, we obtained preliminary evidence to suggest that the EHM is unlikely derived from invagination of the plasma membrane, rather it may be mainly synthesized de novo.
在穿透宿主细胞壁后，白粉菌在被侵染的宿主细胞中形成一种名为吸器的取食结构。伴随着吸器的生物发生，吸器外膜（EHM）形成，将吸器与宿主细胞质分隔开。拟南芥抗性蛋白RPW8.2特异性定位于EHM，在那里它激活针对白粉病的吸器靶向抗性。RPW8.2属于一个小家族，在拟南芥（Arabidopsis thaliana）中有六个成员。RPW8同源物（HR）1至HR4是否也定位于EHM并有助于抗性尚未确定。在这里，我们报告HR1、HR2或HR3的过表达导致对白粉病的抗性增强，而HR2或HR3的基因缺失导致易感性增强，表明这些RPW8同源物有助于基础抗性。有趣的是，我们发现N端带有黄色荧光蛋白（YFP）标签的HR1至HR3也定位于EHM。这表明靶向EHM是RPW8家族的一个祖先特征。事实上，测试的来自甘蓝型油菜的两个RPW8同源物也表现出EHM定位。HR3和RPW8.2之间的结构域交换分析表明，RPW8.2 N端146个氨基酸的序列多样化可能在功能上使其与其他家族成员区分开来。此外，我们发现N端带有YFP标签的HR3除了定位于EHM外，还定位于质膜和真菌穿透位点（乳头）。利用YFP-HR3的这一独特特征，我们获得了初步证据，表明EHM不太可能源自质膜内陷，相反，它可能主要是重新合成的。
