Data_Sheet_1_The Exocyst Complex Subunit EXO70E1-V From Haynaldia villosa Interacts With Wheat Powdery Mildew Resistance Gene CMPG1-V.docx

EXO70 belongs to the exocyst complex subunit that plays a critical role in regulating plant cell polarity establishment and defense response. A previous study proved that the E3 ligase CMPG1-V from Haynaldia villosa, a diploid wheat relative, positively regulates the resistance to wheat powdery mildew (Pm), caused by fungus Blumeria graminis f.sp tritici (Bgt). In this study, a member of EXO70 superfamily named EXO70E1-V was isolated from H. villosa, and EXO70E1-V interacted with CMPG1-V were shown by yeast two-hybrid (Y2H), pull-down assay, bimolecular fluorescence complementation (BiFC) assay, and luciferase complementation imaging (LCI) assay. It is localized in various subcellular organs, i.e., plasma membrane (PM) and endoplasmic reticulum. Co-expression of EXO70E1-V and CMPG1-V showed dot-like structure fluorescence signals that were mainly in PM and nucleus. Expression of EXO70E1-V was relatively higher in leaf and was significantly induced by Bgt infection and exogenous application of hormones such as salicylic acid. Transient or stable overexpression of EXO70E1-V could not enhance/decrease the Pm resistance level, suggesting overexpression of EXO70E1-V alone has no impact on Pm resistance in wheat.

EXO70属于胞泌复合体（exocyst complex）亚基，在调控植物细胞极性建立与防御响应过程中发挥关键作用。此前已有研究证实，来自二倍体小麦近缘种簇毛麦（Haynaldia villosa）的E3泛素连接酶（E3 ligase）CMPG1-V，可正调控由真菌小麦白粉菌（Blumeria graminis f.sp tritici, Bgt）引起的小麦白粉病（powdery mildew, Pm）抗性。本研究从簇毛麦中分离得到EXO70超家族成员EXO70E1-V，并通过酵母双杂交（yeast two-hybrid, Y2H）、pull-down实验、双分子荧光互补（bimolecular fluorescence complementation, BiFC）实验以及荧光素酶互补成像（luciferase complementation imaging, LCI）实验，证实EXO70E1-V与CMPG1-V存在相互作用。该蛋白定位于多种亚细胞细胞器，包括质膜（plasma membrane, PM）与内质网。将EXO70E1-V与CMPG1-V共表达后，可观察到主要分布于质膜与细胞核的斑点状荧光信号。EXO70E1-V在叶片中表达量相对较高，且可被Bgt侵染以及外源施加水杨酸等激素显著诱导。无论是瞬时过表达还是稳定过表达EXO70E1-V，均未显著改变小麦对白粉病的抗性水平，这表明单独过表达EXO70E1-V对小麦白粉病抗性无显著影响。