Abstract：
Revealing the effector-host molecular interactions is crucial for understanding the host immunity against Plasmopara viticola and devising innovative disease management strategies. As a pathogenic oomycete causing grapevine downy mildew, Plasmopara viticola employs various effectors to manipulate the defense systems of host plants. One of these P. viticola derived effectors is necrosis- and ethylene-inducing peptide 1 (Nep1) -like protein (PvNLP7), which has been known to elicit cell death and immune responses in plants. However, the underlying molecular mechanisms remain obscure, prompting the focus of this study. Through yeast two-hybrid screening, we have identified the Vitis rotundifolia ADP-ribosylation factor (VrARF1) as a host interactor of PvNLP7. This interaction is corroborated through bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (Co-IP) assays. Heterologous expression of VrARF1 in Nicotiana benthamiana verifies its accumulation in both the cytoplasm and nucleus, and induction of cell death. Moreover, the VrARF1 gene is strongly induced during early P. viticola infection and upon PvNLP7 transient expression. Overexpression of the VrARF1 gene in grapevine and N. benthamiana enhances resistance to P. viticola and Phytophthora capsici, respectively, via induction of defense related genes PR1 and PR2. Conversely, virus-induced gene silencing (VIGS) of NbARF1 in N. benthamiana, homologous to VrARF1, markedly attenuates PvNLP7-triggered cell death and reduces the expression of four PTI marker genes (PTI5, Acre31, WRKY7 and Cyp71D20) and two defense related genes (PR1 and PR2), rendering plants transiently transformed with PvNLP7 more susceptible to oomycete P. capsici. These findings highlight the role of ARF1 in mediating PvNLP7-induced immunity and indicate its potential as a target for engineering disease-resistant transgenic plants against oomycete pathogens.
摘要：
揭示效应子与宿主的分子相互作用对于了解宿主对变形虫的免疫力和制定创新的疾病管理策略至关重要。作为一种致病卵菌，引起葡萄霜霉病，Plasmoparaviticola使用各种效应子操纵寄主植物的防御系统。这些衍生的P.viticola效应子之一是坏死和乙烯诱导肽1（Nep1）样蛋白（PvNLP7），已知在植物中引起细胞死亡和免疫反应。然而,潜在的分子机制仍然模糊，提示了本研究的重点。通过酵母双杂交筛选,我们已经确定了轮叶葡萄ADP-核糖基化因子（VrARF1）是PvNLP7的宿主相互作用因子。这种相互作用通过双分子荧光互补(BiFC)和共免疫沉淀(Co-IP)测定得到证实。VrARF1在烟草中的异源表达证实了其在细胞质和细胞核中的积累，和诱导细胞死亡。此外，VrARF1基因在早期卵黄杆菌感染期间和PvNLP7瞬时表达时被强烈诱导。VrARF1基因在葡萄和N.benthamiana中的过表达增强了对假单胞菌和辣椒疫霉的抗性，分别,通过诱导防御相关基因PR1和PR2。相反，病毒诱导的NbarF1基因沉默（VIGS）。与VrARF1同源，显着减弱PvNLP7触发的细胞死亡，并减少四个PTI标记基因（PTI5，Acre31，WRKY7和Cyp71D20）和两个防御相关基因（PR1和PR2）的表达，使用PvNLP7瞬时转化的植物对卵菌P.capsici更敏感。这些发现强调了ARF1在介导PvNLP7诱导的免疫中的作用，并表明了其作为针对卵菌病原体工程化抗病转基因植物的靶标的潜力。
