Institute of Horticulture, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China.
Institute of Horticulture, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China.
Plant Sci. 2024 Oct;347:112194. doi: 10.1016/j.plantsci.2024.112194. Epub 2024 Jul 14.
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.
揭示效应子-宿主分子相互作用对于理解宿主对葡萄霜霉病的免疫机制以及设计创新的疾病管理策略至关重要。作为一种引起葡萄霜霉病的致病性卵菌，葡萄生单轴霉利用各种效应子来操纵宿主植物的防御系统。其中一种葡萄生单轴霉衍生的效应子是坏死和乙烯诱导肽 1（Nep1）样蛋白（PvNLP7），已知该蛋白在植物中能引起细胞死亡和免疫反应。然而，其潜在的分子机制尚不清楚，这促使我们开展了本研究。通过酵母双杂交筛选，我们鉴定出葡萄圆叶 ADP-核糖基化因子（VrARF1）是 PvNLP7 的宿主互作蛋白。这种相互作用通过双分子荧光互补（BiFC）和免疫共沉淀（Co-IP）实验得到了证实。在本氏烟中异源表达 VrARF1 验证了其在细胞质和细胞核中的积累以及诱导细胞死亡的能力。此外，在早期葡萄生单轴霉感染和瞬时表达 PvNLP7 时，VrARF1 基因强烈诱导表达。在葡萄和本氏烟中过表达 VrARF1 基因分别通过诱导防御相关基因 PR1 和 PR2 增强了对葡萄生单轴霉和辣椒疫霉的抗性。相反，在本氏烟中沉默同源基因 NbARF1 会显著减弱 PvNLP7 触发的细胞死亡并降低四个 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，从而使瞬时转化了 PvNLP7 的植物更容易受到卵菌病原体辣椒疫霉的侵害。这些发现突出了 ARF1 在介导 PvNLP7 诱导的免疫中的作用，并表明其作为工程抗病转基因植物抵抗卵菌病原体的潜在靶标。
