School of Plant Sciences and Food Security, Tel Aviv University, Tel Aviv, Israel.
Department of Biotechnology Engineering, ORT Braude College, Karmiel, Israel.
PLoS Pathog. 2018 Jan 29;14(1):e1006880. doi: 10.1371/journal.ppat.1006880. eCollection 2018 Jan.
The Gram-negative bacterium Xanthomonas euvesicatoria (Xe) is the causal agent of bacterial spot disease of pepper and tomato. Xe delivers effector proteins into host cells through the type III secretion system to promote disease. Here, we show that the Xe effector XopAU, which is conserved in numerous Xanthomonas species, is a catalytically active protein kinase and contributes to the development of disease symptoms in pepper plants. Agrobacterium-mediated expression of XopAU in host and non-host plants activated typical defense responses, including MAP kinase phosphorylation, accumulation of pathogenesis-related (PR) proteins and elicitation of cell death, that were dependent on the kinase activity of the effector. XopAU-mediated cell death was not dependent on early signaling components of effector-triggered immunity and was also observed when the effector was delivered into pepper leaves by Xanthomonas campestris pv. campestris, but not by Xe. Protein-protein interaction studies in yeast and in planta revealed that XopAU physically interacts with components of plant immunity-associated MAP kinase cascades. Remarkably, XopAU directly phosphorylated MKK2 in vitro and enhanced its phosphorylation at multiple sites in planta. Consistent with the notion that MKK2 is a target of XopAU, silencing of the MKK2 homolog or overexpression of the catalytically inactive mutant MKK2K99R in N. benthamiana plants reduced XopAU-mediated cell death and MAPK phosphorylation. Furthermore, yeast co-expressing XopAU and MKK2 displayed reduced growth and this phenotype was dependent on the kinase activity of both proteins. Together, our results support the conclusion that XopAU contributes to Xe disease symptoms in pepper plants and manipulates host MAPK signaling through phosphorylation and activation of MKK2.
革兰氏阴性细菌黄单胞菌（Xanthomonas euvesicatoria，Xe）是引起辣椒和番茄细菌性斑点病的病原体。Xe 通过 III 型分泌系统将效应蛋白输送到宿主细胞中，以促进疾病的发生。在这里，我们表明，在许多黄单胞菌物种中保守的 Xe 效应子 XopAU 是一种具有催化活性的蛋白激酶，有助于促进辣椒植物的疾病症状的发展。农杆菌介导的 XopAU 在宿主和非宿主植物中的表达激活了典型的防御反应，包括 MAP 激酶磷酸化、病程相关（PR）蛋白的积累和细胞死亡的诱导，这些反应依赖于效应子的激酶活性。XopAU 介导的细胞死亡不依赖于效应触发免疫的早期信号成分，当效应子由野油菜黄单胞菌 pv. campestris 而不是由 Xe 递送到辣椒叶片中时，也观察到这种现象。酵母和体内的蛋白-蛋白相互作用研究表明，XopAU 与植物免疫相关 MAP 激酶级联的成分发生物理相互作用。值得注意的是，XopAU 在体外直接磷酸化 MKK2，并增强其在体内多个位点的磷酸化。与 MKK2 是 XopAU 的靶标的观点一致，沉默 MKK2 同源物或在 N. benthamiana 植物中过表达催化失活的突变体 MKK2K99R 减少了 XopAU 介导的细胞死亡和 MAPK 磷酸化。此外，共表达 XopAU 和 MKK2 的酵母显示出生长减少，这种表型依赖于这两种蛋白的激酶活性。总之，我们的结果支持 XopAU 有助于辣椒植物中 Xe 疾病症状的结论，并通过磷酸化和激活 MKK2 来操纵宿主 MAPK 信号。
