Functional characterization of bacterial type III effectors in plant cells

许多革兰氏阴性致病菌的致病性依赖于一种III型分泌系统，该系统将细菌效应蛋白转移到宿主细胞中。这些III型效应蛋白(T3E)已经进化到在真核环境中发挥作用，以促进宿主的定植。这些T3E改变宿主细胞过程的分子机制还远未被了解。在我们的初步工作中，我们能够证明来自丁香假单胞菌的T3E HopZ1a在植物细胞内与以前未描述的remorin蛋白相互作用。进一步的相互作用研究表明，Remorin蛋白与免疫激酶PBS1结合并被其特异性磷酸化，还与E3连接酶SINA4相互作用。在flg22刺激后，remorin的过表达导致基础PTI标志物基因表达增加，并诱导ROS的产生。我们的发现导致了这样的假设，即在PTI过程中，remorin可能与PBS1一起作用于复合体，因此是HopZ1a操纵免疫信号的靶点。在拟议的项目中，我们将研究HopZ1a干扰remorin功能的生化和分子机制，以及这如何导致免疫抑制。此外，我们可以证明T3E XopS形式的野油菜黄单胞菌与转录因子WRKY40和E3-泛素连接酶组成的蛋白质对在植物细胞核内相互作用。E3连接酶在体外和体外与WRKY40相互作用，并在体外泛素化转录因子。因此，在烟草叶片中的瞬时表达表明WRKY40经历了快速的蛋白酶体蛋白周转。然而，WRKY40蛋白在与XopS共表达时强烈积累，这表明XopS干扰了WRKY40蛋白酶体的周转，从而干扰了需要降解这种植物防御负调控因子的防御基因诱导。该项目的第二个目标是揭示XopS如何改变WRKY40的功能，以及这如何影响宿主植物诱导的防御反应。

Pathogenicity of many Gram-negative pathogenic bacteria depends on a type III secretion system, which translocates bacterial effector proteins into the host cell. These type III effector proteins (T3Es) have evolved to function in a eukaryotic context in order to promote colonization of the host. The molecular mechanisms by which these T3Es alter host cellular processes are far from being understood. In our preliminary work we were able to show that HopZ1a, a T3E from Pseudomonas syringae, interacts with a previously uncharacterized remorin protein inside plant cells. Further interaction studies indicate that the remorin protein binds to and is specifically phosphorylated by the immune kinase PBS1 and also interacts with the E3 ligase SINA4. Overexpression of the remorin leads to increased basal PTI marker gene expression and induced ROS production after a flg22 stimulus. Our findings led to the hypothesis that the remorin might act in a complex together with PBS1 during PTI and hence is targeted by HopZ1a to manipulate immune signaling. Within the proposed project we will investigate the biochemical and molecular mechanism by which HopZ1a interferes with remorin function and how this leads to suppression of immunity. In addition, we could show that the T3E XopS form Xanthomonas campestris interacts inside the plant cell nucleus with a protein pair consisting of the transcription factor WRKY40 and an E3-ubiquitin ligase. The E3 ligase interacts with WRKY40 in vitro and in planta and ubiquitinates the transcription factor in vitro. Accordingly, transient expression in leaves of Nicotiana benthamiana suggests that WRKY40 undergoes rapid proteasomal protein turnover. However, WRKY40 protein strongly accumulates upon co-expression with XopS, indicating that XopS interferes with proteasomal turnover of WRKY40 and thus could interfere with defense gene induction that requires degradation of this negative regulator of plant defense. The second aim of the project is to unravel how XopS alters WRKY40 function and how this impacts on induced defense responses in host plants.