Calcium-dependent and independent phospho-control of plant immunity by integrating kinase signalling with transcriptional regulation.

植物免疫是不同抗性强度的连续体，从局部侵染部位扩散到远端组织，通过效应器触发的免疫(ETI，即识别病原体效应器的能力)和自身免疫(通常由于失去效应器靶标而导致)而得到增强。拟南芥钙依赖蛋白激酶(CDPK)CPK5是植物免疫中一个高度钙敏感的信号枢纽。它通过直接磷酸化NADPH-氧化酶RBOHD产生ROS和磷酸化EXO70B1来调控质膜(PM)的免疫反应，从而调节PM关键免疫成分运输中的外囊功能。在一项合作的努力中，唐和罗密斯的研究小组可以证明，CPK5，而不是其他CDPK亚型，是exo70B1激活和TN2依赖的自身免疫所必需的。此外，CPK5信号触发基于转录的抗性，导致系统免疫(通过SARD1和CAMTA3转录因子)。对cpk5突变系的体内比较磷蛋白质组学分析发现，与防御相关的转录调控因子CAMTA3和MAPK信号级联组分是潜在的cpk5底物蛋白。联合实验证明，CPK5直接磷酸化CAMTA3，两种蛋白共同定位在植物细胞核中。此外，据报道，CPK5-OE系和exo70B1突变体对白粉病的增强抗性在显性Camta3-3D(A855V)等位基因的存在下受到影响。这些数据与已报道的CAMTA3作为植物免疫负转录调节因子的功能是一致的；Lee小组最近证实，CAMTA3的核定位和蛋白质稳定性是通过MAPKs的磷酸化来控制的。在这项联合提案中，我们将协同努力调查：(I)EXO70B介导的免疫成分在PM依赖CPK5的转运。(Ii)通过CPK5和MAPKs的动态磷酸化双重调控核负性免疫调节因子CAMTA3。(3)CPK5酶定位和在质膜和细胞核之间洗牌的力学基础。(Iv)CDPK和MAPK信号在局部信号启动到长期系统防御表现中的交叉干扰鉴于所研究的免疫成分的详细生化、分子和遗传特征，以及从遗传学到体内磷蛋白质组学的广泛技术专长，这一提议提供了一个独特的机会，以揭示长期讨论的免疫信号普遍存在的调节原理的潜在机制--即通路(CDPK/MAPK级联)的相互作用，即聚集在共同关键调节底物(例如CAMTA3)上的多个翻译后修饰的动态调节，以及信号组件的亚细胞重新分布的作用--促进在分子水平上的以前的解决方案。

Plant immunity is a continuum of varying resistance intensities, spreading from local infection sites to distal tissues, which is augmented by effector-triggered immunity (ETI, i.e. the ability to recognize pathogen effectors) and autoimmunity (typically resulting from loss of an effector target). The Arabidopsis thaliana calcium-dependent protein kinase (CDPK), CPK5, is a highly calcium sensitive signalling hub in plant immunity. It controls immune responses at the plasma membrane (PM) via direct phosphorylation of the NADPH-oxidase RBOHD for ROS production and phosphorylation of EXO70B1 to modulate exocyst function in the transport of key PM immune components. In a collaborative effort, the Tang and Romeis groups could show that CPK5, but not other CDPK isoforms, is required for exo70B1-activated and TN2-dependent autoimmunity. Additionally, CPK5 signalling triggers transcription-based resistance leading to systemic immunity (via SARD1 and CAMTA3 transcription factors). Comparative in vivo phosphoproteomics analysis of cpk5 mutant lines identified defence-related transcriptional regulator CAMTA3 as well as MAPK-signalling cascade components as potential CPK5 substrate proteins. Joint experiments verify that CPK5 phosphorylates CAMTA3 directly, and both proteins co-localize in the plant nucleus. Furthermore, enhanced disease resistance to powdery mildew, reported for CPK5-OE lines and for exo70B1 mutants, is compromised in the presence of the dominant camta3-3D (A855V) allele. These data are consistent with the reported CAMTA3 function as a negative transcriptional regulator of plant immunity; as validated by the Lee group recently, CAMTA3 nuclear localization and protein stability is controlled by phosphorylation through MAPKs. In this joint proposal, we will synergize our efforts to investigate:(i) EXO70B-mediated transport of immune components at the PM in dependency of CPK5. (ii) Dual control of nuclear negative immune regulator CAMTA3 by dynamic phosphorylation through CPK5 and MAPKs. (iii) Mechanistic basis for CPK5 enzyme location and shuffling between the PM and the nucleus. (iv) Cross-interference between CDPK and MAPK signalling in local signal initiation to long-term systemic defence manifestationGiven the detailed biochemical, molecular, and genetic characterization of the immune components investigated, and the broad technical expertise spanning from genetics to in vivo phosphoproteomics, this proposal provides a unique opportunity to uncover underlying mechanisms of long discussed regulatory principles prevalent to (but not only) immune signalling – that is the interplay of pathways (CDPK/MAPK cascades), the dynamic modulation of multiple post-translational modifications converging on a common key regulatory substrate (e.g. CAMTA3), and the role of subcellular re-distribution of signalling components - facilitating an unpreceded resolution on the molecular level.