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，从局部感染部位扩散到远端组织，其通过效应子触发的免疫（ETI，即识别病原体效应子的能力）和自身免疫（通常由效应子靶标的丧失引起）增强。拟南芥钙依赖性蛋白激酶（CDPK），CPK 5，是植物免疫中高度钙敏感的信号传导中心。通过NADPH氧化酶RBOHD的直接磷酸化产生ROS和EXO 70 B1的磷酸化调节外囊在运输关键PM免疫组分中的功能，控制质膜（PM）处的免疫应答。在一项合作努力中，Tang和Tongis小组可以证明CPK 5，而不是其他CDPK亚型，是exo 70 B1激活和TN 2依赖性自身免疫所必需的。此外，CPK 5信号传导触发基于转录的抗性，导致全身免疫（通过SARD 1和CAMTA 3转录因子）。比较在体内磷酸蛋白质组学分析的cpk 5突变株确定防御相关的转录调节CAMTA 3以及MAPK信号级联组件作为潜在的CPK 5底物蛋白。联合实验证实，CPK 5直接磷酸化CAMTA 3，两种蛋白质共定位于植物细胞核。此外，CPK 5-OE株系和exo 70 B1突变体对白粉病的增强的抗病性在显性camta 3 -3D（A855 V）等位基因的存在下受到损害。这些数据与报道的CAMTA 3作为植物免疫的负转录调节因子的功能一致;如Lee Group最近所验证的，CAMTA 3核定位和蛋白质稳定性由通过MAPK的磷酸化控制。在这项联合提案中，我们将协同努力研究：（i）依赖CPK 5的PM处EXO 70 B介导的免疫组分转运。(ii)通过CPK 5和MAPK的动态磷酸化对核负性免疫调节剂CAMTA 3的双重控制。(iii)CPK 5酶定位和PM与细胞核之间改组的机制基础。(iv)CDPK和MAPK信号传导在局部信号启动到长期系统防御表现中的交叉干扰考虑到所研究的免疫组分的详细生物化学、分子和遗传表征，以及从遗传学到体内磷酸蛋白质组学的广泛技术专长，这项建议提供了一个独特的机会，以揭示长期讨论的普遍存在的监管原则的潜在机制，（但不仅是）免疫信号传导-即途径（CDPK/MAPK级联）的相互作用、汇聚于共同关键调节底物（例如CAMTA 3）的多种翻译后修饰的动态调节以及信号传导组分的亚细胞再分布的作用-促进分子水平上的前所未有的解决。

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.