Calcium-regulated signalling network meets atypical NLR immune receptor: Control of CPK5-dependent plant defence initiation and immune signal propagation through TN2 and EXO70B1

细胞内钙离子浓度的增加是植物对病原菌攻击最早的细胞内反应之一，在PTI和ETI中，快速的钙变化是激活免疫系统的先决条件。钙依赖蛋白激酶(CPK)是钙离子的感受器和信号转运体，在植物免疫信号转导中起着重要的作用。EXO70B1是胞囊复合体的一个亚单位，其功能丧失会导致自身免疫。CPK5在CPK5-OE植物系中增强的信号转导导致增强的防御反应和增强的病原菌抗性，类似于在exo70B1突变体中观察到的。值得注意的是，这两种情况下的防御表型都需要一个截短的核苷酸结合域和富含亮氨酸的重复序列(NLR)蛋白TIR-NBS2(TN2)。在正向遗传筛查中，唐小组发现，exo70B1激活的免疫反应需要CPK5，而不是其他CPK亚型，这表明CPK5在植物免疫中发挥着独特的作用。Tn2与CPK5的N-末端相互作用，包括可变区和蛋白激酶结构域。Romeis小组可能表明，当在exo70B1原生质体中异位表达时，CPK5显示出增强的结构性蛋白激酶活性，这在TN2背景下是检测不到的。这些数据首次确认了非典型免疫受体和钙调节网络的信号成分之间的直接联系，钙调节网络是早期免疫反应和防御开始所必需的。这份联合提案阐述了CPK5、EXO70B1和TN2在PTI和ETI期间防御信号的启动和传播中的机制、相互要求和功能。目前对CPK5和EXO70B1的理解支持两种模型：(I)CPK5可以被认为是TN2的守护者，各自的全长NLR蛋白尚未确定；(Ii)TN2激活的免疫和exo70B1中的免疫增强是由CPK5和TN2之间的正反馈循环通过TN2稳定活性CPK5而诱导的。我们的研究旨在区分这两种情况。

An increase in the cytoplasmic calcium concentration is one of the earliest intracellular responses of plants exposed to a pathogen attack and rapid calcium changes are prerequisite to the activation of the immune system in both, PTI and ETI. Calcium-dependent protein kinases (CPKs) are calcium sensors and signal transmitters, and are important key players in plant immune signalling. EXO70B1 is a subunit of the exocyst complex and loss-of-function of EXO70B1 leads to autoimmunity. Enhanced signalling by CPK5 in CPK5-OE plant lines leads to enhanced defence responses and increased pathogen resistance similar to those observed in the exo70B1 mutant. Remarkably, the defence phenotypes in both scenarios require a truncated nucleotide binding domain and leucine-rich repeat (NLR) protein TIR-NBS2 (TN2). In a forward genetic screen, the Tang group found that CPK5, but not other CPK isoforms, is required for exo70B1-activated immune responses, indicating that CPK5 plays a unique role in plant immunity. TN2 interacts with the N-terminal part of CPK5 encompassing the variable and protein kinase domain. The Romeis group could show that upon ectopic expression in exo70B1 protoplasts, CPK5 displays enhanced constitutive protein kinase activity, which is not detectable in the tn2 background. These data provide the first identification of a direct link between an atypical immune receptor and a signalling component of the calcium-regulatory network that is required in the early immune response and for the onset of defence. This joint proposal addresses the mechanism, the mutual requirement, and the functions of CPK5, EXO70B1 and TN2 in defence signal initiation and propagation during PTI and ETI. The current understanding in the field allows the projection of two models for the TN2-mediated regulation of CPK5 and EXO70B1: (i) CPK5 can be considered a guardee of TN2 with a respective full-length NLR protein still to be identified, and (ii) TN2-activated immunity and increased immunity in exo70B1 is induced by a positive feedback loop between CPK5 and TN2 through the stabilization of active CPK5 by TN2. Our research aims to distinguish between these two scenarios.