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

细胞质钙浓度的增加是植物受到病原体攻击时最早的细胞内反应之一，而钙的快速变化是PTI和ETI中免疫系统激活的先决条件。钙依赖性蛋白激酶（calcium -dependent protein kinase, CPKs）是钙的传感器和信号传递体，在植物免疫信号传导中起着重要作用。EXO70B1是囊胞复合体的一个亚基，EXO70B1的功能丧失会导致自身免疫。CPK5在CPK5- oe植物系中的信号传导增强，导致防御反应增强，病原体抗性增强，与在exo70B1突变体中观察到的相似。值得注意的是，两种情况下的防御表型都需要截断的核苷酸结合域和富含亮氨酸的重复序列（NLR）蛋白TIR-NBS2 （TN2）。在正向遗传筛选中，Tang小组发现CPK5，而不是其他CPK异构体，是exo70b1激活的免疫应答所必需的，这表明CPK5在植物免疫中起着独特的作用。TN2与CPK5的n端相互作用，包括变量和蛋白激酶结构域。Romeis小组可以证明，当在exo70B1原生质体中异位表达时，CPK5表现出增强的组成蛋白激酶活性，这在tn2背景中是检测不到的。这些数据首次确定了非典型免疫受体与钙调节网络信号成分之间的直接联系，这是早期免疫反应和防御开始所必需的。本联合提案阐述了CPK5、EXO70B1和TN2在PTI和ETI期间防御信号发起和传播中的机制、相互要求和功能。目前对该领域的理解允许对TN2介导的CPK5和EXO70B1的调控提出两种模型：(i) CPK5可以被认为是TN2的监护人，其各自的全长NLR蛋白仍有待鉴定；（ii） TN2激活的免疫和EXO70B1的免疫增加是由CPK5和TN2之间的正反馈回路诱导的，通过TN2稳定活性CPK5。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.