Molecular analysis of leucine rich-repeat receptor protein-mediated immune signaling

富含亮氨酸重复序列(LRR)胞外结构域的PRRs介导了蛋白质免疫原模式的识别。LRR类型的PRRs有两种形式：LRR受体蛋白(LRR-RK)和LRR受体蛋白(LRR-RP)，LRR-RK携带细胞内蛋白激活区，而LRR-RP缺乏细胞内功能区。最近有人假设，在没有配体的情况下组成的LRR-RP/SOBIR1异构体配合物(SOBIR1本身就是LRR-RK)相当于双分子或两部分LRR-RK型PRR。通过LRR-RK或LRR-RP型PRRs介导的免疫激活的分子机制尚未得到证实。事实上，我们最近的发现挑战了这一假说，以至于有理由对信号网络和生理输出进行更深入的比较分析，这些信号网络和生理输出是通过激活任何一种受体介导的。在拟南芥LRR-RK Fls2和拟南芥LRR-RP RLP23介导的细胞反应中观察到了显著的差异。例如，细胞质蛋白激酶BIK1被认为是flg22诱导的ROS爆发的正向调节因子，它是nlp20诱导的ROS爆发和乙烯产生的负调节因子；flg22和nlp20介导的ROS爆发的幅度和动力学都有很大的不同，而nlp20处理的拟南芥Col-0导致了Camalexin的产生，而flg22处理则没有。为了表征和比较LRR-RK和LRR-RP介导的免疫信号，我们建议对模式特异性信号转导以及通过两个LRR-RK型PRR(FLS2，EFR)或通过两个LRR-RP(RLP23，RLP42)介导的免疫反应进行系统分析。这项研究将揭示通过LRR-RK和LRR-RP型激活的信号通路的不同程度，以及通过激活相同类型的受体在信号网络和信号输出方面是否存在差异。我们将进一步测试嵌合受体是否会将flg22的感知转化为nlp20的输出反应，反之亦然。如果是这样的话，这将表明这些受体类型是严格模块化的组成。我们进一步建议评估BIK1介导的nlp20信号负调控的机制基础。在这里，我们将分析这一新功能是否需要BIK1蛋白激酶活性，BIK1是否以配体依赖的方式与SOBIR1物理结合，是否观察到BIK1的nlp20特异性磷酸化，BIK1底物(如RbohD)是否以nlp20特异的方式被磷酸化，或者是否可以确定可能与BIK1相互作用的新的蛋白质(底物)，这些蛋白质(底物)可能解释BIK1在flg22/fls2和nlp20/RLP23介导的免疫信号中的差异参与。

Leucine-rich repeat (LRR) ectodomain-containing PRRs mediate recognition of proteinaceous immunogenic patterns. LRR-type PRRs exist in two forms as LRR receptor kinases (LRR-RK), which carry an intracellular protein kinase domain, and LRR receptor proteins (LRR-RP) that lack a functional intracellular domain. It has recently been hypothesized that LRR-RP/SOBIR1 heteromeric complexes (SOBIR1 is an LRR-RK itself), constitutively formed in the absence of ligands, are equivalent to bi-molecular or bipartite LRR-RK-type PRRs. The proposed identity of molecular mechanisms underlying immune activation mediated through either LRR-RK or LRR-RP-type PRRs has yet to be shown. In fact, our recent findings have challenged this hypothesis to an extent that justifies a more in-depth comparative analysis of signaling networks and physiological outputs mediated through activation of either receptor type. Substantial differences were observed between Arabidopsis LRR-RK FLS2 and Arabidopsis LRR-RP RLP23-mediated cellular responses. For example, the cytoplasmic protein kinase BIK1, known as a positive regulator of flg22-induced ROS burst, acts as a negative regulator of nlp20-induced ROS burst and ethylene production; both amplitude and kinetics of flg22- and nlp20-mediated ROS burst differ substantially, and nlp20-treatment of Arabidopsis Col-0 resulted in the production of camalexin, whereas flg22-treatment did not. To characterize and compare LRR-RK and LRR-RP-mediated immune signaling, we propose to conduct a systematic analysis of pattern-specific signal transduction and of immune responses mediated through two LRR-RK-type PRRs (FLS2, EFR) or through two LRR-RPs (RLP23, RLP42). This research will reveal to which extent signaling pathways activated through LRR-RK and LRR-RP-type differ as well as whether there are differences in signaling networks and signal outputs mediated through activation of the same receptor types. We will further test whether chimeric receptors in which kinase domains of FLS2 and SOBIR1 were replaced with one another would turn flg22 perception into an nlp20 output response and vice versa. If so, this would suggest a strictly modular composition of these receptor types. We further propose to assess the mechanistic basis for BIK1-mediated negative regulation of nlp20 signaling. Here, we will analyze whether this novel function requires BIK1 protein kinase activity, whether BIK1 physically associates with SOBIR1 in ligand (in)dependent fashion, whether nlp20-specific phosphorylation of BIK1 is observed, whether BIK1 phosphorylates substrates (such as RbohD) in an nlp20-specific manner or whether putative novel BIK1-interacting proteins (substrates) can be identified that might explain the differential involvement of BIK1 in flg22/FLS2 and nlp20/RLP23-mediated immune signaling.