Dissecting the Arms Race between Pathogenic Bacteria and the Plant Immune System

病原菌及其宿主为生存而进行共同进化的军备竞赛，这是由病原菌毒力因子和宿主免疫系统介导的。细胞相互作用的第一个层次是宿主模式识别受体(PRRs)对保守的微生物相关分子模式(MAMPs)的识别。一些病原体能够通过III型分泌效应器(T3SE)抑制这种免疫反应。一些植物寄主可以通过核苷酸结合的富含亮氨酸重复序列的抗性蛋白(NLR)来响应T3SE，在识别T3SE活性后触发第二层免疫。**这个提议有两个主要目标：**目标1.剖析T3SE的HopZ家族和它们的寄主目标之间的军备竞赛。*HopZ等位基因由农学上重要的植物病原菌紫丁香假单胞菌携带。这个不同的T3SE家族有四个主要的等位基因亚家族，它们既可以作为毒力因子，也可以作为免疫激发子，这取决于ZAR1 NLR在拟南芥中的存在。我们最近发现，一个HopZ等位基因可以乙酰化多种宿主蛋白，包括假性激酶ZED1和与ZED1密切相关的一系列称为ZRKs的蛋白。ZED1由ZAR1监控/守卫。**-我们的假设是：HopZ T3SE靶向ATP结合域蛋白；不同的HopZ等位基因针对ZED1/ZRK家族的不同成员；ZRK的HopZ乙酰化干扰其激酶活性；抑制ZRK活性抑制免疫信号。**-我们这个目标的具体目标是：1a)确定哪些HopZ等位基因是乙酰转移酶。1b)评估HopZ和ZED1/ZRK家族之间的物理和生化相互作用。1c)确定在HopZ和ZRK家族中对相互作用和翻译后修饰可能重要的残基。1d)确定特定的ZRK家族成员对细菌毒力或宿主免疫是否重要。和1e)确定HopZ或ZRK蛋白的翻译后修饰是否调节毒力或免疫激活。**目标2.剖析细菌MAMP和植物PRR之间的军备竞赛。*我们已经开发了新的筛选来识别新的MAMP及其同源PRR，并想要确定这些相互作用在宿主适应的非常早期阶段起到什么作用。**-我们的假设是：在任何给定的病理系统中，MAMP-PRR的数量比以前描述的要多得多；MAMP-PRR相互作用具有高度的特异性，但大多数相互作用通过相当少量的调节PRR来传递信号；MAMP的变异可以导致宿主特异性的调节；主机切换的第一步涉及MAMP的进化而不是PRR识别。**-我们实现这一目标的具体目标是：2A)识别识别新识别的MAMP的PRR。2b)验证PRR和MAMP之间的直接相互作用。2C)通过实验进化确定与宿主范围扩展相关的第一个变化。以及2)确定MAMP在最近的进化时间内是如何进化的，以及这对免疫诱导的结果。**这些研究继续我们对宿主特异性研究中最好的病理系统之一的比较和功能解剖。通过这些目标，我们将破译不同的细菌T3SE家族和与其相互作用的更不同的宿主蛋白家族之间的一系列功能相互作用。然后，我们将从机械上描述调节细菌毒力和宿主免疫的相互作用。我们还将研究MAMP及其同源PRR的多样性。我们将确定这种变异如何影响宿主特异性，并进行基础研究，这些研究将成为未来专注于将MAMP用作替代抗菌剂的应用工作的基础。

Pathogens and their hosts engage in a co-evolutionary arms race for survival, which is mediated by pathogen virulence factors and the host immune system. The first level of cellular interaction is the recognition of conserved microbe-associated molecular patterns (MAMPs) by host pattern recognition receptors (PRRs). Some pathogens are able to suppress this immune response via type III secreted effectors (T3SEs). Some plant hosts can in turn respond to T3SEs via nucleotide binding leucine rich repeat resistance proteins (NLRs), which trigger a second layer of immunity upon recognition of T3SE activity.**This proposal has two major goals:**Goal 1. Dissect the arms race between the HopZ family of T3SEs and their host targets.* HopZ alleles are carried by the agronomically-important phytopathogenic bacterium Pseudomonas syringae. This diverse family of T3SEs has four major allele sub-families that can act as either virulence factors or immune elicitors depending on the presence of the ZAR1 NLR in Arabidopsis. We have recently shown that one HopZ allele acetylates a variety of host proteins including the pseudokinase ZED1 and a family of kinases closely related to ZED1 called ZRKs. ZED1 is monitored / guarded by ZAR1. ** - Our hypotheses are: HopZ T3SEs target ATP-binding domain proteins; different HopZ alleles target different members of the ZED1/ZRK family; HopZ acetylation of ZRKs interferes with their kinase activity; and inhibition of ZRK activity suppresses immune signaling.** - Our specific aims for this goal are to: 1a) Determine which HopZ alleles are acetyltransferases. 1b) Assess physical and biochemical interactions between the HopZ and ZED1/ZRK families. 1c) Identify residues putatively important for interaction and post-translational modification in the HopZ and ZRK families. 1d) Determine if specific ZRK family members are important for bacterial virulence or host immunity. And 1e) Determine if post-translation modification of HopZ or ZRK proteins modulates virulence or immune activation.**Goal 2. Dissect the arms race between bacterial MAMPs and plant PRRs. * We have developed novel screens to identify new MAMPs and their cognate PRRs, and want to determine what role these interactions play during the very early stages of host adaptation.** - Our hypotheses are: there are many more MAMPs in any given pathosystem than have been previously characterized; MAMP-PRR interactions are highly specific, but most interactions signal through a fairly small number of regulatory PRRs; MAMP variation can result in modulation of host specificity; and the first step in a host switch involves evolution of MAMPs away from PRR recognition.** - Our specific aims for this goal are to: 2a) Identify the PRRs that recognize newly identified MAMPs. 2b) Validate the direct interaction of PRRs and MAMPs. 2c) Identify the first changes associated with host range expansion via experimental evolution. And 2d) Determine how MAMPs evolve over recent evolutionary time and the consequence of this for immune elicitation.**These studies continue our comparative and functional dissection of one of the best established pathosystems for host-specificity research. Through these aims we will decode the range of functional interactions between a diverse family of bacterial T3SEs and the even more diverse family of host proteins with which they interact. We will then mechanistically characterize the interactions that modulate bacterial virulence and host immunity. We will also examine the diversity of MAMPs and their cognate PRRs. We will determine how this variation influences host specificity, and produce fundamental research that will be the foundation for future applied work focusing on the use of MAMPs as alternative antimicrobials.