Elicitor release upon flagellin glycan modification

对植物中病原体的免疫力对农业乃至地球上的生命都是至关重要的。病原菌识别是植物免疫的第一个关键步骤。大多数植物通过它们的鞭毛识别细菌病原体，细菌利用鞭毛移动。不同的植物物种可以识别鞭毛蛋白的不同片段，尽管大多数种子植物识别一个保守的22个氨基酸区域，称为flg22，这是已知的细菌病原体的主要识别元件。Fls2受体样激酶对Flg22在细胞表面的识别作用已被广泛研究。Flg22在大多数植物中都能诱导免疫反应，包括模式植物拟南芥和烟草近缘种烟草。这些免疫反应包括氧化爆发、MAP激酶信号和转录重新编程，建立防御反应，包括加强细胞壁和分泌有毒代谢物和有害蛋白质。适应的细菌病原体都使用效应器来阻止flg22诱导的信号转导，这一事实强调了flg22触发的免疫的相关性。目前，flg22-fls2信号转导系统是目前对植物病原菌最了解和最重要的识别系统。然而，关键的一步仍然悬而未决。鞭毛蛋白激发子，如flg22，深入鞭毛蛋白结构，存在于鞭毛杆内。这些埋藏的激发子如何与Fls2受体结合？显然，它们必须通过胞外水解酶活性来释放，但释放激发子的水解酶甚至自然释放的激发子还没有被鉴定出来。人们可能会认为细菌在感染过程中会抑制这些激发子释放的水解酶，以阻止它们的识别，所以鞭毛蛋白的水解很可能是植物-病原体界面上的一个重要战场。重要的是，我们发现了一种胞外半乳糖苷酶样的GH35糖苷酶，它可以从细菌中释放鞭毛蛋白激发子。一个可疑的掩盖表明了这种GH35酶的相关性：在细菌感染期间，GH35被细菌代谢物特异性地抑制。重要的是，GH35对模式细菌病原体紫丁香假单胞菌的处理引发了本底烟草和拟南芥的氧化爆发。这种氧化爆发依赖于植物中的Fls2受体，以及病原菌中编码鞭毛蛋白的flc基因。缺乏GH35酶的突变株对丁香假单胞菌更敏感，证实了它们在免疫中的作用。弗拉格氏菌与一种独特的三糖聚糖糖化。我们假设GH35作用于这些多糖，并且这种修饰与其他水解酶协同作用，导致鞭毛蛋白来源的激发子与Fls2结合。使用描述良好的鞭毛蛋白糖基化突变株和本氏烟草作为宿主，我们处于独特的位置来测试GH35如何促进免疫。这项建议的目的是阐明鞭毛蛋白来源的激发子是如何被GH35和其他水解酶释放的，并研究这种机制在植物与细菌的相互作用中有多常见。本研究的目的是：1)阐明GH35修饰鞭毛蛋白的作用和机制；2)研究GH35介导的鞭毛蛋白激发子释放在作物和其他病原菌中的更广泛作用；3)阐明鞭毛蛋白在GH35处理过程中的蛋白质加工。本项目将有助于阐明植物识别细菌病原体的一个重要的新机制，该机制可能在植物界普遍存在。类似的水解酶驱动的激发子释放也有望用于识别丝状病原体。这些发现将启发新的作物保护战略，包括引入具有抑制剂弹性的水解酶和农用化学品，阻止鞭毛蛋白糖基化或GH35抑制剂的生物合成。

Immunity to pathogens in plants is essential for agriculture and even for life on earth. Pathogen recognition is the first crucial step in plant immunity. Most plants recognise bacterial pathogens by their flagella, which bacteria use to move. Different plant species can recognise different fragments of the flagellin proteins, though most seed plants recognise a conserved, 22 amino acid region, known as flg22, the major recognition element known for bacterial pathogens. Recognition of flg22 at the cell surface by the FLS2 receptor-like kinase is very well studied. Flg22 perception elicits immune responses in most plants, including the model plant Arabidopsis thaliana and tobacco relative Nicotiana benthamiana. These immune responses include an oxidative burst, MAP kinase signalling and transcriptional reprogramming, mounting a defence response that includes cell wall strengthening and the secretion of toxic metabolites and harmful proteins. The relevance of flg22-triggered immunity is stressed by the fact that adapted bacterial pathogens all use effectors to block flg22-induced signalling. Currently, flg22-FLS2 signalling is the best understood and most important recognition system of plant pathogens. A crucial step, however, is still unresolved. Flagellin-derived elicitors like flg22 are embedded deep within the structure of flagellin protein and reside inside the flagellar rod. How can these buried elicitors bind to the FLS2 receptor? It seems obvious that they must be released by extracellular hydrolase activities, but elicitor-releasing hydrolases and even the naturally-released elicitor have not yet been identified. One may expect that bacteria suppress these elicitor-releasing hydrolases during infection to prevent their recognition, so it is very likely that flagellin hydrolysis represents an important battlefield at the plant-pathogen interface. Importantly, we discovered an extracellular galactosidase-like GH35 glycosidase that releases flagellin-derived elicitors from bacteria. The relevance of this GH35 enzyme was indicated by a suspicious cover-up: GH35 is specifically inhibited during bacterial infection by a bacterial metabolite. Importantly, GH35 treatment of the model bacterial pathogen Pseudomonas syringae triggers an oxidative burst in Nicotiana benthamiana and Arabidopsis thaliana. This oxidative burst is dependent on the FLS2 receptor in the plant, and the flagellin-encoding fliC gene in the pathogen. Mutant N. benthamiana lacking the GH35 enzyme are more susceptible for P. syringae, confirming their role in immunity.Flagella are glycosilated with a unique trisaccharide glycan. We HYPOTHESISE that GH35 acts on these glycans and that this modification, in concert with other hydrolases, results in the release of flagellin-derived elicitors that bind to FLS2. Using well described P. syringae mutants with altered flagellin glycosylation and Nicotiana benthamiana as a host, we are in the unique position to test how GH35 contributes to immunity. The AIM of this proposal is to elucidate how flagellin-derived elicitors are released by GH35 and other hydrolases and to investigate how common this mechanism is in plant-bacteria interactions. The OBJECTIVES are to: i) elucidate the role and mechanism of GH35 modification of flagellin; ii) investigate the broader role of GH35-mediated elicitor release from flagellin in crop plants and from other pathogens; and iii) elucidate protein processing of flagellin upon GH35 treatment.This project will lead to the elucidation of an important novel mechanism in bacterial pathogen recognition by plants that is probably universal in the plant kingdom. Similar hydrolase-driven elicitor release is expected for the recognition of filamentous pathogens. These discoveries will inspire new crop protection strategies, including the introduction of inhibitor-resilient hydrolases and agrochemicals blocking flagellin glycosylation or GH35 inhibitor biosynthesis.