What triggers the production of Lysin-domain (LysM) virulence effectors by plant pathogenic fungi?

全球粮食安全不断受到由植物病原体引起的疾病造成的产量损失的挑战。小麦黑穗病（STB）是英国和西欧小麦最重要的经济病害，由小麦黑穗病（Zymoseptoria tritici）引起。与许多其他植物病原真菌一样，小麦霉依靠分泌蛋白效应物的释放来致病。一类特殊的效应蛋白，Lysin-domain containing （LysM）分泌蛋白，在植物感染期间大量释放，并通过其在阻止植物激活几丁质触发的免疫中的作用作为重要的毒力决定因素（FEMS Microbiol）。评论2015 v39 p171)。然而，目前尚不清楚这些真菌是如何知道何时启动这些重要效应器的生产的。是否有特定的触发因素驱动效应器的产生？同样，在驱动这些基因表达的启动子上游有哪些信号通路？是否可以操纵这些元件来阻止LysM效应物的表达，从而保护许多作物免受真菌疾病的侵害？我们之前已经证明，分泌的LysM效应蛋白“Zt3LysM”通过阻止植物免疫的早期激活，在小麦小麦的毒力中发挥重要作用（plant Physiol. 2011 v156 p756; Mol. plant - microbe interaction . 2014 v27 p236）。在野生型真菌细胞中，该效应蛋白在叶片感染期间强烈表达，在远离植物的无菌培养生长期间低表达。然而，我们最近发现了一种可能影响真菌细胞壁结构的基因（2型糖基转移酶- ZtGT2）的小麦Z. tritici突变株，即使在无菌培养中也构成性地过表达Zt3LysM （PLoS Pathogens 2017 v13 e1006672）。这表明真菌细胞壁的改变可能通过与叶片表面的相互作用而发生，从而影响Zt3LysM的表达水平。本项目旨在确定LysM效应基因表达的“触发器”，并鉴定检测这些“触发器”的真菌蛋白，以及将触发信号传递给效应基因启动子以增强其表达的真菌蛋白。该项目将产生多种转基因小麦轴虫报告菌株，在荧光报告基因GFP和GUS前融合Zt3LysM基因启动子序列，随后将这些菌株用于筛选(1)LysM效应表达的特定触发因素（化学和/或物理）和(2)不能表达LysM效应的突变体。与效应表达缺失相关的候选基因将通过对不表达菌株的全基因组重测序来确定。此外，启动子序列将直接用于筛选，目的是识别调节结合蛋白（转录因子）。与感知LysM效应表达的“触发器”或介导效应启动子信号传导有关的候选基因将通过产生相关基因的真菌敲除（KO）菌株并测试它们在小麦上的效应表达和毒力来进一步表征。最后，将分析验证的靶点在其他致病真菌基因组中的存在/不存在，并探索潜在阻断效应表达的途径。

Global food security is continually challenged by yield losses resulting from diseases caused by plant pathogens. The most economically important disease of wheat in the UK and W. Europe is Septoria tritici blotch (STB) caused by the ascomycete fungus Zymoseptoria tritici. Alike many other plant pathogenic fungi, Z.tritici relies upon the release of secreted protein effectors to establish disease. One particular class of effector proteins, the Lysin-domain containing (LysM) secreted proteins, are released at high levels during plant infections, and function as important virulence determinants through their roles in preventing the activation of chitin-triggered immunity by plants (FEMS Microbiol. Reviews 2015 v39 p171). However, it remains currently unknown as to how these fungi know when to switch on production of these important effectors? Are there specific triggers which drive effector production? Similarly, what signalling pathways lie upstream of the promoters driving expression of these genes? Could these elements be manipulated to prevent the expression of LysM effectors with the potential to protect many crops from fungal diseases? We have previously shown that the secreted LysM effector protein "Zt3LysM" plays a major role in the virulence of Z. tritici on wheat, through preventing the early activation of plant immunity (Plant Physiol. 2011 v156 p756; Mol. Plant-Microbe Interact. 2014 v27 p236) In wild type fungal cells, the effector is strongly expressed during leaf infection with low expression observed during growth in axenic culture, away from the plant. However, we recently identified a Z. tritici mutant strain affected in a gene which likely contributes to fungal cell wall structure (a type 2 glycosyltransferase- ZtGT2), which constitutively overexpresses Zt3LysM, even in axenic culture (PLoS Pathogens 2017 v13 e1006672). This suggests that alterations to the fungal cell wall, possibly occurring through interactions with leaf surfaces, influences Zt3LysM expression levels. This project aims to define the "triggers" of LysM effector expression, and to identify fungal proteins which detect these "triggers", as well as those that transmit the trigger signal to the promoter of the effector gene to enhance its expression. The project will generate various transgenic reporter strains of Z. tritici engineered to possess the Zt3LysM gene promoter sequence fused in front of the fluorescent reporters GFP and GUS, and the subsequent use of these strains in screens to identify (1) specific triggers (chemical and or physical) of LysM effector expression and (2) mutants which fail to express the LysM effector. Candidate genes associated with the loss of effector expression will be identified through whole genome resequencing of the non-expressing strains. In addition, the promoter sequence will be used directly in screens aimed to identify the regulatory binding proteins (transcription factor(s)). Candidate genes implicated in sensing the "trigger(s)" of LysM effector expression, or mediating signalling to the effector promoter, will be further characterised by generating fungal knock-out (KO) strains of the genes in question and testing them for effector expression and virulence on wheat. Finally, the validated targets will be analysed for presence / absence in the genomes of other pathogenic fungi and avenues for potentially blocking effector expression will be explored.