Deconstruction of the phytotoxic secretome to decipher the necrotrophic pathogenesis of Botrytis cinerea

灰霉病菌是世界上最重要的经济植物病原菌之一，在多种作物上引起采前和采后灰腐病。在感染过程中，真菌会分泌一种裂解酶、植物毒素蛋白和代谢物的复杂混合物，从而导致寄主植物细胞的迅速坏死。为了更好地了解这种寄主范围广的嗜死性细胞的关键感染机制，我们正在使用一种消减方法来连续消除所有植物细胞死亡诱导蛋白(CDIP)和代谢物，直到所产生的突变体完全失去对坏死的诱导。为此，我们建立了一种高效的CRISPR/CAS编辑方法，在短时间内产生多个突变体。我们的初步工作表明，由灰霉菌分泌的每个已知CDIP只对感染起到很小的作用。为了充分解决植物毒性化合物的高度冗余问题，我们希望通过研究单个和多个突变体的表型，鉴定尚不清楚数量的剩余CDIP，并分析它们在坏死性感染不同阶段中的作用。我们正在进一步研究CDIP与其植物受体或靶标的相互作用。一些真菌CDIP可以激活细胞表面的模式识别受体(PRR)，一旦激活，就会诱导一种基本的植物防御机制，称为模式触发免疫(PTI)。值得注意的是，由灰霉病菌激活的PTI往往最终导致植物细胞死亡，这是这种基本植物防御的一个相当不寻常的特征。因此，我们想要调查Botrytis是如何利用CDIP在不被植物防御机制阻止的情况下利用PTI为自己谋利的。为了鉴定植物CDIP受体，将用拟南芥PRR突变体以及在其遗传PRR设备中不同的拟南芥和番茄生态型进行筛选。受体-配体对的鉴定将有助于从功能上深入了解植物细胞死亡的诱导，并可能有助于更好地了解灰霉病菌广泛的寄主范围。我们相信，我们提出的项目将阐明微生物致病机制的一个中心方面，并将为全面了解灰霉菌的死亡营养生活方式铺平道路。

Botrytis cinerea is one of the economically most important plant pathogens worldwide, causing pre- and postharvest grey rot on numerous crops. During infection, the fungus secretes a complex mixture of lytic enzymes and phytotoxic proteins and metabolites, thereby inducing rapid necrosis of host plant cells. To better understand the key infection mechanism of this broad host range necrotroph, we are using a subtractive approach to serially eliminate all plant cell death-inducing proteins (CDIPs) and metabolites, until complete loss of necrosis induction of the resulting mutants. To this end, we have established a highly efficient CRISPR/Cas editing method to generate multiple mutants within short time. Our preliminary work has shown that each of the known CDIPs secreted by B. cinerea contributes only to a minor extent to infection. To fully address the high redundancy of phytotoxic compounds, we want to identify the as yet unknown number of remaining CDIPs and analyse their roles in different stages of necrotrophic infection, by investigating the phenotypes of single and multiple mutants. We are further studying the interaction of CDIPs with their plant receptors or targets. Several fungal CDIPs have been shown to activate pattern recognitions receptors (PRR) at the cell surface, which upon activation induce a basic plant defence called PTI (pattern triggered immunity). Notably, PTI-activation by Botrytis often results eventually in plant cell death, a rather unusual feature of this basic plant defence. Therefore, we want to investigate how Botrytis is using CDIPs to exploit PTI for its own benefit without being stopped by plant defence mechanisms. To identify plant CDIP receptors, screenings will be performed with Arabidopsis PRR mutants and with Arabidopsis and tomato ecotypes that vary in their genetic PRR equipment. Identification of receptor-ligand pairs will allow functional insight into plant cell death induction and might lead to a better understanding of the wide host range of Botrytis. We believe that our proposed project will shed light on a central aspect of microbial pathogenesis, and will pave the way towards a comprehensive understanding of the necrotrophic lifestyle of B. cinerea.