The function of effector proteins of Pseudozyma flocculosa in a tritrophic interaction plant-pathogen-biocontrol agent

植物病原体生物防治的实际应用的先决条件是彻底了解这种现象背后的遗传，分子和生态事件。这一挑战一直是申请人实验室在其探索计划背景下开展的研究活动的焦点。最后一部分已经特别有益，因为我们的团队已经取得了一些重大的和意想不到的进展，阐明了复杂的和难以捉摸的因素，控制Pseudozyma flocculosa，一种天然的拮抗剂的白粉病真菌的生物防治特性。这些发现推动我们的团队将进化生物学、三营养相互作用、真菌生态学、真菌基因组学和CRISPR-Cas转化的复杂性融合在一起，以完全重新定义一个持续多年的教条。特别重要的是，我们报道了以下内容：1）对三营养相互作用中涉及的三个主角进行了前所未有的同时转录组学分析; 2）发现了决定相互作用特异性的新因素; 3）提出了一种新的假设，表明生物控制剂（BCA）释放的效应蛋白参与了其对另一种真菌的活性：4）开发了一个管道来识别真菌基因组中的效应基因; 5）首次在BCA中应用CRISPR-Cas敲除系统，以提供证据证明真菌的作用，长期以来一直被认为是BCA的主要作用模式。后一项成就有助于简化一种以前从未在边境管制行动方式领域内描述过的未预见现象。在本申请的背景下，并与我们最近的进展直接相关，目标是：1）应用CRISPR-Cas编辑三个基因，所述三个基因编码絮状单胞菌特有的效应子，并且在絮状单胞菌-禾本科布氏菌-大麦三营养相互作用期间特异性表达; 2）进行基因的功能分析并量化它们对BCA的生物防治活性的影响; 3）通过免疫定位鉴定效应子的靶标; 4）确定BCA效应子是否与病原体（白粉病）、植物或在它们的界面即吸器外基质相互作用; 5）提供一些BCA依赖于效应子发挥其生物防治活性的机制证据。这些目标的实现将产生新的和独特的信息方面：调节P. flocculosa的生物防治活性的分子决定因素，以及这些决定因素的表达（或抑制）如何影响改变白粉病真菌或植物通过干扰脆弱的生物营养相互作用连接它们。这些结果将对植物病理学领域产生深远的影响，因为我们的模型将提供独特的数据，可以完全重新定义BCAs拮抗其宿主的概念。

Practical application of biological control of plant pathogens presupposes a thorough understanding of the genetic, molecular and ecological events underlying such phenomena. This challenge has been the focal point of the research activities carried out in the applicant's laboratory in the context of his Discovery program. The last installment has been particularly rewarding since our team has made some major and unexpected strides toward elucidating the complex and elusive factors governing the biocontrol properties of Pseudozyma flocculosa, a natural antagonist of powdery mildew fungi. These findings have pushed our group to amalgamate the intricacies of evolutive biology, tritrophic interactions, fungal ecology, fungal genomics, and CRISPR-Cas transformation to redefine completely a dogma that had persisted for many years. Of particular significance, we reported the following: 1) the unprecedented simultaneous transcriptomic analysis of the three protagonists involved in a tritrophic interaction; 2) the discovery of new factors dictating the specificity of the interaction; 3) a new hypothesis suggesting that effector proteins released by a biocontrol agent (BCA) were involved in its activity against another fungus: 4) the development of a pipeline to identify effector genes in fungal genomes; and 5) the first application of a CRISPR-Cas knockout system in a BCA to provide evidence against the role of antibiosis, long thought to be the main mode of action of the BCA. This latter achievement was instrumental in streamlining an unforeseen phenomenon never previously described within the realm of modes of action of BCAs. In the context of this application and in direct link with our recent progress, the objectives are: 1) to apply CRISPR-Cas editing of three genes coding for effectors unique to P. flocculosa, and specifically expressed during the tritrophic interaction P. flocculosa-Blumeria graminis-barley; 2) to conduct functional analyses of the genes and quantify their impact on the biocontrol activity of the BCA; 3) to identify the target(s) of the effectors by immunolocalization; 4) to determine if the BCA effectors interact with the pathogen (powdery mildews), the plant or at their interface i.e. the extrahaustorial matrix; 5) to provide mechanistic evidence that some BCAs rely on effectors to exert their biocontrol activity. Fulfillment of these objectives will generate novel and distinctive information with regards to: the molecular determinants that regulate the biocontrol activity of P. flocculosa and how expression (or repression) of these determinants influence alter powdery mildew fungi or the plant through disturbance of the fragile biotrophic interaction linking them. These results will have a far reaching impact into the field of plant pathology as our model will provide unique data that could completely redefine the concepts by which BCAs antagonize their host.