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

植物病原菌生物防治的实际应用前提是彻底了解这些现象背后的遗传、分子和生态事件。这一挑战一直是申请者实验室在其发现计划范围内开展的研究活动的焦点。最后一期特别有意义，因为我们的团队在阐明控制白粉菌天然拮抗剂--簇毛假酵母菌的生防特性的复杂和难以捉摸的因素方面取得了一些重大和意想不到的进展。这些发现促使我们的团队融合了进化生物学、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，以完全重新定义一个持续多年的教条。具有特别重要意义的是，我们报道了以下内容：1)对参与三元互作的三个主角进行了前所未有的同时转录转录分析；2)决定了相互作用的特异性的新因素的发现；3)一种新的假说表明，由生防剂(BCA)释放的效应蛋白参与了它对另一种真菌的活性：4)开发了一条管道来确定真菌基因组中的效应基因；以及5)首次在BCA中应用CRISPR-Cas基因敲除系统来提供证据，以对抗抗生素的作用，长期以来一直被认为是BCA的主要作用模式。后一项成就有助于精简以前在协调机制行动模式领域中从未描述过的一种不可预见的现象。在这一应用的背景下，并与我们最近的进展直接联系，目标是：1)应用CRISPR-CAS编辑编码丛生菌特有的三个效应物基因，并在三元互作中特异表达；2)对这些基因进行功能分析并量化它们对BCA生防活性的影响；3)通过免疫定位确定效应物的靶标(S)；4)确定BCA效应物是否与病原体(白粉病)、植物或它们的界面(即吸器外基质)相互作用；5)提供一些BCA依靠效应器发挥生物防治活性的机制证据。这些目标的实现将产生关于以下方面的新的和独特的信息：调节丛枝疫霉生防活性的分子决定因素，以及这些决定因素的表达(或抑制)如何通过干扰连接白粉菌或植物的脆弱的生物营养相互作用来影响白粉菌或植物。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，可能完全重新定义BCA对抗宿主的概念。

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.