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

絮状假酶效应蛋白在植物-病原体-生物防治剂三营养相互作用中的功能

• 批准号: RGPIN-2022-03326
• 负责人: Bélanger, Richard
• 金额: $ 4.74万
• 依托单位: Université Laval
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=763522
• 关键词: function; effector; proteins; Pseudozyma; flocculosa; function; effector; proteins; Pseudozyma; flocculosa

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.

对植物病原体的生物控制的实际应用前提是对这种现象的遗传，分子和生态事件的彻底理解。这项挑战一直是在他的发现计划中，申请人实验室进行的研究活动的重点。最后一部分特别有意义，因为我们的团队在阐明了Pseudozyma flocculosa的生物防治特性的复杂和难以捉摸的因素方面做出了一些重大和意外的进步，Pseudozyma flocculosa是粉状霉菌真菌的天然拮抗剂。这些发现促使我们的小组将进化生物学，粮食相互作用，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，以完全重新定义了多年来持续多年的教条。特别重要的是，我们报道了以下内容：1）对参与粮食相互作用的三个主角的前所未有的同时转录组分析； 2）发现决定相互作用特异性的新因素； 3）一种新的假设表明，生物防治剂（BCA）释放的效应蛋白参与了其对另一种真菌的活性：4）开发识别真菌基因组中效应基因的管道线的发展； 5）CRISPR-CAS敲除系统在BCA中的首次应用，以提供反对抗生素作用的证据，长期以来，人们认为是BCA的主要作用方式。后一种成就在简化了以前从未在BCA的作用模式领域中描述过的不可预见的现象。在此应用程序的背景下，并与我们最近的进步直接联系，目的是：1）应用CRISPR-CAS编辑三个编码的基因编码为絮状假单胞菌所特有的效应子，并在粮食相互作用中专门表示。 2）进行基因的功能分析并量化其对BCA生物防治活性的影响； 3）通过免疫定位识别效应子的靶标； 4）确定BCA效应子是否与病原体（白粉病），植物或其界面相互作用，即外部austerorial矩阵； 5）提供机械证据，表明某些BCA依靠效应子发挥其生物防治活性。这些目标的实现将对以下方面产生新颖而独特的信息：调节絮状假单胞菌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）如何影响白粉病真菌或植物通过扰动将它们联系起来的脆弱的生物营养相互作用。这些结果将对植物病理学领域产生巨大影响，因为我们的模型将提供独特的数据，这些数据可以完全重新定义BCAS与宿主拮抗的概念。