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

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.

植物病原体生物防治的实际应用预示着对这些现象背后的遗传、分子和生态事件的透彻理解。这一挑战一直是申请人实验室在其发现计划背景下开展的研究活动的焦点。上一篇文章特别有意义，因为我们的团队在阐明控制絮状假酵母（白粉病真菌的天然拮抗剂）的生物防治特性的复杂且难以捉摸的因素方面取得了一些重大且意想不到的进展。这些发现促使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 转化的复杂性结合起来，彻底重新定义了多年来一直存在的教条。特别重要的是，我们报告了以下内容：1）对参与三营养相互作用的三个主角进行了前所未有的同步转录组分析； 2）发现决定相互作用特异性的新因素； 3) 一个新的假设表明生物防治剂 (BCA) 释放的效应蛋白参与了其针对另一种真菌的活性：4) 开发了鉴定真菌基因组中效应基因的管道； 5) 首次在 BCA 中应用 CRISPR-Cas 敲除系统，以提供反对抗生素作用的证据，长期以来，抗生素被认为是 BCA 的主要作用模式。后一项成就有助于简化 BCA 作用模式领域中以前从未描述过的不可预见的现象。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 编辑编码 P. floculosa 独特的效应子的三个基因，并在三营养相互作用期间特异性表达。 Blumeria graminis 大麦； 2) 对基因进行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器的靶点； 4) 确定 BCA 效应子是否与病原体（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依赖效应器发挥生物防治活性的机制证据。实现这些目标将产生以下方面的新颖且独特的信息：调节絮状白粉病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）如何通过扰乱脆弱的生物营养来影响白粉病真菌或植物的改变。将它们联系起来的互动。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 BCA 对抗宿主的概念。