Functional Characterization of Fungal Effectors in the Phytopathogen Fusarium graminearum

植物病原体禾谷镰刀菌中真菌效应子的功能特征

• 批准号: RGPIN-2022-04901
• 负责人: Subramaniam, Rajagopal
• 金额: $ 2.33万
• 依托单位: Carleton University
• 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=745905
• 关键词: Functional; Characterization; Fungal; Effectors; Phytopathogen

Summary Fungi are the main cause of diseases in crops. Fusarium head blight (FHB) is a devastating disease of wheat, maize, and other small grain crops and is caused by several related Fusarium species. The disease reduces grain quality due to mycotoxin deposition and the contaminated grains with deoxynivalenol (DON) mycotoxin can restrict trade and impact the livelihood of producers. To maintain high-quality grain production and reduce DON accumulation, research efforts have produced partially resistant cereal crops and identified many quantitative trait loci (QTL) in wheat. However, the underlying mechanisms involved in partial or complete FHB resistance remain unclear. Colonization of plants by pathogenic fungi is governed by the secretion of effector molecules into the host cell. Effectors can be defined as molecules that alter host cell structure and function, facilitating infection and/or triggering defense responses; the molecules include proteins, peptides, and secondary metabolites (toxins). Our overarching goal is to delineate the mechanisms that allow the fungal effectors to promote virulence or induce defense response in the plant. Although great strides have been made in the identification and characterization of effectors in bacteria and oomycetes, a similar statement cannot be said with respect to studying phytopathogenic fungal effectors. My lab has been studying mechanisms underlying the pathogenesis of F. graminearum. We use genetics (CRISPR/Cas9 editing), and Omics platforms (genomics, transcriptomics, proteomics, and metabolomics) to unravel signalling pathways that regulate the biosynthesis of the mycotoxin DON in the pathogen and understand the infection processes in wheat. This proposal will use datasets from various Omics platforms to prioritize candidate effector proteins (CEPs) in F. graminearum that will be used for functional characterization studies. Prioritization will be based on machine learning algorithms, phenotypic studies, and expression patterns in axenic cultures and infected wheat tissues. Next, the selected CEPs (~ 40) will be monitored for secretion into plant cells using the self-assembling split super-folder fluorescent protein system in Arabidopsis. Localization of CEPs will be assessed based on the split Green fluorescent protein marker in various organelles in Arabidopsis. In parallel, the CEPs function will be assessed based on phenotype (defense or disease) using the well-developed type III AvrRpt2/RPS2 translocation/recognition system. Additionally, CEPs will also be functionally validated in their native host (wheat) using the Fusarium-wheat pathosystem, well-established in my lab. The CEPs will be rendered non-functional either individually or in combination using the CRISPR/Cas9 technique and monitored for disease symptoms on wheat. Finally, those CEPs that display phenotype will be used as baits to identify host targets using the proximal-dependent biotin identification (BioID) system.

真菌是农作物病害的主要原因。镰刀菌头枯病（FHB）是小麦、玉米和其他小谷类作物的毁灭性疾病，并且由几种相关镰刀菌属物种引起。由于真菌毒素沉积，病害降低了谷物质量，并且被脱氧雪腐镰刀菌烯醇（DON）真菌毒素污染的谷物可以限制贸易并影响生产者的生计。为了保持高品质的谷物生产和减少DON积累，研究工作已经产生了部分抗性的谷类作物，并在小麦中鉴定了许多数量性状基因座（QTL）。然而，涉及部分或完全FHB抗性的潜在机制仍不清楚。病原真菌在植物中的定殖受效应分子分泌到宿主细胞中的控制。效应子可以被定义为改变宿主细胞结构和功能的分子，促进感染和/或触发防御反应;这些分子包括蛋白质、肽和次级代谢物（毒素）。我们的首要目标是阐明允许真菌效应子提高毒力或诱导植物防御反应的机制。虽然在细菌和卵菌中效应子的鉴定和表征方面已经取得了很大的进展，但在研究植物病原真菌效应子方面还不能说类似的声明。我的实验室一直在研究F.我们使用遗传学（CRISPR/Cas9编辑）和组学平台（基因组学，转录组学，蛋白质组学和代谢组学）来解开调节病原体中真菌毒素DON生物合成的信号传导途径，并了解小麦中的感染过程。该提案将使用来自各种组学平台的数据集来优先考虑F中的候选效应蛋白（CEP）。禾谷早熟禾，将用于功能表征研究。优先顺序将基于机器学习算法、表型研究以及纯培养物和受感染小麦组织中的表达模式。接下来，使用拟南芥中的自组装分裂超级折叠荧光蛋白系统监测所选CEP（~ 40）分泌到植物细胞中。CEP的定位将基于拟南芥中各种细胞器中的分裂绿色荧光蛋白标记来评估。同时，将使用开发良好的III型AvrRpt 2/RPS 2易位/识别系统基于表型（防御或疾病）评估CEP功能。此外，CEP也将在其天然宿主（小麦）中使用在我的实验室中建立良好的镰刀菌-小麦病理系统进行功能验证。将使用CRISPR/Cas9技术使CEP单独或组合失去功能，并监测小麦的疾病症状。最后，将显示表型的CEP用作诱饵，以使用近端依赖性生物素识别（BioID）系统识别宿主靶标。