Functional characterization of effectors of the Microbotryum complex of fungal phytopathogens

真菌植物病原体 Microbotryum 复合体效应子的功能表征

• 批准号: 2007449
• 负责人: Michael Perlin
• 金额: $ 29.96万
• 依托单位: University of Louisville Research Foundation Inc
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-15 至 2025-03-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2007449&HistoricalAwards=false
• 关键词: Functional; characterization; effectors; Microbotryum; complex

The emergence of new infectious diseases or modification of existing diseases refractive to treatment, or with expanded host range, poses serious threats to human survival. This project will explore genetic and molecular underpinnings of such disease emergence, and may allow better prediction of emerging infectious diseases through host shifts. The fungi, members of the Microbotryum violaceum fungal species complex, that infect a large diversity of wildflower species in the Carnation family (Pinks), provide a unique model compared with organisms that cause disease on more genetically homogeneous agricultural plants. Since shifts of these host-specific fungi to new wildflower species has been observed in nature, understanding the range and limits of such shifts, as well as the genes and proteins involved could provide a unique approach for dealing with ever-increasing diseases that either directly or indirectly affect human health and survival (e.g., by threatening food supply). In the process of investigating such fundamentally and practically important scientific questions, this project will also provide training to a range of students and professional trainees (from undergraduate to graduate student to Postdoctoral), in areas of comparative population genomics and proteomics, and in technical training in state-of-the-art methods used in molecular biological and biotechnological investigations. Concerted recruitment efforts established by the PI target candidates from traditionally under-represented groups in the region of Kentucky and the Southeastern US (specifically, African-Americans, Latinx, women, Appalachians) that enhances their pursuit of an advanced degree. Moreover, a “bridge year” will be established to support recruits as mentors for subsequent minority students recruited. The Microbotryum violaceum complex of fungal plant pathogens is a useful model of emerging infectious diseases through host shifts. As pathogens on genetically more diverse host plant populations, the M. violaceum complex provides an important alternative model to pathogens on agricultural generally homogenous hosts. Pathogenic fungi often secrete effector proteins to manipulate their hosts and these may be an important aspect of host specialization. In Aim 1, populations of Microbotryum on Dianthus host species will be compared to test the hypothesis that fungal small secreted proteins (SSPs) play important roles in localized host adaptation/specificity. Via comparative genomics, we already identified possible Microbotryum SSP effectors across 3 Microbotryum species. Here, comparisons will be added, of “generalist” Microbotryum species on Dianthus hosts with a “specialist” species, limited to one Dianthus host species, to test predictions of local adaptations, reflected in effector function, within the same Microbotryum species. Aim 2 will validate possible effectors in plant hosts. Expression levels in each fungus, modified by over-expression of specific candidate effector genes via “mix and match” experiments, will assess ability of effectors from one fungal species to expand the host range of another species. CRISPR/Cas9 will be used to delete effectors in fungi and test such mutants in plants. Candidate effector genes in host plants, absent the fungus, will test the hypothesis that the candidate effector is responsible for plant pathology, possibly by modifying host development; such experiments will also provide localization in planta, co-localization studies of effectors and predicted host targets, and Co-IP.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

新的传染病的出现或现有疾病无法治疗的改变，或宿主范围的扩大，对人类的生存构成严重威胁。该项目将探索这种疾病出现的遗传和分子基础，并可能通过宿主的变化更好地预测新出现的传染病。这些真菌是堇菜微生物真菌物种复合体的成员，它们感染康乃馨科（Pinks）中多种多样的野花，与在基因更同质的农业植物上致病的生物体相比，它们提供了一种独特的模型。由于在自然界中已经观察到这些宿主特异性真菌向新的野花物种的转变，了解这种转变的范围和限制，以及所涉及的基因和蛋白质，可以为处理直接或间接影响人类健康和生存（例如，通过威胁食物供应）的日益增加的疾病提供一种独特的方法。在研究这些具有根本性和实践性的重要科学问题的过程中，本项目还将为一系列学生和专业学员（从本科生到研究生再到博士后）提供比较群体基因组学和蛋白质组学领域的培训，以及分子生物学和生物技术研究中使用的最先进方法的技术培训。在肯塔基州和美国东南部地区（特别是非洲裔美国人、拉丁裔美国人、女性、阿巴拉契亚人），PI针对传统上代表性不足的群体（特别是非洲裔美国人、拉丁裔美国人、女性、阿巴拉契亚人）开展了协调一致的招聘工作，以提高他们对高等学位的追求。此外，将设立一个“桥梁年”，以支持新招募的学生担任随后招募的少数民族学生的导师。植物真菌病原菌堇菜菌复合体是一种有用的通过寄主转移来研究新发传染病的模型。作为寄主植物种群遗传多样性更高的病原菌，紫芽孢杆菌复合体提供了一种重要的替代模式，以替代寄主植物的病原菌。病原真菌经常分泌效应蛋白来操纵宿主，这可能是宿主特化的一个重要方面。在Aim 1中，我们将比较Dianthus宿主物种上的Microbotryum种群，以验证真菌小分泌蛋白（ssp）在宿主局部适应/特异性中发挥重要作用的假设。通过比较基因组学，我们已经在3种微生物物种中发现了可能的微生物SSP效应物。在这里，将增加对石竹寄主上的“通才型”微腐物种与仅限于一种石竹寄主的“专才型”物种的比较，以检验在同一微腐物种中反映在效应函数中的局部适应预测。目的2将验证植物宿主中可能存在的效应物。通过“混合匹配”实验，通过过度表达特定候选效应基因来修饰每种真菌的表达水平，将评估来自一种真菌物种的效应物扩大另一物种宿主范围的能力。CRISPR/Cas9将用于删除真菌中的效应物，并在植物中测试这些突变体。在没有真菌的情况下，寄主植物中的候选效应基因将检验候选效应基因可能通过改变寄主发育而导致植物病理的假设；这些实验还将提供植物定位、效应物和预测宿主靶点的共定位研究以及Co-IP。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。