Functional characterization of effectors of the Microbotryum complex of fungal phytopathogens

新的传染病的出现或对现有疾病的改造或治疗，或随着宿主范围的扩大，对人类生存构成严重威胁。该项目将探索此类疾病出现的遗传和分子基础，并可能通过宿主转移更好地预测新出现的传染病。这些真菌是薇甘菊真菌物种复合体的成员，它感染康乃馨家族(粉红色)中大量多样性的野花物种，与在基因更同质的农业植物上致病的生物相比，它们提供了一种独特的模式。由于在自然界中已经观察到这些寄主特有的真菌向新的野花物种的转移，了解这种转移的范围和限度以及涉及的基因和蛋白质可以为应对不断增加的直接或间接影响人类健康和生存的疾病(例如，通过威胁食物供应)提供独特的方法。在研究这些根本性和实践性重大科学问题的过程中，该项目还将为一系列学生和专业受训人员(从本科生到研究生到博士后)提供比较人口基因组学和蛋白质组学领域的培训，并提供关于分子生物学和生物技术研究中使用的最新方法的技术培训。PI制定了协调一致的招聘工作，目标是肯塔基州和美国东南部地区传统上代表性不足的群体(特别是非洲裔美国人、拉丁裔、妇女、阿巴拉契亚人)的候选人，这些群体提高了他们对高级学位的追求。此外，还将设立“过渡年”，以支持新招募的学生作为后续招募的少数族裔学生的导师。植物病原菌微孢子菌复合体是研究寄主转移引起的新发传染病的有效模型。作为在遗传上更加多样化的寄主植物种群上的病原菌，Vioraceum复合体为农业同源寄主上的病原体提供了一个重要的替代模式。病原真菌常常分泌效应蛋白来操纵寄主，这可能是寄主特化的一个重要方面。在目标1中，将比较石竹属寄主物种上的微孢子菌种群，以验证真菌小分泌蛋白(SSPs)在局部寄主适应/专一性中发挥重要作用的假说。通过比较基因组学方法，我们已经在3种小盆草中确定了可能的SSP效应子。在这里，将增加对石竹属寄主上的“多面手”小花菊属物种与仅限于一个石竹属寄主物种的“专业”种的比较，以检验对同一小花花属物种内反映在效应器功能上的局部适应的预测。AIM 2将在植物宿主中验证可能的效应器。通过“混合匹配”实验，通过特定候选效应器基因的过度表达，改变每种真菌的表达水平，将评估一个真菌物种的效应器扩大另一个物种宿主范围的能力。CRISPR/CAS9将用于删除真菌中的效应器，并在植物中测试此类突变体。在没有真菌的寄主植物中，候选效应器基因将测试候选效应器对植物病理负责的假设，可能是通过修改寄主发育；这样的实验还将提供植物中的定位，效应器和预测的宿主靶标的共定位研究，以及Co-IP。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.