EAGER Proposal: Bet hedging as a mechanism for pathogenic variation in the rice blast fungus

许多微生物会导致植物、人类和其他动物的严重疾病。植物和动物都拥有免疫系统，能够识别外来生物的存在，从而引发防御反应，防止入侵微生物的进一步传播。识别通常发生在宿主感觉到外来蛋白质时，这些外来蛋白质要么是由微生物在试图确定致病状态时分泌的，要么是构成有机体“外壳”的一部分。不幸的是，病原体已经发展出破坏宿主防御的机制，允许它们生长并导致疾病。例如，众所周知，引起水稻毁灭性疾病稻瘟病的真菌稻瘟菌可以通过突变或丢失编码蛋白质的基因来避免被植物识别，这些蛋白质通常会与水稻中的特定抗性受体结合。研究人员实验室最近的研究表明，存在一种新的机制，通过这种机制，稻瘟病，可能还有其他病原微生物，可能会逃脱宿主的识别。假设的机制包括一种更好的对冲策略，即基因相同的真菌菌落会改变它们分泌的蛋白质类型，从而逃避宿主免疫系统的识别。该项目寻求直接证据表明这种机制在感染期间使用。任何给定的米曲霉菌株都有可能在感染过程中分泌数百种蛋白质，其中许多可能会引发防御反应。然而，目前的数据表明，这种真菌通过在彼此遗传相同的真菌菌落中表达不同的分泌蛋白质亚群来更好地抵御识别。基因表达的这种克隆变异被认为是一种风险传播策略，它增加了在不可预测的植物基因型上感染成功的机会。这个项目的目标是检验这一假说，从而提高对病原体及其宿主之间的识别过程的理解。计划中研究的具体目标是：1)利用单个侵染部位的RNAseq来测试其产品在水稻中诱导抗性的真菌基因的差异表达；2)使用差异RNAseq在中间寄主反应和一个或多个真菌无毒基因的差异表达之间建立因果联系。中心假说具有变革性，因为它为植物-微生物识别建立了一个新的范式，有可能彻底改变该领域目前的思维方式。该项目产生的知识可能会改变植物的抗病育种方式，并可能为人类和其他动物的微生物疾病带来新的治疗策略。该项目将为一名现有的研究生提供研究培训，该研究生还将在项目期间参加下一代测序和生物信息学研讨会。这些培训机会将极大地扩展她的技能集，从而增强未来的职业前景。

Many microbes cause serious diseases of plants, humans and other animals. Both plants and animals possess immune systems that serve to recognize the presence of foreign organisms, which then leads to the initiation of defense responses that prevent further spread of the invading microbe. Recognition usually occurs when the host senses foreign proteins that are either secreted by the microbe as it attempts to establish the pathogenic state, or which form part of the organism's "coat." Unfortunately, pathogens have developed mechanisms that subvert the host's defenses, allowing them to grow and cause disease. For example, it is well known that the fungus Magnaporthe oryzae, which causes a devastating disease of rice known as blast, can avoid recognition by the plant through the mutation or loss of genes that code for proteins that would normally bind to specific resistance receptors in the rice plant. Recent studies in the investigator's laboratory imply the existence of a novel mechanism by which rice blast, and probably other pathogenic microbes, may escape host recognition. The hypothesized mechanism involves a bet hedging strategy in which genetically identical fungal colonies vary the types of proteins they secrete, thereby evading recognition by the host immune system. This project seeks direct evidence that this mechanism is employed during an infection.Any given M. oryzae strain has the potential to secrete hundreds of proteins during the infection process, many of which could elicit a defense reaction. However, current data suggest that the fungus bet-hedges against recognition by expressing different secreted protein subsets in fungal colonies that are genetically identical to one another. Such clonal variation in gene expression is believed to be a risk-spreading strategy that increases the chance of infection success on unpredictable plant genotypes. The goal of this project is to test this hypothesis and thereby improve understanding of the recognition processes between pathogens and their hosts. The specific objectives of the planned research are to: 1) Use RNAseq of individual infection sites to test for variable expression of fungal genes whose products elicit resistance in rice; 2) Use differential RNAseq to establish a causal link between mesothetic host reactions and the variable expression of one or more fungal avirulence genes. The central hypothesis is transformative because it establishes a new paradigm for plant-microbe recognition, with the potential to revolutionize current thinking in the field. The knowledge generated in the project could alter how plants are bred for disease resistance and may lead to novel therapeutic strategies for microbial diseases of human and other animals. The project will provide research training for an existing graduate student, who will also attend a Next Generation Sequencing and Bioinformatics workshop during the term of the project. These training opportunities will greatly expand her skill set and thereby enhance future career prospects.