Yeast-mycelium dimorphism in the Dutch elm disease fungi and its contribution to pathogenicity and overall fitness

自世纪以来，荷兰榆树病（DED）连续两次大流行造成超过10亿榆树死亡。高度侵袭性的子囊菌真菌Ophiostoma novo-ulmi是目前正在进行的DED大流行的原因，并对北美最广泛的本土榆树物种美洲榆的种群产生了巨大的影响。在自然界中，O. novo-ulmi由小蠹引导，并显示酵母-菌丝体二型性。这种特性在真菌病原体中经常遇到，但在DED真菌中仅受到有限的关注。 拟议研究的总体目标是确定DED真菌中酵母-菌丝体二型性的分子途径，并评估该性状对这些病原体中寄生适应性的贡献。我建议调查1）参与氮吸收和代谢的基因，和2）它们与丝裂原活化蛋白激酶（MAPK）和环AMP依赖性蛋白激酶A（cAMP-PKA）途径中基因的可能联系。我还建议3）研究涉及脂肪酸转化为衍生物（包括氧脂素）的基因。短期目标是：i）评估O。ii）在榆树组织中获得O. novo-ulmi; iii）对O. novo-ulmi突变体的表型和遗传;和iv）鉴定与酵母-菌丝体转变相关的基因共表达网络。我建议的一般方法包括分析敏感和抗性美国的汁液和内树皮中的含氮化合物和脂肪酸。americana树苗，以及野生型和突变株的详细分析。新乌尔米。突变体将通过破坏、编辑（CRISPR-Cas9）或通过RNAi干扰参与氮吸收、MAPK和cAMP-PKA信号传导级联或脂肪酸转化的选定基因的敲低来获得。突变体的表型分析将包括酵母-菌丝体转化动力学的观察和金冠苹果和美国的接种。在受控条件下的美国树苗。遗传分析将包括基因表达的互补测试和全基因组RNAseq研究。将进行基因共表达网络分析，以充分评估单个突变诱导的转录修饰的程度，并确定在给定的实验条件下是否以协调的方式激活不同的途径。**拟议工作的短期意义在于，它将导致控制DED真菌中酵母菌丝体二型性的途径的遗传鉴定，并为（或反对）与寄生适应性的联系提供实验证据。从长远来看，我们的研究结果可能为有效控制导致DED，其他树木疾病或人类疾病的二型Ophiostomatoid真菌提供新的方向。

More than one billion elms were killed by two successive Dutch elm disease (DED) pandemics since the early 20th century. The highly aggressive ascomycete fungus Ophiostoma novo-ulmi is responsible for the current, ongoing DED pandemic and has had a dramatic impact on populations of Ulmus americana, the most widespread native elm species in North America. In nature, O. novo-ulmi is vectored by bark beetles and displays a yeast-mycelium dimorphism. This trait is frequently encountered in fungal pathogens but has received only limited attention in the DED fungi. The general objective of the proposed research is to identify molecular pathways underlying yeast-mycelium dimorphism in the DED fungi and assess the contribution of this trait to parasitic fitness in these pathogens. I propose to investigate 1) genes involved in nitrogen uptake and metabolism, and 2) their possible link with genes in the Mitogen-Activated Protein Kinase (MAPK) and cyclic AMP-dependent Protein Kinase A (cAMP-PKA) pathways. I also propose 3) to study genes involved in the conversion of fatty acids into derivatives including oxylipins. Short-term objectives are to : i) assess the chemical environment faced by O. novo-ulmi in elm tissue; ii) obtain knockout and/or knockdown mutants for targeted genes in O. novo-ulmi; iii) characterize O. novo-ulmi mutants both phenotypically and genetically; and iv) identify gene coexpression networks associated with yeast-mycelium transition.******The general approach which I propose includes the analysis of nitrogen compounds and fatty acids in the sap and inner bark of sensitive and resistant U. americana saplings, as well as detailed analyses of wild-type- and mutant strains of O. novo-ulmi. The mutants will be obtained by disruption, editing (CRISPR-Cas9) or knockdown by RNAi interference of selected genes involved in nitrogen uptake, MAPK and cAMP-PKA signaling cascades, or fatty acid conversion. Phenotypic analysis of the mutants will include the observation of yeast-mycelium transition kinetics and inoculations of Golden Delicious apples and U. americana saplings under controlled conditions. Genetic analyses will include complementation tests and genome-wide RNAseq studies of gene expression. Gene coexpression network analyses will be conducted to fully assess the extent of transcriptional modifications induced by a single mutation and establish if different pathways are activated in coordinated fashion under a given experimental condition.******The short-term significance of the proposed work is that it will lead to the genetic identification of pathways controlling yeast-mycelium dimorphism in DED fungi and provide experimental evidence for (or against) a link with parasitic fitness. In the longer term, our findings may provide new directions for efficient control of dimorphic Ophiostomatoid fungi causing DED, other tree diseases, or diseases to humans.