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

自20世纪初以来，连续两次荷兰榆病(DED)大流行导致超过10亿只榆树死亡。高度侵袭性的子囊菌Ophiostoma novo-ulmi是目前正在进行的DED大流行的罪魁祸首，并对北美分布最广泛的本土榆树--美洲榆树的种群产生了巨大影响。在自然界中，Onovo-ulmi是由树皮甲虫传播的，并表现出酵母-菌丝体二型性。这一特征经常出现在真菌病原体中，但在DED真菌中只受到有限的关注。这项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，并评估这一特性对这些病原菌的寄生适合性的贡献。我建议研究1)与氮的吸收和代谢有关的基因，2)它们与丝裂原激活的蛋白激酶(MAPK)和环磷酸腺苷依赖的蛋白激酶A(cAMP-PKA)途径中的基因的可能联系。我还建议研究与脂肪酸转化为包括氧脂在内的衍生品有关的基因。短期目标是：i)评估O.novo-ulmi在榆树组织中所面临的化学环境；ii)获得针对O.novo-ulmi中目标基因的敲除和/或敲除突变；iii)鉴定O.novo-ulmi突变体的表型和遗传特征；以及iv)鉴定与酵母-菌丝体转换相关的基因共表达网络。*我提出的一般方法包括分析敏感和抗性美洲根癌菌幼树树液和内树皮中的氮化合物和脂肪酸，以及野生型和突变株的详细分析。这些突变体将通过中断、编辑(CRISPR-Cas9)或通过RNAi干扰参与氮吸收、MAPK和cAMP-PKA信号级联或脂肪酸转换的选定基因而获得。突变株的表型分析将包括观察酵母-菌丝转化动力学，以及在受控条件下接种金冠苹果和美洲豆苗。遗传分析将包括互补测试和基因表达的全基因组RNAseq研究。基因共表达网络分析将被用来全面评估单个突变引起的转录修饰的程度，并确定在给定的实验条件下，不同的途径是否以协调的方式被激活。拟议工作的短期意义是，它将导致对控制DED真菌中酵母-菌丝体二态的途径的遗传鉴定，并提供实验证据，支持(或反对)与寄生适合度的联系。从长远来看，我们的发现可能为有效控制引起DED、其他树木疾病或人类疾病的二形性蛇口类真菌提供新的方向。

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.