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

荷兰榆树病真菌中的酵母菌丝体二态性及其对致病性和整体适应性的贡献

• 批准号: RGPIN-2018-06607
• 负责人: Bernier, Louis
• 金额: $ 4.81万
• 依托单位: Université Laval
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=765389
• 关键词: Yeast; mycelium; dimorphism; Dutch; elm

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.

自20世纪初期以来，两种连续的荷兰榆树疾病（DED）大流行病杀死了超过十亿的榆树。高度侵略性的comcomycete真菌Novo-Ulmi负责当前的DED大流行，并对北美最广泛的本地ELM物种Ulmus Americana的种群产生了巨大影响。在自然界中，O。Novo-Ulmi被树皮甲虫进行了培训，并显示出酵母肌二态性。这种特征经常在真菌病原体中遇到，但在DED真菌中仅受到有限的关注。 拟议的研究的一般目标是确定DED真菌中酵母肌二晶的分子途径，并评估该性状对这些病原体中寄生型适应性的贡献。我建议研究1）参与氮摄取和代谢的基因，以及2）它们可能与有丝分裂原激活的蛋白激酶（MAPK）（MAPK）和环状AMP依赖性AMP依赖性蛋白激酶A（CAMP-PKA）途径的基因联系。我还建议3）研究涉及脂肪酸转化为衍生物在内的基因。短期目标是：i）评估O. Novo-Ulmi在榆树组织中面临的化学环境； ii）在O. ulmi中获得靶向基因的基因敲除和/或敲除突变体； iii）在表型和遗传上以表型和遗传学的特征来表征O. novo-ulmi突变体；和iv）确定与酵母膜过渡相关的基因共表达网络。我建议的一般方法包括对敏感和抗性的美国苗条的氮化合物和脂肪酸的分析，以及对野生型和突变型Novo-ulmi的野生型和突变菌株的详细分析。突变体将通过破坏，编辑（CRISPR-CAS9）或通过RNAi干扰涉及氮摄取，MAPK和CAMP-PKA信号级联反应或脂肪酸转化的RNAi干扰来获得突变体。突变体的表型分析将包括观察酵母 - 乳纤维过渡动力学，以及在受控条件下的黄金美味苹果和美国美洲苗的接种。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引起的转录修饰的程度，并在给定的实验条件下确定不同的途径是否以协调的方式激活不同的途径。拟议的工作的短期意义是，它将导致遗传识别型杂种型在专用拟合型和实验性方面的遗传识别（或提供实验性的依赖性）（或提供实验性链接）（或提供实验性的链接）。从长远来看，我们的发现可能会提供新的方向，以有效控制二态性ophiostomotoid真菌，从而导致DED，其他树疾病或对人类的疾病。