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: $ 2.4万
• 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: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=645310
• 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 世纪初以来，连续两次荷兰榆树病 (DED) 大流行导致超过 10 亿棵榆树死亡。高度侵袭性的子囊菌 Ophiostoma novo-ulmi 是当前持续的 DED 大流行的罪魁祸首，并对北美最广泛分布的本土榆树种美洲榆的种群产生了巨大影响。在自然界中，O. novo-ulmi 由树皮甲虫传播，并表现出酵母菌丝体二态性。这一特征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。 本研究的总体目标是确定 DED 真菌中酵母菌丝体二态性的分子途径，并评估该性状对这些病原体寄生适应性的贡献。我建议研究 1) 参与氮吸收和代谢的基因，以及 2) 它们与丝裂原激活蛋白激酶 (MAPK) 和环 AMP 依赖性蛋白激酶 A (cAMP-PKA) 途径中的基因的可能联系。我还建议3）研究参与脂肪酸转化为包括氧脂素在内的衍生物的基因。短期目标是： i) 评估榆树组织中 O. novo-ulmi 面临的化学环境； ii)获得O. novo-ulmi中目标基因的敲除和/或敲低突变体； iii) 表征 O. novo-ulmi 突变体的表型和遗传特征； iv) 识别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洲树苗的树液和内皮中的氮化合物和脂肪酸，以及 O. novo-ulmi 野生型和突变株的详细分析。突变体将通过破坏、编辑 (CRISPR-Cas9) 或通过 RNAi 干扰敲低参与氮吸收、MAPK 和 cAMP-PKA 信号级联或脂肪酸转化的选定基因来获得。突变体的表型分析将包括观察酵母菌丝体转变动力学以及在受控条件下接种金冠苹果和美国树苗。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，以全面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。******所提出的短期意义其工作在于，它将导致对 DED 真菌中控制酵母菌丝体二态性的途径进行遗传鉴定，并为支持（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引起干眼症、其他树木疾病或人类疾病的二形性蛇口真菌提供新的方向。