精氨酸调节菰黑粉菌二型态转换的分子机制研究

Arginine was important for promoting most fungi to switch from unicellular yeast growth forms to multicellular filamentous growth forms (dimorphism) to succeed in survival and environmental adaptation. Previous research results demonstrated that Ustilago esculenta grows within the flowering stem of the aquatic grass Zizania latifolia and induces swelling or enlargement of the tissue near the base of the host plant and forming edible galls by dimorphism. However, in U. esculenta, exogenous arginine prevented filamentous forms formation obviously, which was distinct from other reported positive regulatory role on fungi dimorphism regulation，indicating that the regulation mechanism of arginine involved in fungi dimorphism was diversity, but there was little conclusive evidence. In the present study, two compatible haploid U. esculenta strains with different mating type genes and with ability to fuse to hypha in plates were used as research objects and transgenic strains of knocking out or over-expressing genes involved in arginine biosynthesis and metabolism were constructed, in order to clear the key factor directly function on fungi dimorphism in cells and analysis the molecular pathway which the key factor was involved in, based on the MAPK/PKA signaling pathway defective mutants; meanwhile to analysis whether the environment factors involved in fungi dimorphism (exogenous arginine or reactive oxygen species (ROS) ) participate in endogenous arginine synthetic metabolism, based on stable isotope labeling and other technique; finally to identify the importance of arginine regulated dimorphism involved in the interaction between U. esculenta and Z. latifolia, based on the artificial inoculation and tissue slice technique. Our expected findings will help to uncover the underlying mechanism of arginine involved in fungi dimorphism in U. esculenta, and the related plant signals, further to provide theoretical guidance for edible galls formation. Also it is a supplement mechanism of arginine involved in fungi dimorphism.

精氨酸是促进多数病原真菌由酵母型向菌丝型转换（二型态转换）以实现侵染的重要因子。项目组前期研究发现菰黑粉菌二型态转换是茭白形成的关键，其中精氨酸起重要调节作用。但是在菰黑粉菌中，外源精氨酸显著抑制菌丝形成，与其在真菌中被广泛证实的正向调控作用相反，表明精氨酸调节真菌二型态转换机制存在多样性。本项目通过菰黑粉菌中精氨酸合成代谢途径基因的敲除和回补，结合MAPK/PKA途径缺陷突变体，明确细胞内直接作用于二型态转换的关键因子及其作用的分子途径；同时结合稳定同位素标记技术明确调节二型态转换的环境因子（外源精氨酸或活性氧）与细胞内精氨酸合成代谢的关系；最后结合人工接种及组织切片技术验证精氨酸调节的二型态转换在菰黑粉菌与茭白植株互作中的作用。预期结果不仅可揭示精氨酸调节菰黑粉菌二型态转换的分子机制及可能参与的植物信号，为开展茭白孕茭机制研究提供理论基础，亦是对精氨酸调节真菌二型态转换机制的补充。

精氨酸是促进多数病原真菌由酵母型向菌丝型转换（二型态转换）以实现侵染的重要因子。项目组前期研究发现菰黑粉菌二型态转换是茭白形成的关键，其中精氨酸起重要调节作用。本项目通过菰黑粉菌中精氨酸合成代谢途径基因的敲除表型分析结合精氨酸含量动态变化，明确了精氨酸是直接作用于二型态转换的关键因子；进一步观察不同浓度精氨酸处理MAPK途径基因（UeKpp2和UeKpp6）以及PKA途径基因（UePkaC）缺失突变体后的表型变化以及分析相关基因的表达情况，明确精氨酸参与调节UeKpp2调控的菰黑粉菌二型态转换；同时用不同浓度稳定同位素标记的外源精氨酸或不同水平的过氧化氢处理分析野生型菌株二型态转换表型差异，结合内源精氨酸合成量的变化及精氨酸合成代谢途径基因的表达分析，明确外源精氨酸和活性氧是影响精氨酸合成代谢从而调节菰黑粉菌二型态转换的重要环境因子，其中外源精氨酸通过精氨酸的反馈调节途径作用于精氨酸合成从而影响菰黑粉菌二型态转换，而活性氧通过正向调控UeArg8的表达来促进精氨酸合成从而抑制菰黑粉菌二型态转换；最后通过人工接种试验验证了精氨酸调节的二型态转换机制在菰黑粉菌与茭白植株互作中的重要性，明确了互作过程中活性氧作为互作信号分子起重要作用。预期结果揭示了精氨酸调节菰黑粉菌二型态转换的分子机制及可能参与的植物信号，为明晰二型态转换在菰黑粉菌致病过程中的作用及相关调控因子奠定基础，同时研究优化的菰黑粉菌人工接种体系为茭白与菰黑粉菌的互作机制研究提供了保障。通过项目研究，共在Fungal Genetics & Biology、BMC Microbiology 等期刊上发表论文 10 篇，其中 SCI 收录论文6篇，授权发明专利4项。培养硕士研究生 3 名。