Development of forward and reverse genetic methods of ectomycorrhizal fungi Lyophyllum shimeji

Research Project

Project/Area Number	17K15287
Research Category	Grant-in-Aid for Young Scientists (B)
Allocation Type	Multi-year Fund
Research Field	Forest science
Research Institution	The University of Shiga Prefecture
Principal Investigator	Izumitsu Kosuke 滋賀県立大学, 環境科学部, 講師 (20579263)
Project Period (FY)	2017-04-01 – 2020-03-31
Project Status	Completed (Fiscal Year 2019)
Budget Amount *help	¥4,290,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥990,000) Fiscal Year 2019: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000) Fiscal Year 2018: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000) Fiscal Year 2017: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Keywords	外生菌根菌 / 遺伝子ノックアウト / KU80 / MAPキナーゼ / オートファジー / アグロバクテリウム法 / 遺伝子破壊 / ホンシメジ / アカマツ / 逆遺伝学
Outline of Final Research Achievements	The purpose of this study was to develop forward and reverse genetic methods to establish a basis for molecular genetic research for the ectomycorrhizal fungus Honshimeji (Lyophyllum shimeji). First, we generated a disrupted strain of the KU80 gene, which is involved in non-homologous recombination. Using this KU80 disrupted strain as a parent strain, three MAP kinase genes (KSS1, (MPK1, HOG1) and autophagy-related gene ATG8 were successfully disrupted by homologous recombination. Homologous recombination efficiency was about 92% when the KU80 disruption strain was used as the parent strain, which was a dramatic increase compared to the wild strain as the parent strain.
Academic Significance and Societal Importance of the Research Achievements	外生菌根共生や子実体形成の分子メカニズムを研究していくための分子基盤が構築できた。これにより，今後は遺伝子発現解析などを用いて菌根共生または子実体形成に関与することが予測された遺伝子群について，速やかに遺伝子破壊株を作出し，機能解析を行うことが可能となる。長期的には，外生菌根菌の応用利用やマツタケ，トリュフなどの人工栽培が困難な外生菌根菌の栽培手法構築にも貢献できる可能性がある。