Marker-free genome editing in the edible mushroom, Pleurotus ostreatus, using transient expression of genes required for CRISPR/Cas9 and for selection

In a previous study, we reported a transient transformation system using repeated screening for hygromycin B (Hyg) resistance in the basidiomycete Ceriporiopsis subvermispora. In the present study, by combining this technique with CRISPR/Cas9, we demonstrated successful marker-free genome editing in Pleurotus ostreatus, which is one of the most economically important cultivated mushrooms as well as a model white-rot fungus. At first, transformant selection mediated by the transient expression of marker genes was demonstrated using a plasmid harboring the Hyg resistance gene (hph) in P. ostreatus. Then, genome editing of fcy1, which confers 5-fluorocytosine (5-FC) resistance to the host cell, was performed by the transient expression of Cas9, gRNA, and hph and strains with 5-FC resistance and Hyg sensitivity were isolated. Additionally, genome editing of fcy1 in these strains was confirmed by Sanger sequencing. To our knowledge, this is the first report of marker-free genome editing through the transient expression of Cas9, gRNA, and hph in agaricomycetes, which opens the door for repeated genome editing in these fungi

植物病原糸状菌の高浸透圧ストレス適応と殺菌剤感受性に関わる二成分制御シグナル伝達経路に関する研究

京都大学0048新制・課程博士博士(農学)甲第15420号農博第1805号新制||農||978(附属図書館)学位論文||H22||N4519(農学部図書室)27898京都大学大学院農学研究科地域環境科学専攻(主査)教授 二井 一禎, 教授 舟川 晋也, 教授 渡邊 隆司学位規則第4条第1項該当Doctor of Agricultural ScienceKyoto UniversityDA

Two-Component Response Regulators Ssk1p and Skn7p Additively Regulate High-Osmolarity Adaptation and Fungicide Sensitivity in Cochliobolus heterostrophus

Filamentous ascomycetous fungi possess many histidine kinases and two conserved response regulators, Ssk1p and Skn7p, in their two-component signaling systems. We previously reported that the fungus unique group III histidine kinase regulates high-osmolarity adaptation and iprodione/fludioxonil fungicide sensitivity by controlling the phosphorylation of Hog1-type mitogen-activated protein kinase (MAPK) in filamentous ascomycetes. Here, we have characterized the response regulator genes ChSsk1 and ChSkn7 in the southern corn leaf blight fungus Cochliobolus heterostrophus. Both ChSsk1- and ChSkn7-disrupted mutants showed little sensitivity to high-osmolarity stress and moderate resistance to the iprodione/fludioxonil fungicides. The phosphorylation of Hog1-type MAPK BmHog1p induced by high-osmolarity stress and fungicide treatments was only regulated by ChSsk1p, indicating that ChSkn7p has roles in high-osmolarity adaptation and fungicide sensitivity that are independent from the activation of BmHog1p. The Chssk1 Chskn7 double mutants clearly showed higher sensitivity to osmolar stress and higher resistance to fungicides than the single mutants. The dose responses of the double mutants fit well with those of the group III histidine kinase-deficient strain. These results suggest that in filamentous ascomycetes, the Ssk1- and Skn7-type response regulators control high-osmolarity adaptation and fungicide sensitivity additively with differential mechanisms under the regulation of the group III histidine kinase. This study provides evidence that filamentous fungi have a unique two-component signaling system that is different from that of yeast and is responsible for high-osmolarity adaptation and fungicide sensitivity