Serine protease CrKP43 interacts with MAPK and regulates fungal development and mycoparasitism in Clonostachys chloroleuca

Serine proteases are a group of important hydrolytic enzymes that play vital roles in various cellular processes in fungi. In this study, the S8 serine protease-encoding gene CrKP43 was identified in the highly efficient Clonostachys chloroleuca 67-1 (formerly C. rosea 67-1) strain, which was markedly upregulated when parasitizing Sclerotinia sclerotiorum. The function of CrKP43 was investigated using gene deletion and complementation, and the results indicated that the lack of CrKP43 resulted in deformed fungal hyphae and cell morphology, inhibition of conidiation, and decreased antagonistic activity toward the pathogenic fungus Fusarium oxysporum f. sp. cucumerinum. Moreover, the mutants displayed much weaker mycoparasitic ability to S. sclerotiorum sclerotia and lower control efficiency against soybean Sclerotinia rot compared with the wild-type strain. All biological characteristics and biocontrol activities were recovered when the CrKP43 gene was reinserted into the fungus. Using qRT-PCR analysis and protein-protein interaction assays, it was further proved that the CrKP43 protein interacted with the mitogen-activated protein kinase (MAPK) Crmapk, suggesting serine proteases might be involved in the mycoparasitism of C. chloroleuca with the regulation of Crmapk. The findings improve our knowledge of serine proteases and their regulation in mycoparasites and help to illuminate the mechanisms underlying mycoparasitism of C. chloroleuca.IMPORTANCE Mycoparasites play important roles in the biocontrol of plant fungal diseases, during which they secret multiple hydrolases such as serine proteases to degrade their fungal hosts. In this study, we demonstrated that the serine protease CrKP43 was involved in C. chloroleuca development and mycoparasitism with the regulation of Crmapk. To the best of our knowledge, it is the first report on the functions and regulatory mechanisms of serine proteases in C. chloroleuca. Our findings will provide new insight into the regulatory mechanisms of serine proteases in mycoparasites and contribute to clarifying the mechanisms underlying mycoparasitism of C. chloroleuca, which will facilitate the development of highly efficient fungal biocontrol agents as well.