Screening and characterisation of proteins interacting with the mitogen-activated protein kinase Crmapk in the fungus Clonostachys chloroleuca

Clonostachys chloroleuca 67-1 (formerly C. rosea 67-1) is a promising mycoparasite with great potential for controlling various plant fungal diseases. The mitogen-activated protein kinase (MAPK)-encoding gene Crmapk is of great importance to the mycoparasitism and biocontrol activities of C. chloroleuca. To investigate the molecular mechanisms underlying the role of Crmapk in mycoparasitism, a high-quality yeast two hybrid (Y2H) library of C. chloroleuca 67-1 was constructed, and proteins interacting with Crmapk were characterised. The library contained 1.6 × 10(7) independent clones with a recombination rate of 96%, and most inserted fragments were > 1 kb. The pGBKT7-Crmapk bait vector with no self-activation or toxicity to yeast cells was used to screen interacting proteins from the Y2H library, resulting in 60 candidates, many linked to metabolism, cellular processes and signal transduction. Combined bioinformatics and transcriptome analyses of C. chloroleuca 67-1 and ΔCrmapk mutant mycoparasitising Sclerotinia sclerotiorum sclerotia, 41 differentially expressed genes were identified, which might be the targets of the Fus3/Kss1-MAPK pathway. The results provide a profile of potential protein interactions associated with MAPK enzymes in mycoparasites, and are of great significance for understanding the mechanisms of Crmapk regulating C. chloroleuca mycoparasitism.