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 × 107 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.
Clonostachys rosea is an important mycoparasite, with great potential for controlling numerous plant fungal diseases. Understanding the mechanisms and modes of action will assist the development and application of this biocontrol fungus. In this study, the highly efficient C. rosea 67-1 strain was marked with the green fluorescent protein (GFP), and the transformant possessed the same biological characteristics as the wild-type strain. Fungal interactions with Botrytis cinerea during co-culture and encounter on tomato leaves were assessed by fluorescence confocal and electron microscopy. The results indicated that once the two fungi met, the hyphae of C. rosea grew alongside those of B. cinerea, then attached tightly to the host and developed special structures, via which the biocontrol fungus penetrated the host and absorbed nutrients, eventually disintegrating the cells of the pathogen. Mycoparasitism to B. cinerea was also observed on tomato leaves, suggesting that C. rosea can colonize on plants and act following the invasion of the pathogenic fungus.
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