| Summary: | <i>Clonostachys rosea</i> is an excellent biocontrol fungus against numerous fungal plant pathogens. The cAMP signaling pathway is a crucial signal transduction pathway in fungi. To date, the role of the cAMP signaling pathway in <i>C. rosea</i> mycoparasitism remains unknown. An adenylate cyclase-encoding gene, <i>crac</i> (an important component of the cAMP signaling pathway), was previously screened from <i>C. rosea</i> 67-1, and its expression level was dramatically upregulated during the <i>C. rosea</i> mycoparasitization of the sclerotia of <i>Sclerotinia sclerotiorum</i>. In this study, the function of <i>crac</i> in <i>C. rosea</i> mycoparasitism was explored through gene knockout and complementation. The obtained results show that the deletion of <i>crac</i> influenced the growth rate and colony morphology of <i>C. rosea</i>, as well as the tolerance to NaCl and H<sub>2</sub>O<sub>2</sub> stress. The mycoparasitic effects on the sclerotia of <i>S. sclerotiorum</i> and the biocontrol capacity on soybean Sclerotinia stem rot in ∆<i>crac-6</i> and ∆<i>crac-13</i> were both attenuated compared with that of the wild-type strain and complementation transformants. To understand the regulatory mechanism of <i>crac</i> during <i>C. rosea</i> mycoparasitism, transcriptomic analysis was conducted between the wild-type strain and knockout mutant. A number of biocontrol-related genes, including genes encoding cell wall-degrading enzymes and transporters, were significantly differentially expressed during <i>C. rosea</i> mycoparasitism, suggesting that <i>crac</i> may be involved in <i>C. rosea</i> mycoparasitism by regulating the expression of these DEGs. These findings provide insight for further exploring the molecular mechanism of <i>C. rosea</i> mycoparasitism.
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