Colletotrichum lentis is a serious foliar and stem pathogen causing anthracnose in lentil, which can lead to major losses in crop productivity. This fungal pathogen is asexual under field conditions but can undergo sexual reproduction in the laboratory, where two mating incompatibility groups (mIG-1 and mIG-2) were identified. Based on the absence of MAT1-1 and the presence of MAT1-2 in isolates of both mIGs, it was previously determined that C. lentis does not follow the classical mating system of heterothallic ascomycetes, which is governed through the mating type (MAT) locus. Bulk segregant analysis of an ascospore-derived population had revealed one major QTL, qClMAT3, on chromosome 3 of the C. lentis genome associated with mating incompatibility, but it is devoid of classical MAT genes. It was therefore hypothesized that genes other than the classical MAT genes may be regulating compatibility of two isolates as the first step in mating and the development of sexual reproductive structures. Using Colletotrichum gene-specific parameters, a total of 106 genes were predicted at QTL qClMAT3 through the gene prediction tool FGENESH. Blasting (BLASTx) against the genera Colletotrichum, Neurospora and Podospora to identify homologous genes, and a search of the literature for evidence of an involvement in various pathways regulating mating identified 28 genes with a role in mating. As C. lentis appears to follow a heterothallic incompatibility system the status of these 28 genes in isolates of the two mIGs was analyzed in terms of presence or absence and sequence polymorphisms. Presence or absence of genes was not correlated with mIG, nor were there polymorphisms that were characteristic for specific mIGs. To study the temporal dynamics of candidate genes in vegetative mycelium and at several sexual stages, four developmental stages of perithecia were determined based on morphological characters such as the shape and color of perithecia as well as the development of asci and ascospores. Gene expression studies in individual mycelia, a vegetative co-culture of two compatible isolates and in two developmental stages of perithecia through RT-qPCR of 11 most promising candidate genes revealed that two genes, Cl_Nop2 and Cl_IDC1, showed differential expression in the vegetative mycelia of CT-21 and CT-30. Cl_IDC1 plays a role in mRNA trafficking during chemotropic interactions in model species P. anserina and N. crassa, and Cl_Nop2 plays a role in cell viability. Four genes (Cl_HP3, Cl_SART1, Cl_Hpt and Cl_KH) associated with signaling pathways during chemotropic interactions showed upregulation in the co-culture compared to other stages, except for Cl_HP3, which was also upregulated in stage 3 perithecia. Expression of Cl_BING4CT associated with vegetative incompatibility and Cl_HP9 involved in cell cycle was downregulated in the co-cultures. The expression of Cl_Cro1, a gene expressed mainly during the dikaryotic stage and ascospore maturation in P. anserine, and mutation of which resulted in defects in septum formation in that species, was downregulated in stage 2 perithecia as compared to other stages. Expression of Cl_BING4CT, Cl_HP9 and Cl_IDC1, specifically in the mycelial co-cultures, identified them as the most promising candidates for regulating mating incompatibility, which requires further validation through a gene silencing approach or the creation of mutants followed by mating experiments
