Functional Analysis Of Melanins Associated Gene CmMR1 In Coniothyrium Minitans

Coniothyrium minitans is a sclerotial parasite,and is explored to control crops diseases caused by Sclerotinia sclerotiorum commercially.Previously we obtained a T-DNA insertional mutant ZS-1TN24363 which could not produce melanin during conidiation.To understand the possible function of melanin for C.minitans,mutant ZS-1TN24363 was studied.The results were as follows:1.We cloned the gene disrupted by T-DNA insertion,and we found that this gene,we called CmMR1,encoded putative protein with 1011 aa which is a homologue of transcription factor MR.The full-length of CmMR1 is 3167 bp with three exons and two introns.We found that the protein which CmMR1 contained three conserved domain,Zn-clus,Med3 and fungal-TF-MHR.This protein is conserved in fungi,phylogenetic analysis revealed that this protein was closely related to Clohesyomyces aquaticus Cmr1.2.To confirm this disrupted gene is response to the melanin-deficiency of mutant,this gene was targeted disrupted,and three targeted knockout mutants were obtained.Biological assay showed that the phenotype of targeted knockout mutants is similar to that of T-DNA insertional mutant.Colonies of mutant ZS-1TN24363 and knockout transformants lacked melanin and acquired an orange color,but they could form pycnidia and conidia as wildtype strain ZS-1 did.Furthermore,gene complementary experiment results further confirmed that the CmMR1 gene is responsible to the phenotype change of mutant.ZS-1TN24363,knockout transformants and ZS-1,complementary transformants had no significant difference in growth rate,sporulation,biomass,parasitic capacity and tolerance to osmotic.But,interestingly,the accumulation of ROS in mutants was increased and anti-UV light ability was significantly decreased.3.In mutant ZS-1T24363,the expression of Cm4 HNR,CmSCD,Cm3 HNR and CmLAC1 were significantly down-regulated,suggesting that these genes were regulated by CmMR1.To summarise,disruption of CmMR1 gene could not affect the hyphal growth,conidiation and parasitzation of C.minitans,but is closely related to the formation of melanin.