Screening And Functional Analysis Of The Genes Related To Mycoparasitism In Clonostachys Rosea 67-1

Clonostachys rosea has significance potential for biocontrol of fungal plant pathogens.However,little studies of mycoparasitism genes were reported.Six genes with remarkably up-regulated expression level（sodium P-type ATPase encoding gene crena,heterokaryon incompatibility protein encoding gene crhip,ethanolamine utilization protein encoding gene cruet and creutq,short-chain dehydrogenase protein encoding gene crsdr and Unigene494）were isolated from C.rosea 67-1 transcriptome during mycoparasitism of Sclerotinia sclerotiorum.Full DNA sequence of crena was obtained from the whole genome of C.rosea 67-1.The coding region of crena was 1419 bp,which encoding a protein consist of 472 amino acids.Phylogeny anslysis showed that crena had 73.8%similarity with sodium P-type ATPase from Acremonium chrysogenum.Phenotypes analysis showed that the growth rate and ofΔcrena was significantly lower than that of wild type and the complemented strain.The parasitic ability ofΔcrena to sclerotia was reduced from 4^th grade to 2^nd grade compared with the WT and complemented mutant.The control efficacy ofΔcrena to Fusarium wilt was decreased by 48.5%compared with the WT and complemented mutant.The results indicated that crena might be involved in the mycoparasitism of C.rosea 67-1.Bioinformatics analysis showed that the coding region of crhip was 1749 bp,encoding a protein consist of 597 amino acids.Phylogeny anslysis showed that crhip was closest to heterokaryon incompatibility protein from Colletotrichum orbicular with a homology of 43.1%.Phenotypes analysis showed that the growth rate ofΔcrhip was significantly lower than that of wild type and the complemented mutant.The parasitic ability ofΔcrhip to sclerotia was reduced from 4^th grade to 3^rd grade compared with WT and complemented strain.The control efficacy ofΔcrhip to Fusarium wilt was decreased by 24.5%.The results showed that crhip might be involved in the mycoparasitism of C.rosea 67-1.Bioinformatics analysis showed that the coding region of creut was 363 bp,encoding a protein consist of 120 amino acids.Phylogeny anslysis showed that creut had 81%similarity with heterokaryon incompatibility protein from Colletotrichum higginsianum.Gene knockout was conducted by PEG-Ca Cl₂mediated transformation method,and a creut deleted mutant?creut was obtained.The bioassay showed that?creut was able to inhibit the germination of sclerotia of S.sclerotiorum,however,its parasitic ability to sclerotia was reduced from 4^th grade to 2^nd grade than that of WT.Under a microscope it coud see that the mycelia of the wild strain 67-1 adhered,wrapped and penetrated the mycelia of R.solani.In addition,the control efficiency of?creut against Fusarium wilt decreased by 41%compared to the wild type.The results indicated that creut might be involved in the mycoparasitism and biocontrol of C.rosea and provided insight into the molecular mechanisms underlying C.rosea mycoparasitism on plant pathogenic fungi.The coding region of crsdr was 495 bp,and was closest to short-chain dehydrogenase protein from Colletotrichum gloeosporioides with a homology of 56.1%.We constructed the knock-out vector by homologous recombination and obtaind the deletion mutantΔcrsdr by PEG-Ca Cl₂ mediated protoplast transformation.Δcrsdr showed a significantly decreased in parasitic.The control efficacy ofΔcrsdr against Fusarium wilt was decreased by 42.4%compared with WT.The results demonstrated that crsdr might be involved in the mycoparasitism of C.rosea 67-1.This study provided insight into the molecular mechanisms underlying C.rosea mycoparasitism on plant pathogenic fungi.