Functional Research Of Conidiation Associated Genes In Coniothyrium Minitans

Coniothyrium minitans is a mycoparasite of Sclerotinia sclerotiorum.The conidial production and parasitism are two important aspects for commercialization of this biological control agent.To understand the mechanism of conidiation and parasitism at the molecular level,we constructed a T-DNA insertional library with the wild-type strain ZS-1.In this research,three conidiation-deficient mutants ZS-1N22225,ZS-1TN1961 and ZS-1TN5012 were screened from the T-DNA insertional library and studied.The results were as follows:Mutant ZS-1N22225 showed lighter pigmentation and significantly reduced hyphal growth,conidiation as well as mycoparasitism.Only one copy of T-DNA was inserted into the genome of mutant ZS-1N22225.Through inverse PCR technique,we found CmVps39,a homologue of Vam6/Vps39 was disrupted by a T-DNA insertion.CmVps39 is predicted to consist of 3315 bp,including a 3216-bp open reading frame and two introns,and to encode a protein of 1071 amino acids.CmVps39 in the mutant ZS-1N22225 was disrupted by T-DNA insertion at the nucleotide position 442,before the translational start codon(ATG),which was predicted to be the promoter region and the insertion event in the promoter region reduced the expression of CmVps39 in ZS-1N22225.The deduced amino acid sequence of CmVps39 contains citron homology and clathrin homology domains in the N-and C-terminal regions respectively,and shares moderate similarity with Vam6p/Vps39 p homologs from various fungi.Targeted gene replacement and gene complementation tests confirmed that a null mutation of CmVps39 was responsible for the phenotype of ZS-1N22225.Further analysis showed that CmVps39 was localized to vacuoles and abnormal fragmented vacuoles were observed in ΔCmVps39 mutant.We also found CmVps39 not only affected the vacuolar morphology,but also affected the osmotic and pH homeostasis.Further analysis showed CmVps39 was involved in the autophagy pathway.The disruption of CmVps39 impaired autophagy pathway and thus resulted in defective conidiation and germination as well as mycoparasitism.Mutant ZS-1TN1961 exhibited normal hyphal growth and lighter pigmentation.Southern blot analysis confirmed there was only one copy of T-DNA inserted into the genome of mutant ZS-1TN1961.Through hiTAIL-PCR technique,we found a gene coding a hypothetical protein which contains a conserved Aim24 domain,was disrupted by T-DNA insertion in this mutant.Therefore,we named this gene as CmAim24.CmAim24 is predicted to consist of 1327 bp including a 1131-bp open reading frame and three introns.CmAim24 in the ZS-1TN1961 mutant was disrupted by T-DNA insertion at the nucleotide position 46 nt after the translational start code(ATG).Gene replacement and complementation experiments confirmed that CmAim24 was essential for conidiogenesis and conidia germination as well as mycoparasitism of C.minitans.Furthermore,cellular localization assays showed that CmAim24 was localized to mitochondria and abnormal mitochondria were observed in ΔCmAim24 mutant.ΔCmAim24 mutant exhibited significantly increased accumulation of reactive oxygen species(ROS)in the hyphal and a growth defect on non-fermentable medium.Conidiation-deficient mutant ZS-1TN5012 exhibited lighter pigmentation and significantly reduced hyphal growth as well as mycoparasitism.Southern blot analysis confirmed there was only one copy of T-DNA inserted into the genome of mutant ZS-1TN5012.hiTAIL-PCR technique was used to amplify the T-DNA flanking genomic DNA sequence in ZS-1TN5012.The obtained flanking sequence contained the left border of the inserted T-DNA and an incomplete open reading frame.The coding region was obtained by comparing to C.minitans genome.A gene sharing moderate similarity with HMGR from various fungi was disrupted by a T-DNA insertion,thus named CmHmgr.CmHmgr is predicted to consist of 1170 bp and to encode a protein of 389 amino acids.CmHmgr in ZS-1TN5012 mutant was disrupted by T-DNA insertion at the nucleotide position 166 nt after the translational start code(ATG).HMGR is the rate-limiting enzyme of mevalonate pathway and statins are well-known inhibitors of HMGR.The colony morphology of ZS-1 was similar to mutant ZS-1TN5012 when atorvastatin was added into the medium and ZS-1TN5012 showed more sensitive to atorvastatin.Futhermore,cellular localization assays showed that CmHmgr was localized to mitochondria.Gene replacement and complementation experiments confirmed that CmHmgr was essential for growth,conidiogenesis and mycoparasitism of C.minitans.In summary,three genes CmVps39,CmAim24 and CmHmgr were cloned and identified from C.minitans.Through a series of experiments,we proved that these three genes play important roles in conidiogenesis and mycoparasitism.These indicate that conidial production of C.minitans is a complicated process and regulated by multi-genes.