Function Study Of Conidiation,Parasitism And Penetration Associated Gene CmSMT1 In Coniothyrium Minitans

Coniothyrium minitans is an important sclerotial parasitic fungus of Sclerotinia sclerotiorum,it has significant biological control effects on crop Sclerotinia diseases,and C.minitans agents have been developed commercially.Further understanding the mechanism of growth,sporulation and parasitism of C.minitans will be beneficial to improve the biological control efficiency.In the previous study,a T-DNA insertional mutant ZS-1TN11990 which showed stunt growth,conidial defect and weak parasitic ability had been screened,and further found that the T-DNA marker was inserted on a gene encoding for sterol C-24 methyltransferase gene（CmSMT1）.This study aimed to further investigate the characteristics and biological functions of the gene CmSMT1 of C.minitans.The results were as follows:CmSMT1 is 1220 bp in length and contains two introns and three exons by comparing the genome database of C.minitans.It encodes protein with 358 amino acids.By aligning the NCBI protein database,two functional domains were discovered,there were Methyltransf₂5 conserved domain and a Sterol_MT_C conserved domain at the C-terminus.CmSMT1 is closely related to a gene of Paraphaeosphaeria sporulosa which encodes a putative tocopherol O-methyltransferase.Real-time PCR showed that CmSMT1 was at the highest expression at 72 h.Knockout mutants of CmSMT1 were obtained by split mark method.The CmSMT1-knockout mutants showed similar phenotype of the T-DNA insertional mutant,but significantly different from wildtype strain ZS-1.Themutants colonies formed on the PDA were abnormal with white clour,no conidia were produced,the growth rate,biological mass decreased,and the parasitic ability also were significantly decreased.Furthermore,the knockout of CmSMT1 led to the accumulation of reactive oxygen species.The knockout mutants was sensitive to sorbitol,glycerol and sodium chloride hypertonic medium indicating that the gene is associated with cellular osmotic balance..Complementary mutants of CmSMT1 were obtained by Agrobacterium tumefaciens-mediated technique using a complementary vector,and the complementary mutants could recover the above phenotype.The results indicated that CmSMT1 was associated with abnormal phenotype.The secondary metabolites of wildtype strain ZS-1 and knockout mutants were compared by LC-MS.Total ion chromatorgraphy of the two CmSMT1-knockout mutants showed the similar profiles.KO-CmSMT1-1 and KO-CmSMT1-2 had 6550 and 6835 differential metabolic components comparing to strain ZS-1 with no restrict conditions,respectively.While,under the conditions of p-value ≤ 0.01 and fold change ≥ 2,there were 534 significant differential metabolic components in KO-CmSMT1-1 comparing to ZS-1,among then 361 metabolites were upregulated and 173 metabolites were downregulated;and 1128 significant differential metabolites in KO-CmSMT1-2,among them 975 metabolites were upregulated,and 153 metabolites were downregulated.Two Group data of knockout mutants were further compared,379 differential metabolic components were shared in the two knockout mutants.The metabolic data of KO-CmSMT1-1 and KO-CmSMT1-2 were further predicted by systematic biological analysis,and the significantly downregulated metabolites,（2E,6E）-farnesyl diphosphate and dimethylallyl diphosphate were found,both were closely related to the ergosterol biosynthesis pathway.In addition,pyridoxal 5’ phosphate and 4-amino-2-methyl-5-diphosphate methylpyrimidine were significantly downregulated and riboflavin was significantly upregulated in knockout mutants.Our finding indicated that CmSMT1 is essential for the growth,development,parasitic ability of C.minitans.Disruption of CmSMT1 led to decrease of（2E,6E）-farnesyl diphosphate and dimethylallyl diphosphate that could negatively affect the synthesis of ergosterol which is an essential indispensable component of fungal cell membrane;and the highly accumulation ribooflavin in hyphae led to the increase of reactive oxygen species.