Functional Analysis Of Two-component Histidine Kinase Gene CmHK03 In Coniothyrium Minitans

Sclerotinia sclerotiorum（Lib.）de Bary is a worldwide plant pathogenic fungus,which causes sclerotinia disease of many crops and results in serious economic losses.Coniothyrium minitans is a mycoparasite of S.sclerotiorum,which can be used to control sclerotinia diseases.Two-component histidine kinases（HKs）are involved in the regulation of various life activities in fungi.However,the effects of HKs on the growth and development,mycoparasitism and fungicide resistance of C.minitans are still unclear.In this study,we investigated the function of the two-component histidine kinase gene Cm HK03 in C.minitans by the deletion,complemention and over-expression of this gene using the PEG mediated protoplast transformation.The role of Cm HK03 in growth and development,mycoparasitism and fungicide resistance was analyzed.The major results of this study are summarized below:1. The deletion mutant of Cm HK03 showed increased resistance to dicarboximides and phenylpyrrole fungicides,while exhibited no difference with wild type in sensitivity to triazole,pyrimidine,imidazole and methoxyacrylate fungicides.2. Sensitivity to osmotic stress showed that the Cm HK03 deletion mutant significantly increased its sensitive to neutral osmotic stress（1 M sorbitol,or 1 M glucose）and ionic osmotic stress（0.6 M Na Cl,or 0.6 M KCl）.The deletion mutant of Cm HK03 revealed no difference with wild type in response to other abiotic stresses,including 0.2 M Ca Cl₂,8 m M H₂O₂,150μg/m L CFW,and 50μg/m L CR.The results of NBT staining showed that deletion or over-expression of Cm HK03 had no significant effect on the concentration of ROS in the mycelia.3. The results showed that the concentration of glycerol in mycelia of wild type and Cm HK03 deletion mutant were increased significantly when treated with sorbitol（1 M）for 5 h,but the increase of glycerol in Cm HK03 deletion mutant was significantly lower than that in wild type.However,when treated with fludioxonil（5.0μg a.i./m L）for 5 h,the concentration of glycerol in the mycelia of wild type and Cm HK03 deletion mutant did not change significantly.It is suggested that Cm HK03might involve in regulation of glycerol synthesis in response to osmotic stress.4. RT-q PCR results showed that the expression of Cm HK03,Sho1 and Hog1were up-regulated by 6. 9,1.83 and 2.06-fold in wild type after treatment with fludioxonil（5.0μg a.i./m L）for 12 h,respectively.While Sho1 and Hog1 were down-regulated by 0.34 and 0.72-fold in Cm HK03 deletion mutants after treatment with fludioxonil（5.0μg a.i./m L）,respectively.These results suggested that Cm HK03is involved in the regulation of fungicide resistance,which may be related to Hog1signaling pathway.5. Sensitivity to oxalic acid showed that deletion or over-expression of Cm HK03had no significant effect on the response to oxalic acid in C. minitans.6. Deletion or over-expression of Cm HK03 had no significant effect on mycelial growth,conidiation,and mycoparasitism in C. minitans.In conclusion,results of this study indicated that deletion of Cm HK03 resulted in increased resistance to dicarboximides and phenylpyrrole fungicides,and Cm HK03might also be involved in the regulation of glycerol synthesis in response to osmotic stress.Moreover,deletion of Cm HK03 had no significant effect on mycelial growth,conidiation and mycoparasitism in C.minitans.Therefore,Cm HK03 deletion mutant is expected to be mixed with dicarboximides or benzopyrrole fungicides to control the resistant strains of S.sclerotiorum in the field.