Studies Of The Biological Functions Of MfGh27A,MfMel1 And MfSR Genes In Monilinia Fructicola

Peach brown rot mainly caused by Monilinia fructicola in China,which is one of the most important diseases in peach production.According to the transcriptomic data during the infection of M.fructicola on peach fruits in our previous study,it was found that two genes MfMel1 and MfGh27A encoding galactosidase in glycoside hydrolase family were significantly down-regulated.Therefore,it is necessary to explore whether these genes are related to the pathogenicity.At the same time,chemical control is widely used to control brown rot,among which DMI fungicides are the most effective ones.DMIs play the function to suppress the sterol synthesis.Studying the mechanisms of DMI fungicide resistance and preventing the development of fungicide resistance are very important for the sustainable management of peach brown rot in practice.The homologous gene of SR related to synthesis of sterols,MfSR,was investigated in M.fructicola.The research results are as follows.The expression of MfMel1 gene was significantly down-regulated at early stage of infection,especially was down-regulated by 16 times at 1 h after inoculation.Compared with the original strain Bmpc7,the growth rate and spore germination rate of knockout transformants were not significantly different,but the sporulation was significantly decreased,indicating that the MfMel1 gene is essential for the spore production.In terms of cell wall osmotic pressure sensitivity,knocking out the MfMel1 gene enhanced the sensitivity of knockout transformants（△MfMel1 strains）to 200 g/L glucose.The△MfMel1 strains did not show significant difference compared with Bmpc7 on the media of 0.01%SDS,600μg/ml Congo red,150 g/L glycerol,200 g/L sucrose,6 m M H₂O₂,4%Na Cl,1.2 m M sorbitol.Pathogenicity test showed that the virulence of knockout transformants to peach fruit was significantly reduced.At the same time,theα-galactosidase activity of the△MfMel1 strains decreased significantly.These results indicated that lacking of the MfMel1 gene led to the decreasedα-galactosidase activity,reduced sporulation ability and decreased virulence in M.fructicola strains.The expression of the MfGh27A gene was significantly down-regulated at early stage of infection,and the expression level at 12 h was down-regulated by 13 times.△MfGh27A strains did not show significant difference with Bmpc7 on 0.01%SDS,600μg/ml Congo red,150 g/L glycerol,200 g/L glucose,4%Na Cl media.Also,no significant difference was observed for the virulence between△MfGh27A strains and Bmpc7.However,in the 200 g/L sucrose and 1.2 m M sorbitol tolerance experiment,the growth of knockout transformants was promoted,indicating that MfGh27A gene knockout affected the osmotic regulation pathway.On PDA with 6 m M H₂O₂,the sensitivity of the knockout strains decreased,indicating that MfGh27A was involved in the redox pathway.Finally,it was shown that theα-galactosidase activity in knockout transformants was significantly lower than that in the wild-type strain Bmpc7.These results demonstrated that the MfGh27A gene was involved in the osmotic transduction pathway and redox pathway of M.fructicola,and played an important role in the synthesis ofα-galactosidase.In M.fructicola,DMI fungicide resistance is mainly caused by overexpression of the CYP51 gene encoding lanosterol demethylase which is important for the biosynthesis of the ergosterol.The MfSR gene is a homologous gene of SR genes in M.fructicola which encode the transcription factor SR for regulating the synthesis of sterol.In order to make sure if the MfSR gene is involved in the regulating the resistance to DMI fungicides,the function of the MfSR was investigated based on the genetic transformation.Two knockout homozygotes and corresponding complementary transformants were obtained.Results showed that no difference was observed for the growth of wild-type strain Bmpc7,knockout strains and complementary strains on DMI fungicide amended medium.At the same time,MfSR gene knockout did not affect the pathogenicity of M.fructicola.Under exogenous stress,△MfSR strains did not show significant difference with Bmpc 7 on PDA with 0.01%SDS,600μg/ml Congo red,150 g/L glycerol,200 g/L sucrose,200 g/L glucose,4%Na Cl,or 1.2 m M sorbitol.However,the growth of the△MfSR strains on the6 m M H₂O₂ amended medium was inhibited,indicating that the MfSR was related to the redox stress response.These results demonstrated that the MfSR played a role in the oxidation response in M.fructicola.It should be noted that it is the first time to obtain the pure homogenous knockout transformants in multinucleate fungus M.fructicola,representing the more accurate evaluation of gene functions.