Studies Of The Biological Functions For The Transcription Factor Encoding Genes MfSre1,MfADR1 And Mfsre1n In Monilinia Fructicola

Lanosterol demethylation inhibitors?DMIs?,such as propiconazole,tebuconazole and triazolone,play an important role in controlling peach brown rot caused by Monilinia spp.worldwide.DMI fungicides inhibit the formation of fungal ergosterol by binding to cytochrome P450 family protein CYP51 to affect the structure,fluidity and protein function of fungal cell membrane.In previous studies,it was reported that the over-expression of the target gene CYP51 was the main reason for pathogenic fungi to acquire the resistance to DMI fungicides.Therefore,the screening of transcription factors regulating the expression of CYP51 gene would be crucial to elucidate the molecular mechanism of DMI fungicide resistance.However,little is known about the function of transcription factors related to ergosterol biosynthesis in plant pathogenic fungi.In this study,the biological functions of three transcription factors related to sterol biosynthesis pathway were carried out in Monilinia fructicola to determine whether they were involved in regulating the sensitivity to DMI fungicides.In this project,three putative transcription factors encoding genes MfSre1,MfADR1and MfSre1N,which were homologous with those regulating the ergosterol biosynthesis in fission yeast,were selected.To investigate their functions,gene knock-out was performed in the DMI resistant isolate Bmpc7 by protoplast transformation mediated by PEG.The main results are as follows:1.MfSre1 was the homologous gene of sterol regulatory element-binding protein?SREBP?in M.fructicola.The knockout of MfSre1 had no significant effect on mycelial growth,sporulation and pathogenicity.There was no significant difference between Bmpc7 and knockout transformants in the sensitivity to DMI fungicides,high concentration glycerin and H₂O₂ solution.However,compared with the resistant isolate Bmpc7,the sensitivity to metal ions,saccharides compounds,cell wall and membrane disruptors was significantly decreased in knockout transformants,and the level of the resistance to DCF fungicide was increased.2.The results of previous studies showed that the insertion of the 65bp fragment?Mona?in promoter region of MfCYP51 gene led to the overexpression of this gene,from which the resistance of M.fructicola to DMI fungicides was developed.There were two recognition sites of transcription factor ADR1 in Mona fragment.MfADR1 was the homologous gene of ADR1 in M.fructicola.The MfADR1 knockout transformants did not show significant difference with wild type isolate Bmpc7 in mycelial growth,sporulation,pathogenicity and the sensitivity to DMI fungicides,metal ions and H₂O₂ pressure.However,the sensitivity of knockout transformants to high concentration of glycerol was increased,indicating that MfADR1 may be related with the hypertonic glycerol pathway.3.Sre1N could specifically bind to the sterol regulatory element.MfSre1N knockout transformants showed no significant changes in mycelial growth rate,pathogenicity and the sensitivity to DMI fungicides and osmotic pressure.However,the sensitivity of knockout strains to high concentration of glycerol was also increased,which indicate that MfSre1N gene may affect the hypertonic glycerol pathway in M.fructicola.