Functional Characterization Of A Phlorizin Hydrolase Gene And Two Bleomycin Resistance Genes In Valsa Mali

Apple Valsa canker,caused by the weakly parasitic ascomycete Valsa mali,resulting in severe necrosis on tree bark and even death to trees,has always been key objects of prevention and treatment in apple production.Although the research on V.mali has been deep into the molecular level,the pathogenic mechanism of this pathogen still remains elusive.V.mali can produce toxic metabolites by hydrolyzing phlorizin,which will cause the canker symptom on apple trees.z Moreover,an array of saprophytes could colonized in the dead tissues,where horizontal gene transfer(HGT)between V.mali and these microbes can take place.Based on the genome of V.mali,this study identified one candidate gene encoding lactase phlorizin hydrolase(LPH),designated as Vmlph1,and two HGT genes which may confer V.mali bleomycin(Bm)resistance,designated as Vmhgt14 and Vmhgt32.Characterization of the functions of Vmlph1,Vmhgt14 and Vmhgt32 during infection of V.mali could help to understand and mechanism about how V.mali colonizes on apple trees,providing scientific basis for targeted control strategies for apple Valsa canker in future.The mutants were obtained by Double-joint PCR and PEG-mediated transformation of protoplast,and were further validated by PCR and Southern Blot.The research of the mutants led to the results as follows:1.Using PCR and Southern blot,one deletion mutant of Vmlph1(?Vmlph1-4)was obtained.Compared with the wild type 03-8,?Vmlph1-4 showed a whiter colony,and the growth rate of mycelium together with sporulation was significantly decreased.In addition,the pathogenicity of ?Vmlph1-4 on apple leaves and twigs were reduced by 22.0% and11.0%,respectively.However,the phlorizin hydrolase activity of ?Vmlph1-4 showed no significant difference when comparing with the wild type.By qRT-PCR,we found that under the induction of phloridzin,Cmr1,a transcription factor regulating melanin synthesis of V.mali,was significantly down-regulated in ?Vmlph1-4,compared with the wild type strain.2.The capacity of Bm resistance of V.mali,Aspergillus nidulans and Fusarium graminearum was examined under three levels of Bm stress(10,50 and 100 ? g/ml).Compared wiht A.nidulans and F.graminearum,V.mali was significantly more resistant.The relative expression of Vmhgt14 and Vmhgt32 were up-regulated,and it was Vmhgt32 but not Vmhgt14 was induced at a high level(50 ? g/ml)of Bm stress.After knockdown of Vmhgt32 gene,the Bm resistance of ?Vmhgt32-1 was no significant change.Vmhgt14 of the Vmhgt32 null mutant ?Vmhgt32-1 was induced under Bm stress(50 ?g/ml)at 36 hpi.Thus,we generated the double deletion mutant of Vmhgt14 and Vmhgt32,which showed a significantly reduced resistance compared with the resistance of the wild type strain 03-8.However,compared with wild-type 03-8,the pathogenicity of ?Vmhgt32-1,?Vmhgt32-37 and ?Vmhgt32 / 14-60 did not change significantly.In conclusion,these results suggest that,the phlorizin hydrolase gene Vmlph1 may be related to vegetative growth,participate in pathogenic process,conidiation and melanin biosynthesis of V.mali,and Vmhgt14 together with Vmhgt32,contributes to Bm-resistance.