Pathogenicity-Associated Factors BcNop53 And BcSnf2 Regulate Growth And Pathogenesis Of Botrytis Cinerea

Botrytis cinerea is a typical necrotrophic fungal pathogen that causes gray mold on over 1400 plant species.The gray mold has a great impact on agricultural production and annually causes enormous economic losses worldwide.In recent years,as the release of its genome sequences,the molecular mechanisms of the pathogenesis in the gray mold fungus B.cinerea have been extensively studied,which constantly update our understanding of the interaction process between B.cinerea and its hosts.Although many pathogenicity-related genes have been identified,the pathogenic regulatory mechanisms remain largely unexplored.Construction of a mutant library is an effective way to discover novel genes and to conduct functional genomics research.In this study,we generated a B.cinerea T-DNA insertion mutant library,containing ~ 9000 transformants,via Agrobacterium tumefaciens-mediated transformation(ATMT)method and obtained over 400 non-pathogenicity or virulence-attenuated mutants after three rounds of pathogenicity screening with two hosts(tomato and strawberry).The T-DNA integration site was identified by thermal asymmetric interlaced polymerase chain reaction(TAIL-PCR)analysis.Three T-DNA labeled genes(named as BcNOP53,BcRAB-6A and BcSNF2 respectively)that may correspond to the pathogenicity attenuation of the mutants were selected to perform functional analysis.To investigate the biological functions of these genes,we gernerated their gene knockout mutants and genetic complementary transformants.After validation of the virulence of the mutants ?Bcnop53,?Bcrab-6a,and ?Bcsnf2,genes BcNOP53 and BcSNF2 were identified as pathogenicity-associated genes that were responsible for virulence loss or attuenuation of the corresponding T-DNA insertion mutants.We thus systematically analyzed their biological functions and mechanisms that regulate B.cinerea development and pathogenesis process.The ribosome is an organelle for cellular protein biosynthesis.However,little is known about how the ribosome mediate microbial pathogenesis.In this work,we demonstrate that Nop53,a late-acting factor for 60 S ribosomal subunit maturation,is crucial for the pathogen's development and virulence.Bc Nop53 is functionally equivalent to the yeast Nop53 p.Complementation of Bc NOP53 completely restored the growth defect of the yeast ?Scnop53 mutant.Bc Nop53 is located in the nuclei and disruption of BcNOP53 also dramatically impaired pathogen growth.Deletion of BcNOP53 blocked infection structure formation and abolished virulence of the pathogen,possibly due to reduced production of reactive oxygen species(ROS).Moreover,loss of BcNOP53 impaired pathogen conidiation and stress adaptation,altered conidial and sclerotial morphology,retarded conidium and sclerotium germination as well as reduced the activities of cell-wall degradation-associated enzymes.Sclerotium production was,however,increased.Complementation with the wild-type BcNOP53 allele rescued the defects found in the?Bcnop53 mutant.Snf2 proteins are a family of ATP-dependent helicase-like proteins that direct energy obtained from ATP hydrolysis into modulating chromatin dynamics.They alter the contact between histones and DNA,facilitate the process of replication,transcription,recombination or reparation.BcSnf2 is identified as a Snf2 family protein based on bioinformatics analysis.However,its biological function in pathogenic fungi has not been reported.In this study,we demonstrate that Bc Snf2 plays an important role in the development and pathogenesis as well as regulation of the transcription of specific genes in B.cinerea.Our findings suggest that BcSnf2 may play a role in pathogenicity through transcriptional regulation of some virulence factors.Disruption of BcSNF2 gene in B.cinerea resulted in a significant attenuation of the pathogen virulence and a reduction in the pathogen growth rate,conidiation as well as a retardation of conidium germination.Loss of BcSNF2 also altered the mutant colony and sclerotial morphology.Comparison of the differential expressed genes in the ?Bcsnf2 mutant and wild type strain B05.10 was performed via analysis of the transcriptome sequencing data.Our findings demonstrate that in addition to the differential genes related to growth and development,some pathogenicity-related genes were also enriched in the comparision.Among these genes,we found some B.cinerea virulence-related factors which have been previously reported.A systematic analysis of the mechanism of Bc Snf2 and/or other Snf2 factors that regulate the pathogen virulence and interaction with its hosts will constitute a valuable direction for our future research.Taken together,we gernerated a T-DNA insertion mutant library using B.cinerea as a model pathogen in this study.Two novel pathogenicity-related genes BcNOP53 and BcSNF2 were identified and the biological functions of the two genes were systematically analyzed.Importantly,the mechanisms of ribosomal processing factor BcNop53 regulating development and virulence of pathogenic microorgansims were systematically investigated.Our findings demonstrate that the growth,pathogenic development and penetration of B.cinerea depend on BcNop53 mediatating protein synthesis and endogenous ROS production;and that Snf2 family protein BcSnf2 regulates transcription of specific genes,affects the development and pathogenesis.This study provides novel insights into the pathogenic mechanism mediated by ribosome formation and epigenetic regulation of ATP-dependent chromatin remodeling complex.Our work may open up a new target for the treatment of fungal diseases and provide a theoretical basis for the scientific control of plant fungal diseases.