| RNA acetyltransferase Kre33 uses acetyl coenzyme A as an acetyl donor to catalyze the formation of N4-acetylcytosine nucleoside(ac4C)modification of RNA in various organisms,thereby regulating the level of RNA acetylation in organisms and affecting their growth and development.So far,the function and pathogenic mechanism of RNA acetyltransferase BcKre33 in Botrytis cinerea have not been reported.In this study,bioinformatics technology was used to analyze the homology of Botrytis cinerea Kre33,and the coding gene of Botrytis cinerea RNA acetyltransferase Kre33 was identified as BcKre33.The BcKre33 gene knockout mutants ΔBcKre33 was constructed by homologous recombination.The phenotype and pathogenicity of the ΔBcKre33 mutants were analyzed to clarify the function of the BcKre33 gene in its growth and development and pathogenicity.The cell wall degrading enzyme activity and acid production ability ofΔBcKre33 mutants were analyzed to explore the mechanism of BcKre33 gene regulating the pathogenicity of pathogens.The effect of RNA acetyltransferase BcKre33 on the level of RNA acetylation modification in Botrytis cinerea was determined by detecting the RNA acetylation modification regulated by BcKre33 using Dot Blot technology.RNA-Seq was used to sequence the transcriptome of Botrytis cinerea B05.10 and ΔBcKre33 mutants,and the regulatory mechanism of BcKre33 gene was analyzed.The results lay a foundation for elucidating the mechanism of BcKre33 gene regulating the growth,development and pathogenicity of Botrytis cinerea.The main results are as follows:1.Bioinformatics identification of RNA acetyltransferase BcKre33 in Botrytis cinerea.Through bioinformatics methods,the phylogenetic analysis and conserved domain analysis of RNA acetyltransferase Kre33 in Botrytis cinerea,Fusarium graminearum,Fusarium oxysporum,Magnaporthe grisea,Saccharomyces cerevisiae,Neurospora crassa,Aspergillus flavus and Aspergillus nidulans were carried out.It was found that the RNA acetyltransferase Kre33 in fungi was highly conserved and had a close homology,and all contained conserved TmcA-N,Helicase,GNAT and tRNA-bind domains.The gene encoding RNA acetyltransferase Kre33 was identified as BcKre33.2.The function of BcKre33 gene in the development and pathogenicity of Botrytis cinerea.The knockout vector of BcKre33 gene was constructed by homologous recombination technology.The knockout transformants of BcKre33 gene were obtained by PEG-mediated protoplast transformation.The mutants ΔBcKre33-1,ΔBcKre33-2 andΔBcKre33-3 were obtained by purification and molecular identification of the transformants.The phenotype and pathogenicity of ΔBcKre33 mutants were analyzed.It was found that the growth rate of ΔBcKre33 mutants was slowed down,no sclerotium was produced,the mycelium was thinner,the amount and size of conidia were significantly reduced,the number and size of infection pads were reduced,and the pathogenicity to tomato fruit and tobacco leaves was weakened.It indicated that BcKre33 gene positively regulated the growth,development and pathogenicity of Botrytis cinerea.3.The mechanism analysis of BcKre33 gene regulating pathogenicity in Botrytis cinerea.By measuring the intracellular and extracellular cell wall degradation enzyme activity,related gene expression and acid production ability of ΔBcKre33 mutants,it was found that the intracellular and extracellular pectinase and cellulase activities,the expression of cell wall degradation enzyme related genes and acid production ability ofΔBcKre33 mutants were significantly reduced.It was confirmed that the BcKre33 gene positively regulated the cell wall degrading enzyme activity and acid production ability of Botrytis cinerea.4.The function of BcKre33 gene in response to stress.By measuring the sensitivity ofΔBcKre33 mutants to stress,it was found that the sensitivity of ΔBcKre33 mutants to NaCl,KCl,sorbitol in osmotic stress and Congo red,fluconazole and SDS stress,which were used to test the integrity of cell wall,was significantly enhanced,and the sensitivity to oxidative stress of H2O2 and menadione was significantly reduced.The results showed that BcKre33 gene was involved in the response of pathogens to osmotic stress,oxidative stress and the regulation of cell integrity.5.Functional analysis of RNA acetyltransferase BcKre33 from Botrytis cinerea on regulating the level of RNA acetylation.Analysis of RNA acetylation levels in wild-type and ΔBcKre33 mutants revealed a decrease in the acetylation modification level of ac4C RNA in the ΔBcKre33 mutants.It is speculated that the RNA acetyltransferase BcKre33 is positively regulating the acetylation modification of ac4C RNA.6.Analysis of the regulatory mechanism of BcKre33 gene in Botrytis cinerea.The transcriptome data were used to analyze the differentially expressed genes regulated by BcKre33 gene.It was found that compared with Botrytis cinerea B05.10,ΔBcKre33 mutants produced 1991 significantly up-regulated genes and 2189 significantly down-regulated genes.The genes whose transcription levels were significantly down-regulated in GO analysis showed that ribosome biosynthesis,cell wall formation,conidia formation and development,rRNA processing,maturation and metabolism,and transmembrane transport processes were mainly present in biological processes,molecular functions,and cellular components.KEGG analysis of differentially expressed genes showed that most of the proteins encoded by down-regulated genes were involved in galactose metabolism,glycolysis,arginine,proline and cysteine metabolism,tRNA and ribosome synthesis.It was preliminarily determined that RNA acetyl transferase BcKre33 positively regulates the transcription of these genes and affects the growth and pathogenicity of Botrytis cinerea. |
