Functional Analysis Of A Single Nucleotide Mutation Of Adenylate Cyclase Gene In The Growth,Development And Pathogenicity Of Botrytis Cinerea

Botrytis cinerea is a typical necrotrophic pathogen with a broad host range and complex pathogenic strategies,causing considerable pre-and postharvest economic losses.In addition to the broad host range and flexible pathogenic strategies,the high intraspecific variability of this fungus increases the challenge of controlling this fungus.The intraspecific variation among B.cinerea strains causes them to show high variability in reproduction structure,pathogenicity and drug resistance.The research on the genetic mechanism of intraspecific natural variation in B.cinerea is still scarce.In this thesis,the natural variant strain B05.10^M isolated from wild type strain B05.10was studied,and the role of a single nucleotide mutation of adenylate cyclase gene bac in the growth,development and virulence was analyzed by whole genome resequencing technology and molecular genetic methods.The transcriptome sequencing and molecular genetics were conducted to investigate the regulatory mechanism of bac point mutation.The main results obtained are summarized as follows:1.Phenotypic analysis of B05.10 and B05.10^M strains revealed that B05.10^M had significant defects in growth rate,conidiation and sclerotia formation,and pathogenicity,whereas red light could significantly promote its conidiation,showing enhanced red light response compared with B05.10.Using whole-genome resequencing,a single nucleotide mutation(T to C)at locus 4,646 of the adenylate cyclase gene bac that results in a change in an amino acid residue(from serine to proline,S1407P)was speculated to be the cause of various defects in the B05.10^Mstrain.Furthermore,the in situ point mutants B05.10^M:bac^P1407S and B05.10:bac^S1407Pwere obtained in B05.10^M and B05.10 by homologous recombination strategy,respectively.The phenotypes analysis confirmed that the phenotypic difference between B05.10^M and B05.10 was caused by the S1407P point mutation in bac.The results indicated that the S1407P point mutation in bac led to the decrease of the growth rate and conidiation,sclerotia formation defect,and reduced pathogenicity,but an increased sensitivity to red light.2.To explore the mechanism of S1407P point mutation in bac mediated defects in growth,development and virulence,we analyzed the differentially expressed genes between point mutation strain B05.10:bac^S1407P and wild type B05.10 in dark and light by transcriptome sequencing.The results showed that the S1407P point mutation of bac changed the expression patterns of a large number of genes affected by continuous light,and affected the expression levels of some light receptor genes,reproductive differentiation related transcription factors,melanin synthesis related genes,including the red light receptor gene bcphy2 and melanin synthesis gene bcscd1.In addition,the S1407P mutation in bac also affected the expression of genes related to toxin synthesis and reactive oxygen species production and clearance.And the expression levels of related genes were tested by RT-q PCR.It suggested that the S1407P mutation of bac may participate in the production of conidia and sclerotia by regulating the expression level of light receptor genes and related transcription factors,participate in the pathogenicity by affecting the balance of reactive oxygen species and toxin synthesis,and may change the red light phenotype by regulating the expression level of the red light receptor gene bcphy2.3.To investigate the relationship between the enhanced red light response in the S1407P point mutant and the expression level changing of the red light receptor gene bcphy2,the deletion mutants of bcphy2 were obtained in B05.10 and B05.10^M,respectively,and the in situ point mutants of bac were further generated.The phenotype analysis showed that the knockout of bcphy2 did not cause significant phenotypic changes in B05.10.However,in B05.10^M,the knockout of bcphy2 resulted in reduced growth rate and pathogenicity,and increased conidiation.Importantly,their phenotypic differences were caused by the S1407P point mutation in bac.The results indicated that the S1407P mutation of bac affected the expression of the red light receptor gene bcphy2,and the adenylate cyclase(BAC)mediated-c AMP signaling pathway regulated the red light receptor bcphy2 mediated-light signaling pathway.4.To investigate the effect of bac point mutation on melanin synthesis genes,the experiments were conducted as the same as that used for the bcphy2 gene.The results showed that the absence of bcscd1 in B05.10 only affected melanin accumulation,but in B05.10^M,the deletion of bcscd1 also caused reduced growth rate and pathogenicity.It indicated that bac point mutation affects the expression of bcscd1,resulting in the phenotypic difference of?bcscd1 mutants in B05.10 and B05.10^M.5.To further explore the regulatory mechanism of the S1407P mutation,in situ point mutation strains B05.10:bac^S1407D and B05.10:bac^S1407A were constructed using homologous recombination strategy,respectively.Phenotypic analysis and protein structure prediction of BAC revealed that the S1407P mutation may affect protein conformation of BAC and thus affect development and virulence.Further studies showed that the S1407P mutation affected the intracellular c AMP content and cell wall integrity.The results indicated that the S1407P mutation may affect the protein structure of BAC,which lead to the enzyme activity changing,the intracellular c AMP level decreasing,thereby regulating the development and virulence in B.cinerea.In summary,the study clearly analyzed that the S1407P mutation of adenylate cyclase gene bac caused severe growth and pathogenic defects,changed the sensitivity to red light response,and affected the balance between light signal transmission and downstream metabolic regulation,and preliminarily proved that the mutation may affect protein conformation and thus affect enzyme activity.Furthermore,this study found that BAC mediated c AMP signaling pathway regulates the red light signal response triggered by bcphy2,which can not only provide a new idea for the further exploration of BAC function and light signal pathway,but also provide a theoretical basis for the control of B.cinerea.