Investigation Of The Interaction Mechanisms Of Penicillium Expansum And Postharvest Pears Based On Proteomics And Transcriptomics Technology

Pear is one of the most important fruits in China.Blue mold decay caused by Penicillium expansum is one of the most common diseases in pear storage and transportation,causing huge economic losses.With the expansion of pear cultivation area and the increase of yield,the occurrence of postharvest diseases in pears has serious impacts on the commodity value and market supply of pears,and restricts the healthy development of the pear industry.Clarification of the interaction mechanism between P.expansum and pear has become a key issue in developing new control strategies for pear blue mold.However,the research on the interaction mechanism between P.expansum and pear is still in the early stage,and a large number of unknown pathogenic and disease resistant factors have yet to be excavated and studied,which severely limits the breakthrough progress of pear blue mold control strategy.In this work,self-selected P.expansum was selected as a research object to study its pathogenicity to pear.Transcriptome technology was used to study the expression of its own genes and to find out the key pathogenic genes in the process of infection of pears by P.expansum.The suspected pathogenic gene mutant was generated and its function was studied using Agrobacterium-mediated homologous recombination technology;the gene and protein expression of pear in response to P.expansum infection was studied using transcriptomics and proteomics technology,and the key genes and proteins related to disease progression were identified;analysis of the relationship between the regulatory network between P.expansum gene and pear genes and proteins in the process of pear infection with P.expansum,so that the interaction mechanism between P.expansum and pear can be fully elucidated.The findings may provide a theoretical basis for the development of new methods for postharvest pear disease control.The main conclusions of this study are as follows:(1)A pathogenic strain A1 was isolated and purified from the surface of decayed pear.It was identified as P.expansum based on morphological characteristics,molecular biology assay,and Koch’s verification.It was inoculated into the pear wound,the decay of the wound was observed,and the rot diameter was measured,and it was found to have strong pathogenicity to pear.9 h,24 h,and 36 h after pear infection are the most important time points in the infection process.(2)The gene expression levels of P.expansum at the key infection time points were studied by transcriptome sequencing and bioinformatics.During the course of pear infection,P.expansum completed its invasion of pear by altering gene expression encoding extracellular protease and regulating gene expression involved in glutathione metabolism,polysaccharide metabolism and cell response to reactive oxygen species(ROS).Genes encoding plant cell wall degrading enzymes(CWDEs)account for the majority of extracellular protease genes.Furthermore,the proteins(polygalacturonase(PG)and exo-1,4-β-glucanase(Cx))encoded by key genes were determined.It was found that the activities of PG and C_X increased with the extension of the time that P.expansum infects pear,which was consistent with the transcriptome results of P.expansum showing that P.expansum supports successful infection of pear fruit by secreting plant CWDEs.(3)Analysis of the genes involved in the infection of pears by P.expansum revealed that the expression level of the gene encoding necrosis inducing protein(NIP)was significantly up-regulated with the extension of infection time,and the up-regulated expression multiple was up to 8.34 fold at 24 h,which was closely related to the gene encoding plant CWDEs.To investigate the effect of NIP on P.expansum,a NIP knockout mutant(ΔNIP)and a complement mutant(ΔNIP-C)were generated.NIP is related to P.expansum growth rate,mycelial formation,and pathogenicity of infected pear.On the one hand,the absence of NIP slowed colony formation of P.expansum and reduced mycelial yield of P.expansum,decreasing spore yield and patulin(PAT)yield,but had no effect on its basic morphology and structure.In contrast,the absence of NIP,reduced the pathogenicity of P.expansum infecting pears.Moreover,PAT has a certain toxic effect on pears and increases the pathogenicity of P.expansum andΔNIP.Conversion of NIP toΔNIP restored growth,spore yield,PAT yield,and pathogenicity to the level of wild type P.expansum.(4)The gene expression levels of pear in the process of responding to P.expansum were studied by transcriptome sequencing and bioinformatics.Pear up-regulated genes were mainly related to pear defense,while down-regulated genes were mainly involved in pear growth and development.Moreover,in the process of P.expansum infecting pear,P.expansum would trigger corresponding signal transduction in pear,leading to the significant up-regulation of the coding genes and proteins of some secondary metabolites(such as terpenoids,flavonoids,and alkaloids)in pear.It is speculated that pear synthesized secondary metabolites to respond to the infection.(5)The protein expression levels of pear in the process of responding to P.expansum were studied by proteomics technology.The sequence alignment of total genes and total proteins in pear fruit revealed that 96%of the proteins were associated with pear fruit total genes.The associated genes and proteins were screened and analyzed,and the activity changes of enzymes involved in pear resistance were measured during P.expansum infection.Resistance-related enzyme activity revealed that catalase(CAT),superoxide dismutase(SOD),and phenylalanine ammonia lyase(PAL)were involved in the defense response of pear to P.expansum infection.Moreover,SOD and PAL were significantly up-regulated at the gene and protein levels.Combined with the results of bioinformatics analysis of the associated genes/proteins,it was found that when P.expansum infects pear,it triggered corresponding signal transduction,such as ROS bursts and plant hormone signal transduction and leads to further activation of secondary regulatory networks in pear,including regulation of transcription factors and expression changes of genes encoding protein kinases,and transmits signals for pathogen protein recognition to downstream defense pathways,leading to the significant up-regulation of the coding genes and proteins of some secondary metabolites(such as terpenoids,flavonoids,and alkaloids)in pear,and finally changes some physiological indicators of pear,making it resistant to P.expansum infection.