Investigation Of The Molecular Mechanisms Of Apples Infection By Penicillium Expansum Using Transcriptome

Apple is rich in nutrition and sweet in taste.It is a kind of fruit that people often eat.Postharvest apple is easy to be infected by pathogens,which causes serious economic losses.Blue mold disease caused by Penicillium expansum is the main cause of postharvest loss of apple.At present,researches on P.expansum infecting apple are still shallow and the most important pathogenic factors in the process of P.expansum infecting apple are not clear.That seriously obstruct people's understanding the mechanisms by which the pathogen infects the apple fruit.In this paper,the pathogen was screened from rotten apple and its infection process was observed.Transcriptome sequencing was used to study the transcription level,changes,and interaction between the pathogen and apple at the critical time point of infection.The possible key genes in the infection process were screened out.The key genes were knocked out and supplemented by homologous recombination for further verification.The main research outputs are the following:(1)A suspected pathogen strain K1 was isolated and purified from rotten apple.According to Koch's rule,four steps: coexistence observation,isolation,inoculation,and re-separation were used to identify the suspected K1 pathogen.It was found that 1,3 and 6 h were the key time points in the process of K1 infection.Through molecular biological identification and morphological observation,the pathogen K1 and K2 were identified as P.expansum.(2)The gene expression levels of P.expansum K1 at 1,3 and 6 h infection were studied by transcriptome sequencing.In the process of infecting apple,the P.expansum K1 mainly passed through the following three aspects: 1)P.expansum K1 regulated the expression of glucose oxidase related genes to regulate the environmental p H value,so as to promote spore germination and mycelial growth and provide a good environment for gene expression of cell wall degrading enzymes;2)The expression of glutathione S-transferase(GST)and catalase(CAT)related genes help P.expansum K1 respond to the oxidative stress;3)P.expansum K1 secreted infection related effectors to help infect the apple.(3)In response to P.expansum K1 infection,apple mainly responded to P.expansum K1 infection by activating PTI(1 h)and ETI(3 h);when P.expansum K1 was inoculated into apple 1 h,P.expansum K1 regulated the p H of its microenvironment through the expression of glucose oxidase to regulate the spore germination.The expression of germination,cell wall degrading enzymes(CWDEs)and mycelial development related genes were detected.The expression of CWDEs in P.expansum K1 activated the PTI defense system of apple at the same time.After P.expansum K1 was inoculated into apple,Reactive oxygen spices(ROS)was produced in apple inoculated with P.expansum K1 at 3 h,which damaged the spores of P.expansum K1 and regulated the PTI in apple.P.expansum K1 regulates the expression of antioxidant stress related genes through GST and CAT,and activates plant hormone signal transduction pathway to regulate PTI and ETI in apple.(4)Through multiple screening of the key genes of P.expansum K1 infecting apple,a gene GHS(P.expansum alkali-sensitive linkage protein 1,GHS)with a large number of interaction genes and up-regulated expression in 1,3 and 6 h of apple was obtained.The results showed that the relative expression of GHS increased first(0-3h)and then decreased(3-6 h)at 0-6 h.The expression level of GHS gene was the highest at 3 h post inoculation;Bioinformatics analysis showed that GHS protein contained 265 amino acids,the molecular weight of GHS protein is 28 k Da.GHS protein was classified as stable and hydrophobic protein according to the prediction;Subcellular localization predicted that GHS protein was a secretory protein and contained a signal peptide;Phylogenetic tree analysis showed that it was highly homologous with Glycoside hydrolase superfamily protein of P.expansum;Database comparison and analysis showed that GHS protein encoded a precursor of alkali sensitive linkage protein and contained a conservative glycosyl hydrolase catalytic core;the secondary and tertiary structure of GHS protein is predicted.(5)The GHS gene knockout mutants ?GHS-T5,?GHS-T6,and GHS-C5 and GHS-C6 were constructed by homologous recombination.GHS gene knockout did not affect the normal growth of the strain.By comparing the decay rate,decay diameter,and decay rate of P.expansum K1 and ? GHS-T5,? GHS-T6 and GHS-C5 and GHS-C6 in apple wounds,it was found that the ? GHS-T5,? GHS-T6 mutant significantly decreased the pathogenicity in apple,but the pathogenicity was restored after the GHS gene was complemented.Thus,it was determined that GHS gene was the key gene controlling the pathogenicity of P.expansum K1 in apple wounds.This research was started with pathogen isolation,K1 was identified as P.expansum according to Koch's postulates and the key time point of P.expansum K1 infecting apple was determined;the changes of P.expansum K1 and apple during the infection process was detected by transcriptome technology;the possible key pathogenic gene GHS was screened from the perspective of pathogen and host,and then the bioinformatics of GHS gene was analyzed;the GHS gene knockout mutants and complemented strains were constructed by homologous recombination,which proved that GHS gene is the key gene which controlled the pathogenicity of P.expansum K1 in apple wound tissues.These results revealed the molecular mechanisms of apples infection by P.expansum K1 preliminarily.This study can provide a theoretical basis for the control postharvest blue mold in apple fruit.