The Study Of Molecular Mechanism In Spore Germination And Virulence Mediated By PeHsf1 In Penicillium Expansum

Penicillium expansum is an important pathogen in the fruit industry.P.expansum produces patulin which results in serious contamination in food and vegetable products,leading to huge economic losts in postharvested fruits.Mining the pathogen-related virulence factors involved in postharvested fruits' infection is meaningful for pathogen control and decreasing the economic losts.Heat shock transcription factors(Hsfs)are important regulators,which mediate the stress response in eukaryotes.Hsfs are also veridied to be involved in the pathogenicity of pathogens.In this study,the RNA-seq of wild-type spores cultured at 4 and 25 ? for 8 h and 12 h was performed.The analysed data showed that low temperature stress not only inhibited the spore germination of P.expansum,but also affected the biosynthesis pathway of amino acids,fatty acids and steroids,glucose metabolism pathway,pentose phosphate pathway,protein synthesis and degradation pathway of P.expansum.q RT-PCR furether supported nine cell-growth related genes were significantly down-regulated in P.expansum at 4 ?.Based on transcriptome data,694 stably expressed genes were screened.The expression stabilities of 14 randomly selected candidate genes were evaluated.Finally,two,Isy1 and spt5,were confirmed to be suitable for stable and reliable internal reference genes,which providing well reference genes for the subsequent gene expression analyses in P.expansum.In this study,three Hsf genes,Pe Hsf1,Pe Hsf2,and Pe Hsf3,were identified in P.expansum genome.The phylogenetic analyses demonstrated that three Pe Hsf genes were distributed in three different clades,and might play differential roles in P.expansum.Through homologous recombination method,and the knockout mutant of Pe Hsf1(?Pe Hsf1)was obtained.Compared to thoses in wild-type,the spores germination in ?Pe Hsf1 were inhibited,showing significant shorter germ tube.Compared to wild-type,the ?Pe Hsf1 also showed delayed growth rate at 25 ?on PDA and Cazpek medium,respectively.Additionally,the back of PDA plates of?Pe Hsf1 represented yellow color.Furthermore,the decay expansion of apple and pear fruits infected by ?Pe Hsf1 was also slower than that infected by wild-type.The decreased ROS level was also observed in ?Pe Hsf1.At 4 ?,?Pe Hsf1 showed faster growth rate than wild-type on PDA plates,representing its tolerance against low temperature.At 4 ?,on Cazpek medium,the?Pe Hsf1 also showed slower growth rate than wild-type.During infecting apple and pear fruits at 4 ?,the decay expansion caused by ?Pe Hsf1 was also slower than that of wild-type.The RNA-seq was also performed using wild-type and ?Pe Hsf1,cultured on PDA plate at 25 ? for 3 days.The transcriptome data analyses showed that amino acid synthesis and metabolism,starch and sucrose metabolism,MAPK signaling pathway,pentose phosphate pathway,pentose and glucuronic acid conversion were significantly affected in ?Pe Hsf1.In addition,both RNA-seq data and q RT-PCR confirmed 15 patulin-biosynthesis genes were significantly down-regulated.Further q RT-PCR analyses also showed that 15 and 14 patulin-biosynthesis genes were significantly down-regulated in ?Pe Hsf1 cultured at 4 ? and during infecting apple fruits,respectively.Furthmore,q RT-PCR verified nine virulence-related effector genes in ?Pe Hsf1 during the process of infecting apple fruits showed significantly down-regulated expression levels.As mentioned above,the deletion of Pe Hsf1 inhibited the spore germination,delayed the mycelium growth rate,and decrease the virulence.The Pe Hsf1 might mediate the patulin-biosynthesis genes and virulence-related effector genes,to regulate the virulence.Our results also could provide the well candidate genes and though for constructing genetically engineered fungi,which produce high-content patulin.