Isolation And Functional Characterization Of PeSCP And PeASP Effector Genes In Blue Mold Penicillium Species Of Apple Fruit

Apple is very popular because of its rich nutrition and sweet taste.Penicillium expansum,which causes blue mold decay,is one of the most devastating postharvest pathogens in apple fruits.In addition to causing great economic loss,this pathogen produces toxic patulin that pose a significant threat to human health.Despite its significant economic loss,little is known about the pathogenicity determinants of P.expansum,and tracking the fundamental biology of this fungal pathogen would provide the foundation for new alternative disease management approaches.Our previous results showed that PeSCP and PeASP,which encoded two secreted proteins,were up-regulated significantly during the infection of P.expansum on apple fruits,suggesting their potential roles in the fungal virulence.In this work,PeSCP and PeASP genes were isolated and their possible biological functions were further characterized.The main results were obtained as follows:(1)The genomic sequence of PeSCP gene with 1761 bp of length was isolated,which encodes a protein of 533 amino acid residues and is interrupted by three introns.Meanwhile,the full length of PeASP gene is 1310 bp,which contains two introns and encodes a protein of 396 amino acid residues.Bioinformatic analysis revealed that PeSCP has no conserved domain,while PeASP protein contains a conserved Aspergillopepsin-like domain.Both of them are secreted proteins and closely related to the homologous sequences of P.italianum,P.camemberti and P.griseofulvum.(2)Deletion mutants of?PeSCP and?PeASP were constructed with the method of homologous recombination and PEG-mediated protoplast transformation.Compared with the wild-type of P.expansum,the mutants show some phenotypic differences,including the decreased mycelium density,sporulation ability and germination rate,but the increased mycelium diameter,spore diameter,radial growth rate and sensitivity to Na~+.(3)In vivo pathogenicity tests on apple fruits revealed that the lesion diameters on apples inoculated with?PeSCP and?PeASP were much smaller than those inoculated with wild type P.expansum,with a reduction of 61.9%and 44.3%respectively.Further scanning electron microscopy observation of inoculation site of fruit epidermal layer revealed that the conidia of mutants could germinate and form hyphae,but the hyphae failed to penetrate through the fruit cuticles as examined at 9 h after inoculation,suggesting their potential roles at the early penetration and invasion stage of P.expansum on apple fruits.