System Discovery And Functional Analysis Of The Aspergillus Ochraceus Effector Of Pears

Aspergillus ochraceus is one of the most important postharvest pathogens in perat fruits.In addition to causing great economic loss,this pathogen produces mycotoxin ochratoxin A(OTA),which poses a serious threat to human health.During the pathogen-host interaction,a great diversity of virulence factors was secreted by pathogens to overcome the plant innate immune defense,modulate host plant physiology to accommodate microbial invaders and ultimately cause disease.However,identification of effectors in A.ochraceus and functional characterization of their potential roles in pathogen colonization and disease development have not been accomplished.In this study,an appracch combined RNA-Seq analysis with in silico prediction was used to identify the effector candidates from A.ochraceus followed by biological function characterization.The main results of the study are as follows:(1)A total of 4618 and 2842 differentially expressed genes were identified by comparative transcriptome analysis of A.ochraceus grown in pear juice,a medium used to mimic the natural in planta condition,and Czapek medium revealed after 24 h and 72 h of incubation,respectively.Further in silico prediction revealed that 272 and 214 of them were effector candidate genes,and most of which are glycoside hydrolase,peptidase,oxidoreductase,esterase,and so on.(2)AoEGH gene,one of the effector candidate genes,was isolated successfully.This gene encodes a protein of 345 amino acid residues,which is predicted to be a secreted extracellular protein.Blast alignment revealed that AoEGH contains a Glyco-hydro-26 non-conserved functional domain.(3)Knockout mutant of AoEGH(?AoEGH)was successfully constructed by homologous recombination and PEG-mediated protoplast transformation.Deletion of AoEGH gene leads to changes in colony growth and hyphal morphology,reduced spore yield and delayed spore germination,indicating that AoEGH was involved in the regulation of A.ochraceus growth.(4)In vivo pathogenicity tests on pear fruits revealed that the lesion diameter on pears inoculated with ?AoEGH was much smaller than those inoculated with wild type A.ochraceus,with a reduction of 83.76%.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 A.ochraceus on pear fruits.