Molecular Mechanism Of Alternaria Alternata Pre-penetration Induced By Cuticular Wax Of Pingguoli Pear Based On Transcriptome Analysis

ABI STEP ONE PLUS sequencing method was used to study the transcriptomes of A.alternata infective structure differentiation induced by waxy treatment.The GO function,COG classification and KEGG metabolic pathway network of differentially expressed genes of A.alternata treated with pear cuticular wax and beeswax were analysed through bioinformatics methods,and major differentially expressed genes were further validated by qPCR technique.In addition,regulatory role of calcium signal pathway in pear cuticular wax inducing infection structure differentiation of A.alternata were studied through different inhibitors treatment.The results showed as follows:1.Through high-throughput sequencing and bioinformatics analysis,there were 56(33 up-regulations,23 down-regulations)and 62 differentially expressed genes(35 up-regulations,27 down-regulations)after pear cuticular wax treatment compared to the control and beeswax treatment groups at the spores germination stage of A.alternata(6h).During appressorium formation of A.alternata period(12h),the pear cuticular wax treatment had 127(64 up-regulation,63 down-regulation)and 260 differentially expressed genes(140 up-regulation,120 down-regulation)compared to the control and beeswax group.There were 626(486 up-regulated,140 down-regulated)genes differentially expressed in A.alternata at the appressorium formationstage(12h)compared to the spore germination stage(6h)after pear cuticular wax treatment.In general,during the process of A.alternata infection structure differentiation,the number of differential expressed genes of A.alternata response to pear cuticular wax was significantly lower than those of beeswax.2.The function annotation and enrichment analysis of the GO database showed that the A.alternata differentially expressed genes after pear cuticular wax and beeswax treatment were mainly enriched in three major categories of cellular components,molecular functions and biological process.The differential genes in the pear cuticular wax treatment group were lower than those in the beeswax group and the control group in GO analysis.COG classification database analysis showed that the differentially expressed genes were mainly enriched in process of primary metabolism,secondary metabolism,cell division and intracellular signal transduction of A.alternata.The primary metabolism,secondary metabolism,cell division,and intracellular signal transduction of A.alternata after the pear cuticular wax treatment were weaker than that after beeswax treatment.Analysis of KEGG metabolic pathway revealed that ratio of differential gene of A.alternata in the peroxidase pathway and fatty acid metabolism in the pear cuticular wax treatment were significantly higher than the beeswax treatment during the spore germination and appressorium formation process.3.Quantitative PCR validating of the 8 key differentially expressed genes associated with fatty acid metabolism,peroxisomes,and calcium signaling pathways showed that except the phospholipase C gene,long-chain fatty acid coenzyme A ligase,acyl-CoA oxidase,acyl-CoA dehydrogenase,3-ketoacyl-CoA thiolase,peroxisome gene PEX2,and calmodulin genes of A.alternata were all up-regulated after pear cuticular wax treatment.The differentially expressed genes in the sequencing results were up-regulated similarly,indicating that the sequencing results in this study were reliable.4.Spores germination and appressorium formation of A.alternata were significantly inhibited after calcium ion chelating agent(EGTA),phospholipase C inhibitor(neomycin),and cells intracellular calcium channel blockers(TMB-8),calmodulin antagonists(W-7),calcineurin kinase inhibitors(cyclosporin A),which are Ca2+ signal pathway inhibitors,and the inhibitory effects were in dose-dependent manner.At the same time,the inhibitory effect of each inhibitor treatment on the formation of appressoria induced by pear cuticular wax was greater than that of spore germination;the results showed that calcium signal pathway plays an important role in regulating the formation of appressoria of A.alternata induced by pear cuticular wax.In summary,molecular mechanisms of A.alternata infection structure formation induced by Pingguoli pear cuticular wax were comprehensively analyzed through the transcriptomics method,and main induction-related metabolic pathways and signaling pathways were found.These findings might provide important theoretical basis for further elucidating molecular mechanism of infection structure formation and pathogenicity in A.alternata response to fruit cuticular wax cues.