Molecular Regulatory Mechanism Of CAMP-PKA Signaling Pathway On Infection Structure Differentiation Of Alternaria Alternata Induced By Physichemical Signals From Pear Fruit Cuticular Wax

The cuticle of the fruit epidermis has a dual role in the interaction between the pathogen and the host.On the one hand,it acts as a physical barrier for pathogen infection,on the other,the physicochemical cues from the cuticle facilitates to pathogen infection,especially for compatible fungi.However,its detailed regulatory mechanism needs to be further revealed.In this study,the effects of atropine,a cAMP specific inhibitor,on the growth,infection structure and pathogenicity of A.alternata induced by wax extract of pear peel were firstly studied by pharmacological methods.Based on the cloning and bioinformatics analysis of PKAc and PKAr genes in A.alternata,the casual agent of black rot in pear fruit,the PKAc and PKAr deletion mutants were constructed,and the effects of?AaPKAc and ?AaPKAr on vegetative growth,stress response,infectious structures formation and pathogenicity of A.alternata were systematically studied on the molecular level The results are as follows:1.cAMP inhibitor atropine significantly inhibited the spore germination and appressorium formation of A.alternata induced by cuticular physiochemical signals.4h after inhibitor treatment,the appressorium formation rate on the hydrophobic surface and fruit wax extract coated gelbond film surface decreased by 75.3% and 63.7%,respectively.The application of exogenous cAMP partially restored its inhibitory effect,After 4 h of incubation,appressorium formation rate of A.alternata on the high hydrophobic surface and fruit wax extract coated gelbond film surface treated with exogenous cAMP+atropine was respectively 2.4 times and 1.6 times higher than that treated with atropine alone.2.Through NCBI blast analysis and gene cloning,the catalytic and regulatory subunits of cAMP-dependent protein kinase PKA were identified from the A.alternata genome and named PKAc and PKAr.The ORFs of the PKAc and PKAr genes of A.alternata are 1571 bp and 1737 bp,encoding 488 and 461 amino acids,respectively.The homology of each functional domain and other fungal PKA kinase catalytic and regulatory subunits was higher,both PKAc and PKAr contain their conserved domains,RXSY and RRXS.The expression level of PKAc gene during appressorium formation of A.alternata on the surface of pear fruit waxy film was significantly higher than that on hydrophobic membrane alone,while the expression level of PKAr gene was significantly up-regulated during the process of spore germination induced by hydrophobic surface.3.Homologous recombination strategy and agrobacterium transformation method were used to knock out the PKA kinase catalytic subunit PKAc and regulatory subunit PKArof A.alternata to obtain hygromycin-resistant transformants.After DNA level and RNA level double identificating,PKAc and PKAr gene deletion mutants were finally obtained.Compared with A.alternata wild type,?AaPKAc significantly inhibited the colony growth,biomass,and spore germination of A.alternata.However,the loss of PKAc increased spore production compared to the wild type,and ?AaPKAr lost the ability for conidia production.Deletion of PKA kinase leads to increased intracellular cAMP content and reduced PKA content.Mutant ?AaPKAc increased tolerance to oxidative and osmotic stress and sensitivity to cell wall stress.On the contrary,mutant ?AaPKAr increased sensitivity to oxidative and osmotic stress.Compared with wild-type strains,the black rot development in pear fruit wounded inoculated with ?AaPKAc decreased.Compared with wild-type strains,?AaPKAc mutants have higher level melanin content,lower levels of toxins TEN,ALT,AME,and AOH,and higher enzyme activities of Cx and ?-glucanase.4.Higher hydrophobic surface and fruit wax extract coated Gelbond film surface significantly promoted spore germination rate and appressorium formation rate of A.alternata.?AaPKAc significantly inhibited spore germination and appressorium formation induced by cues from hydrophobi and pear wax coated surface compared to wild-type strains,and more significant inhibition was found on appressorium formation,4 h after incubation,appressorium formation rate of ?AaPKAc on the high-hydrophobic(108 °)and fruit wax coated surface decreased by 31.6% and 20%,respectively,compared with that of the wild-type strain.Pear wax coated dewaxing onion epidermis significantly promoted the appressorium and infective hyphae formation of A.alternata.In summary,the cAMP-PKA signaling pathway plays an important role in the growth,pathogenicity,and biological stress of A.alternata,The findings suggested that the cAMP signal cascade pathway might affect the recognition and response of A.alternata to the physicochemical signal of pear fruit peel through regulating the formation of infection structures.