Regulation Role Of Hog1 And Slt2 MAP Kinases On Infection Structural Differentiation Of Alternaria Alternata Induced By Pear Cuticular Wax And Hydrophobicity

Mitogen activated protein kinase?MAPK?cascade signaling pathway has a variety of physiological functions such as pathogenic fungal growth,morphogenesis,infection structure differentiation,secondary metabolism,pathogenicity and osmotic response,and its regulatory effect varies with the types and interaction system of pathogens.In order to further clarify whether Hog1 and SLT2 MAP kinases are involved and how to regulate Alternaria alternata responsing to the epidermal hydrophobic and waxy signals of pear epidermis and initiating the infection process,the MAPK genes Hog1 and Slt2 of pathogenic fungi of pear fruit A.alternata?JT-03?were identified,cloned,and bioinformatics analyzed based on pharmacological test,and then the molecular mechanisms of Hog1 and Slt2 on A.alternata in response to the waxy physiochemical signal of pear peel to initiate infection were incovered through constructing deletion mutant and complementary strain.The results are as follows:1.Pharmacological tests showed that the MAPK specific inhibitor SB203580 could significantly inhibit the formation of A.alternata spore germination and appressorium formation induced by wax and hydrophobicity,and also decrease the pathogenicity of A.alternata on Zaosu pear.2.1615 bp and 1747 bp of AaHog1 and AaSlt2,respectively,were cloned from A.alternata?JT-03?.Homologous sequence alignment revealed that both the AaHog1 and AaSlt2 proteins contained unique phosphorylation motif"TGY"and a self-phosphorylation site"TEY"which is required for hypertonic response.The main elements of the secondary structure are?-helix and random curl,and the tertiary structure has stable spatial structure,both of which are hydrophilic stable proteins.Phylogenetic tree showed that AaHog1 and AaSlt2 genes had 99%and 91%homology with Hog1 of Alternaria longipes and Slt2 genes of Alternaria arborescens,respectively.3.?AaHog1,?AaSlt2,?AaHog1-C and?AaSlt2-C mutants were constructed by homologous recombination.Compared with the wild type,the spore morphology and size of?AaHog1 and?AaSlt2 have changed,the colony growth was slower,and the spore production was significantly reduced by 12.53%and 68.13%?p<0.05?.Spores germination,appressorium and infecting hyphae formation of?AaHog1 and?AaSlt2 on the wax-coated surface and hydrophobic surface were significantly lower than those of the wild type.Among them,?AaSlt2 significantly inhibited the infection structure differentiation on the wax-coated surface of the onion epidermis.The relative inhibition rate of the formation of appressorium and infected hyphae of?AaSlt2 were 67.76%and 93.33%,respectively,after 8 h incubation?P<0.05?.4.?AaSlt2 is more sensitive to cell wall interfering agents?Congo red,SDS?and oxidative stress?H₂O₂?,while?AaHog1 is sensitive to various external stresses including osmotic stress?NaCl,sorbitol?,cell wall interfering agents?Congo red,SDS?and oxidative stress?H₂O₂?.The cell wall degrading enzyme activity?PG,PMG,Cx,and?-glucosidase?,melanin and ALT toxin content of?AaHog1 and?AaSlt2 were significantly lower than those of the wild type.The lesion inhibition rates of Zaosu pear wounded inoculated with?AaHog1and?AaSlt2 mutants were 57.07%and 57.48%after 3 days,incubation respectively?p<0.05?,and with the extension of storage time,the black spot of pear fruit did not further expand.In summary,AaHog1 and AaSlt2 are required for spore morphology,infection structure,growth,cell wall integrity,and pathogenicity of A.alternata.Among them,AaSlt2 plays a more important role in regulating the process of the recognition and response of A.alternata to pear wax and hydrophobicity.