Biological Function Analysis Of Phospholipase C In Alternaria Alternata And Its Molecular Regulatory Mechanism On Appressorium Formation Induced By Peel Physicochemical Signals

Phospholipase C,a key enzyme in the calcium signaling pathway,can influence cell development and other physiological functions by regulating intracellular Ca²⁺concentration,and plays an important role in the response of pathogenic fungi to external stimuli.Our previous studies revealed a positive correlation between pear cuticular wax hydrophobicity and appressorium formation in Alternaria alternata.How PLC regulates the formation of A.alternata infection structure induced by the physicochemical signals of pear peel by Ca²⁺signaling pathway has not been reported.Based on the previous cloning and bioinformatics analysis of AaPLCs,the aim of this study was to systematically evaluate the biological functions of the AaPLCs and the regulation of A.alternata infectious structures formation induced by physicochemical signals of pear peel through pharmacological experiments and molecular biology.The results are as follows:1.During the formation of hydrophobic-induced infection structure,the intracellular calcium distribution in A.alternata changed with the infection structure.Neomycin treatment significantly inhibited the spore germination and appressorium formation of A.alternata.10?m neomycin treatment significantly reduced appressorium formation rates by 47.4%compared to controls after 4 h of incubation on the fruit wax-coated surface,while exogenous Ca Cl₂only partially reversed neomycin impairment on spore germination.Neomycin also affected mycotoxin production2.Fruit wax extracts significantly upregulated the expression of all three AaPLC genes.Compared with the wild type,the growth of?AaPLC1 was slow and decreased by 80.8%at 3days.The deletion of AaPLC2 leads to changes in colony morphology,while the growth of?AaPLC3 was the same as the wild type.The regulation of AaPLCs on the response to exogenous stress was varied.The?AaPLC1 mutant increased sensitivity to salt and cell wall synthesis inhibitors,while the deletion of AaPLC2 increases tolerance to salt stress,and the?AaPLC3 has increased sensitivity to SDS.In addition,the?AaPLC1mutant reduced virulence,decreased toxin synthesis and increased melanin content compared to the wild-type.AaPLC2 and AaPLC3 have similar functions and have little effect on virulence of A.alternata,but could affect toxin synthesis.3.Infection structure formation experiments on wax coating,onion epidermis surface and pear peel found that fruit wax-coated surfaces significantly induced spore germination and appressorium formation in A.alternata and mutant strains,and?AaPLCs spores all germinate normally,but appressorium and infected hyphae formation were varied.The deletion of AaPLC1 led to the decrease of appressorium formation and infected hyphae.Appressorium formation of?AaPLC1 strain was significantly reduced by 50.1%compared to wild-type 4 h after incubation,while?AaPLC2 and?AaPLC3 could form infected hyphae normally as wild-type strain.In summary,the AaPLCs plays an important regulatory role on the growth,pathogenicity and biological stress of A.alternata.The PLC-mediated calcium signaling pathway is involved in the regulation of A.alternata in response to pear peel physicochemical signals and initiating infection.