The Role Of PMA1 In The Growth And Pathogenesis Of Penicillium Digitatum And Preliminary Study On Its Regulatory Mechanism

Green mold of citrus caused by Penicillium digitatum is one of the most important causes of postharvest rot of citrus fruit.At present,control methods are mainly dependent on chemical fungicides,but long-term,heavy use of chemical fungicides has caused the emergence of many resistant strains,leading to a reduction in the efficacy of chemical fungicides and a shortening of the service period.Therefore,there is an urgent need to develop fungicides with new target,low toxicity and high efficiency.Plasma membrane H⁺-ATPase（PMA1）plays a key role in fungal growth and pathogenicity by maintaining fungal cytoplasmic p H balance and regulating nutrient uptake,while PMA1 is a potential drug target due to its fungal structural specificity.PMA1 plays an important role in several stages of plant and yeast growth,but its function and regulatory mechanisms in plant pathogenic fungi are not yet clear.In this experiment,we used RNA interference（RNAi）and overexpression techniques to obtain PMA1 mutant strains,and measured the effects of PMA1 on mycelial growth,spore germination,spore viability,pathogenicity and tolerance to acid-base stress,ionic stress and proton pump inhibitors in P.digitatum.We further analyzed the mechanism of PMA1 in regulating the growth and pathogenicity of P.digitatum and explored the potential of PMA1 as a novel fungicide target for citrus green mold.The main results were as follows:（1）PMA1 gene silencing and overexpression vector construction and its mutant strains acquisition:The experiment successfully constructed the PMA1 gene silencing vector p-PS-A-H-3300 and overexpression vector p-H-PP-3300 using RNAi and overexpression techniques,and screened a series of transformers with different expression levels of PMA1gene,and finally selected si57（53%silencing efficiency）,si194（84%silencing efficiency）and oe9（about 100%upregulation of expression）for the follow-up experiments.（2）The Effect of PMA1 gene silencing on the growth characteristics of P.digitatum.Compared with the wild-type strain（WT）of P.digitatum,the colony diameters of si57 and si194 decreased by 33.3%and 11.7%,respectively,while oe9 increased by 16.7%;compared with the WT,the mycelial biomass of si57 decreased by 58.12%,and the biomass of overexpression strain was 1.03±0.0678 g,which was 1.65 times that of WT strain.The spore germination rate,spore production and spore viability of PMA1 silenced strains were significantly lower than those of the WT strain,and the overexpression strain showed the opposite trend;the pathogenicity of citrus fruit infested with si57 strain was significantly weaker,and the spot diameter（47.28±2.69 mm）was observably less than that of WT strain（94.30±1.83 mm）,and the degree of citrus fruit rot inoculated with the oe9 strain showed a more serious development trend,with a spot diameter of 107.0±0.707 mm.Under acidic and alkaline conditions,the inhibition rate of mycelial growth of PMA1-silenced strains was significantly higher than that of WT strains;compared with WT,si57 was sensitive to high concentrations of Ca²⁺,Cu²⁺and Fe²⁺;in addition,si57 was significantly more sensitive to proton pump inhibitors than WT strains,and oe9 strains were decreased.（3）Study of the action mechanism of PMA1 gene silencing on the growth and pathogenicity of P.digitatum.The morphological observation showed that compared with WT,the cell morphology of si57 was significantly changed,with a significantly thickened cell wall,broken cell membrane at polar growth sites,leakage of contents,and the formation of a large number of autophagosomes inside the cell;consistent with the changes in cell wall morphology,si57 mycelium cell wall chitin and glucan content was significantly reduced,accompanied by increased chitinase and glucanase activities and upregulated expression levels of related genes,respectively.Compared with WT,the ergosterol content of si57 mycelium decreased and the cell membrane permeability increased,leading to the leakage of cytoplasmic contents（ions,reducing sugars and soluble proteins）,which was further evidenced by the enhanced fluorescence of PI staining;in addition,the activity of si57 cell wall degrading enzymes was generally lower than that of WT strains under both in vitro and in vivo conditions,and the relative expression of related degrading enzyme genes were down-regulated.The relative expression of Atg1,Atg15 and metacaspase-1,important genes regulating the autophagic pathway,were significantly higher than that of WT strain,indicating that the silencing of PMA1 gene may activate the autophagic and apoptotic programs in P.digitatum cells.In summary,the PMA1 gene silencing affected the growth rate,spore viability,spore production and pathogenicity of P.digitatum by affecting cell wall and cell membrane components and reducing cell wall degrading enzyme activity,while the sensitivity of strain si57 to acidic and alkaline environments,high metal ion stress and proton pump inhibiting fungicides was increased.This indicates that PMA1 plays an important role in the cell growth and pathogenicity of P.digitatum,which can be used as a potential target for the development of new fungicides for citrus green mold.