Study On The Interaction Mechanism Between Penicillium Expansum And Apple Based On Proteomics

Apple(Malus domestica)is a common fruit packed with vitamins and minerals,among other nutrients.At present,China has the largest apple planting area and the highest output in the world,and the development of apple industry has an important influence on China’s agricultural economy.However,apple is susceptible to many kinds of pathogens during storage and transportation in postharvest,causing serious losses to the apple industry.Among them,blue mold disease caused by P.expansum is the most important disease causing postharvest loss of apples.Therefore,prevention and control of the postharvest disease should be solved urgently.With the development of modern biological technology,research from the perspective of molecular biology is increasing.Therefore,the current study aimed to determine the P.expansum apple infection mechanism,so as to provide a better theoretical basis for the prevention and treatment of postharvest diseases of apples.Previous studies on the interaction mechanism of P.expansum and apple mostly focused on the gene level,while few focused on proteins.Based on the proteomics analysis,this study explored the interaction mechanism of P.expansum and apple;and screened and analyzed the key proteins.The main research results are as follows:1.The germination of P.expansum spores at different time points of infection was observed by microscope.After 1 h of infection,the spore began to change from oval shape to tip shape;At 3 h,the spore deformation was obvious and several bud tubes appeared.At 6 h,spores invaded apple tissue and formed appressorium.At 9 h,the bud tube extended to form infection nails and successfully invaded the interior of the apple.Therefore,9 h was the key time point for inoculation of P.expansum spores.After inoculation of P.expansum spores for 9 h,proteomic sequencing analysis and PRM verification were performed on P.expansum samples.The results showed that P.expansum could infect apples by three ways:(1)The up-regulated expression of proteins related to spore germination and growth and development of fungi improved the infection ability of P.expansum in apple;(2)Activate the redox reaction of P.expansum to remove reactive oxygen species(ROS)produced by apple defense;(3)Activation of the peroxisome pathway,MAPK signaling pathway,and p53 signaling pathway related to the pathogenesis of P.expansum can improve the pathogenicity of P.expansum.2.9 h after inoculation with P.expansum spores,the wound site of apple was sampled and protein extraction,omics sequencing analysis and PRM verification were carried out.The results showed that:(1)defense-related proteins such as ACBP and CPR were significantly up-regulated in apple;(2)The infection of P.expansum activated MAPK signaling pathway,plant-pathogen interaction metabolic pathway and phenylpropane biosynthesis pathway,which were related to stress response,and regulated ROS generation and oxidative stress,and improved the resistance of apple.(3)Promoted the synthesis of lignin,flavonoids and other secondary metabolites,improved the mechanical strength of apple cell wall,and enhanced the defense ability of apple against P.expansum.(4)The synthesis of pathogenesis-related protein helps apples directly defend against P.expansum infection.3.Through the analysis of the proteomics results of apple,two potential key proteins in the defense process of apple were screened: acyl-Co A binding protein(MdACBP),which has high binding ability for acyl-co A and phospholipids.Pectin lyase(MdPL)regulates cell wall permeability.MdACBP and MdPL may play important roles in regulating growth and development and stress response.Basic physicochemical properties and protein structure analysis indicate that MdACBP belongs to the ACBP1 family and MdPL belongs to the PL1 family.The results of RTq PCR showed that the expression levels of potential key proteins increased at different periods of P.expansum infection,suggesting that MdACBP and MdPL played a continuous role in the defense process of apple.The results of Agrobacterium-mediated subcellular localization of onion epidermis showed that MdACBP protein was located in the nucleus and MdPL protein was located in the cytoplasm.4.Y2 H system was used to explore the proteins of P.expansum interacting with apple proteins during infection.The results showed that there was no self-activation of apple bait protein and no toxic effect on Y2 H cells.Pe-A0A0A2IR3 and MdPL,PeA0A2IS21 and MdACBP could grow normally in SD/-Trp-Leu nutrient deficiency medium and SD/-Leu/-Trp/-His/-Ade.Note Pe-A0A0A2IR3 and Pe-A0A0A2IS21 interact with apple protein during P.expansum infection.The confidence level of the interaction between two pairs of proteins was analyzed,and the results showed that the confidence score of MdPL Pe XR was 0.8299;The confidence score of MdACBPPe Ppda is 0.9028,confirming the results of Y2 H.