Action Mechanisms Of Jelleine-Ⅰ And P235 Against Clavibacter Michiganensis Subsp. Michiganensis

Tomato bacterial canker disease,caused by the Clavibacter michiganensis subsp.michiganensis（Cmm）,is one of the devastating diseases of tomatoes and poses a serious threat to global tomato production.The pathogenic bacterium spreads quickly and is highly pathogenic,which seriously affects the yield and quality of tomatoes.Although chemical control can be used to control tomato canker disease,over-reliance on chemicals can lead to problems such as pathogen resistance,environmental pollution and food safety.Therefore,novel antimicrobial agents are urgently needed for controlling Cmm.Antimicrobial peptides（AMPs）have attracted much attention because of their high antimicrobial activity,difficulty in developing resistance and environmental friendliness,and are considered to be ideal antimicrobial agents.In view of this,antimicrobial peptides Jelleine-Ⅰ and the designed peptide P235 as the research objects,their antibacterial activities against Cmm were measured.Then their cell membrane and intracellular target mechanisms against Cmm were studied.Finally,the antibacterial effects of both peptides against Cmm in tobacco leaves were evaluated.The main results are as follows:（1）The MICs of Jelleine-Ⅰ,P235 and agricultural streptomycin（AS）against Cmm were25μg/m L,12.5μg/m L and 1000μg/m L,respectively.The EC₅₀and IC₅₀of Jelleine-Ⅰ against Cmm were 4.62μg/m L and 4.98μg/m L,respectively.The EC₅₀and IC₅₀of P235against Cmm were 2.47μg/m L and 2.36μg/m L,respectively.The EC₅₀and IC₅₀of AS against Cmm were 214.83μg/m L and 210.96μg/m L,respectively.These results indicated that the antimicrobial activities of both peptides were superior to that of commercial AS,and the designed P235 was superior to that of Jelleine-Ⅰ.The dynamic antibacterial activity results showed that Jelleine-Ⅰ and P235 could significantly inhibit the growth of Cmm at concentrations of 2 MIC and 1 MIC.The synergistic results showed that Jelleine-Ⅰ and P235 at lower concentrations（1/4 MIC-1/256 MIC）have a synergistic effect with EDTA（1/4 MIC-1/8 MIC）.（2）The results of cell membrane permeability showed that Jelleine-Ⅰ and P235treatments could increase the permeability of Cmm cell membrane.The permeability of Cmm cell membrane after treatment with Jelleine-Ⅰ and P235 increased from 4.60%to9.37%and 11.78%at 4 h,respectively.Calcium ion leakage experiment showed that after treatment with Jelleine-Ⅰ and P235 for 8 h,the concentrations of extracellular calcium ions increased to 0.096 m M and 0.117 m M,respectively.PI staining results showed that when treated with P235 for 1 h and 2 h,the permeability of Cmm cell membranes was 2.29%and8.93%,respectively.The results of cell membrane depolarization showed that both peptides act on the cell membrane,and cause membrane depolarization.（3）The results of scanning electron microscopy showed that the Cmm cells showed cell surface depression,abnormality and membrane damage after treatment with Jelleine-Ⅰ and P235 for 4 h.The results of transmission electron microscopy showed that after Jelleine-Ⅰ and P235 treatments,the Cmm cell membrane was damaged,the edges were blurred,and the intracellular substances were leaked.These results indicated that both peptides could destroy the Cmm cell membrane and lead to cell death.（4）The results of FITC-P235 and DAPI co-staining showed that P235 and DAPI were co-localized in Cmm cells,indicating that P235 could interact with the DNA of Cmm.DNA and RNA gel electrophoresis showed that both Jelleine-Ⅰ and P235 could bind to genomic DNA and RNA of Cmm,and delay DNA and RNA migration in a concentration-dependent manner.RT-q PCR results showed that compared with the control,after treatment with Jelleine-Ⅰ and P235 for 12 h,the expression of DNA replication-related genes（dna A）of Cmm was significantly upregulated,and the expression of DNA repair-related genes（rec A）of Cmm was downregulated.（5）ROS detection showed that Jelleine-Ⅰ and P235 induced the accumulation of ROS in Cmm in a dose-dependent manner.With the addition of ROS scavengers,the MICs of both peptides were increased and the activities of both peptides were decreased.The results of non-specific esterase activity assay showed that the intracellular fluorescence intensity of Cmm was significantly reduced after treatment with both peptides compared with the control,indicating that both peptides could affect the activity of intracellular esterase.（6）The HR response results showed that compared with the untreated group,the degree of leaf damage was significantly reduced after Jelleine-Ⅰ and P235 treatments,indicating that Jelleine-Ⅰ and P235 could reduce the toxicity of Cmm in tobacco leaves.This study theoretically contributes to understand the mechanism of action of AMPs against Cmm,and can provide a theoretical basis for the development of AMPs into new bactericides.