Prediction And Activity Of An Anitmicrobail Peptide ZM-804 From Maize (Zea Mays L.Inbred Line B73)

Antimicrobial peptides（AMPs）are small molecules consisting of less than fifty residues of amino acids.Plant AMPs establish the first barrier of defense in innate immune system,in response to invading pathogens.The purpose of this study was to isolate new AMPs from Zea mays L.inbred line B73 and investigate their antimicrobial activities and mechanisms against certain essential plant pathogenic bacteria.In silico,the Collection of Anti-Microbial Peptides(CAMP_R3),a computational AMP prediction server,was used to screen a cDNA library for AMPs.A ZM-804 peptide,isolated from Z.mays L.inbred line B73 cDNA library,was predicted as a new cationic AMP with high prediction values.APD3 server predicted the physicochemical properties of ZM-804 along with other servers,ZM-804 peptide is composed of 20 amino acids（MW:2569.131 Da）,net charge（+4）with high hydrophobicity ratio（70%）,isoelectric point12.29,and predicted to be an amphipathicα-helix AMP.The ZM-804 was tested against eleven pathogens of Gram-positive and Gram-negative bacteria and exhibited high antimicrobial activities as determined by the Minimal Inhibitory Concentrations（MICs）,the Minimum Bactericidal Concentrations（MBCs）,and the Minimum Lethal Concentration（MLC）.The lowest MIC values of ZM-804 against Gram-positive bacterial indicators were 8μM and 4μM for Gram-negative bacteria while the lowest MBC values were 16μM for Gram-positive and 8μM Gram-negative bacteria,respectively.The MLC value of ZM-804 that caused complete death was≤4μM against all bacterial indicators in sodium phosphate buffer.A confocal laser-scanning microscope observation showed that the ZM-804 AMP targets bacterial cell membranes.The FITC tag has decreased the antimicrobial activity of ZM-804 which was determined by the antimicrobial activity assay of four bacterial indicators.SEM and TEM images revealed the disruption and damage of the cell membrane morphology of Clavibacter michiganensis subsp.michiganensis and Pseudomonas syringae pv.tomato（Pst）DC3000 caused by the ZM-804.In planta,the ZM-804 demonstrated antimicrobial activity and prevented the infection of tomato plants by the Pst DC3000.Moreover,four virulent phytopathogenic bacteria were prevented from inducing hypersensitive response（HR）on tobacco leaves,in response to low ZM-804 concentrations.The ZM-804exhibits low hemolytic activity against mouse red blood cells（RBCs）and is relatively safe for mammalian cells.In conclusion,the ZM-804 peptide has a strong antibacterial activity and provides an alternative tool for plant disease control.Also,the ZM-804peptide is considered a promising candidate for human and animal drug development.