Expression and Antimicrobial Activity Assessment of CBD-alfAFP Recombinant Peptide Produced in Tobacco Hairy Roots Against Plant Pathogens

Expression of antimicrobial peptides (AMPs) in plants to resist plant pathogens as well as to produce novel AMPs for pharmaceutical applications has recently received much consideration. alfAFP, a defensin cationic peptide synthesizing in alfalfa seeds, exhibits a strong antimicrobial activity. In order to facilitate alfAFP access to the pathogen’s membrane and increase the activity of the alfAFP peptide, the alfAFP encoding sequence was fused to the C-terminal of a chitin-binding domain (CBD) from a rice chitinase encoding gene. First, the antimicrobial properties of the recombinant peptide were assessed using bioinformatics tools. Next, the pGSA1285 expression vector harboring the CBD-alfAFP heterologous DNA was transformed into Agrobacterium rhizogenes for hairy root (HR) production in tobacco. The presence of transgene, transcription, and the expression of recombinant peptide in the HRs were confirmed by PCR and semi-quantitative RT-PCR analysis, respectively. Bioinformatic analysis was used to predict the antimicrobial activity of the alfAFP recombinant peptide. The results of the 3D structure analysis revealed a β-sheet and an α-helix structure that corresponded well with the structure of plant defensins. A Knottin functional domain was also recognized, suggesting that the recombinant peptide retains its antimicrobial activity. The results of the in vitro antimicrobial activity of the alfAFP recombinant peptide using CFU test showed that the recombinant peptide had significant inhibitory effects on Pseudomonas syringae pathogen. Therefore, the chitin-binding domain provided a better access of the recombinant peptide to the pathogenic bacterial cell wall through binding to peptidoglycan, and probably the recombinant peptide was able to target the plasma membrane with better efficiency. The results of this study suggested that the expression of the CBD-alfAFP recombinant peptide in crop plants and HRs can be a promising approach to producing pathogen-resistant plants as well as to produce new recombinant pharmaceutical AMPs.