Abstract
Dermaseptin B1 (DrsB1), an antimicrobial cationic 31 amino acid peptide, is produced by Phyllomedusa bicolor. In an attempt to enhance the antimicrobial efficacy of DrsB1, the DrsB1 encoding 93 bp sequence was either fused to the N or C terminus of sequence encoding chitin‐binding domain (CBD) of Avr4 gene from Cladosporium fulvum. Tobacco leaf disk explants were inoculated with Agrobacterium rhizogenes harboring pGSA/CBD‐DrsB1 and pGSA/DrsB1‐CBD expression vectors to produce hairy roots (HRs). Polymerase chain reaction (PCR) was employed to screen putative transgenic tobacco lines. Semi‐quantitative RT‐PCR and western blotting analysis indicated that the expression of recombinant genes were significantly higher, and recombinant proteins were produced in transgenic HRs. The recombinant proteins were extracted from the tobacco HRs and used against Pectobacterium carotovorum, Agrobacterium tumefaciens, Ralstonia solanacearum, and Xanthomonas campestris pathogenic bacteria and Alternaria alternata and Pythium sp. fungi. Two recombinant proteins had a statistically significant (p < 0.01) inhibitory effect on the growth and development of plant pathogens. The CBD‐DrsB1 recombinant protein demonstrated a higher antibacterial effect, whereas the DrsB1‐CBD recombinant protein demonstrated greater antifungal activity. Scanning electron microscopy images revealed that the structure of the fungal mycelia appeared segmented, adhered to each other, and crushed following the antimicrobial activity of the recombinant proteins. Due to the high antimicrobial activity of the recombinant proteins against plant pathogens, this strategy can be used to generate stable transgenic crop plants resistant to devastating plant pathogens.
Highlights
Plant pests and diseases are among the main factors reducing the production of agricultural products and diminishing their quality and yield as well as threatening food safety (Oerke, 2006)
We showed that fusion of dermaseptin B1 (DrsB1) antimicrobial peptide to the chitin‐binding domain (CBD) of Avr4 protein from C. fulvum enhanced the antibacterial activity of Dermaseptin B1 (DrsB1) peptide, suggesting that CBD might facilitate DrsB1 peptide access to the fungal plasma membrane, leading to cell membrane rupture and deformation
Expression of fungal and bacterial cell wall‐degrading en‐ zymes, expression of pathogenesis‐related proteins, in‐ crease in production of host proteins and metabolites involved in plant defense pathways, and expression of plant antimicrobial pro‐ teins and peptides have been reported (Punja, 2001)
Summary
Plant pests and diseases are among the main factors reducing the production of agricultural products and diminishing their quality and yield as well as threatening food safety (Oerke, 2006). Given the negative effects of chemical control on human health and the envi‐ ronment, and emergence of resistance by pathogens, it is necessary to employ safer and more sophisticated methods to cope with plant pathogens (Vidaver, 2002) Plants activate their immune‐defense system when pathogens attack (Nguyen, Haney, & Vogel, 2011; Zasloff, 2002, 2006). Chitin is one of the main structural oligosaccharides of the cell wall in vari‐ ous pathogenic fungi, playing a role of an important elicitor of innate defense responses in plants (Sánchez‐Vallet, Mesters, & Thomma, 2015) Through their hydrolytic activities, plant chitinases hydrolyze cell wall chitin eventually leading to cell death (Latgé, 2010; Latgé & Beauvais, 2014; Thomma, Nürnberger, & Joosten, 2011). We showed that fusion of dermaseptin B1 (DrsB1) antimicrobial peptide to the chitin‐binding domain (CBD) of Avr protein from C. fulvum enhanced the antibacterial activity of DrsB1 peptide, suggesting that CBD might facilitate DrsB1 peptide access to the fungal plasma membrane, leading to cell membrane rupture and deformation
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