Antimicrobial activity of Epsilon-Poly-L-lysine against phytopathogenic bacteria.

Antimicrobial peptides (AMPs) are components of immune defense in many organisms, including plants. They combat pathogens due to their antiviral, antifungal and antibacterial properties, and are considered potential therapeutic agents. An example of AMP is Epsilon-Poly-L-lysine (EPL), a polypeptide formed by ~ 25 lysine residues with known antimicrobial activity against several human microbial pathogens. EPL presents some advantages such as good water solubility, thermal stability, biodegradability, and low toxicity, being a candidate for the control of phytopathogens. Our aim was to evaluate the antimicrobial activity of EPL against four phytobacterial species spanning different classes within the Gram-negative phylum Proteobacteria: Agrobacterium tumefaciens (syn. Rhizobium radiobacter), Ralstonia solanacearum, Xanthomonas citri subsp. citri (X. citri), and Xanthomonas euvesicatoria. The minimum inhibitory concentration (MIC) of the peptide ranged from 80 µg/ml for X. citri to 600 µg/ml for R. solanacearum and X. euvesicatoria. Two hours of MIC exposure led to pathogen death due to cell lysis and was enough for pathogen clearance. The protective and curative effects of EPL were demonstrated on tomato plants inoculated with X. euvesicatoria. Plants showed less disease severity when sprayed with EPL solution, making it a promising natural product for the control of plant diseases caused by diverse Proteobacteria.

Generation and In-planta expression of a recombinant single chain antibody with broad neutralization activity on Bothrops pauloensis snake venom.

The main systemic alterations present in bothropic envenomation are hemostasis disorders, for which the conventional treatment is based on animal-produced antiophidic sera. We have developed a neutralizing antibody against Bothrops pauloensis (B. pauloensis) venom, which is member of the genus most predominant in snakebite accidents in Brazil. Subsequently, we expressed this antibody in plants to evaluate its enzymatic and biological activities. The ability of single-chain variable fragment (scFv) molecules to inhibit fibrinogenolytic, azocaseinolytic, coagulant and hemorrhagic actions of snake venom metalloproteinases (SVMPs) contained in B. pauloensis venom was verified through proteolytic assays. The antibody neutralized the toxic effects of envenomation, particularly those related to systemic processes, by interacting with one of the predominant classes of metalloproteinases. This novel molecule is a potential tool with great antivenom potential and provides a biotechnological antidote to snake venom due to its broad neutralizing activity.

The plant-based chimeric antimicrobial protein SlP14a-PPC20 protects tomato against bacterial wilt disease caused by Ralstonia solanacearum.

Cecropin-B (CecB) is a peptide with well-established antimicrobial properties against different phytopathogenic bacteria. Despite modest action against Ralstonia solanacearum, its animal source limits the acceptance in transgenic applications. To overcome this, we selected eight alpha-helical (AH) cationic peptides derived from plant protein sequences and investigated their antimicrobial properties against R. solanacearum. Remarkably, PPC20 (a linear AH-peptide present in phosphoenolpyruvate carboxylase) has a three-fold lower lethal dose on R. solanacearum than CecB and lower toxicity to human intestinal epithelial cells. Linking PPC20 to SlP14a (part of a pathogenesis-related protein) established an apoplast-targeted protein providing a means of secreting and stabilizing the antimicrobial peptide in the plant compartment colonized by the pathogen. SlP14a is also a potential antimicrobial, homologous to a human elastase which likely targets outer membrane proteins in Gram-negative bacteria. Recombinant SlP14a-PPC20 showed antibacterial activity against R. solanacearum in vitro, making it a promising candidate for plant protection. This was confirmed with genetically-modified tomato plants engineered to express SlP14a-PPC20, in which bacterial populations in stems were reduced compared to inoculated wild-type control plants. Disease symptoms were also markedly less severe in SlP14a-PPC20-expressing plants, demonstrating a viable strategy to improve resistance against bacterial wilt in tomato.

The Type II Secreted Lipase/Esterase LesA is a Key Virulence Factor Required for Xylella fastidiosa Pathogenesis in Grapevines.

Pierce's disease (PD) of grapevines is caused by Xylella fastidiosa (Xf), a xylem-limited gamma-proteobacterium that is responsible for several economically important crop diseases. The occlusion of xylem elements and interference with water transport by Xf and its associated biofilm have been posited as the main cause of PD symptom development; however, Xf virulence mechanisms have not been described. Analysis of the Xf secretome revealed a putative lipase/esterase (LesA) that was abundantly secreted in bacterial culture supernatant and was characterized as a protein ortholog of the cell wall-degrading enzyme LipA of Xanthomonas strains. LesA was secreted by Xf and associated with a biofilm filamentous network. Additional proteomic analysis revealed its abundant presence in outer membrane vesicles (OMVs). Accumulation of LesA in leaf regions associated positively with PD symptoms and inversely with bacterial titer. The lipase/esterase also elicited a hypersensitive response in grapevine. Xf lesA mutants were significantly deficient for virulence when mechanically inoculated into grapevines. We propose that Xf pathogenesis is caused by LesA secretion mediated by OMV cargos and that its release and accumulation in leaf margins leads to early stages of observed PD symptoms.