title: Antimicrobial Activity of Bacteriophage Endolysin Produced in Nicotiana benthamiana Plants.
abstract: The increasing spread of antibiotic-resistant pathogens has raised the interest in alternative antimicrobial treatments. In our study, the functionally active gram-negative bacterium bacteriophage CP933 endolysin was produced in Nicotiana benthamiana plants by a combination of transient expression and vacuole targeting strategies, and its antimicrobial activity was investigated. Expression of the cp933 gene in E. coli led to growth inhibition and lysis of the host cells or production of trace amounts of CP933. Cytoplasmic expression of the cp933 gene in plants using Potato virus X-based transient expression vectors (pP2C2S and pGR107) resulted in death of the apical portion of experimental plants. To protect plants against the toxic effects of the CP933 protein, the cp933 coding region was fused at its Nterminus to an N-terminal signal peptide from the potato proteinase inhibitor I to direct CP933 to the delta-type vacuoles. Plants producing the CP933 fusion protein did not exhibit the severe toxic effects seen with the unfused protein and the level of expression was 0.16 mg/g of plant tissue. Antimicrobial assays revealed that, in contrast to gram-negative bacterium E. coli (BL21(DE3)), the gram-positive plant pathogenic bacterium Clavibacter michiganensis was more susceptible to the plant-produced CP933, showing 18% growth inhibition. The results of our experiments demonstrate that the combination of transient expression and protein targeting to the delta vacuoles is a promising approach to produce functionally active proteins that exhibit toxicity when expressed in plant cells.
authors.affiliation: Molecular Plant Pathology Laboratory, U.S. Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705, USA.
authors.forename: Natalia
authors.initials: N
authors.lastname: Kovalskaya
authors.affiliation: Animal Biosciences and Biotechnology Laboratory, U.S. Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705, USA.
authors.forename: Juli
authors.initials: J
authors.lastname: Foster-Frey
authors.affiliation: Animal Biosciences and Biotechnology Laboratory, U.S. Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705, USA.
authors.forename: David M
authors.initials: DM
authors.lastname: Donovan
authors.affiliation: Electron and Confocal Microscopy Unit, U.S. Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705, USA.
authors.forename: Gary
authors.initials: G
authors.lastname: Bauchan
authors.affiliation: Molecular Plant Pathology Laboratory, U.S. Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705, USA.
authors.forename: Rosemarie W
authors.initials: RW
authors.lastname: Hammond
mesh_terms: D039903:Actinobacteria / Q000187:drug effects / Q000254:growth & development; D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000494:pharmacology*; D001435:Bacteriophages / Q000201:enzymology* / Q000235:genetics; D010450:Endopeptidases / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology*; D004926:Escherichia coli / Q000187:drug effects / Q000254:growth & development; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology*
chemical_list: D000900:Anti-Bacterial Agents; D014764:Viral Proteins; D010450:Endopeptidases; C001143:endolysin
keywords: Nicotiana benthamiana; Potato virus X; endolysin; potato proteinase inhibitor I; transient expression; vacuolar targeting