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United States Department of Agriculture

Agricultural Research Service

Research Project: NOVEL DISEASE CONTROL STRATEGIES FOR CELLULAR AND SUB-CELLULAR PATHOGENS

Location: Molecular Plant Pathology

Title: Bacteriophage endolysin production in Nicotiana benthamiana plants

Authors
item Kovalskaya, Natalia
item Foster Frey, Juli
item Donovan, David
item Hammond, Rosemarie

Submitted to: Mid Atlantic Plant Molecular Biology Society Conference
Publication Type: Abstract Only
Publication Acceptance Date: June 24, 2013
Publication Date: N/A

Technical Abstract: The increasing spread of antibiotic resistant microorganisms is a growing concern for both modern animal husbandry and medicine. In recent years, peptidoglycan hydrolases (lysins) have acquired significant attention in the fight against bacterial diseases. The main advantages of lysins versus antibiotics are: 1) exogenous lysin application leads to rapid lysis of the bacterial cell wall avoiding such intracellular resistance mechanisms as efflux pumps, thereby making microbial resistance development difficult and 2) endolysins possess narrow species specificity, without effecting aboriginal (normal) microflora. In an effort to examine the antimicrobial efficacy of a Gram-negative phage endolysin, we attempted to produce the endolysin encoded by a cryptic prophage CP-933P [Escherichia coli O157:H7 str. EDL933] (GenBank Acc.# NP_287988). The endolysin gene was subcloned into the pET21a inducible prokaryotic expression vector and introduced into E.coli strains BL21 (DE3), C43 (DE3) pLysS, C43 (DE3), C41 (DE3) pLysS and C41 (DE3). Expression of the cp933 gene in bacterial cells led to growth inhibition and lysis of the host cells (strains BL21 (DE3), C43 (DE3) pLysS, C41 (DE3) pLysS) or production of trace amounts of the CP933 endolysin (strains C43 (DE3) and C41 (DE3)). To overcome the expression problems in E. coli, we attempted to produce the lysin in Nicotiana benthamiana tobacco plants using a Potato virus X (PVX)-based transient expression vector. Cytoplasmic expression of cp933 resulted in death of the apical region of experimental plants 10 days after viral transcript infection. To protect plants against the detrimental impact of CP933, the cp933 gene containing a 6xHis-tag at its C-terminus was fused at its N-terminus to an N-terminal signal peptide from potato proteinase inhibitor I (PPI-I) to direct CP933to delta type vacuoles. Plants producing PPI-I/CP933 fusion protein did not exhibit the severe toxic effect observed with cytoplasmic expression of cp933. At this stage of our studies, the results of our experiments demonstrated that targeting of proteins to the delta vacuoles is a promising approach for the production of proteins that exhibit toxicity when expressed in plant cells.

Last Modified: 4/19/2014
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