Production of Antimicrobial Defensin in Nicotiana benthamiana with a Potato Virus X Vector

Abstract

A recombinant plasmid, pTXS.TH, was constructed to express the gene-encoding wasabi (Wasabia japonica) defensin with the potato virus X (PVX) vector. pTXS.TH allows the expression of defensin in the host Nicotiana benthamiana, and the defensin protein WT1 can be purified from virus-infected leaves by heat treatment and affinity chromatography. WT1 exhibits strong antifungal activity toward the phytopathogenic fungi Magnaporthe grisea(50% inhibitory concentration [IC50] = 5 μg/ml) and Botrytis cinerea (IC50 = 20 μg/ml) but is weakly active against the phytopathogenic bacterium Pseudomonas cichorii. This virus-mediated expression system is a rapid and efficient method to produce and characterize antimicrobial proteins in plants. It is particularly useful for the study of proteins that are difficult to produce with other expression systems.

Keywords

• degenerate primer
• His6
• immunoblot analysis
• PCR
• SDS-PAGE