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

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 [IC 50 ] = 5 μg/ml) and Botrytis cinerea (IC 50 = 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.