A rapid method for the production and characterization of recombinant insecticidal proteins in plants

Development and evaluation of transgenic plants containing genes that confer insecticidal activity can be a labor-intensive and time-consuming process. Transformed plants require months of growth before they can be analyzed. Transient gene expression by plant viral vectors offers an alternative. Using this approach, infected plants can be screened after only two weeks. Genes coding for the enhanced green fluorescent protein (EGFP), the Bacillus thuringiensis (Bt) Cry1Ac toxin and a wound-induced (Kunitz-type) proteinase inhibitor from poplar (WIN3) were introduced into a potato virus X (PVX) expression vector, pPC2S. Infectious full-length RNA transcripts synthesized in vitro or infectious viral particles were used to inoculate plants. Western blots demonstrate expression of the proteins in Lycopersicon esculentum cv. Rutgers and Nicotiana benthamiana leaves. Fluorescence microscopy was also used to monitor EGFP expression. To demonstrate the effectiveness of the PVX expression system, N. benthamiana expressing Cry1Ac or WIN3 was used and growth of Heliothis virescens, tobacco budworm was assayed. Leaves that expressed Cry1Ac were almost completely resistant to larval feeding. Tobacco budworm fed on leaves expressing WIN3 grew to only 77% of the weight of insects fed with leaves expressing PVX alone. These results illustrate that the PVX expression system allows rapid and reliable production of recombinant plant material for characterization of insecticidal genes in insect bioassays.