title: Agroinfiltration as a technique for rapid assays for evaluating candidate insect resistance transgenes in plants.
abstract: Functional analysis of candidate transgenes for insect resistance in stably transformed plants is a time-consuming task that can take months to achieve in even the fastest of plant models. In this study, a rapid screening technique is described, which employs candidate transgene transient expression using agroinfiltration in Nicotiana benthamiana combined with a simple insect bioassay. Using this system the known insecticidal protein Cry1Ac is demonstrated to effectively control Helicoverpa zea. Insects fed tissue with synthesized GFP (green fluorescent protein) as a positive control were shown to have enhanced growth and development. Additionally, a Brassica oleracea proteinase inhibitor (BoPI), a less characterized insect resistance candidate, demonstrated effectiveness to decrease the growth and development of H. zea at high levels of transient expression. Bioassays performed on stable transformants showed that BoPI had a low level of insect resistance at the more typical levels of gene transcription found in stably transformed plants. This agroinfiltration-insect bioassay procedure can give a rapid assessment of insect resistance significantly decreasing the time needed for evaluation of candidate genes.
authors.affiliation: Department of Plant Sciences, University of Tennessee, 2431 Joe Johnson Dr, 252 Ellington Plant Sciences Bldg, Knoxville, TN 37996-4561, USA.
authors.forename: Brian Michael
authors.initials: BM
authors.lastname: Leckie
authors.forename: C
authors.initials: C
authors.lastname: Neal Stewart
mesh_terms: D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics*; D004731:Endotoxins / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006460:Hemolysin Proteins / Q000235:genetics*; D009036:Moths / Q000187:drug effects / Q000472:pathogenicity*; D030821:Plants, Genetically Modified / Q000235:genetics; D011480:Protease Inhibitors / Q000494:pharmacology; D018749:RNA, Plant / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012231:Rhizobium; D014026:Nicotiana / Q000235:genetics*; D014170:Transformation, Genetic; D019076:Transgenes
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D011480:Protease Inhibitors; D018749:RNA, Plant; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D049452:Green Fluorescent Proteins