Use of a Plant Virus Vector for Screening of Gene Constructs with Potential Insecticidal Activity Musarrat Shaheen 2017

Abstract

Extending the novel implications of plant viruses as invaluable genetic tools in the field of transgenic plant technology, Potato Virus X (PVX) based transient gene expression is a simple rapid, inexpensive methodology employed for preliminary screening and evaluation of insecticidal gene constructs in plant sucking insect species. In this study, PVX vector was employed to achieve high throughput expression of two-candidate insecticidal proteins viz. Hvt, (Hydronyche versuta spider) and ACA Lectin (Allium cepa agglutinin) in tobacco plants. The Reverse transcriptase (RT-PCR) revealed a high throughput expression of 117 bp Hvt, 325 bp ACA Lectin in tobacco were bioassayed in Phenacoccus solenopsis Tinsley (P. solenopsis Tinsley), Aphis gossypii Glover, (A. gossypii Glover), M. persicae Sulzer (M. persicae Sulzer) and herbivorous insect species of Helicoverpa virescens (H. virescens), Spodoptera littoralis (S. littoralis). The results revealed severe insecticidal and antagonistic effect of Hvt in terms of significant insect mortality p<0.01%, reduced population survival and nymph production in P. solenopsis Tinsley, A. gossypii Glover and M. persicae Sulzer. ACA Lectin also exerted significant anti-feedent and insecticidal characteristics at p<0.01% on colonization and population survival in tested insects and larval mortality and lower dry weight gain in herbivorous insect species of H. virescens and S. littoralis. The practical application of PVX vector was extended through PVX- based VIGS mediated production of dsRNAs of two partial insect gene transcripts i.e. Voltage Gated Calcium Channel (PSCaVα1) and Elongation Factor (PSEF-1α) in tobacco plants and RNAi response in P. solenopsis Tinsley. The Reverse Transcriptase (RT-PCR) revealed a high throughput expression of 117 bp Hvt, 325 bp ACA Lectin and plants dsRNAs of 152 bp PSCaVα1 and 325 bp PSEF-1α in tobacco used in plant feeding bioassays. The results revealed significant insect mortality 96% PSCaV-α1 and 46% PSEF-1α and several allied phenotypic deformities like stiffing, melanization and shedding of cuticle, arrested growth and reduced fecundity in P. xviii solenopsis Tinsley were consonant with declined expression of targeted genes in insects feeding corresponding dsRNAs duly elaborated by a semi-quantitative RT-PCR. The specificity of dsRNAs 152 bp PSCaV-α1 and 325 bp PSEF-1α dsRNAs derived from P. solenopsis Tinsley and 402 bp Arginine kinase (AK) from A. gossypii Glover was checked through feeding bioassays in non-target insect species like M. persicae Sulzer, H. virescens and S. littoralis. The results revealed non-significant effect of 152 bp PSCaV-α1 and 325 bp PSEF-1α and significant effect of AK in adult aphid in terms of mortality, nymphs produced and survived, larval mortality and dry weight gain in tested insect species. The cumulative results of this study highlighted the significance of PVX vector as valuable genetic tool for preliminary screening and evaluation of candidate insecticidal proteins and RNAi gene constructs. This study will enhance the efforts of biologists in a way to screen the candidate insect genes and design the complementary transgenic crop protection strategies.