G. Sharath Chandra

Diet-delivered RNAi in Helicoverpa armigera – progresses and challenges

Helicoverpa armigera (the cotton bollworm) is a significant agricultural pest endemic to Afro-Eurasia and Oceania. Gene suppression via RNA interference (RNAi) presents a potential avenue for management of the pest, which is highly resistant to traditional insecticide sprays. This article reviews current understanding on the fate of ingested double-stranded RNA in H. armigera. Existing in vivo studies on diet-delivered RNAi and their effects are summarized and followed by a discussion on the factors and hurdles affecting the efficacy of diet-delivered RNAi in H. armigera.

Evaluation of reference genes for quantitative real-time PCR normalization in cotton bollworm Helicoverpa armigera

Reverse-transcription quantitative real-time PCR (RT-qPCR), a sensitive technique is being extensively employed in quantification of gene expression. However this requires normalization with suitable reference gene (RG) which is crucial in minimizing inter sample variations. Information regarding suitable RG is scarce in general and more so in insects, including the cotton bollworm, Helicoverpa armigera, an economically important pest. In management of this pest RNA interference (RNAi) is perceived as a potential tool, which is achieved by double-stranded RNA (dsRNA) delivery. These studies demand accurate quantification of gene silencing. In this study we assessed the suitability of five RGs viz. β-actin (ACTB), 18S rRNA (18S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-tubulin (TUB) and elongation fator-1-alfa (EF1-α) for gene expression studies in dsRNA treatment and across different developmental stages of H. armigera and ranked using geNorm, NormFinder and BestKeeper software programs. Data analysis revealed that best ranked RGs were varied in dsRNA treatment and in developmental stages. Under dsRNA treatment, 18S and GAPDH were more stable whereas, TUB and GAPDH were more stable across developmental stages. We also demonstrate that inappropriate selection of RG led to erroneous estimation of the target gene, chymotrypsin expression. These results facilitate accurate quantification of gene expression in H. armigera.

Molecular diversity of Helicoverpa armigera Hubner (Noctuidae: Lepidoptera) in India

Molecular diversity of genes, namely actin, glutathione-S-transferase (gst), cytochrome P450, chymotrypsin and serine protease from the interspecific populations of the fruit borer Helicoverpa armigera was studied to understand the sequence polymorphism. These genes were cloned, sequenced and analysed employing the sequence editor BioEdit and MEGA 4.0. Sequence comparisons showed variation of the genes as follows: Actin – 35 positions, 3.09%, gst – 60 positions, 9.66%, cytochrome P450 – 64 positions, 8.11%, chymotrypsin – 36 positions, 4.02%, and serine protease – 119 positions, 15.31%. Phylogenetic analysis concluded that these intraspecific populations from India formed a major clade. Comparison of deduced amino acid sequences concluded that there were no major differences except for serine protease. This analysis will be useful for delineating genetic relationships amongst the intraspecific populations and estimating genetic diversity, thereby gaining insight into genetic structure of populations.

One-step DNA fragment assembly for expressing intron-containing hairpin RNA in plants for gene silencing

Double-stranded RNA-mediated RNA interference in plants involves generating a construct expressing intron-containing hairpin RNA (ihpRNA), which usually is a cumbersome, multistep process. Here, we describe a simplified method involving single steps of PCR, restriction, ligation, and transformation for assembling an ihpRNA construct for plant transformation. Our method has several advantages over the currently available ones, viz., wider choice of restriction sites and facility for rapid screening of positive clones, among others. We demonstrate the utility of this approach in assembling the tomato phytoene desaturase gene. This simplified DNA fragment assembly strategy for ihpRNA construction facilitates high-throughput gene silencing in plants.

Tyrosine Hydroxylase, a Potential Target for the RNAi-Mediated Management of Diamondback Moth (Lepidoptera: Plutellidae)

Abstract Cruciferous vegetables are severely damaged by infestation of the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae). Successful utilization of RNA interference (RNAi) in insect pest management programs depends on various factors, such as target gene, mode of double-stranded RNA (dsRNA) delivery, frequency of application, and development stage of the target insect. Among these factors, selection of the target gene is crucial to the success of RNAi-based programs. In the present investigation, an attempt was made to assess the potential of tyrosine hydroxylase (TH) as a target gene, which is a key regulator in the biosynthesis of 3,4-dihydroxyphenylalanine (DOPA). DOPA is a precursor for agents that function in neurotransmission, melanization, sclerotization of the cuticle, and immune responses in insects. Here, we provided to diamondback moth larvae 3 concentrations (1.04, 2.08, and 3.12 μg/cm2) of cognate dsRNA coated on discs of cabbage leaf, the natural diet for the larvae. We recorded the influence of the dietary dsRNA on TH transcript levels, larval growth, and larval survival rate. The dietary dsRNA led to reduced target gene transcript level and larval feeding level, and caused larval mortality in a concentration-dependent manner. These results demonstrate that the TH gene has potential as a target gene for RNAi-mediated management of P. xylostella.