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Double-Stranded RNA-Mediated Suppression of Trypsin-Like Serine Protease (t-SP) Triggers Over-Expression of Another t-SP Isoform in Helicoverpa armigera

Abstract

High diversity of digestive proteases is considered to be the key factor in the evolution of polyphagy in Helicoverpa armigera. Serine proteases (SPs) contribute ~85% of the dietary protein digestion in H. armigera. We investigated the dynamics of SP regulation in the polyphagous pest, H. armigera using RNA interference (RNAi). HaTry1, an isoform of SP, expressed irrespective of the composition of the diet, and its expression levels were directly proportional to the larval growth rate. Therefore, HaTry1 was silenced by delivering 10 and 20 μg concentrations of double-stranded RNA through semi-synthetic diet. This led to a drastic reduction in the target gene transcript levels that manifested in a significant reduction in the larval weight initially, but the larvae recovered in later stages despite continuous dsRNA treatment. This was probably due to the compensatory effect by over-expression of HaTry13 (31-folds), another isoform of SP. Phylogenetic analysis of H. armigera SPs revealed that the over-expressed isoform was closely related to the target gene as compared to the other tested isoforms. Further, silencing of both the isoforms (HaTry1 and HaTry13) caused the highest reduction in the larval weight and there was no larval growth recovery. These findings provide a new evidence of the existence of compensatory effect to overcome the effect of silencing individual gene with RNAi. Hence, the study emphasizes the need for simultaneous silencing of multiple isoforms.

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Acknowledgements

We are grateful to ICAR, New Delhi, for funding this study under the NAIP sub project “Potential of RNAi in insect pest management: A model in silencing genes specific to tomato fruit borer, Helicoverpa armigera Hübner (Lepidoptera: Noctuidae). We sincerely thank The Director, IIHR, Bengaluru, for facilities and encouragement. We also acknowledge BCRL, (PCI) for providing H. armigera larvae. We sincerely thank the Division of Entomology, ICRISAT, for providing facilities and H. armigera larvae.

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Correspondence to G. Sharath Chandra or R. Asokan.

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Electronic Supplementary Material

figure6

High resolution image (GIF 20 kb)

figure7

High resolution image (GIF 143 kb)

Fig. S1

Effect of HaTry13 dsRNA on the expression of HaTry1. Error bars indicate standard error of the four biological replicates. (TIFF 137 kb)

Fig. S2

Effect of pyramided dsRNA on the expression of the other isoforms of SPs. Error bars indicate standard error of the four biological replicates. (TIFF 401 kb)

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Sharath Chandra, G., Asokan, R., Manamohan, M. et al. Double-Stranded RNA-Mediated Suppression of Trypsin-Like Serine Protease (t-SP) Triggers Over-Expression of Another t-SP Isoform in Helicoverpa armigera . Appl Biochem Biotechnol 184, 746–761 (2018). https://doi.org/10.1007/s12010-017-2584-3

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Keywords

  • RNA interference
  • Helicoverpa armigera
  • Serine protease
  • Double-stranded RNA
  • Insect bioassay
  • RT-qPCR