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Screening of a multi-virus resistant RNAi construct in cowpea through transient vacuum infiltration method

  • K. Prasad BabuEmail author
  • Manamohan MaligeppagolEmail author
  • R. Asokan
  • M. Krishna Reddy
Original Article
  • 23 Downloads

Abstract

Plant viruses are the most devastating pathogens causing substantial economic losses in many crops. Current viral disease management relies on prophylactics, roguing and insect vector control, since in most crops resistant gene pools for resistance breeding are unavailable. RNA interference, a sequence dependent gene silencing mechanism holds great potential in imparting virus resistance. In this study, the efficacy of a RNAi gene construct developed against four viruses commonly infesting tomato and chilli viz., capsicum chlorosis virus, groundnut bud necrosis virus, cucumber mosaic virus and chilli veinal mottle virus was evaluated. A 3546 bp dsRNA-forming construct comprising sense-intron-antisense fragments in binary vector pBI121 (hpRNAi-MVR) was mobilized into Agrobacterium tumefaciens. Cowpea (Vigna unguiculata) was used as an indicator plant for GBNV agroinfiltration to evaluate the efficacy of hpRNAi-MVR construct in conferring GBNV resistance. The type of agroinfiltration, bacterial concentration and incubation-temperatures were optimized. Vacuum infiltration of three pulses of 20–30 s at 66.66 kPa were effective than syringe infiltration. Of the five Agrobacterial concentrations, OD600 0.5 was more efficient. Incubation temperature of 31 ± 1 °C was favorable for development of disease symptoms than 20 ± 1 °C and 26 ± 1 °C. ELISA revealed a 35% decline in viral load in hpRNAi-MVR infiltrated plants compared to vector control plants. Quantitative real time PCR results have shown a viral gene silencing to the extent of 930–990 folds in hpRNAi-MVR infiltrated plants compared to vector control. This approach is simple, rapid and efficient to screen the efficacy of RNAi constructs developed for the RNAi mediated plant virus management.

Keywords

Agroinfiltration Tospovirus Cowpea GBNV Bacterial concentrations 

Notes

Acknowledgements

We greatly thankful to ICAR-IIHR, for providing the facility to carry out research work and thankful to the Division of Plant Pathology, ICAR-IIHR for providing the space for virus maintenance/screening. This is the part of Ph.D. dissertation work of first author.

Supplementary material

13337_2018_509_MOESM1_ESM.docx (1.4 mb)
Supplementary material 1 (DOCX 1472 kb)

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Copyright information

© Indian Virological Society 2019

Authors and Affiliations

  1. 1.Division of BiotechnologyICAR-Indian Institute of Horticultural ResearchBangaloreIndia
  2. 2.Division of Plant PathologyICAR-Indian Institute of Horticultural ResearchBangaloreIndia
  3. 3.Department of BiotechnologyCentre for Post-graduate StudiesBangaloreIndia

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