Engineered Disease Resistance in Cotton Using RNA-Interference to Knock down Cotton leaf curl Kokhran virus-Burewala and Cotton leaf curl Multan betasatellite Expression

Viruses. 2017 Sep 14;9(9):257. doi: 10.3390/v9090257.


Cotton leaf curl virus disease (CLCuD) is caused by a suite of whitefly-transmitted begomovirus species and strains, resulting in extensive losses annually in India and Pakistan. RNA-interference (RNAi) is a proven technology used for knockdown of gene expression in higher organisms and viruses. In this study, a small interfering RNA (siRNA) construct was designed to target the AC1 gene of Cotton leaf curl Kokhran virus-Burewala (CLCuKoV-Bu) and the βC1 gene and satellite conserved region of the Cotton leaf curl Multan betasatellite (CLCuMB). The AC1 gene and CLCuMB coding and non-coding regions function in replication initiation and suppression of the plant host defense pathway, respectively. The construct, , was transformed into cotton plants using the Agrobacterium-mediated embryo shoot apex cut method. Results from fluorescence in situ hybridization and karyotyping assays indicated that six of the 11 T₁ plants harbored a single copy of the Vβ transgene. Transgenic cotton plants and non-transgenic (susceptible) test plants included as the positive control were challenge-inoculated using the viruliferous whitefly vector to transmit the CLCuKoV-Bu/CLCuMB complex. Among the test plants, plant Vβ-6 was asymptomatic, had the lowest amount of detectable virus, and harbored a single copy of the transgene on chromosome six. Absence of characteristic leaf curl symptom development in transgenic Vβ-6 cotton plants, and significantly reduced begomoviral-betasatellite accumulation based on real-time polymerase chain reaction, indicated the successful knockdown of CLCuKoV-Bu and CLCuMB expression, resulting in leaf curl resistant plants.

Keywords: Begomovirus; Rep protein; cotton leaf curl disease; siRNA; transgenic resistance.

MeSH terms

  • Begomovirus / genetics*
  • DNA, Satellite / genetics
  • DNA, Viral / genetics
  • Disease Resistance / genetics
  • Gene Knockdown Techniques*
  • Genetic Engineering / methods
  • Gossypium / genetics*
  • Gossypium / virology*
  • In Situ Hybridization, Fluorescence
  • Phylogeny
  • Plant Diseases / virology*
  • Plants, Genetically Modified / genetics
  • Plants, Genetically Modified / virology
  • RNA Interference*
  • Real-Time Polymerase Chain Reaction
  • Satellite Viruses / genetics*
  • Sequence Analysis, DNA


  • DNA, Satellite
  • DNA, Viral

Supplementary concepts

  • Cotton leaf curl Kokhran virus