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Viruses 2017, 9(9), 257; doi:10.3390/v9090257

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

1
Center of Excellence in Molecular Biology, University of the Punjab, 87 W Canal Bank Road, Thokar Niaz Baig, Lahore 53700, Pakistan
2
Institute of Agricultural Sciences (IAGS), University of the Punjab, Lahore 54590, Pakistan
3
Jamie Whitten Delta States Research Center, United States Department of Agriculture (USDA), Stoneville, MS 38776, USA
4
School of Plant Sciences, University of Arizona, Tucson, AZ 85721, USA
*
Authors to whom correspondence should be addressed.
Academic Editor: Gian Paolo Accotto
Received: 20 June 2017 / Revised: 8 August 2017 / Accepted: 30 August 2017 / Published: 14 September 2017
(This article belongs to the Special Issue Geminiviruses)
View Full-Text   |   Download PDF [4565 KB, uploaded 14 September 2017]   |  

Abstract

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. View Full-Text
Keywords: Begomovirus; cotton leaf curl disease; Rep protein; siRNA; transgenic resistance Begomovirus; cotton leaf curl disease; Rep protein; siRNA; transgenic resistance
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Ahmad, A.; Zia-Ur-Rehman, M.; Hameed, U.; Qayyum Rao, A.; Ahad, A.; Yasmeen, A.; Akram, F.; Bajwa, K.S.; Scheffler, J.; Nasir, I.A.; Shahid, A.A.; Iqbal, M.J.; Husnain, T.; Haider, M.S.; Brown, J.K. 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, 9, 257.

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