Development of transgenic okra (Abelmoschus esculentus L. Moench) lines having RNA mediated resistance to Yellow vein mosaic virus (Geminiviridae)

• Published in: Journal of virological methods 2022-03, Vol.301, p.114457-114457, Article 114457
• Main Authors: Ganesh, Kelkar Vipul, Mathew, Deepu, Augustine, Rehna, K.B., Soni, Alex, Swapna, M.R., Shylaja, Cherian K., Anita
• Format: Article
• Language: English
• Subjects: Abelmoschus esculentus; Begomovirus; Genetic engineering; In planta transformation; RNAi; YVMV; βC1
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2022.114457

•Interference with viral βC1 ORF offers YVMV resistance in transgenic okra lines.•ihpRNA cassette with sense and antisense strands was cloned in pRNAi-LIC vector.•Ligation independent cloning is an easier, PCR based strategy.•Agrobacterium mediated in planta transformation is an efficient strategy in okra.•Artificial screening with infected vectors in containment has proven the resistance. Begomovirus Yellow vein mosaic virus causes severe yield losses in okra and even the resistant lines developed through conventional breeding show susceptibility at various levels. This paper describes the development of YVMV resistant lines through RNAi strategy. A universal ihpRNA construct harbouring βC1 ORF from the β-satellite of the begomovirus was designed using pRNAi-LIC plasmid. Complementarity checks in sequence databases had shown no off-target effects by the target region and the success of siRNA in interference was proven using Custom Dicer-Substrate siRNA analysis. The βC1 ORF of the begomovirus was PCR amplified and sequenced using the primer combination designed. The pRNAi-LIC vector, a derivative of pCAMBIA2300 containing duplicated CaMV 35S promoter and Nos terminator from pYL44, was SmaI digested and the amplified sense and antisense strands of the βC1 region were cloned. E. coli transformed with the plasmid were screened for antibiotic resistance, and the plasmids confirmed for the sense and antisense regions through sequencing, were transferred to Agrobacterium tumefaciens strain GV3101. In planta transformation strategy was followed to transform a highly susceptible okra cv. Salkeerthi with ihpRNA-βC1 cassette. Transformation success, confirmed by the amplification of sense strand using the primers VLIC1 and VLIC5, was 11.42 %. Transcription of siRNA from the βC1 ORF in the transgenic lines was confirmed by its PCR amplification from the cDNA, using the stem loop primers designed (68 bp). When the transformed and healthy wild-type plants were co-grown with infected wild-type plants, inside an insect cage released with whiteflies and maintained within a containment facility, three of the four transgenic plants remained completely healthy throughout the crop span.