RNA mediated resistance to Yellow vein mosaic virus in okra

Abstract

Okra (Abelmoschus esculentus L. Moench, Malvaceae) is one of the leading vegetable crops in hot and humid tropics. Unfortunately, this climate is conducive for many of the pests and diseases. Okra is susceptible to viruses such as Yellow vein mosaic virus (YVMV) and Enation leaf curl virus (ELCV), belonging to the genus Begomovirus (family Geminiviridae). Because of the favourable conditions prevailing in the coastal region, the losses in Kerala state are 60-100%, depending upon the stage of plant growth and the severity of infection. RNAi is one of the promising molecular biology approach against the viral diseases. Keeping the above facts in view, the present study “RNA mediated resistance to Yellow vein mosaic virus in okra” was taken up at the Centre for Plant Biotechnology and Molecular Biology, CoA, Thrissur from September 2017 to May 2021. The high yielding and YVMV susceptible popular okra cv. Salkeerthi was selected for the development of resistance using RNAi mechanism. Total DNA was isolated from the YVMV infected plant and part of the βC1 gene (187 bp) of the virus was amplified using primers VβC1F and VβC1R. Sequence information of PCR product has revealed that the gene is 90-95% identical with the Indian isolates. The βC1 gene sequence was analysed using IDT software and 10 siRNAs were found at three different position (19-44, 34-59, 99-124 bp). Through Restriction Mapper, it was confirmed that the sequence selected for the preparation of sense and antisense strand, do not possess recognition sites for SmaI, HindIII and MauBI restriction enzymes which are present in the pRNAiLIC vector. The output of VSupPred revealed that the fragment does not contain any Viral Suppressor Regions (VSRs), with a high prediction score (0.625). The hairpin RNAi construct harbouring the region of βC1 gene of β satellite of Begomovirus of okra was generated using pRNAi-LIC (CD3-1285) vector. The SmaI digested plasmid produced three fragments, vector backbone (9842 bp), Pdk intron (1641 bp) and ccdB gene (614 bp) and the digested plasmid was treated with dTTP. Product-1 was PCR amplified (215 bp) with VLIC1 and VLIC2 primers, using the DNA from YVMV infected plant as template. Product-2 was PCR amplified (243 bp) with VLIC3 and VLIC4 primers using product-1 as template. Product-1 and product-2 were eluted from the gel and treated with dATP. The dATP treated PCR products and dTTP treated SmaI digested plasmid were mixed together and ligated by incubation at 65ºC for 5 min. followed by 22ºC for 15 min. Ligated product was successfully transformed in competent cells of E. coli (DH5α) and incubated on LB medium containing Kanamycin and Chloramphenicol. Colony PCR was performed, the transformation efficiency was found to be 80%. Plasmid was isolated from the positive DH5α colony and sequenced using the primers VLIC5 and VLIC6. The sequence data had shown that both sense and antisense strands are at right position and direction. Plasmid containing ihpRNA-βC1 cassette was successfully transformed into the competent cells of Agrobacterium (GV3101) and incubated on LB medium containing Kanamycin, Chloramphenicol and Rifampicin. Colony PCR was performed, the transformation efficiency was found to be 100%. Plasmid was isolated from the positive GV3101 colony and sequenced using the primers VLIC5 and VLIC6. Sequence data has further confirmed that both sense and antisense strands are at right position and direction. The ihpRNA-βC1 cassette was successfully transformed into okra cv. Salkeerthi using in planta method of Agrobacterium mediated transformation. The transformation efficiency observed was 11.42% and the transformation was confirmed by the amplification of sense strand using the primers VLIC1 and VLIC5. cDNA was prepared from the total RNA isolated from transformed and control plants. siRNA synthesis was confirmed using the primers VLIC1 and VLIC5 (400bp) and Ubiquitin gene was confirmed using the primer UBQ7 (187 bp). Silencing potential of the RNA interference of βC1 gene and the development of resistance was evaluated by keeping the 15-day old transformed and control plants along with YVMV infected plants inside containment facility, with whiteflies released into insect cage for infection. All the control plants and one transgenic plant have shown the YVMV symptoms after 10 days. Three transgenic plants were healthy with no symptoms. The present investigation was successful in the development of YVMV resistant okra plants carrying ihpRNA-βC1 using pRNAi-LIC (CD3-1285) plasmid vector. The further evaluation is needed in the coming generations for the identification of stable transgenic lines.