HAIRPIN RNA INTERFERENCE CONSTRUCTS AGAINST Tomato Leaf Curl Sudan Virus: TRANSFORMATION, AND EVALUATION IN Nicotiana benthamiana PLANTS - Mahmoud Ahmed Amer; Zaheer Khalid; Muhammad Taimoor Shakeel; A Sabra; Muhammad Amir; Muhammad Zaman; Khadim Hussain; Ibrahim Mohammed Al-Shahwan; Mohammed Ali Al-Saleh (Journal of Animal and Plant Sciences, 2026)
Tomato Leaf Curl Sudan Virus (ToLCSDV) is a begomovirus that severely damages tomato plants, causing growth retardation, yellowing, and curling of the leaves. Hairpin RNA interference (hpRNAi) is a defense mechanism against viruses and other infectious nucleic acids has been successfully exploited for engineering virus resistance in Nicotiana benthamiana plants. In this study, we developed a hpRNAi construct on the conserved region of capsid protein (CP) gene of ToLCSDV to offer protection against tomato-infecting ToLCSDV as a begomoviruses. In order to create the hairpin RNAi construct, the primer pairs CPS-F and CPS-R then CPAS-F and CPAS-R primers were used to amplify the CP gene fragment in sense and antisense orientation. The hairpin RNAi construct (hpCP-pFGC5941) was created by ligating both sense and antisense orientation into pFGC5941 as a binary vector in MCS1 and MCS2, respectively. XhoI and NcoI restriction of the pFGC5941 vector and CPS PCR result reveals the distinct bands for CP sense and plasmid vector. The amplified CP anti-sense segment was restricted and ligated into the vectCPS-pFGC5941 using BamHI and XbaI restriction sites. For confirmation of recombinant clones, restriction analysis displayed required bands at a certain size: pFGC5941 vector (11.406 bp), CPAS (~686 bp), and CPS-pFGC5941 vector (~12.1 Kb). Following PCR validation of CP sense and antisense clones using 35S-Pro-F/chsA-intr-R and chsA-intr-F/OCS-term, respectively, three clones for sense and antisense (1Kb) were obtained. In each experiment, 10 N. benthamiana plants were inoculated: 5 plants received ToLCSDV (infectious clone) + hpCP-pFGC5941 (hpRNAi construct) and 5 plants received ToLCSDV (infectious clone) alone. These experiments were performed in triplicates. Plants that were inoculated with the hpRNAi construct showed encouraging resistance when compared to those that were inoculated with the ToLCSDV infectious clone alone. In this study, hpRNAi plants exhibited resistance compared to the controls hpRNAi targeting CP of ToLCSDV reduced infection from 100% to 27% in treated plants. The results of RNAi resistance and their importance in development of RNAi based resistance in elite tomato cultivars are discussed
Keywords: journal article; hpRNAi; ToLCSDV; begomovirus; viral resistance; capsid protein; Nicotiana benthamiana