Molecular cloning, a crucial prerequisite for engineering plasmid constructs intended for functional genomic studies, relies on successful restriction and ligation processes. However, the lack of unique restriction sites often hinders construct preparation, necessitating multiple modifications. Moreover, achieving the successful ligation of large plasmid constructs is frequently challenging. To address these limitations, we present a novel PCR strategy in this study, termed ‘long-fragment circular-efficient PCR’ (LC-PCR). This technique involves one or two rounds of PCR with an additional third-long primer that complements both ends of the newly synthesized strand of a plasmid construct. This results in self-circularization with a nick-gap in each newly formed strand. The LC-PCR technique was successfully employed to insert a partial sequence (210 nucleotides) of the phytoene desaturase gene from Nicotiana benthamiana and a full capsid protein gene (770 nucleotides) of a begomovirus (tomato leaf curl New Delhi virus) into a 16.4 kb infectious construct of a tobamovirus, cucumber green mottle mosaic virus (CGMMV), cloned in pCambia. This was done to develop the virus-induced gene silencing vector (VIGS) and an expression vector for a foreign protein in plants, respectively. Furthermore, the LC-PCR could be applied for the deletion of a large region (replicase enzyme) and the substitution of a single amino acid in the CGMMV genome. Various in planta assays of these constructs validate their biological functionality, highlighting the utility of the LC-PCR technique in deciphering plant-virus functional genomics. The LC-PCR is not only suitable for modifying plant viral genomes but also applicable to a wide range of plant, animal, and human gene engineering under in-vitro conditions. Additionally, the LC-PCR technique provides an alternative to expensive kits, enabling quick introduction of modifications in any part of the nucleotide within a couple of days. Thus, the LC-PCR proves to be a suitable ‘all in one’ technique for modifying large plasmid constructs through site-directed gene insertion, deletion, and mutation, eliminating the need for restriction and ligation.

Watermelon production is significantly affected by watermelon bud necrosis virus (WBNV), an economically important orthotospovirus in the Indian subcontinent. RNA interference (RNAi)-based approaches offer promising strategies for developing broad-spectrum antiviral resistance in susceptible plants. In this study, we evaluated the efficacy of foliar application of double-stranded RNA (dsRNA) and hairpin RNA (hpRNA) derived from the full-length NSs gene of WBNV against WBNV infection in two host plants, Nicotiana benthamiana and cowpea (Vigna unguiculata). Fluorescein-labeled dsRNA of NSs gene (dsNSs) was tracked in N. benthamiana plants, where dsNSs translocated from the treated leaf to adjacent leaves, stem, and roots. Naked dsNSs was no longer detectable in treated leaves at 5 days post-inoculation (dpi), whereas mesoporous silica nanoparticle-conjugated dsNSs (dsNSs-MSNP) remained detectable in the treated leaves beyond 5 dpi, indicating enhanced stability. Following WBNV inoculation, N. benthamiana plants treated with hairpin of NSs gene (hpNSs), dsNSs, and dsNSs-MSNP exhibited 112, 303, and 370 fold reduction in viral RNA accumulation, respectively. Although these treatments delayed symptom development, all inoculated plants eventually showed the viral symptoms in N. benthamiana. Similarly, treatment of Vigna unguiculata cv. Pusa Komal (cowpea) plants with hpNSs, dsNSs, and dsNSs-MSNP resulted in reduced viral load, where dsNSs-MSNP provided the maximum reduction up to 208 fold. The dsNSs provided 100% reduction in development of systemic symptoms of bud necrosis disease in the treated cowpea plants. Collectively, these findings demonstrate that foliar application of nanoparticle-conjugated dsRNA targeting the full-length NSs gene can effectively suppress WBNV infection and provide enhanced protection against disease development in cowpea.