Replicase gene mediated RNA silencing mechanism confers resistance against groundnut bud necrosis virus in tomato (Lycopersicon esculentum Mill.)

Groundnut bud necrosis virus (GBNV), a member of the genus Orthotospovirus, is the most devastating pathogen causing bud blight of tomato and causes substantial crop losses in India. Current management strategies rely upon the use of virus tolerant cultivars, control of insect vectors, and other cultural practices. Under field conditions, these methods are ineffective in reducing the disease. Control can be achieved with RNA silencing, which regulates the homologous specific degradation of targeted genes, resulting in reduced virus multiplication. In the present study, virus infected tomato plant samples were collected from different parts of Tamil Nadu, and infection was confirmed through DAC-ELISA using a polyclonal antibody specific to GBNV. The virus inoculum was propagated on the local lesion host, cowpea (Vigna unguiculata), resulting in the production of chlorotic and necrotic spots on inoculated primary leaves. Our results demonstrated that the complete nucleotide sequence of the replicase gene identified using PCR shared an identity of 94.7 to 97.7% with other isolates of GBNV. To investigate the virus suppression mechanism, an effective RNAi construct was developed with the conserved sequence of the replicase gene for GBNV. A 3674 bp hpRNA cassette, comprising the sense and antisense fragments of 357 bp along with the flanking sequence, inserted in a pHANNIBAL vector, generated transgenic tomato plants using shoot apical meristem explants through Agrobacterium harboring the gene construct. The presence of the transgene in the developed putative transformants was assessed by PCR analysis using nptII and Rep genes and dot blot hybridization using a DIG luminescent detection kit. The expression of the replicase hpRNA construct revealed reduced symptom development upon artificial inoculation of GBNV. Further analysis of the transgenic tomato plants using DAC-ELISA confirmed the reduced level of virus titer. We propose that the RNAi construct, established with a conserved sequence of the replicase gene, showed a gene silencing mechanism as evidenced by reduced virus accumulation in putative transgenic lines, and this could be used as an effective strategy in the management of GNBV in tomato.