Mutational study of radish leaf curl betasatellite to understand the role of the non-coding region in begomovirus pathogenesis

Diverse disease complexes formed by the association of betasatellites with multiple helper begomoviruses result in enhanced symptom severity, breakdown of disease resistance, and eventual yield loss in several dicot crops like cotton, tomato, and tobacco. The roles of the protein beta C1 encoded by several betasatellites in pathogenicity have been well characterized. In this study, we examined the role of the non-coding region (NCR) of radish leaf curl betasatellite (RaLCB) in the symptom development in Nicotiana benthamiana. Deletion mutation study revealed that mutant RaLCB molecules lacking satellite conserved region (1190-1320 nt) and downstream sequences of SCR (1091-1190 nt) were unable to replicate and failed to produce betasatellite mediated symptoms. Deletion mutant RaLCB683-1090 with truncated A-rich region replicated at a very low level and exhibited milder symptoms compared to the wild RaLCB. Deletion of beta C1 completely abolished the disease symptoms and betasatellite replication. In silico studies of NCR predicted the presence of several Cis-active elements and host factor binding sites responsible for stress signaling, light response, phytohormone signaling, defence responses, and regulatory sequences essential for beta C1 transcription. Mutant RaLCB683-1090 lacking G-box (-364 nt) exhibited symptoms similar to RaLCB1190-1320 and RaLCB1091-1190. Taken together, these results suggested that the noncoding region plays a significant role in betasatellite replication and beta C1 expression, facilitating a successful infection process.