Functional characterization of a new ORF ßV1 encoded by radish leaf curl betasatellite.

Whitefly-transmitted begomoviruses infect and damage a wide range of food, feed, and fiber crops worldwide. Some of these viruses are associated with betasatellite molecules that are known to enhance viral pathogenesis. In this study, we investigated the function of a novel ßV1 protein encoded by radish leaf curl betasatellite (RaLCB) by overexpressing the protein using potato virus X (PVX)-based virus vector in Nicotiana benthamiana. ßV1 protein induced lesions on leaves, suggestive of hypersensitive response (HR), indicating cell death. The HR reaction induced by ßV1 protein was accompanied by an increased accumulation of reactive oxygen species (ROS), free radicals, and HR-related transcripts. Subcellular localization through confocal microscopy revealed that ßV1 protein localizes to the cellular periphery. ßV1 was also found to interact with replication enhancer protein (AC3) of helper virus in the nucleus. The current findings suggest that ßV1 functions as a protein elicitor and a pathogenicity determinant.

Tobacco curly shoot virus C3 protein enhances viral replication and gene expression in Nicotiana benthamiana plants.

Geminiviruses are single-stranded DNA viruses that cause devastating diseases in many crops worldwide. The replication enhancer proteins (REn), encoded by the C3 (AC3, and AL3) ORFs of geminiviruses, have critical roles in viral DNA accumulation and symptom development in infected plants. In the current study, we have constructed an infectious clone of the Tobacco curly shoot virus (TbCSV) C3 mutant, TbCSVΔC3, that contains two start codon mutations that abrogated C3 ORF expression, but did not alter the amino acid sequence of the C2 ORF. As predicted, the absence of the C3 protein reduced TbCSV DNA accumulation, and over-expression of the C3 protein enhanced TbCSV DNA accumulation in infected leaves of Nicotiana benthamiana. The C3 mutation reduced the expression levels of both virion- and complementary-sense TbCSV genes whereas over-expression of the C3 protein increased TbCSV gene expression. Furthermore, the expression of the wild-type and site-directed mutants of C3 proteins using the potato virus X (PVX) system showed that Y93A mutation reduced the replication enhancement activity of the C3 protein in N. benthamiana. All the available evidence demonstrates that the C3 protein is tightly coupled with TbCSV DNA accumulation. However, the TbCSVΔC3 mutant was nearly as infectious in N. benthamiana as TbCSVWT and only had slightly delayed and attenuated symptom expression. Our findings demonstrate that TbCSV C3 protein enhances viral replication and gene expression, but has only moderate effects on symptom development in N. benthamiana.