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, TbCSV Delta C3, 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 TbCSV Delta C3 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.