Characterization of Small Interfering RNAs Derived from the Geminivirus/Betasatellite Complex Using Deep Sequencing

BACKGROUND: Small RNA (sRNA)-guided RNA silencing is a critical antiviral defense mechanism employed by a variety of eukaryotic organisms. Although the induction of RNA silencing by bipartite and monopartite begomoviruses has been described in plants, the nature of begomovirus/betasatellite complexes remains undefined. METHODOLOGY/PRINCIPAL FINDINGS: Solanum lycopersicum plant leaves systemically infected with Tomato yellow leaf curl China virus (TYLCCNV) alone or together with its associated betasatellite (TYLCCNB), and Nicotiana benthamiana plant leaves systemically infected with TYLCCNV alone, or together with TYLCCNB or with mutant TYLCCNB were harvested for RNA extraction; sRNA cDNA libraries were then constructed and submitted to Solexa-based deep sequencing. Both sense and anti-sense TYLCCNV and TYLCCNB-derived sRNAs (V-sRNAs and S-sRNAs) accumulated preferentially as 22 nucleotide species in infected S. lycopersicum and N. benthamiana plants. High resolution mapping of V-sRNAs and S-sRNAs revealed heterogeneous distribution of V-sRNA and S-sRNA sequences across the TYLCCNV and TYLCCNB genomes. In TYLCCNV-infected S. lycopersicum or N. benthamiana and TYLCCNV and βC1-mutant TYLCCNB co-infected N. benthamiana plants, the primary TYLCCNV targets were AV2 and the 5′ terminus of AV1. In TYLCCNV and betasatellite-infected plants, the number of V-sRNAs targeting this region decreased and the production of V-sRNAs increased corresponding to the overlapping regions of AC2 and AC3, as well as the 3′ terminal of AC1. βC1 is the primary determinant mediating symptom induction and also the primary silencing target of the TYLCCNB genome even in its mutated form. CONCLUSIONS/SIGNIFICANCE: We report the first high-resolution sRNA map for a monopartite begomovirus and its associated betasatellite using Solexa-based deep sequencing. Our results suggest that viral transcript might act as RDR substrates resulting in dsRNA and secondary siRNA production. In addition, the betasatellite affected the amount of V-sRNAs detected in S. lycopersicum and N. benthamiana plants.