A diverse set of miRNAs responsive to begomovirus-associated betasatellite in Nicotiana benthamiana

BACKGROUND: Roles of microRNAs (miRNAs) and short interfering RNAs (siRNAs) in biotic stress responses, e.g., viral infection, have been demonstrated in plants by many studies. Tomato yellow leaf curl China virus (TYLCCNV) is a monopartite begomovirus that can systemically infect Solanaceae plants, and induces leaf curling, yellowing and enation symptoms when co-inoculated with a betasatellite (TYLCCNB). The released genome sequence of Nicotiana benthamiana provides an opportunity to identify miRNAs and siRNAs responsive to begomovirus-associated betasatellite in N. benthamiana. RESULTS: miRNAs were identified in three small RNA libraries generated using RNA isolated from N. benthamiana plants systemically infected with TYLCCNV (Y10A) alone, co-infected with Y10A and its betasatellite TYLCCNB (Y10β) or a TYLCCNB mutant (Y10mβ) that contains a mutated βC1, the sole betasatellite-encoded protein. A total of 196 conserved miRNAs from 38 families and 197 novel miRNAs from 160 families were identified. Northern blot analysis confirmed that expression of species-specific miRNAs was much lower than that of conserved miRNAs. Several conserved and novel miRNAs were found to be responsive to co-infection of Y10A and Y10β but not to co-infection of Y10A and Y10mβ, suggesting that these miRNAs might play a role unique to interaction between Y10β and N. benthamiana. Additionally, we identified miRNAs that can trigger the production of phased secondary siRNAs (phasiRNAs). CONCLUSIONS: Identification of miRNAs with differential expression profiles in N. benthamiana co-infected with Y10A and Y10β and co-infected with Y10A and Y10mβ indicates that these miRNAs are betasatellite-responsive. Our result also suggested a potential role of miRNA-mediated production of phasiRNAs in interaction between begomovirus and N. benthamiana.