Host-specific accumulation and temperature effects on the generation of dimeric viral RNA species derived from the S-RNA of members of the Tospovirus genus

Polygonum ringspot virus (PolRSV) is a recently characterized Tospovirus reported in Italy. Northern blot analyses of PolRSV infections in Nicotiana benthamiana and tomato plants showed that a viral RNA species with nearly twice the length of the Small genomic RNA (S-RNA) accumulated abundantly in the former host, but was not detected in the latter. Additional assays confirmed that biogenesis of this novel RNA species was common to all PolRSV isolates tested and also to an isolate of Tomato spotted wilt virus (TSWV). Given its size, we hypothesized that the novel RNA species was a dimer molecule and we confirmed this hypothesis by RNA sequencing (RNAseq) analysis and reverse transcription (RT)-PCR of putative predicted dimer junction sites in RNA extracts of N. benthamiana challenged with PolRSV isolates Plg6 and Plg13/2. We also confirmed that these molecules are derived from head-to-tail dimers and often contain deletions at their junction sites. We named these novel molecules imperfect dimer RNAs (IMPD-RNAs). PolRSV IMPD-RNAs systemic accumulation in a range of host plants was restricted to N. benthamiana and Nicotiana occidentalis. Notably, IMPD-RNAs accumulation was modulated by temperature and their generation was restricted to late stages of systemic infection (12 days post-inoculation) in N. benthamiana. Differently from all other PolRSV isolates used in this study, Plg13/2 generated more IMPD-RNAs coupled with low amounts of genomic S-RNA and maintained them even at 18 °C, besides having lost the ability to infect tomato plants. This is the first characterization of S-RNA dimers for Tospovirus, and of occurrence of dimers of genomic segments at the whole organism level for Bunyaviridae.