RESUMO
Ty-1 presents an atypical dominant resistance gene that codes for an RNA-dependent RNA polymerase (RDR) of the gamma class and confers resistance to tomato yellow leaf curl virus (TYLCV) and other geminiviruses. Tomato lines bearing Ty-1 not only produce relatively higher amounts of viral small interfering (vsi)RNAs, but viral DNA also exhibits a higher amount of cytosine methylation. Whether Ty-1 specifically enhances posttranscriptional gene silencing (PTGS), leading to a degradation of RNA target molecules and primarily relying on 21-22 nucleotides (nts) siRNAs, and/or transcriptional gene silencing (TGS), leading to the methylation of cytosines within DNA target sequences and relying on 24-nts siRNAs, was unknown. In this study, small RNAs were isolated from systemically TYLCV-infected leaves of Ty-1 encoding tomato plants and susceptible tomato Moneymaker (MM) and sequence analyzed. While in susceptible tomato plants vsiRNAs of the 21-nt size class were predominant, their amount was drastically reduced in tomato containing Ty-1. The latter, instead, revealed elevated levels of vsiRNAs of the 22- and 24-nt size classes. In addition, the genomic distribution profiles of the vsiRNAs were changed in Ty-1 plants compared with those from susceptible MM. In MM three clear hotspots were seen, but these were less pronounced in Ty-1 plants, likely due to enhanced transitive silencing to neighboring viral genomic sequences. The largest increase in the amount of vsiRNAs was observed in the intergenic region and the V1 viral gene. The results suggest that Ty-1 enhances an antiviral TGS response. Whether the elevated levels of 22 nts vsiRNAs contribute to an enhanced PTGS response or an additional TGS response involving a noncanonical pathway of RNA dependent DNA methylation remains to be investigated.
RESUMO
Tomato yellow leaf curl virus (TYLCV), a begomovirus, causes large yield losses and breeding for resistance is an effective way to combat this viral disease. The resistance gene Ty-1 codes for an RNA-dependent RNA polymerase and has recently been shown to enhance transcriptional gene silencing of TYLCV. Whereas Ty-1 was earlier shown to also confer resistance to a bipartite begomovirus, here it is shown that Ty-1 is probably generic to all geminiviruses. A tomato Ty-1 introgression line, but also stable transformants of susceptible tomato cv. Moneymaker and Nicotiana benthamiana (N. benthamiana) expressing the Ty-1 gene, exhibited resistance to begomoviruses as well as to the distinct, leafhopper-transmitted beet curly top virus, a curtovirus. Stable Ty-1 transformants of N. benthamiana and tomato showed fewer symptoms and reduced viral titres on infection compared to wild-type plants. TYLCV infections in wild-type N. benthamiana plants in the additional presence of a betasatellite led to increased symptom severity and a consistent, slightly lowered virus titre relative to the high averaged levels seen in the absence of the betasatellite. On the contrary, in Ty-1 transformed N. benthamiana viral titres increased in the presence of the betasatellite. The same was observed when these Ty-1-encoding plants were challenged with TYLCV and a potato virus X construct expressing the RNA interference suppressor protein ßC1 encoded by the betasatellite. The resistance spectrum of Ty-1 and the durability of the resistance are discussed in light of antiviral RNA interference and viral counter defence strategies.
