Tobacco RING E3 Ligase NtRFP1 Mediates Ubiquitination and Proteasomal Degradation of a Geminivirus-Encoded βC1.

The βC1 protein encoded by the Tomato yellow leaf curl China virus-associated betasatellite functions as a pathogenicity determinant. To better understand the molecular basis whereby βC1 functions in pathogenicity, a yeast two-hybrid screen of a tobacco cDNA library was carried out using βC1 as the bait. The screen revealed that βC1 interacts with a tobacco RING-finger protein designated NtRFP1, which was further confirmed by the bimolecular fluorescence complementation and co-immunoprecipitation assays in Nicotiana benthamiana cells. Expression of NtRFP1 was induced by βC1, and in vitro ubiquitination assays showed that NtRFP1 is a functional E3 ubiquitin ligase that mediates βC1 ubiquitination. In addition, βC1 was shown to be ubiquitinated in vivo and degraded by the plant 26S proteasome. After viral infection, plants overexpressing NtRFP1 developed attenuated symptoms, whereas plants with silenced expression of NtRFP1 showed severe symptoms. Other lines of evidence showed that NtRFP1 attenuates βC1-induced symptoms through promoting its degradation by the 26S proteasome. Taken together, our results suggest that tobacco RING E3 ligase NtRFP1 attenuates disease symptoms by interacting with βC1 to mediate its ubiquitination and degradation via the ubiquitin/26S proteasome system.