Mechanism Of The Interaction Between TYLCCNB βC1and Tobacco Host Factor NtRFP1

Tomato yellow leaf curl China virus (TYLCCNV) is a typical monopartite geminivirus, accompanying a betasatellite DNA (TYLCCNB) which is necessary for causing disease symptoms on host plants. The complementary strand of TYLCCNB can encode a βC1protein which functions as a suppressor of RNA silencing and a important pathogenicity determinant.To better understand the mechanisms of mutual interaction between geminivirus pathogenicity and host plant defense response during the infection of TYLCCNV/TYLCCNB, we carried out a study on the interaction between TYLCCNB βC1and Nicotiana tabacum host factor.βC1can interact with a RING finger type E3ligase which is named NtRFP1. Their interaction was demonstrated by yeast two-hybrid (Y2H) and bi-molecular fluorescent complementation (BiFC) assays in vivo and vitro. Sequence analysis showed the full-length of NtRFPl cDNA had1455bp encoding485amino acids. NtRFPl shared high homology with CaRFP1in Capsicum annuum and S1RFP1in Solanum lycopersicum (86.4%and83.4%, respectively). InterProScan online software was used to analyze the conservative functional domains in NtRFP1. NtRFPl contains a conserved VWA (von Willebrand factor (vWF) type A domain) and a RING-finger domain. Four defective mutants of NtRFP1were then designed to determine the binding region of NtRFP1to the TYLCCNB βC1. Results indicate that the intact RING-finger region in NtRFP1(443-475aa) is the binding site to interact with the βC1protein.We analyzed the expression pattern of NtRFP1mRNA in various plant tissues by means of real-time RT-PCR. Results showed the expression level of NtRFPl mRNA was the highest in the seed, followed in the root and flower, and the lowest level of NtRFP1mRNA was detected in the leaf and stem. In addition, the expression of NtRFP1gene at the transcriptional level was increased markedly during the infection of TYLCCNV/TYLCCNB, indicating that βC1contributed to the accumulation of NtRFPl mRNA. The analysis of subcellular localization showed that NtRFPl distributed both in cytoplasm and nucleus of plant cells.To further investigate the biological significance of the interaction between NtRFP1and βC1in vivo, we obtained kanamycin-resistant transgenic N. tabacum plants by the technique of agrobacterium-mediated genetic transformation. We generated TO transgenic plants overexpressing NtRFP1[NtRFP1(+)] and transgenic plants silencing NtRFP1[NtRFP1(-)]. The positive transgenic NtRFP1plants were then screened by PCR amplification. NtRFPl mRNA levels in transgenic plants NtRFPl(+), NtRFP1(-) and nontransgenic tobacco plants were analyzed by real-time RT-PCR. To investigate the effect of NtRFP1on symptoms induced by TYLCCNV/TYLCCNB, we inoculated NtRFP1(+), NtRFP1(-) and nontransgenic plants with TYLCCNV/TYLCCNB and observed symptom appearance. NtRFP1(+) plants showed mild disease symptoms, but increased the accumulation of viral and betasatellite DNA; NtRFPl(-) plants developed more severe symptoms, but accumulated less viral DNA, suggesting that NtRFPl in plants can attenuate symptoms induced by βC1and contribute to enhancing disease susceptibility to geminivirus.We constructed the recombinant expression vectors containing NtRFP1, E3ligase-dead mutant (NtRFP1H459Y) and GFP by the Potato virus X vector (pGR106), respectively. Then we co-expressed βC1protein together with NtRFP1, NtRFP1H459Y or GFP in the leaves of N. benthamiana and observed symptom appearance. Plants co-expressing βC1and NtRFP1showed mild symptoms, while plants co-expressing βC1together with NtRFP1H459Y or GFP developed more severe symptoms. These results indicate that NtRFP1attenuates βC1symptom. The immunoblot analysis indicated that there was significantly less βC1protein in the tissue coexpressing βC1and NtRFP1than the control where βC1and GFP were coexpressed, while the samples coexpressing βC1and NtRFP1H459Y did not showed any obvious change in the βC1protein level when compared with the control.These results indicate that NtRFP1can attenuate βC1symptom by promoting the degradation of the βC1protein in plant defense response...