Identification Of Promoters From Geminivirus Associated Satellites And Analysis Of Genetic Determinants Of Symptoms On Viral DNA Satellites

The Geminiviridae is a family of plant ssDNA viruses, which have caused destructive diseases in many commercial crops all over the world. Recently, most monopartite begomoviruses have been shown to be associated with betasatellites and in many cases, alphasatellites. All characterized betasatellites contain a single gene called βC1on the complementary-sense strand, while alphasatellite encodes a replication-associated protein (Rep) on the virion-sense strand. Using Agrobacterium-mediated transient expression and stable transformation system, putative promoters of Malvastrum yellow vein betasatellite (MYVB), Tomato yellow leaf curl China betasatellite (TYLCCNB) and Tobacco curly shoot alphasatellite (TbCSA) were studied in this research.A991nucleotide (nt) fragment upstream of the translation start site of the βC1open reading frame (ORF) of the MYVB was studied using fluorometric assay, and β-glucuronidase (GUS) expression level of the991nt showed approximately29%of that observed in tissues infiltrated with the Cauliflower mosaic virus (CaMV)35S promoter. By construction of deletion mutants, a28nt region (between390nt and418nt upstream of MYVB βC1), which includes a5UTR Py-rich stretch motif, was important for promoter activity. Deletion of the5UTR Py-rich stretch resulted in a remarkable reduction in promoter activity compared with wild type construct. Replication assays using Nicotiana benthamiana leaf discs showed that deletion of the5UTR Py-rich stretch impaired accumulation of satellite DNA to61%that of wild type. However, there was no significant differences between symptoms induced by the deletion mutant and the wild type construct, while the mutational satellite replication declined to62%that of wild type.The entire non-coding region (982nt) upstream of TYLCCNB-Y25βC1gene was studied using fluorometric and histochemical assay. The results showed that GUS activity of the982nt fragment exhibit about44%that of observed in leaf tissues infiltrated with the CaMV35S promoter. Histochemical assay revealed that the982nt fragment confers a constitutive GUS expression in the transformed tobacco plants. A tandem repeats of the982nt increased fluorometric GUS activity to59%of that provided by CaMV35S promoter, which was significantly different from that of the 982nt. Previous study has been shown that TYLCCNB-Y10βC1promoter confers a phloem specific expression pattern. Our results indicate that βC1promoters of different satellites associated with the same helper virus might show different activity and display distinct expression pattern.Using the TYLCCNV as the helper virus, TYLCCNB-Y25could induce leaf curling in N. benthamiana, N. glutinosa, N. tabacum and Solarium lycopersicum, while TYLCCNB-Y10induced enations, yellow vein and shoot distortions as well as leaf curling. Therefore, we presume symptoms co-segregated with the TYLCCNB-Y10component. To determine whether the expression pattern of promoter is responsible for symptom induction, a overlap extension PCR method was used to precisely exchange the entire non-coding region (putative full-length promoter region) and the part promoter fragment (PP, approximately200nt upstream of translation start site of βC1gene) of the two betasatellites. Infectious clones of hybrid satellites were inoculated into N. benthamiana plants using TYLCCNV as helper virus, respectively. The results showed that a TYLCCNB-Y10hybrid with TYLCCNB-Y25full-length promoter induced similar symptoms with TYLCCNB-Y10, including enations and shoot distortions; while a TYLCCNB-Y25hybrid having the TYLCCNB-Y10promoter fragment displayed only leaf curling, which was similar to TYLCCNB-Y25. Similarly, exchange of the PP fragment failed to influence symptoms induced by the two different wild type betasatellite. The results indicate that the βC1protein is the major symptom determinant, and promoters of betasatellites along with TYLCCNV have little influence on the symptom production. Using the overlap extension PCR method, the C-terminus, middle part and N-terminus of the two βC1were exchanged, respectively. Using TYLCCNV as helper virus, infectious clones of hybrid satellites were inoculated into N. benthamiana plants. The results showed that TYLCCNB-Y25hybrid with TYLCCNB-Y10C-terminus fragment acquired the ability to induce similar symptoms with TYLCCNB-Y10, including enations and shoot distortions, though the size of enations was decreased and their numbers was reduced. The above results indicate that TYLCCNB-Y10C-terminus play an important role in symptom induction, but that the other part of the βC1can also have influence on the symptom production.Using the Agrobacterium-mediatcd transient expression and stable transgenic systems, respectively, we demonstrated that a420nt fragment located upstream of the Rep gene of TbCSA was capable of initiating transcription of the GUS gene in tobacco plants, and the promoter activity was about18%that of the CaMV35S promoter. Using GUS and the green fluorescent protein (GFP) as the reporter gene, respectively, deletion analysis indicated that3’-terminus deletion of the420nt fragment was not functional, while the5’-terminus deletions exhibited varying promoter activities. Furthermore, the227nt fragment located upstream of the Rep gene showed the same promoter activity as the longest420nt fragment and this should be considered as the minimal promoter sequence.