In this study, a 991 nt fragment upstream of the translation start site of βC1 of MYVB was identified as the promoter and the 214 nt fragment from the 3′ end of the 991 nt fragment, which contains a G-box, was shown to be involved in basic promoter activity. Promoter activity was almost abolished in the 138 nt fragment from the 3′ end of the 991 nt without a G-box. These results suggested that the G-box acts as a positive regulatory element in the control of MYVB βC1 transcription. Previous reports have indicated that G-box elements present in promoter regions of several geminiviruses and some plant genes bind to host factors involved in activating transcription
[14, 19]. Recently Eini et al.
 also identified a 68 nt fragment containing a G-box upstream of the βC1 gene associated with CLCuMB that was found to be important in the regulation of promoter activity. Furthermore, the G-box motif was shown to bind specifically to proteins in nuclear extracts from tobacco leaf tissues. We postulate that MYVB βC1 shares a similar transcription regulation mechanism with other organisms although further investigations are required to elucidate the interaction of the MYVB G-box motif with host nuclear factors.
Previous evidence has shown that the 5′UTR Py-rich stretch motifs are highly transcription level-related sequence elements regulating the activity of various promoters
[16–18]. As shown in Figure
4A, our results also demonstrated that site-directed deletion of the 5′UTR Py-rich stretch within the 991 nt βC1 promoter sequence resulted in a 60% reduction in promoter activity compared with the intact βC1 promoter, indicating the involvement of this element in the transcriptional regulation of βC1. In both fungi and animals, although transcription and DNA replication are divided into different biological processes, they frequently share the same regulatory elements
[20–23]. Sequences/motifs involved in transcription or DNA replication have been detected in some geminiviruses
[9, 10, 24–26]. Tu and Sunter
 identified a conserved binding site within the TGMV AL-1629 promoter, which is necessary for efficient viral DNA replication. Previous studies that showed that betasatellites depend on the helper begomoviruses for replication
[5, 15]. However, up to date, the mechanism of interaction of begomovirus-encoded Rep with betasatellites to initiate satellite replication was not fully understood as well as betasatellites lack the iteron sequences encoded by their helper viruses. In this study, infectious assays in leaf discs showed that the 5′UTR Py-rich stretch motif also has an important role in MYVB replication.
Mutagenesis of the TYLCCNB and Tobacco Curly Shoot betasatellite (TbCSB) showed that the βC1 protein is the symptom determinant, although the promoter of βC1 has some influence on symptom induction
. In our experiments, despite the production of low levels of betasatellite accumulation, the truncated MYVBΔUTR had no marked effect on the viral symptoms compared with the wild-type MYVB. Taken together, it is suspected that 5′UTR Py-rich stretch motif is involved in regulating the replication of betasatellite but is indispensable for viral symptom development.
Among these characterized geminivirus promoters, some are able to drive constitutive gene expression in transgenic plants, while others have more specific patterns of expression
[8, 14, 28, 29]. Histochemical staining assays revealed that the 991 nt fragment and the 214 nt fragment containing a G-box conferred a constitutive expression pattern. Previous studies have indicated that a 955 nt fragment upstream of the translation start site of the βC1 gene from TYLCCNB is a phloem-specific promoter
. Sequence analysis showed that the two putative promoters encompassing the entire non-coding region upstream of the βC1 open reading frame of MYVB and TYLCCNB shared only 42% nucleotide sequence identity. An ASL box and a TATGAAC motif, which are thought to be responsible for the phloem-specific expression
, were absent in the promoter region of MYVB. It can be speculated that sequence differences result in the different tissue expression patterns driven by betasatellite promoters.
In conclusion, the MYVB βC1 promoter directs a constitutive expression pattern in tobacco plants and might be suitable for special plant genetic engineering studies of low-level gene expression.