Characterization of sequence elements from Malvastrum yellow vein betasatellite regulating promoter activity and DNA replication
© Zhang et al.; licensee BioMed Central Ltd. 2012
Received: 8 March 2012
Accepted: 1 October 2012
Published: 11 October 2012
Many monopartite begomoviruses are associated with betasatellites, but only several promoters from which were isolated and studied. In this study, the βC1 promoter from Malvastrum yellow vein betasatellite (MYVB) was characterized and important sequence elements were identified to modulate promoter activity and replication of MYVB.
A 991 nucleotide (nt) fragment upstream of the translation start site of the βC1 open reading frame of MYVB and a series of deletions within this fragment were constructed and fused to the β-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes, respectively. Agrobacterium-mediated transient expression assays showed that the 991 nt fragment was functional and that a 28 nt region (between −390 nt and −418 nt), which includes a 5′UTR Py-rich stretch motif, was important for promoter activity. Replication assays using Nicotiana benthamiana leaf discs and whole plants showed that deletion of the 5′UTR Py-rich stretch impaired viral satellite replication in the presence of the helper virus. Transgenic assays demonstrated that the 991 nt fragment conferred a constitutive expression pattern in transgenic tobacco plants and that a 214 nt fragment at the 3'-end of this sequence was sufficient to drive this expression pattern.
Our results showed that the βC1 promoter of MYVB displayed a constitutive expression pattern and a 5′UTR Py-rich stretch motif regulated both βC1 promoter activity and MYVB replication.
The Geminiviridae are a family of plant DNA viruses whose members are classified into four genera: Mastrevirus, Begomovirus, Curtovirus and Topocuvirus. The majority of geminiviruses belong to the genus Begomovirus. Begomoviruses are either monopartite or bipartite in the organization of their genome and many monopartite begomoviruses are associated with betasatellites (formerly called DNAβ) [1–3]. Betasatellites depend on the helper begomoviruses for replication, encapsidation and insect transmission as well as spread within and between plants [2, 4]. Comparison of the nucleotide sequences from all known betasatellite molecules reveals three conserved features: a highly conserved region called the satellite conserved region (SCR), a single gene (known as βC1) that is conserved in both position and size and is a determinant of symptoms, and an A-rich region [5–7].
Using Agrobacterium-mediated transient expression and stable transformation system, many motifs/sequences have been identified to be involved in regulation of geminivirus transcription [8–10]. For example, Shung et al.  identified two elements located upstream of AL1935 and AL1629, important for transcription of complementary sense RNAs derived from Tomato golden mosaic virus (TGMV). A region located between −125 nt and −60 nt from the transcription start site in the TGMV CP promoter was reported to be involved in both activation and derepression by TrAP [12, 13]. However, few promoters from betasatellites have been isolated and studied since Guan and Zhou  first reported the characterization of the βC1 promoter of the Tomato yellow leaf curl China betasatellite (TYLCCNB) and subsequently Eini et al.  identified sequence elements which regulated βC1 transcription associated with the Cotton leaf curl Multan betasatellite (CLCuMB). Malvastrum yellow vein virus (MYVV) is a typical monopartite geminivirus. Previous reports have shown that the betasatellite associated with MYVV (MYVB) is involved in symptom induction and it is required for enhancing the accumulation of helper virus in tobacco plants . In order to further elucidate the transcriptional regulation and replication of the MYVB, in this study, we have characterized the putative promoter of the βC1 gene of MYVB using both transient and stable transgenic expression approaches. Furthermore, we have identified a motif consisting of a 5′UTR Py-rich stretch important for MYVB replication.
Analysis of the putative promoter sequence of the MYVB βC1 gene
Identification of cis-elements regulating βC1 expression
In order to further identify the cis-elements involved in the transcriptional control of βC1, different promoter deletion sequences were inserted individually upstream of the GFP reporter gene within the expression vector pCHF3:GFP. The results revealed that 64 h after infiltration, significant differences in the intensity of GFP fluorescence were observed among the various constructs. As illustrated in Figure 3B, compared with other constructs, pβC1 and pβC1-418 produced relatively high levels of fluorescence, but much lower levels compared with the positive control pCHF3:GFP. GFP fluorescence was also observed to be produced from constructs pβC1-389 to pβC1-214, while the fluorescence of pβC1-138 was almost identical to that of the negative control pCHF3. Calculation of the fluorescence intensity revealed that the sequence within a 214 nt region upstream of the translation start site was fundamentally required for βC1 promoter activity (Figure 3A). These results were consistent with those of the fluorometric GUS assay.
A 5′UTR Py-rich stretch motif regulates βC1 promoter activity
Figure 3 showed that deletion of the region from −991 to −390 nt in the pβC1-389 resulted in a remarkable reduction in promoter activity compared with pβC1-418, which indicated the presence of a positive cis-element in the region between −390 to −418 nt of the MYVB βC1 promoter. Further sequence alignment analysis revealed the presence of a 5′UTR Py-rich stretch in this region. Therefore, the entire non-coding region promoter construct excluding the 5′UTR Py-rich stretch motif (pβC1ΔUTR) was obtained. Sixty-four hours after infiltration into leaves of N. benthamiana plants, fluorometric assays revealed that relative GUS activity of the pβC1ΔUTR declined to 11% of that driven by the CaMV 35S promoter, which differed significantly from that of pβC1 (P < 0.01).
Roles of the 5′UTR Py-rich stretch motif in MYVB replication and pathogenicity
Evaluation of the expression pattern in tobacco
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.
Construction of plant expression vectors
Sequences of primers used for the PCRs
Underlined restriction site
Position on MYVB
962-953 plus 943-924
934-943 plus 953-976
The 5′UTR Py-rich stretch motif (βC1ΔUTR) was deleted from the entire non-coding region of the βC1 gene using an overlap-extension PCR strategy . Two independent PCRs were conducted with primer pairs, Y47βp-F/Y47βΔUTR-R and Y47βΔUTR-F/Y47βp-R and PCR products were subsequently added to the standard PCR system and the flanked primer pair Y47βp-F/Y47βp-R was added to amplify the completed βC1ΔUTR. The resulting fragments were digested with HindIII/BamHI and inserted into the corresponding sites within the binary vector pINT121 to produce the expression construct, pβC1ΔUTR. Using the same overlap-extension PCR strategy, the full-length MYVB sequence with deletion of the 5′UTR Py-rich stretch (MYVBΔUTR) was obtained. An infectious clone containing MYVBΔUTR was produced as described in . The complete monomeric sequence of MYVBΔUTR was amplified using primers β01/β02. The fragment was then inserted into the pGEM-T easy vector (Promega) to produce the clone, pGEM-MYVBΔUTR. Subsequently, another copy of the complete MYVBΔUTR sequence was amplified using primers β03/β02 to produce pGEM-MYVBΔUTR′. The pGEM-MYVBΔUTR clone was digested with KpnI and inserted into the unique KpnI site of pGEM-MYVBΔUTR′ to produce pGEM-2MYVBΔUTR. Then pGEM-2MYVBΔUTR was digested with EcoRI and inserted into the binary vector pBINPLUS to produce pBIN-2MYVBΔUTR, which contains a tandem dimeric repeat of MYVBΔUTR molecules. Infectious clones of MYVV and MYVB were produced previously .
Expression vectors were introduced individually into Agrobacterium tumefaciens strain EHA105 as described previously .
Transient expression assay
Transient expression analysis by Agrobacterium-mediated delivery into plants was carried out as described previously . Three independent experiments were carried out for each construct.
Fluorometric GFP assay
Leaves of 4 week-old N. benthamiana plants were infiltrated with the A. tumefaciens harboring the various expression constructs fused to the GFP marker gene. Approximately 64 h after infiltration, 1 cm2 leaf fragments were excised and GFP fluorescence was examined in epidermal cells by confocal laser scanning microscopy (CLSM, Leica TCS SP5, Mannheim, Germany).
Analysis of replication in leaf discs and infected plants
A. tumefaciens strain EHA105 harboring either helper virus or betasatellite infectious clones was used for infection of N. benthamiana leaf discs  or the whole plants  as previously described. Total DNA was extracted using the CTAB method from leaf discs after 6 days or from co-inoculated plants after 30 days. Approximately 10 μg of total DNA was blotted and hybridized with 32P-dCTP randomly labeled DNA probes specific for MYVV or MYVB . The band intensities were quantified using Image J software .
Agrobacterium-mediated transformation of Nicotiana tabacum leaf discs was conducted according to a previously published procedure . Transformants were selected on Murashige and Skoog medium containing 100 μg/ml kanamycin and 500 μg/ml carbenicillin. Regenerated kanamycin-resistant plants were grown on a rooting medium and then transferred to soil after confirmation by PCR using specific primers for GUS gene (5′-ATGTTACGTCCTGTAGAAACC-3′/5′-TCATTGTTTGCCTCC CTGC-3′).
Fluorometric GUS assay and histochemical staining of GUS
N. benthamiana leaves were sampled 64 h after infiltration and ground in Passive Lysis Buffer (Promega) using a pestle and mortar. Supernatants obtained after centrifugation were used for fluorometric assays. Protein content of the samples was determined by an Eppendorf BioPhotometer (Eppendorf, Hamburg, Germany) using BSA as a standard. Quantitative GUS fluorometric assays were conducted essentially as described by Jefferson et al.  and using a Perkin-Elmer LS50B luminescence spectrometer (excitation at 365 nm and emission at 455 nm) to measure the fluorescence of 4-methylumbelliferone (4-MU), which is formed as a result of the cleavage of 4-methylumbelliferyl-β-D-glucuronide (MUG). GUS activity was calculated as the production of 4-MU from MUG in picomoles per minute per microgram of protein. The mean GUS activity from the CaMV 35S promoter of pINT121 was arbitrarily assigned as 100% and used to standardize the activities for all of the other constructs. The resulting data were analyzed using the LSD method of SPSS v12.0 software (SPSS, Chicago, IL, USA).
For the histochemical detection of GUS activity, fresh plant tissue from several transgenic N. tabacum was incubated for 3 to 12 h in a 5-bromo-4-chloro-3-indolyl β-D-glucuronide staining solution at 37°C as described by Jefferson et al. . The stained samples were cleared by several washes with 70% ethanol and then embedded as described previously . A 11800 Pyramitome (LKB-BROMMA, Stockholm, Sweden) was used for slicing tissue into semi-thin sections. Images of stained sections were photographed with an OLYMPUS BH-2 stereomicroscope (OLYMPUS, Japan).
This work was supported by grants from the National transgenic Research Projects of China (No. 2009ZX08009-134B) and the National Natural Science Foundation of China (No. 30770092).
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