Department of Plant Sciences, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel 69778
Correspondence
Bernard L. Epel
blepel{at}post.tau.ac.il
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ABSTRACT |
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INTRODUCTION |
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In preliminary studies involving a TMV-based expression vector, or replicon, in which the ORF for the green fluorescent protein (GFP) replaced the complete CP ORF, measurable GFP levels were not detected in inoculated Nicotiana tabacum leaves, although replication ability was conserved, as determined by the formation of necrotic lesions following inoculation of susceptible host plants with replicon-derived in vitro transcripts. This result led us to assume that, along with the deletion of the CP coding region, regulatory elements that are crucial to transcription and/or translation of the CP sgRNA were also eliminated. It was assumed that if regulatory elements do exist, they would most likely be situated in the 5' region of the CP ORF due to its proximity to the CP promoter and/or within the 3' region of the CP ORF due to its proximity to the 3'UTR, which is a regulatory sequence by itself. This hypothesis was supported by previous studies which showed that replacing the CP ORF with a foreign ORF resulted in diminished levels of the foreign protein in comparison with the native CP (Donson et al., 1991). Our hypothesis was also supported by the results of Shivprasad et al. (1999)
and Grdzelishvili et al. (2000)
, which were published during the course of our work and presented qualitative evidence for the presence of regions within the CP ORF of TMV that function as part of the CP sgRNA promoter. In the present study, we have extended their findings and have presented a quantitative statistical analysis testing the contribution of different regulatory elements situated within the CP coding region to sg transcription and protein expression.
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METHODS |
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In vitro transcription and inoculation.
After linearization of the replicons with KpnI, in vitro transcripts were synthesized using T7 RNA polymerase, according to the protocol of the mMessage mMachine Transcription kit (manual version 0004; Ambion). Transcripts were inoculated directly onto the adaxial surfaces of carborundum-dusted (330 grit; Fisher Scientific) N. tabacum MP+ leaves (leaves 3 and 4 from the bottom) in 6- to 7-week-old plants (inoculated leaves were not fully expanded). Immediately after inoculation, leaves were rinsed with water and the plants placed in a growth chamber.
RNA inoculation and Northern blotting.
Total RNA was purified at 7 days post-infection (p.i.) from fluorescent areas of leaves inoculated with in vitro transcripts of GFP-expressing replicons. RNA (10 µg per lane) was analysed by Northern blotting using standard methods (Sambrook et al., 1989). Blots were probed with a PCR-synthesized digoxigenin-labelled probe complementary to the GFP sequence and exposed to X-ray film to record the chemiluminescent signal, following the protocol of the digoxigenin system user's guide for filter hybridization (Boehringer). Bands were quantified by scanning and densitometry with the ImageMaster 1D prime program, version 3.01 (Amersham Pharmacia Biotech).
Protein extraction and analysis.
Total soluble proteins were extracted 7 days p.i. from fluorescent areas of leaves inoculated with in vitro transcripts produced from GFP-expressing replicons: infected leaf tissue (60 mg) was frozen in liquid nitrogen, pulverized while frozen and then ground with a pestle and pre-chilled mortar in 4 ml ice-cold extraction buffer (final concentrations: 20 mM Tris/HCl, pH 8·5, 0·25 M sucrose, 0·02 % sodium azide, 4 mM EDTA, 10 mM EGTA plus the protease inhibitors 1·5 µM aprotonin, 0·01 U -macro globulin ml1, 2·5 mM orthophenanthroline, 36 µM leupeptin, 2 mM PMSF and 14·5 µM pepstatine). Extract was cleared of particulate matter by centrifugation at 34 000 g for 15 min at 4 °C and relative GFP levels were quantified in the clear supernatant using a Perkin-Elmer spectrofluorometer model LS50B with excitation at 485 nm (5 nm slit) and emission at 509 nm, as described by Epel et al. (1996)
. Relative GFP fluorescence values were calculated by subtracting background autofluorescence. Fluorescence intensity was measured in the linear zone where fluorescence is directly correlated to protein level.
Construction of expression constructs for in vitro translation.
To investigate a possible effect of the regulatory elements on translation independent of their effect on transcription, expression constructs (ECs) for in vitro translation were synthesized to produce similar transcription levels for all constructs tested from a T7 promoter rather than from the CP sgRNA promoter. Downstream of the T7 promoter, the 17 nt region upstream of the CP transcription start codon was inserted followed by (i) the native CP transcript leader, defined as the first 9 nt downstream of the transcription start site (Guilley et al., 1979); (ii) the putative regulatory elements from the CP cistron, created as a UTR by mutating the ATG start codon to ACG; (iii) the GFP ORF; or (iv) the 3'UTR. Each EC was based on its equivalent expression replicon (Fig. 1C
), resulting in a series of ECs in which the GFP ORF was flanked by different putative regulatory elements from the CP cistron (see description of ECs below): each EC fragment was amplified by PCR using the corresponding transcriptional replicon as template, a forward primer containing a SacII site followed by the nucleotides corresponding to TMV nt 56865703 and a T3 (Stratagene) reverse primer corresponding to the T3 promoter flanking the KpnI site at the 3' terminus of the replicon (see Fig. 1A
). The obtained PCR product was digested with SacII and KpnI and ligated into the SacII/KpnI-digested KS plasmid (Stratagene) downstream of the T7 promoter. Equal samples of transcripts synthesized from the T7 promoter in each EC were subsequently tested for their translational capacity using an in vitro translation system.
The ECs were delineated as follows: EC 5' 56-GFP-22 3': T7 promoter : : (TMV 56865767)*GFP(TMV 61706395); EC 5' 20-GFP-50 3': T7 promoter : : (TMV 56865731)*GFP(TMV 61426395); EC 5' 20-GFP-22 3': T7 promoter : : (TMV 56865731)*GFP(TMV 61706395); EC 5' 56-GFP-50 3': T7 promoter : : (TMV 56865767)*GFP(TMV 61426395); and EC 5' 0-GFP-22 3': T7 promoter : : (56865711)GFP(TMV 61706395). The asterisk indicates that nt 5713 has been mutated from U to C.
In vitro translation.
Translation of equal amounts (600 ng) of in vitro transcripts from ECs was performed using a rabbit reticulocyte lysate in vitro translation system, according to the manufacture's protocol (Promega). To allow non-radioactive detection of proteins synthesized in vitro, a transcend non-radioactive translation detection tRNA composed of a biotinylated lysine (Promega) was added to the translation reaction as a precharged -labelled biotinylated lysinetRNA complex (transcend tRNA) rather than a free amino acid. After translation, lysate samples (1 µl) were subjected to SDS-PAGE followed by transfer to a PVDF membrane. The biotinylated proteins were visualized by binding with streptavidinalkaline phosphatase, followed by colorimetric (BCIP/NBT) detection, according to the protocol for the transcend non-radioactive translation detection system (Promega).
Fluorescence microscopy.
Infected leaf tissues were analysed using a Leica DMBRE fluorescence microscope equipped with a BP 450490 (FITC) excitation filter, an RKP dichromatic mirror and a BP 515560 suppression filter. Images were recorded using a Sensicam 12 bit cooled imaging camera and processed using the PMIS software package (Photometrics).
Computer prediction of the RNA secondary structure.
The MFold program followed by the PlotFold program from the GCG (Genetics Computer Group, University of Wisconsin) package was used for secondary structure prediction of the sgRNA promoter region followed by the CP ORF in TMVMP and of the modified CP sgRNA promoter regions followed by the GFP ORF in various GFP-expressing replicons.
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RESULTS |
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Fig. 2. shows the mean fluorescence±SD measured using a one-way ANOVA test (P<0·001). A Tukey test (P<0·05) was used to divide replicons further into statistically significant homogeneous groups according to mean fluorescence values. Three subsets were obtained: replicons 5' 56-GFP-22 3' and 5' 81-GFP-22 3' were grouped into a subset producing high GFP expression levels; replicons 5' 56-GFP-50 3' and 5' 23/(55
25)-GFP-50 3' were grouped into a subset producing moderate GFP expression levels; and the rest of the replicons were grouped into a subset producing low GFP expression levels. Subsequently, representative replicons were selected from each of these three distinct subsets.
Regulatory elements derived from the CP coding region affect the accumulation of subgenomic RNA containing the GFP ORF in N. tabacum MP+ leaves inoculated with in vitro transcripts of replicons
Leaves of N. tabacum MP+ were inoculated with in vitro transcripts of each replicon. Total RNA was extracted from tissue expressing GFP at 7 days p.i. and analysed by Northern blot hybridization. Fig. 4 shows a representative Northern blot analysis. Genomic and sgRNA levels were quantified with scanning and densitometry, and results were normalized against the constitutive expression of the rRNA gene (Fig. 4
). The absorbance values (in arbitrary units) of the bands containing the genomic RNAs of replicons 5' 20-GFP-22 3', 5' 56-GFP-22 3', 5' 56-GFP-50 3' and 5' 23/(55
25)-GFP-50 3' were 2860, 2434, 2473 and 2720, respectively, suggesting similar levels of genomic RNA accumulation. The absorbance values of the bands containing the sgRNAs derived from the CP sgRNA promoter for the replicons indicated above were 389, 2713, 1689 and 1597, respectively. The level of sgRNA accumulation of replicon 5' 56-GFP-22 3' containing the first 56 nt of CP ORF was higher than that of replicon 5' 20-GFP-22 3' containing only the first 20 nt of the CP ORF (Fig. 4
, lanes 3 and 2, respectively), indicating that the complete CP sgRNA promoter extends further than the first 20 nt of the CP ORF (but not beyond the first 56 nt, as the addition of the sequence between positions +56 and +81, upstream of the GFP ORF, did not further increase GFP accumulation, as described above). This elevated sgRNA transcription level was also consistent with higher protein levels (Fig. 2
). Our previous assumption that the sgRNA promoter activity is stimulated by an enhancer element located between nt +25 and +55 was additionally confirmed by Northern blot analysis, which revealed similar transcript accumulation levels for replicons 5' 56-GFP-50 3' and 5' 23/(55
25)-GFP-50 3' (Fig. 4
, lanes 4 and 5, respectively); i.e. nt +25 to +55 were shown to function to the same degree in stimulating transcription, irrespective of orientation. Inclusion of the last 50 nt of the CP ORF downstream of the GFP ORF reduced sgRNA accumulation of replicon 5' 56-GFP-50 3' in comparison with replicon 5' 56-GFP-22 3', as shown in Fig. 4
, lanes 4 and 3, respectively. Thus, it is reasonable to assume that the reduced GFP accumulation level produced by replicon 5' 56-GFP-50 3' in comparison with replicon 5' 56-GFP-20 3' (Fig. 2
) is a reflection of its lower transcription level.
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DISCUSSION |
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To analyse the correlation between the TMV CP sgRNA promoter activity and its putative secondary structure, we used the MFold program (Zuker, 1989). Secondary structure models were generated for the native CP sgRNA promoter followed by the CP ORF, as in replicon TMV
MP (Fig. 8
A), as well as for altered sgRNA promoter regions followed by the GFP ORF in GFP-expressing replicons (Fig. 8B
E). Computer analysis for the minus strand sequence of the native CP sgRNA promoter starting at position 157 with respect to the transcription start site (TSS) and extending into the CP ORF predicted multiple SLs (Fig. 8A
). The promoter region folds into two SL structures: SL1 encompasses the sequence between 157 and 74 and SL2 encompasses the sequence between 70 and +34, with respect to the TSS (position +34 corresponds to nt +25 of the CP ORF with respect to the translation start site). The sequence between nt +34 and +64 with respect to the TSS containing the putative enhancer element is outside SL2 (indicated by the dashed line in Fig. 8A
). The resulting secondary structure presented here differs from that previously suggested by Grdzelishvili et al. (2000)
, which predicted that the sgRNA promoter folded into one long SL structure. Their analysis was based on a short sequence between 100 and +52 with respect to the TSS, although they mapped the beginning of the fully active promoter to position 157 with respect to the TSS. In contrast, our computer-generated model was based on a much longer sequence, starting at position 157 and extending to the end of the CP ORF. By testing the biological activity of differently altered CP sgRNA promoters situated within various GFP-expressing replicons, we were able to predict a direct correlation between the native SL2 sequence, structure and promoter activity. The predicted secondary structure for the modified sgRNA promoter in replicon 5' 0-GFP-0 3', which produced undetectable levels of GFP, revealed that the stem in SL2 was shortened at its base (Fig. 8B
). Inclusion of the 20 nt from the 5' terminus of the CP ORF, which had previously been shown to be part of the extended CP sgRNA promoter (Grdzelishvili et al., 2000
), elevated to some extent promoter activity in replicons 5' 20-GFP-22 3' and 5' 20-GFP-0 3'. Folding analysis for the modified sgRNA promoter followed by the GFP ORF in these replicons predicted a partial restoration of the stem in SL2 (Fig. 8C
). Full sgRNA promoter activity in our system was gained with the addition of the first 56 nt from the CP ORF upstream of the GFP ORF. In replicon 5' 56-GFP-22 3', SL1 and SL2 were predicted to fold as for the native pattern (Fig. 8D
). The GFP accumulation level for replicon 5' (55
1)-GFP-50 3' in which the first 55 nt of the CP were positioned in an inverted-sense orientation upstream of the GFP ORF was negligible. This loss of promoter activity may have derived from a total disruption of SL2 structure, as predicted by computer analysis (data not shown). The extended 3'UTR containing the terminal 50 nt from the CP ORF apparently had no effect on the secondary structure of the CP sgRNA promoter according to computer modelling (data not shown). The SL2 structure could be important for the recognition of the viral replicase with its cognate promoter template of specific sequence and/or structure. Destabilization of SL2 could abolish this process, leading to inhibition of sgRNA synthesis. In contrast, restricted inversion of the sequence between nt +34 and +64 with respect to the TSS (+25 to +55 with respect to the CP translation start site), which encompasses the putative enhancer sequence in replicon 5' 23/(55
25)-GFP-50 3', retained the same promoter activity as that produced by replicon 5' 56-GFP-50 3' and the same putative folding pattern of SL1 and SL2 (Fig. 8E and D
).
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Replicon 5' 56-GFP-22 3' was the most effective of all those tested in our system, although it performed poorly in comparison with the control replicon, TMVMP (Figs 6 and 7
). The impaired genomic RNA accumulation of replicon 5' 56-GFP-22 3' in comparison with replicon TMV
MP (Fig. 7
) could explain the diminished GFP accumulation level produced by this replicon relative to the much higher CP accumulation level produced by TMV
MP. In the absence of the CP, levels of accumulation of positive-strand RNA are sometimes reduced and this has been ascribed to degradation of the RNA in the absence of the protective capsid (e.g. Beet western yellow vein virus and Cucumber necrosis virus). In other cases, there appears to be little or no effect (e.g. Brome mosaic virus) (Buck, 1996
). However, in our system the lack of CP is probably not accountable for the poor functioning of the TMV-based replicon. In a concurrent study, we have assessed different attributes of the GFP-expressing replicon that affect genomic replication. It was found that the cause of the impaired genomic replication of GFP-expressing replicons derived from the presence of the GFP coding sequence inserted into the viral genome, whereas the foreign protein product and the lack of CP had no effect on genomic replication capabilities (unpublished results). Understanding the factors that inhibit the productivity of replicons could lay the foundations for the design of an efficient TMV-based vector as a tool for foreign gene expression in the future.
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ACKNOWLEDGEMENTS |
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Received 23 November 2003;
accepted 23 January 2004.
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