1 Miyagi Prefectural Agriculture and Horticulture Research Center, Takadate-kawakami, Natori, Miyagi 981-1243, Japan
2 Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan
3 Laboratory of Plant Pathology, Faculty of Agriculture, Niigata University, Ikarashi, Niigata 950-2181, Japan
4 National Institute of Agro-Biological Sciences, Kannondai, Tsukuba, Ibaraki 305-8602, Japan
Correspondence
Shigeo Nakamura
nakamura-sh894{at}pref.miyagi.jp
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ABSTRACT |
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Graphs showing transient GUS expression of MDV-derived promoters in pea and tobacco leaves (Supplementary Fig. S1) and GUS expression by MDV-derived promoters in E. coli and A. tumefaciens (Supplementary Fig. S2) are available as supplementary material in JGV Online.
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INTRODUCTION |
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The promoter activity associated with some members of the family Nanoviridae, BBTV, SCSV and CFDV, has been characterized. In transgenic tobacco and banana plants, the promoters derived from the BBTV C1C6 intergenic regions generally gave weak, tissue-specific expression patterns restricted to phloem-associated cells and at least one of them was highly expressed in actively dividing, undifferentiated cell types (Dugdale et al., 1998, 2000
). In SCSV, the promoter activity varies between the seven components and appears to be primarily vascular-associated (Surin et al., 1998
). SCSV C4 (DNA-N) and C7 (DNA-U1) showed substantial activity in transgenic tobacco plants. SCSV C2 and C6, which encode satellite Reps, had almost undetectable activity. SCSV C1 (DNA-M) conferred weak vascular expression, but its activity increased in callus tissue. The promoter associated with the intergenic region of CFDV had weak phloem-specific activity in transgenic tobacco and substantial activity in Escherichia coli (Rohde et al., 1995
; Hehn & Rohde, 1998
).
In this study, the activities of predicted promoter sequences derived from MDV C1C11 have been assessed to understand the control of gene expression in MDV. Bacterial cells and tobacco plants were transformed with chimeric genes consisting of MDV-derived promoters fused to a -glucuronidase (GUS) reporter gene to examine their expression profiles. The results indicate that expression of the individual components is differentially regulated.
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METHODS |
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GUS reporter-gene construction.
The promoter fragments of C1, C2, C3, C8 and C10 were each excised from pBluescriptII SK by digestion with HindIII and BamHI, whereas those of C4, C5, C6, C7 and C11 were excised by digestion with SalI and BamHI. These promoter fragments were cloned in the binary vector pBI101.3 (Clontech) as transcriptional GUS gene fusions. The resulting chimeric plasmids were named PMC1PMC11 : : GUS, respectively. The plasmid pBI121 (Clontech), referred to as P35S : : GUS in this report and containing the Cauliflower mosaic virus 35S RNA promoter, was included as a positive control. Deletions of the MDV C5 and C8 promoter fragments were excised from the vector pGEM-T by digestion with HindIII and BamHI and cloned into pBI101.3.
Plasmid DNA was extracted from overnight cultures of E. coli JM109 (TOYOBO) by using a Quantum Prep Kit (Bio-Rad) according to the supplier's recommendations. All promoter fusions and the control were introduced into Agrobacterium tumefaciens strain LBA4404 (Ooms et al., 1982; Hoekema et al., 1983
) by direct transformation.
Transformation of tobacco plants.
Tobacco (Nicotiana tabacum cv. Samsun NN) plants were co-cultivated with Agrobacterium by the leaf disc-infection method (Horsch et al., 1985) and transformants were selected on MS medium (Murashige & Skoog, 1962
) supplemented with 100 mg kanamycin l1 and 500 mg carbenicillin l1. Regenerated plants were analysed for integration of the promoter : : GUS fusion genes into the plant genome by PCR. For detection of the GUS region, a sense primer sequence from the GUS coding region, 5'-GCAACGTCTGGTATCAGC-3' (corresponding to nt 194211), and an antisense primer sequence from the nopaline synthase gene terminator region, 5'-TCTAGTAACATAGATGATGACA-3' (corresponding to nt 20972079), were used. For the promoter region, each of the primer sets in Table 1
was used. The reactions were run with an initial denaturation at 94 °C for 1 min, then 94 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min for 30 cycles, and a final extension at 72 °C for 8 min.
Measurement of GUS activity.
GUS activity was assayed by the method of Kosugi et al. (1990). Calli or leaf discs from transgenic tobacco plants were homogenized in lysis buffer [50 mM sodium phosphate (pH 6·8), 10 mM EDTA, 10 mM 2-mercaptoethanol, 0·1 % Triton X-100 and 0·1 % sarcosyl] in an Eppendorf tube with a glass rod and carborundum (600 mesh; Nakalai Tesque). The homogenate was centrifuged at 10 000 g for 15 min and the supernatant was assayed for GUS activity in the presence of 20 % methanol. Fluorescence levels were determined by using a Hitachi F-2500 spectrometer.
Histochemical analysis.
Leaf discs, longitudinal half-cut stems, including side buds, and intact roots from the transgenic tobacco plants containing each promoter : : GUS construct were subjected to GUS staining. Tissue sections from transgenic tobacco plants were cut at 80100 µm with a microslicer (model DTK-1000; Dosaka). Histochemical staining for GUS activity was performed at 37 °C as described previously (Ohshima et al., 1990) with a modified reaction mixture; 50 mM phosphate buffer (pH 7·0) containing 1 mM 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc), 5 % methanol, 10 µg cycloheximide ml1 and 1 mM dithiothreitol. The reaction was stopped by the addition of ethanol.
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RESULTS |
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DISCUSSION |
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The low level of promoter activity by Rep-encoding DNAs in transgenic tobacco plants and calli might have been expected, given the previous results for other members of the family Nanoviridae. BBTV C1 (DNA-R) and SCSV C2 and C6, which encode satellite Reps, have been reported to confer negligible activity in assessments of transient and stable expression using tobacco cells and plants (Dugdale et al., 1998; Surin et al., 1998
), whereas BBTV C1 and SCSV C2 have been shown to be able to replicate autonomously (Chu et al., 1995
; Horser et al., 2001
). This indicates that the basal activity of Rep promoters is extremely low, but sufficient to express Rep and initiate DNA replication.
The MDV-derived, non-Rep promoters conferred a higher level of GUS expression in calli than in plants, whereas P35S conferred the same level. This is consistent with the reported activities of non-Rep promoters of BBTV and SCSV. Dugdale et al. (1998) pointed out the possibility that the CATGACGTCA sequence in BBTV, which contains the ASF1 motif (TGACG; Lam et al., 1989
; Benfey & Chua, 1990
) and a hexamer motif characteristic of plant histone promoters (ACGTCA; Mikami et al., 1987
; Nakayama et al., 1992
; Morozov et al., 1994
), contributed to the strong promoter activity in undifferentiated, actively dividing cell types. The TGACGTCA sequence has been reported as a palindromic C box that is recognized by two bZIP proteins with a zinc-finger motif, STF1 and STF2, from soybean apical hypocotyl (Cheong et al., 1998
). This sequence is found in all MDV-derived non-Rep promoters and MDV-C11 (Fig. 1b
). However, PMC11 conferred weak promoter activity in transgenic tobacco plants, as well as other additional Reps (Figs 2 and 3
). Furthermore, the deletion of the palindromic C element, as well as the SL domain, in the deletions of PMC5 and PMC8 had no significant effect on promoter activity in transgenic tobacco plants (Fig. 5
).
Dof proteins are members of a major family of transcription factors unique to plants and have a similar DNA-binding specificity for the sequence AAAG or CTTT (Yanagisawa, 2002). Dof proteins have diverse roles in gene expression associated with plant-specific phenomena. A tobacco Dof protein, NTBBF1, is involved in auxin-inducible expression of a plant oncogene, rolB, in apical meristem and vascular tissues (Baumann et al., 1999
). An investigation of cis elements in the predicted promoter regions of MDV by PLACE showed four to eight Dof domains and zero to four NTBBF1 domains 200 bp upstream of each TATA box in the non-Rep promoters, and two to four Dof domains scattered in the Rep promoters (Fig. 1b
). There is a possibility that these Dof domains determine the difference in expression profiles between non-Rep and Rep promoters.
Promoters derived from non-Rep-encoding MDV DNAs were active in phloem and meristimatic tissue. Expression of PMC8 was not limited to the phloem and meristem, but was also observed in mesophyll and cortex cell types. Of other nanovirus promoters derived from DNA-M, BBTV C4 did not appear to be confined to the leaf vascular tissues, with visible green fluorescent protein expression in stomata and mesophyll cells (Dugdale et al., 2000), and SCSV C1 conferred a weak vascular expression (Surin et al., 1998
). The factor responsible for the tissue-specific expression conferred by MDV promoters could not be determined in this study.
PMC5 differed in terms of the strength of the activity it conferred in transgenic tobacco plants and calli, despite its similarity to PMC4 and PMC7. PMC5 has a unique 90 bp region downstream of the TATA box. This region contains the CT-rich sequence, TCATCTTCTTCTTTCTCACAAACAAC, near the presumed transcription-initiation site. This sequence resembles the CT-rich sequences in 5'-untranslated leaders of genes for thylakoid proteins, which are essential for transcription (Bolle et al., 1994) and may function as a plant-specific regulatory DNA sequence. The result that PMC5 conferred almost no activity in A. tumefaciens (see Supplementary Fig. S2, available in JGV Online) also suggests that these CT-rich sequences function as a plant-specific initiation region.
In the experiment with the deletion of PMC5, C5d1 (495 bp) showed a high level of promoter activity compared to PMC5 (532 bp) (Fig. 5b). From this result, a 37 bp region from the 5' end of PMC5 may contain a downregulatory element. Dugdale et al. (1998)
examined the transient activity in tobacco cells with deletions of BBTV C6 and obtained similar results. However, common sequences were not found in these regions between BBTV and MDV.
PMC9 conferred a relatively low level of activity in transgenic plants, compared with other promoters derived from non-Rep-encoding MDV DNAs. CP will be required in large amounts later in the infection cycle. There is a possibility that other viral proteins, which are generated earlier in the replication of MDV, activate expression of the CP promoter. Studies with the family Geminiviridae, a related ssDNA virus group, have revealed that promoter activity is influenced by the interaction of virus-encoded gene products (Hanley-Bowdoin et al., 2000). In Tomato golden mosaic virus, which is a member of the genus Begomovirus, the product of the AL2 gene is necessary for efficient transcription from the CP promoter (Sunter & Bisaro, 1991
). No information is available concerning which elements are involved in MDV CP expression, yet it is assumed that some regulatory elements may control a transcriptional cascade to warrant an efficiently timed gene expression in the family Nanoviridae.
As the activity levels of MDV non-Rep promoters were higher than that of P35S in transgenic tobacco calli, these promoters would be useful for foreign gene expression in proliferating tissues. The GUS expression levels in individual transgenic plant lines with MDV-derived promoter : : GUS varied greatly (Fig. 3). The observed variation in expression is probably due to a positional effect and a different number of integrated copies of the transgenes in transgenic plants (Hobbs et al., 1990
, 1993
; Peach & Velten, 1991
). Nevertheless, PMC8 confers efficient gene expression and may serve as an alternative to P35S, which has been used widely for genetic manipulation (Shirasawa-Seo et al., 2005
).
As each MDV-derived promoter has unique expression characteristics, further study of MDV-derived promoters will provide valuable understanding of not only the regulatory elements that determine specificity, but also the mechanisms that regulate nanovirus gene expression in host plants.
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ACKNOWLEDGEMENTS |
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Received 29 November 2004;
accepted 8 March 2005.
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