Novel Plant Transformation Vectors Containing the Super-promoter

We developed novel plasmids and T-DNA binary vectors that incorporate a modified and more useful form of the super-promoter. The super-promoter consists of a trimer of the 3 octopine synthase ( ocs ) transcriptional activating element affixed to the mannopine 4 synthase 2’ ( mas2’ ) transcriptional activating element plus minimal promoter. We tested 5 a super-promoter/ gusA fusion gene in stably transformed tobacco and maize plants, and 6 in transiently transformed maize BMS protoplasts. In both tobacco and maize, super- 7 promoter activity was much greater in roots than in leaves. In tobacco, super-promoter 8 activity was greater in mature leaves than in young leaves, whereas in maize activity 9 differed little among the tested aerial portions of the plant. When compared with other 10 commonly used promoters (CaMV 35S, mas2’ , and maize ubiquitin), super-promoter 11 activity was approximately equivalent to those of the other promoters in both maize BMS 12 suspension cells and in stably transformed plants. The addition of a maize ubiquitin 13 intron downstream of the super-promoter did not enhance activity in stably transformed 14 maize.


Introduction 1
The availability of convenient vectors harboring a strong promoter that is active in 2 all or most cells of different plant species would be useful for a variety of applications in 3 plant molecular biology. We previously described a novel synthetic promoter consisting 4 of a trimer of the octopine synthase transcriptional activating element (ocs activator) 5 linked to the mannopine synthase 2' (mas2') activator/promoter region (Ni et al., 1995). 6 Initial studies in tobacco indicated that this promoter, called the "super-promoter", could 7 direct expression of GUS activity to a level 2-to 20-fold higher than the commonly used 8 "enhanced" double Cauliflower Mosaic Virus (CaMV) 35S promoter (Ni et al., 1995). 9 The activity of the super-promoter was highest in roots, but was also high in leaves and 10 stems. 11 The super-promoter was originally created by ligating three ocs activator 12 fragments (positions -333 to -116 relative to the transcription start site [Leisner and 13 Gelvin, 1988]) from the octopine synthase gene to the mas2' activator/promoter region (-14 318 to +65 relative to the transcription start site; [Ellis et al., 1984]), all from the 15 Agrobacterium tumefaciens Ti-plasmid pTiA6. The construction of the original super-16 promoter resulted in the repeated presence within the promoter of several commonly used 17 restriction endonuclease sites (BamHI, EcoRI, HindIII), as well as the presence of the 18 restriction endonuclease sites PstI, and XhoI. This feature precluded one from easily 19 linking genes to the promoter. In addition, it complicated further analysis of T-DNA 20 insertions in the plant genome. We therefore modified the super-promoter, eliminating 21 most of these internal restriction endonuclease sites. This modified super-promoter 22 formed the basis for the construction of several novel plant expression and T-DNA binary 23 vectors. Here, we describe these vectors and the use of the super-promoter to promote 24 reporter gusA gene expression in tobacco and maize. 25 26

Results and Discussion 27
Construction of super-promoter cassettes in pUC119 28 To provide researchers with convenient tools for various applications in plant 29 expression in dicot plant species); (3) a multiple cloning site with 8-16 unique restriction 23 endonuclease sites (depending upon the vector) downstream of the promoter for cloning 24 of genes and for more facile analysis of T-DNA insertions in the plant genome; (4) the 25 agropine or nopaline synthase polyA addition signals; (5) markers for binary vectors for 26 selection in plants using kanamycin, hygromycin, or phosphinothricin/Basta/Bialophos. 27 28 Activity of the super-promoter in transgenic tobacco and maize 29 We previously showed that in transgenic tobacco, a super-promoter-gusA 30 construction was ~5-fold more active in roots than in leaves (Ni et al., 1995). We carried 31 out a more detailed investigation of the activity of the super-promoter in various organs 1 of stably transformed tobacco and maize plants containing a super-promoter-gusA 2 expression cassette. These tests were conducted before construction of the current series 3 of super-promoter vectors described above; the constructs therefore did not have the 4 exact same configuration as do the current vectors. In addition, our original constructions 5 contained (when applicable) the maize ubiquitin intron, rather than the maize adh1 intron 6 incorporated into the current set of vectors. However, tests in transgenic tobacco 7 indicated that the current super-promoter configuration behaves in a manner almost 8 identical to that of the older version of the super-promoter (data not shown). Figure 3  9 shows that in tobacco, GUS activity was greatest in older (lower) leaves than in younger 10 (upper) leaves. Interestingly, this is opposite to the expression pattern directed by the 11 CaMV 35S promoter, which directs expression of transgenes most strongly in younger, 12 meristematic tissues (Williamson et al., 1989). Conversely, GUS activity in young root 13 tips was much higher than that in older portions of the roots. The pattern of expression in 14 transgenic maize, however, was different. There was relatively little difference in GUS 15 activity among younger or older leaves. As with the situation in transgenic tobacco, GUS 16 activity in transgenic maize roots was considerably greater than that in leaves. 17

Comparison of the activities of the super-promoter and other promoters in maize 19
We previously showed that the super-promoter was as strong as or stronger than 20 several commonly used promoters in transgenic tobacco (Ni et al., 1995). We therefore 21 asked how the activity of the super-promoter compared with that of other promoters in 22 monocots. Figure 4 shows a comparison of the relative strengths of the super-promoter, 23 the mas2' promoter, the CaMV 35S promoter (-800 from pBI121; Jefferson et al., 1987), 24 and the maize ubiquitin promoter in transgenic maize. Within a factor of two, all of these 25 transcriptional regulatory sequences promoted approximately the same level of GUS 26 activity in leaves, and also in roots. These data indicate that the super-promoter functions 27 as well as other commonly used promoters in these maize tissues. 28 29

Response of the super-promoter and other promoters to the presence of introns 30
The presence of introns enhances the activity of promoters for gene expression, 1 especially in monocots (Cornejo et al., 1993;Mascarenhas et al., 1990). We therefore 2 tested whether the presence of introns would enhance the activity of the super-promoter 3 and several commonly used promoters in maize. Figure 5 shows the constructions that 4 we tested. Each set of constructions contains either the super-promoter, the mas2' 5 promoter, the CaMV 35S promoter, or the maize ubiquitin promoter. Set I contains no 6 intron in the gusA gene, Set II contains the potato ST-LS1 intron (Vancanneyt et al., 7 1990) in the gusA coding sequence, and Set III contains a maize ubiquitin intron 8 preceding the gusA coding sequence. As a control, we also generated a promoter-less 9 gusA construction. 10 We first tested these constructions in transiently transformed maize BMS 11 protoplasts. Within a factor of 2.5, all of these constructions (except the control, 12 promoterless gusA gene which yielded only a background level of activity) elicited 13 approximately the same amount of GUS activity. Figure 6 shows the average data from 14 four independent experiments. Thus, in transient expression assays, all of these 15 promoter/intron combinations functioned equally well in maize suspension cells. 16 Promoters may have different activities depending upon whether they direct 17 expression from non-integrated transgenes in transiently transformed cells, or from 18 integrated transgenes that may be under transcriptional constraints of assembled 19 chromatin (see, e.g., Frisch et al., 1995). Strong expression of a linked chloramphenicol 20 acetyltransferase (CAT) reporter gene directed by a maize ubiquitin promoter plus 21 ubiquitin leader intron in electroporated maize BMS cells has previously been described 22 by Christensen et al. (1992). Enhancement of gene expression, resulting from increased 23 stability of cytoplasmic mRNA, has also been reported as a result of incorporating the 24 maize Adh1 intron into reporter constructions (Callis et al., 1987;Luehrsen and Walbot, 25 1994). We therefore tested, in stably transformed maize plants, the response of each of 26 four promoters to the presence of the maize ubiquitin intron. Interestingly, the activity of 27 only the ubiquitin promoter increased in maize leaves in the presence of the ubiquitin 28 intron ( Figure 7). This response was up to 55-fold, indicating that, amongst the eight 29 promoter/intron combinations tested, the strongest transcriptional regulatory sequence in 30 maize is clearly the maize ubiquitin promoter followed by the maize ubiquitin intron.
Our results contrast with those of Callis et al. (1987) who demonstrated that 1 introduction of the Adh1 first intron into constructions containing either the CaMV 35S 2 or nopaline synthase (nos) promoters increased activity of a linked CAT gene 8-and 170-3 fold, respectively. Addition of a maize Bronze1 (Bz1) intron to the CaMV 35S promoter 4 also stimulated CAT activity. This stimulation was associated with increased steady-state 5 levels of CAT mRNA. However, these authors utilized Adh1 and Bz1 introns, rather than 6 the ubiquitin intron used in our study, and they performed their analyses in transiently 7 transfected maize BMS protoplasts. It is possible that the maize ubiquitin promoter plus 8 intron combination functions optimally in stably transformed maize tissues, a system not 9 tested by Callis et al. (1987). 10 11

Use of the super-promoter in various plant species 12
We re-configured the original super-promoter (Ni et al., 1995) such that it could 13 easily be utilized for gene expression in a number of plant species. Over the past ~10 14 years, our laboratory has distributed the various super-promoter constructions to dozens 15 of laboratories. In our laboratory, the super-promoter has routinely and effectively been 16 used to drive both transient and stable transgene expression in tobacco plants and BY-2 17 cell suspensions (Ni et al., 1995;He et al., 1996;Narasimhulu et al., 1996;Kononov et 18 al., 1997;Mysore et al., 1998;Veena et al., 2003;Lee and Gelvin, 2004), Arabidopsis 19 plants and cell suspensions (Nam et al., 1997(Nam et al., , 19981999;Mysore et al., 2000;Yi et al., 20 2002;Zhu et al., 2003;Gaspar et al., 2004;Hwang et al., 2004;Hwang and Gelvin, 2006;21 Crane and Gelvin, 2007;Kim et al., 2007), maize plants and BMS cell suspensions 22 (Narasimhulu et al., 1996) The original super-promoter contains a trimer of the ocs activator sequence 12 (Aocs), cloned as a HindIII fragment, upstream of the mas2' activator plus promoter in 13 pE1120. We modified the original super-promoter as follows: 14 We removed the super-promoter from pE1120 by partial digestion with HindIII 15 plus complete digestion with XbaI. We cloned this fragment into the HindIII and XbaI 16 sites of pBluescriptKS(-) to generate the plasmid pE1037. Digestion of pE1037 with 17 HindIII removed the three ocs activator sequences from the super-promoter. The single 18 HindIII site within this resulting plasmid (pE1048) was converted into a BamHI site by 19 filling in the overhanging nucleotides using Klenow fragment of DNA polymerase 20 followed by annealing a BamHI linker, generating pE1049. We likewise converted the 21 HindIII sites flanking Aocs into BamHI sites. We cloned this new Aocs fragment into 22 pE1049. Screening of the resulting colonies yielded insertions of a single Aocs fragment 23 (in either orientation), insertions of a dimer of the Aocs fragment (both in the correct or 24 both in inverted orientation), and a trimer of the Aocs fragment (all three in inverted 25 orientation; pE1054). 26 We continued to modify pE1054, first by removing a PstI site within the super-27 promoter, and subsequently transferring the newly modified super-promoter region into 28 the SalI and XbaI sites of pUC119, generating pU∆P (pE1466). We next removed a XhoI 29 site from the super-promoter region of pE1466 by filling in the XhoI site using Klenow 30 fragment of DNA polymerase, generating pU∆P∆X (pE1467). We digested pE1467 with 31 generating pU∆P∆X-1. 3 We digested pU∆P∆X-1 with EcoRI, filled in the overhanging ends using Klenow 4 fragment, and ligated into this site a 401 bp HincII-EcoRV fragment containing the 5 agrocinopine synthase (ags) terminator (ags-ter) from pTiA6, generating pU∆P∆X-2 6 (pE1694). We digested pU∆P∆X-2 with XbaI and SmaI, filled in the overhanging ends 7 using Klenow fragment, and self-ligated the molecules, removing the XbaI, BamHI, and 8 SmaI sites and generating the plasmid pU∆P∆X-3 (pE1695). 9 We digested pBluescript KS(-) with EcoRV and inserted a linker containing BglII 10 and BclI sites. This plasmid, called pBB (pE1505) contains a new multiple cloning site 11 region. We digested pU∆P∆X-3 with KpnI and SacI and inserted the multiple cloning 12 site region from pBB using KpnI and SacI, generating pMSP-1 (pE1578). 13 Constructions in E. coli were generated in strain DH10B. T-DNA binary vectors 14 were introduced into A. tumefaciens EHA101 (Hood et al., 1986) or EHA105 (Hood et 15 al., 1993). E. coli strains were grown in Luria-Bertani (LB) medium containing the 16 appropriate antibiotics (ampicillin, 100 µg/ml; kanamycin, 25 µg/ml). A. tumefaciens 17 was grown in YEP rich or AB minimal medium (Lichtenstein and Draper, 1986) 18 containing the appropriate antibiotics (rifampicin, 10 µg/ml; kanamycin, 100 µg/ml for 19 plates, 25 µg/ml for liquid growth). 20 21