Construction and characterization of the sul I plasmid vectors
Several novel vectors have been constructed that utilize a sul I selectable marker cassette. The pK-Sul I cloning vector [GenBank:HQ593859] contains a double enhanced CaMV 35S promoter and terminator sul I expression fragment flanked by convenient restriction sites (Hind III, Sph I and Sbf I) that facilitate subcloning (Figure 1A). Four Agrobacterium binary vectors useful for plant transformation have also been generated. The pCS4-BASK vector [GenBank:HQ593861] contains the 35S sul I cassette from pK-Sul I and a Solanum bulbocastanum Ubi 409s promoter, intron and ubiquitin monomer [19] separated by a multiple cloning site from a nos terminator sequence (Figure 1C). This construct is useful for the strong over-expression of a gene of interest as a translational fusion to the 409s ubiquitin monomer.
The pSUNG binary vector [GenBank:HQ593863] was also constructed. This plasmid is well suited for investigating the function of candidate promoter sequences. The pSUNG construct contains a promoterless GUSPlus reporter gene with a large multiple cloning site upstream and a nos promoter-sul I-nos terminator selectable marker (Figure 1E). The nos promoter was utilized in this vector to minimize undesirable interactions between the selectable marker promoter and the candidate promoter being tested. The CaMV 35S promoter/enhancer is known to promiscuously interact with nearby promoters in transgenic Arabidopsis, confounding reporter gene expression characterization studies [26–28]. Further analysis has demonstrated that the nos promoter, in contrast, does not alter the expression conferred by nearby promoters [27, 28] and thus was chosen as the promoter to control sul I expression in pSUNG.
In addition to the construction of the pCS4-BASK and pSUNG vectors, we also developed the precursor vectors pCS [GenBank:HQ593860] and pSUN [GenBank:HQ593862], respectively. These binary plasmids carry only a sul I selectable marker cassette and a multiple cloning site within the T-DNA. The maps of these adaptable 'empty' vectors are shown in Figures 1B and 1D. Although these vectors are already useful tools for generating plant transformation constructs, additional utility may be added in the future by generating Gateway®-compatible (Invitrogen) versions, which will enable high throughput cloning applications.
Validation of the sul I selection constructs
To demonstrate the utility of these novel vectors, numerous transgenic Arabidopsis plants were generated via Agrobacterium-mediated floral dip transformation. Initially the functionality of the sul I selectable marker was examined. Sulfadiazine screening was performed in tissue culture and multiple independent transgenic plant lines were identified for each construct. The results demonstrate that both the 35S expression cassette of the pCS-derived vectors and the nos promoter expression cassette present on the pSUN-derived constructs successfully generate transgenic plants resistant to 5 to 50 mg/L sulfadiazine when germinated in culture. To determine how effectively the pCS- and pSUN-derived vectors confer resistance, the initially isolated lines were subjected to a concentration gradient of 0 - 200 mg/L sulfadiazine. As expected from previous results [12], the pCS-derived plants exhibited resistance and grew well in media containing up to 200 mg/L sulfadiazine. The pSUN-derived plants however were less tolerant, exhibiting good growth at levels up to 50 mg/L, with a gradual loss of fitness at higher levels of selection (data not shown). Even though the pSUN plants exhibit a more modest level of resistance than the pCS-derived plants, the observed resistance in the nos-sul I transgenics is sufficient to provide a clearly distinct resistance phenotype compared to wildtype plants at levels from 5 to 50 mg/L sulfadiazine.
Southern blot analysis examining the T-DNA copy number of seven independent pCS4-BASK transgenic plants using the sul I sequence as a probe was performed and the result is shown in Figure 2. Four of the seven plants exhibit a single uniquely sized band suggesting that they each contain a distinct T-DNA insertion, while three other plants exhibited two to four bands and likely carry two or more T-DNAs. Taken together, these results support the conclusion that a single copy of the sul I selection marker in the pCS- and pSUN-derived vectors is sufficient for selecting transgenic plants.
Sulfadiazine foliar application
Although our results and those of others have established that a tissue culture germination screen is an effective approach for identifying sul I transgenic Arabidopsis, a potential alternative screening method was also investigated. The use of foliar application of a sulfadiazine solution supplemented with 0.03% of the surfactant L-77 silwet was assessed. Col-0 plants were grown directly in soil and sprayed with 50, 100 and 500 mg/L sulfadiazine solution every three days for a total of four applications. Plants from the 50 and 100 mg/L treatment were partially stunted but continued to grow normally once the treatment ceased (data not shown). However, the Col-0 wildtype plants that experienced four applications at 500 mg/L were severely stunted (i.e. true leaves never emerged) and did not recover once the treatment was discontinued (data not shown). To further examine whether the selective pressure of multiple applications of 500 mg/L sulfadiazine was necessary to inhibit growth, wildtype plants were sprayed with the sulfadiazine solution either one, two, three or four times at three day intervals. As shown in Figure 3A, the wildtype Col-0 plants exhibited stunted growth and subsequent chlorosis even after a single treatment, although a few plants continued to grow and develop. However, the results from two to four applications demonstrate that multiple foliar treatments with sulfadiazine are extremely effective at inhibiting the growth of Arabidopsis seedlings in soil.
The resistance to the foliar application of sulfadiazine was examined in several confirmed sul I transgenic plants. Seed from three independent pCS4-BASK and pSUN homozygous transgenic lines were sown in soil and treated with a 500 mg/L sulfadiazine foliar spray. Figure 3B and 3C illustrates that the pCS4-BASK transgenic plants were fully resistant to sulfadiazine and exhibited normal growth at 11 and 21 days after the initial treatment. The initial sign of sulfadiazine resistance under these conditions is the emergence of the first set of true leaves, as is visible at day 11 (Figure 3B). Similar to the more modest level of resistance observed in tissue culture for the pSUN-derived plants, we observed that some of the pSUN plants exhibited less resistance to the foliar application of sulfadiazine as well. We speculate that this is likely due to the nos promoter driving lower levels of sul I expression in the transgenic plants than is typically observed for the double enhanced CaMV 35S promoter. Although we believe this is a likely explanation, other potential causes for this difference are also possible.
These results demonstrate that repeated foliar application of the sulfadiazine/silwet solution does not cause tissue damage to sul I resistant plants and can be successfully used to identify transgenic Arabidopsis. Under this foliar selection procedure, wildtype plants exhibit stunted growth but remain viable up to 14 days after the initial application, but by the end of three weeks, these plants are chlorotic and severely under-developed. In contrast, sul I transgenic plants continue to grow throughout subsequent treatments and are robust and healthy after three weeks. In fact, the sul I transgenic plants were frequently larger and healthier than even unselected wildtype plants grown side-by-side under the same conditions in the greenhouse.
A method of three foliar applications was chosen to screen a dense population of Arabidopsis seedlings (approximately 4,000 per flat) germinated in soil containing a mixture of nontransgenic plants with candidate pCS4-GFP T1 individuals generated from Agrobacterium-mediated floral dip transformation. Following germination, the seedlings were sprayed three times at three day intervals with a 500 mg/L sulfadiazine/0.03% L-77 silwet solution. Plant growth at day 21 is shown in Figure 4A. Clearly, there are numerous plants that exhibit tolerance to sulfadiazine, while the majority are substantially stunted and chlorotic. The frequency of resistant plants (approximately 1.0%) is well within the range expected for a typical transformation and selection. Genomic DNA was isolated from the plants that were judged sulfadiazine resistant and subsequently screened with sul I-specific primers. Thirty-eight of the 39 individuals screened were confirmed to contain the sul I sequence verifying that the foliar screening of soil grown plants is effective and allows few if any non-transgenic plants to escape selection (Figure 4C). The nontransgenic plant that survived selection appeared only weakly resistant to sulfadiazine and was one of the smallest surviving plants. If desired, the stringency of the selection could be heightened by increasing the number of foliar applications and/or by choosing only the largest healthiest plants, although this strategy would likely overlook transgenic plants with marginal levels of sulfadiazine resistance. To examine how the sul I-based selection compared to the npt II and hpt II selectable markers, we germinated T1 seed on media containing 50 μg/ml kanamycin and 20 μg/ml hygromycin, respectively. Based on several independent experiments, we observed selection escape rates that ranged from 0% to 36% for kanamycin and 2% to 23% for hygromycin (data not shown). Taken together, these results illustrate that sul I foliar selection in soil performs at least as well as selection with other frequently used screening methods.
Validation of the overexpression and promoter testing capabilities of the binary vectors
To characterize the pCS4-BASK and pSUNG vectors, we examined the functionality of the other components within the T-DNA. An enhanced GFP coding sequence was translationally fused downstream of the ubiquitin monomer in pCS4-BASK and transformed into Arabidopsis. As shown in Figure 5A, B and 5C, the 409s promoter conferred strong GFP expression that is clearly visible in dry seeds, in young seedlings and in the trichomes of mature leaves. This demonstrates that the pCS4-BASK construct offers a viable alternative to using the 35S promoter for the over-expression of genes in Arabidopsis and likely other dicot plant species.
To validate the utility of the reporter gene in pSUNG, we fused a japonica rice Cytochrome c (OsCc1) promoter to GUSPlus to generate the pSUNG-OsCc1 vector. The indica OsCc1 promoter confers constitutive reporter gene expression in transgenic rice [23]. Figure 5D illustrates the GUSPlus reporter expression controlled by the OsCc1 promoter in a transgenic Arabidopsis seedling. OsCc1 transgenic plants exhibited strong GUS histochemical staining in all the tissues of young seedlings including both the root and the shoot. Weaker staining was observed in mature leaves (data not shown). These results demonstrate that the reporter gene portion of the pSUNG vector functions as expected and validates the utility of this novel construct for promoter testing. In addition, it provides evidence that the rice OsCc1 promoter not only controls strong expression in rice, but Arabidopsis seedlings as well and thus may be a useful promoter for expression in a diverse array of species.
The rapid development of genetics and the desire to answer more complicated questions often demand the simultaneous use of two or more selectable markers. We investigated the feasibility of stacking sul I with other selectable markers. As such, we chose to cross sul I transgenic plants with a previously generated Arabidopsis line that was homozygous for both the npt II and hpt II selectable markers [29]. Additional file 1 shows germinated seeds from the crossed plants screened on media supplemented with sulfadiazine (5 mg/L), hygromycin (10 mg/L), and kanamycin (50 mg/L). The triple transgenic seedlings grew normally, but the Col-0 wildtype failed to survive past the emergence of the radicle. These results indicate that simultaneous expression of the sul I, npt II and hpt II genes is not detrimental to the health of the transgenic plants, and that a triple selection screen is feasible, since the three antibiotics can be successfully used together in selection media.