Evaluation of the Diphtheria Toxin A Subunit Gene (DT-A) as a Non-conditional Negative Selectable Marker in Tobacco and Rice
Pachamuthu Kannan*, Bharat Bhusan Majhi, Karuppannan Veluthambi
Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai, Tamil Nadu, India
To cite this article:
Pachamuthu Kannan, Bharat Bhusan Majhi, Karuppannan Veluthambi. Evaluation of the Diphtheria Toxin A Subunit Gene (DT-A) as a Non-conditional Negative Selectable Marker in Tobacco and Rice. Journal of Plant Sciences. Vol. 4, No. 5, 2016, pp. 106-112. doi: 10.11648/j.jps.20160405.13
Received: July 21, 2016; Accepted: August 1, 2016; Published: August 29, 2016
Abstract: The diphtheria toxin A subunit gene (DT-A) from Corynebacterium diphtheriae inhibits protein synthesis in eukaryotes. In this study, toxicity of the DT-A gene was evaluated by a transgenic approach in tobacco and rice. The DT-A gene was cloned under transcriptional control of the CaMV 35S promoter and transformed into tobacco. Similarly, CaMV 35S and the maize Ubi1 promoter-driven DT-A gene constructs were transformed into rice. The deployment of the DT-A gene in both tobacco and rice drastically reduced the recovery of transgenic plants in comparison to pCAMBIA1301 (without DT-A). Southern blot analyses of the transgenic plants were done using the hph- and DT-A gene-specific probes to check the presence of the hph and DT-A genes. All the tobacco and rice transgenic plants showed hybridization to junction fragments upon using the hph gene probe. Southern blotting with the DT-A probe revealed that all the transgenic plants either did not have the DT-A gene or harboured truncated DT-A gene in the integrated T-DNAs. None of the transgenic plants carried the complete DT-A gene. The results showed that the DT-A gene can be used as a good non-conditional negative selectable marker in both tobacco and rice. Both CaMV 35S promoter- and Ubi1 promoter-driven DT-A genes were effective as non-conditional negative selectable markers in rice.
Keywords: Agrobacterium tumefaciens, Diphtheria Toxin, Negative Selectable Marker, Rice, Tobacco
Expression of the negative selectable marker (NSM) genes in transgenic plants causes immediate or conditional cell death by interfering with normal growth and development. It allows selection of cells, which lack the NSM gene . Based on the mode of action, NSMs are classified as non-conditional (substrate-independent) NSM and conditional (substrate-dependent) NSM. Expression of the non-conditional NSM genes leads to immediate cell death after transformation. It does not require any other substrate for its action. Examples of non-conditional NSM are Corynebacterium diphtheriae toxin A-chain fragment (DT-A) , Pseudomonas aeruginosa exotoxin A , barnase from Bacillus amyloliquefaciens , anti-nptII gene  and DIANTHIN gene from Dianthus caryophyllus [6,7]. In contrast, expression of conditional NSMs does not directly cause lethality to the cells. Instead, it converts a non-toxic substrate to a cytotoxic compound. Cytosine deaminase (codA), indoleacetamide hydrolase (tms2), dehalogenase (dhlA), cytochrome P450 monooxygenase (P450), alcohol dehydrogenase (cue), D-amino acid oxidase (dao1) and herpes simplex virus thymidine kinase (HSVtk) [7,8] are the examples of conditional NSM. For example, codA conditional NSM converts non-toxic 5-fluorocytosine (5-FC) into toxic 5-fluorouracil (5-FU), and has proved to be an efficient conditional NSM in both tobacco and rice [8,9,10].
NSMs have many applications in plant biology research. The DT-A and codA genes have been used in homologous recombination based ‘gene targeting’ experiments to eliminate the random T-DNA integration events [10,11,12], which frequently occur. Selectable marker-free transgenic rice line was developed by using the Mungbean yellow mosaic virus (MYMV) transcriptional activator protein gene (TrAP) as a non-conditional NSM . Male sterile transgenic plants were developed using barnase as an NSM  and also NSMs have been used to eliminate vector backbone sequence in Agrobacterium-mediated plant transformation .
The diphtheria toxin A-chain fragment gene (DT-A) from Corynebacterium diphtheriae causes cell death by NAD+-dependent ADP-ribosylation of elongation factor 2 and inhibits protein synthesis [12,16]. Diphtheria toxin B chain fragment is required for recognition of cell membrane receptors and is responsible for cell to cell movement of the DT-A protein . Since the DT-A gene does not encode the B chain fragment, the cell to cell movement of the DT-A toxin is restricted. The DT-A protein exhibited a high level of toxicity to tobacco  and rice cells .
In this study, we evaluated the usefulness of the DT-A gene as a non-conditional NSM in tobacco and rice. First, a CaMV 35S promoter-driven DT-A transgenic tobacco plants were developed. Next, CaMV 35S promoter- and Ubi1 promoter-driven DT-A transgenic rice plants were generated. Transformation of plants with the NSM gene should reduce the recovery of transgenic plants and the recovered transgenic plants are expected to show truncated T-DNAs either without NSM or with truncated NSM gene [8,11,17]. This approach was used to study the toxicity of the DT-A gene in both tobacco and rice. The results showed that CaMV 35S promoter-driven DT-A served as a good non-conditional NSM in tobacco. Both CaMV 35S promoter- and Ubi1 promoter-driven DT-A genes served as good non-conditional NSMs in rice.
2. Materials and Methods
2.1. Construction of Binary Vectors
The binary vector pCam-P35S-DT-A-35S 3’ was constructed as follows: The DT-A gene (585-bp) was amplified from the plasmid pLMY101  as per the following PCR conditions: Initial denaturation at 94°C for 2 min, 30 cycles at 94°C for 40 s, at 58°C for 40 s and at 72°C for 45 s and a final extension at 72°C for 7 min with primers 5’- TACCATGGATCCTGATGATGTTGTTGATTCTTC-3’ (the start codon and the codons for two modified amino acids are shown in italic) and 5’-TCACAAAGATCGCCTGACACGATTTCCTGC-3’ (the stop codon and the codons of the two introduced amino acids are shown in italic). The amplified fragment was cloned in pGEM-T (Promega, Madison, USA). The primers were designed to introduce the start (ATG) and stop (TGA) codons in the DT-A gene. The first two aminoacids of the native DT-A gene, glycine (GGC) and alanine (GCT), were modified to aspartate (GAT) and proline (CCT) and two additional aminoacids, serine (TCT) and leucine (TTG), were introduced before the stop codon, as reported previously [2,19]. The DT-A ORF was checked and verified by sequencing. The DT-A gene was cloned in the MCS of pJIC35S  to yield the P35S-DT-A-35S 3’ cassette. The 1.3-kb P35S-DT-A-35S 3’ EcoRV fragment was cloned in the SmaI site of pCAMBIA1301 to yield pCam-P35S-DT-A-35S 3’. The binary vector was mobilized into Agrobacterium tumefaciens LBA4404 (pSB1) .
The binary vector pCam-PUbi1-DT-A-35S 3’ was constructed as follows: A 0.9-kb EcoRV/SacII fragment containing the DT-A coding sequence with the 35S 3’ was cloned in the respective sites of pBS(II)KS+ (Stratagene, West Cedar, USA). The 0.9-kb DT-A gene with 35S 3’ was excised as an EcoRV/SacI fragment and cloned downstream of the Ubi1 promoter. The 2.9-kb PUbi1-DT-A-35S 3’ cassette was taken as a StuI/EcoRV fragment and subcloned in the SmaI site of pUC18. The 2.9-kb PUbi1-DT-A-35S 3’ cassette was excised as a HindIII/KpnI fragment and cloned in the corresponding sites of pCAMBIA1301 to yield the binary plasmid pCam-PUbi1-DT-A-35S 3’, which was mobilized into the A. tumefaciens strain LBA4404 (pSB1) by triparental mating.
2.2. Transformation of Tobacco and Rice
The A. tumefaciens LBA4404 (pSB1, pCamP35S-DT-A-35S 3’) strain was used for tobacco (Nicotiana tabacum L. cv. Wisconsin 38) transformation. Tobacco leaf discs (8 mm diameter) were infected with the Agrobacterium strain as described by Sunilkumar et al. (1999) . Agrobacterium-infected tobacco leaf discs were maintained on the shoot-induction medium [Murashige and Skoog (MS) salts , B5 vitamins, 0.5 μM NAA, 4 μM BAP, 3% (w/v) sucrose, 0.8% (w/v) agar, pH 5.7] which contained 50 mg L-1 hygromycin (Hyg) and 250 mg L-1 cefotaxime. The HygRtransgenic shoots were kept for root induction on the BGS medium (MS salts, 1 mg L-1 folic acid, 100 mg L-1 myoinositol, 0.4 mg L-1 thiamine, 0.057 μM indole-3-acetic acid, 0.14 μM kinetin, 3% [w/v] sucrose, 0.9% [w/v] agar, pH 5.7) supplemented with 250 mg L-1 cefotaxime and 50 mg L-1 hygromycin. Scutellum-derived rice (Oryza sativa L. subsp. indica cv. Pusa Basmati 1) calli were used for Agrobacterium-infection (pCam-P35S-DT-A-35S 3’ or pCam-PUbi1-DT-A-35S 3’). Callus induction, Agrobacterium-mediated transformation, selection and regeneration were done as described by Sridevi et al. (2003) . GUS histochemical staining was done as described earlier by Sunilkumar et al. (1999) .
2.3. Southern Blot Analysis
Total DNA was extracted from tobacco and rice leaves . DNA was estimated using the Hoechst dye 33258 in the DyNa Quant 200 fluorometer (Hoefer Scientific Instruments, San Francisco, USA). For Southern blot analysis, 2.5 μg of rice DNA and 10 μg of tobacco DNA was digested with appropriate restriction enzymes and electrophoresed in 0.8% agarose gels in 1x Tris-borate-EDTA (TBE) buffer. The DNA was transferred onto Zeta probe nylon membrane (Bio-Rad, Hercules, USA). The [α-32P]dCTP-labelled hph and DT-A gene fragments were used as probes.
3. Results and Discussion
3.1. Transformation of Tobacco with the DT-A Gene Under the Transcriptional Control of CaMV 35S Promoter
Tobacco leaf discs were used for transformation with the binary vector pCAMBIA1301 (without the DT-A gene) and pCam-P35S-DT-A-35S 3’. pCAMBIA1301 yielded 100% (60 out of 60) transformation of tobacco leaf discs. However, pCam-P35S-DT-A-35S 3’ yielded only four HygR plants out of 100 leaf discs (Table 1). Four HygR tobacco plants obtained with pCam-P35S-DT-A-35S 3’ were subjected to Southern blot analysis to study the T-DNA integration. Plant DNA was digested with EcoRI. The blot was first probed with the [α-32P]dCTP labelled-hph gene. All four HygR tobacco plants displayed hybridization of junction fragments longer than 2.1 kb, which confirmed the integration of T-DNA (Fig. 1a, b). If the complete DT-A gene is integrated in the transgenic tobacco plants, an internal T-DNA fragment of 0.6-kb is expected to hybridize upon EcoRI + HindIII digestion of plant DNA (Fig. 1a). Control and transgenic tobacco DNA was digested with EcoRI + HindIII and the blot was probed with the DT-A gene. The Southern blot results showed that transgenic tobacco plant 2 and 4 did not harbour the DT-A gene. Transgenic tobacco plant 1 showed hybridization close to the 0.6-kb position (Fig. 1c), but the PCR analysis with the DT-A gene-specific primers did not amplify the 0.6-kb DT-A gene (Fig. 1d). The result showed that the DT-A gene in tobacco plant 1 is truncated and did not have an annealing site of one of the two primers. Transgenic tobacco plant 3 showed hybridization to a 2.8 kb junction fragment which was longer than the expected 0.6-kb internal T-DNA fragment (Fig. 1c). The results indicated that none of the four transgenic tobacco plants carried the complete DT-A gene. Thus, DT-A can be effectively used as a non-conditional NSM in tobacco.
|S. No||Binary plasmid||Host plant||No. of tobacco leaf discs/rice calli used for transformation||No. of leaf discs/rice calli which yielded HygR plants||Transformation efficiency|
3.2. Transformation of Rice with the DT-A Under the Transcriptional Control of CaMV 35S and the Ubi1 Promoters
To evaluate the toxicity of the DT-A gene in rice, the binary plasmids pCam-P35S-DT-A-35S 3’ (the DT-A gene under CaMV 35S promoter) and pCam-PUbi1-DT-A-35S 3’ (the DT-A gene under maize ubiquitin promoter) were constructed and mobilized into A. tumefaciens LBA4404 (pSB1). First, pCam-P35S-DT-A-35S 3’ was used for rice transformation along with pCAMBIA1301 (without DT-A gene) as a control. Agrobacterium-mediated transformation with pCam-P35S-DT-A-35S 3’ yielded three HygR rice plants out of 280 calli (1.1% transformation efficiency) but transformation with the control binary vector pCAMBIA1301 yielded 28 plants (out of 200 calli) with 14% transformation frequency (Table 1). Upon Southern blot analysis with the hph gene, all three EcoRI digested pCam-P35S-DT-A-35S 3’-transformed rice plants showed junction fragments longer than 2.1 kb (Fig. 2a). Presence of the complete DT-A gene in the T-DNA of the transgenic rice plants was studied by internal T-DNA fragment analysis with the DT-A probe. Control and transgenic rice plant DNA was digested with EcoRI + HindIII and the blot was probed with the DT-A gene. Transgenic plants 2 and 3 did not display hybridization of the expected 0.6-kb internal T-DNA fragment (Fig. 2b). Plant 1 did not hybridize to the expected 0.6-kb fragment but hybridized to a 2.2 kb fragment, indicating the presence of an incomplete DT-A gene which formed a junction fragment (Fig. 2b). The Southern blotting result conclusively showed that none of the three transgenic rice plants harboured the complete DT-A gene. Toxicity of the DT-A gene is responsible for eliminating the events which contained complete T-DNA with an intact copy of the DT-A gene.
The CaMV 35S promoter is most frequently used in plants for expressing transgenes in a constitutive manner . It has been reported that CaMV 35S promoter showed high level transgene expression in dicotyledonous plants compared to monocotyledonous plants . Different promoters are needed for multiple transgene expression in a single transgenic plant in order to avoid homology-based gene silencing [28,29]. Therefore, the toxicity of the DT-A gene in rice was also evaluated in this study under the control of the Ubi1 promoter. The 21-day-old rice scutellum-derived callus was used for transformation with the binary vectors pCAMBIA1301 (without the DT-A gene) and pCam-PUbi1-DT-A-35S 3’. pCAMBIA1301 yielded 11 HygR plants out of 80 calli (13.7%) and pCam-PUbi1-DT-A-35S 3’ construct yielded only one HygR plant out of 150 calli (0.6%, Table 1). GUS histochemical analysis was done in control, pCAMBIA1301- and pCam-PUbi1-DT-A-35S 3’- transformed rice plants. The pCam-PUbi1-DT-A-35S 3’-transformed plant did not show GUS activity but the pCAMBIA1301-transformed plant displayed GUS staining (Fig. 3a). This result indicated that the T-DNA integrated in the pCam-PUbi1-DT-A-35S 3’-transformed plant did not have the complete T-DNA.
Southern blot analysis was done to study T-DNA integration in the HygR transgenic rice plants. Total DNA (2.5 μg) from transgenic and control rice plants was digested with EcoRI and used for junction fragment analysis. Junction fragments longer than 2.1 kb are expected to hybridize to the hph probe (Fig. 3b). One HygR rice plant obtained with pCAMBIA1301 and one HygR rice plant obtained with pCam-PUbi1-DT-A-35S 3’ were taken for the analysis. Southern blotting with the hph probe displayed hybridization to junction fragments longer than 2.1 kb (Fig. 3c) in pCAMBIA1301- and pCam-PUbi1-DT-A-35S 3’-transformed plants. An internal T-DNA fragment of 0.6-kb was expected to hybridize upon PstI digestion, if the complete DT-A gene is integrated in the transgenic rice plant (Fig. 3b). Hybridization of the 0.6-kb fragment was observed in the positive control lane with the binary vector pCam-PUbi1-DT-A-35S 3’, but the pCam-PUbi1-DT-A-35S 3’-transformed HygR transgenic plant did not show the hybridization to the DT-A probe (Fig. 3d). The result confirmed that the integrated T-DNA did not harbour the DT-A gene. These observations correlate well with the previous reports of rice transformation with P35S-DT-A-35S 3’ and PUbi1-DT-A-35S 3’ .
An NSM gene may show toxicity to one particular plant species and may not show toxicity to others. Expression of the DIANTHIN gene caused lethality in tobacco cells but not in rice . Similarly, the ABRIN-A gene showed toxicity in tobacco but not in rice . In this study, the DT-A gene under the control of constitutive promoters was transformed in both tobacco and rice. Agrobacterium-mediated transformation with the DT-A gene constructs profoundly reduced the recovery of transgenic plants compared to pCAMBIA1301 (without the DT-A gene) transformation (Table 1). All the DT-A transformed transgenic tobacco and rice plants displayed truncated T-DNAs with either the truncated DT-A gene or without the DT-A gene. The results conclusively show that the DT-A gene can be effectively used as a non-conditional NSM in both tobacco and rice. Both CaMV 35S promoter- and maize Ubi1 promoter-driven DT-A genes are effective in rice.