pSiM24 Is a Novel Versatile Gene Expression Vector for Transient Assays As Well As Stable Expression of Foreign Genes in Plants

We have constructed a small and highly efficient binary Ti vector pSiM24 for plant transformation with maximum efficacy. In the pSiM24 vector, the size of the backbone of the early binary vector pKYLXM24 (GenBank Accession No. HM036220; a derivative of pKYLX71) was reduced from 12.8 kb to 7.1 kb. The binary vector pSiM24 is composed of the following genetic elements: left and right T-DNA borders, a modified full-length transcript promoter (M24) of Mirabilis mosaic virus with duplicated enhancer domains, three multiple cloning sites, a 3′rbcsE9 terminator, replication functions for Escherichia coli (ColE1) and Agrobacterium tumefaciens (pRK2-OriV) and the replicase trfA gene, selectable marker genes for kanamycin resistance (nptII) and ampicillin resistance (bla). The pSiM24 plasmid offers a wide selection of cloning sites, high copy numbers in E. coli and a high cloning capacity for easily manipulating different genetic elements. It has been fully tested in transferring transgenes such as green fluorescent protein (GFP) and β-glucuronidase (GUS) both transiently (agro-infiltration, protoplast electroporation and biolistic) and stably in plant systems (Arabidopsis and tobacco) using both agrobacterium-mediated transformation and biolistic procedures. Not only reporter genes, several other introduced genes were also effectively expressed using pSiM24 expression vector. Hence, the pSiM24 vector would be useful for various plant biotechnological applications. In addition, the pSiM24 plasmid can act as a platform for other applications, such as gene expression studies and different promoter expressional analyses.


Introduction
The transfer of foreign genes into higher plants mediated either by Agrobacterium tumefaciens or by employing a biolistic process is the core technique used in genetic engineering-based plant modification. Many useful and versatile vectors have been constructed since the birth of the concept and the first generation of binary vectors for plant transformation [1][2][3][4][5][6]. The general trend in the binary vector development has been to increase the plasmid stability during a long co-cultivation period of A. tumefaciens with the target host plant tissues and also to understand the molecular mechanism of broad host-range replication, and to use it to reduce the size of plasmid for ease in cloning and for higher plasmid yield in Escherichia coli [7,8]. A number of large (.10 kb), firstgeneration binary vectors have been constructed for plant transformation, including Ti plasmid [2], pBin19 [1], pKYLX7 [4] and other expression vectors [3]. One of the binary vectors, pBin19 [1], has been modified to pBI121 and pIG121Hm [9,10] to use the b-glucuronidase (GUS) reporter gene in plant transformation. Binary vectors include pKYLX expression vectors containing 35S and rbcS promoters that are suitable for constitutive or light-regulated expression of foreign genes [4]. These vectors and their derivatives were soon widely distributed among plant scientists. In addition, another widely used series of vectors includes pPZP vectors [11] and their modified form, pCAMBIA vectors (www.cambia.org).  constructed a pCB mini-binary vector series [12] from the relatively large, first-generation binary plasmid pBin [1]. Over time, vector technology evolved, and new generations of plant transformation vectors with improved cloning and delivery strategy were introduced, for example, pGreen vectors [13]; pGD or pSITE vectors, which are suitable for the stable integration or transient expression of various autofluorescent protein fusions in plant cells [14,15]; the pCLEAN binary vector system [16]; the pHUGE binary vector system [17]; and binary bacterial artificial chromosome BIBAC vectors [18]. The TMV RNA-based vector pJLTRBO [19]and its derivative pPZPTRBO [20] were reported to produce recombinant proteins in plants without using the RNA-silencing inhibitor P19. Similar expression levels were provided by the pEAQ-HT vector which has an integrated P19 expression cassette [21]. A bean yellow dwarf virus single-stranded DNA-based vector, pBY030-2R was reported to produce high amount of recombinant proteins [22] while the pMAA-Red vector was known for easy production of transgenic Arabidopsis overexpression lines with strong expression levels of the gene of interest [23].
The binary vectors widely used for plant transformations vary in size, origin of replication, bacterial selectable markers, T-DNA borders and overall structure. Recent modifications of binary vectors provide a number of user-friendly features, such as a wide selection of cloning sites, high copy numbers in E. coli, improved compatibility with strains of choice, a wide pool of selectable markers for plants and a high frequency of plant transformation. Although recent improvements are very useful, the classic vector configuration still appears to be good enough in many occasions. Plasmid manipulations are also easier if the vector replicates in E. coli to high copy numbers. Moreover, the efficiency of in vitro recombination procedures is inversely proportional to the size of the vector DNA [24]. With an increased requirement for the transfer of large pieces of DNA into plants, the size of binary vectors should be kept to a minimum. The availability of lowmolecular-weight, versatile plant expression vectors is currently insufficient in plant molecular biology. For these reasons, we designed a smaller binary vector, pSiM24, which offers a wide selection of cloning sites, high copy numbers in E. coli and is fully functional in the transient (using both the gene-gun or Agroinfiltration methods) as well as stable transformation of plants.
In vitro cloning procedures and DNA sequencing All in vitro recombination techniques were employed using previously described standard methods [27,28]. For DNA sequencing, a dye terminator labeling procedure was followed using a Genome Lab DTCS-Quick Start kit (Beckman Coulter, USA), and an automated sequencing machine (Beckman Coulter CEQ 8000 Genetic Analysis System, USA) was used in accordance with the manufacturer's instructions.
A 642-bp fragment of the replicon OriV of pRK2 was PCRamplified using appropriately designed forward and reverse primers to insert an ApaI site at the 59-end and a SalI site at the 39-end. The gel-purified PCR fragment 59-ApaI-OriV-SalI-39 was inserted into the corresponding site of pBtrfA to form the plasmid pB-oriV-trfA. A fragment of 1803 bp containing the AmpR gene and the ColE1 replicon in pMA (GeneArt vector, Registry part no. K157000), a pUC derivative, was PCR-amplified using appropriately designed forward and reverse primers to insert the KpnI site at the 59-end and the ApaI site at the 39-end. The PCR fragment was digested with KpnI and ApaI, and the gel-purified fragment 59-KpnI-ApaI-39 was cloned into the corresponding site of pB-oriV-trfA to generate the plasmid pBAmpR-ColEI-oriV-trfA.

Tobacco plant transformation
The plant expression constructs pSiM24-GUS and pSiM24 were introduced into the A. tumefaciens strain GV3850 by the freeze-thaw method [30]. Tobacco plants (Nicotiana tabacum cv. SamsunNN) were transformed with Agrobacterium harboring pSiM24-GUS and pSiM24 constructs as described previously [31] or by the gene-gun method using pSiM24-GUS and pSiM24 constructs [32]. Tobacco shoots and then roots were regenerated from kanamycin-resistant calli derived from independent leaf discs. Ten independent kanamycin-resistant plant lines (R 0 generation, 1 st progeny) were generated for the constructs pSiM24-GUS and pSiM24 and were maintained under greenhouse conditions (3065uC with both natural and supplementary lighting of minimum photon flux density, 300 mmole/m 2 /s, 17 h day/7 h night cycle). Seeds were collected from self-pollinated primary transformants. Transgenic tobacco seeds (R1 progeny, 2 nd generation) were germinated in the presence of Kanamycin (250 mg/L). Positive transformants with a KanR:KanS ratio of 3:1 progeny segregation were selected for further analysis. Transgenic lines (R 1 and R 2 progeny, second and third generation) were screened for gene integration, transcription and translation by polymerase chain reaction (PCR), reverse transcriptase-PCR (RT-PCR), real-time quantitative RT-PCR (qRT-PCR), enzymatic assays and GUS histochemical analysis.

Generation of transgenic Arabidopsis plants
The pSiM24 and pSiM24-GUS plasmids introduced into A. tumefaciens GV3850 were used to transfer each of these constructs into Arabidopsis (Arabidopsis thaliana ecotype Columbia-0) by the floral dip method [33]. The transgenic Arabidopsis plants were selected and maintained as described previously [34].

Transient Agro-infiltration assay of pSiM24-GUS in tobacco leaves
Suspensions of the A. tumefaciens strain GV3850 bearing pSiM24 and pSiM24-GUS constructs were infiltrated into leaves of Nicotiana benthamiana as described previously [35]. After two days of agro-infiltration, the transient GUS expression was evaluated by the histochemical GUS staining method [9].

Transient expression analysis in tobacco protoplasts
The isolation of tobacco protoplasts from the suspension cell cultures of N. tabacum L. cv Xanthi-Brad and electroporation of tobacco protoplasts with supercoiled plasmid pBTRKM24-GUS/ GFP and pBTRKM24 constructs were performed as described previously [36]. After 20 h, protoplasts were harvested for fluorometric GUS enzymatic assay [9]. GUS expression levels were within 610% for a given construct in this study. All constructs were tested in at least five independent experiments.

Biolistic-onion peel transient assay
Onion tissues were prepared and bombarded with pBTRKM24, pBTRKM24-GUS, pSiM24 and pSiM24-GUS plasmids following a standard protocol [37]. After two days, transient GUS expression was detected by a histochemical method [9] and visualized under an Olympus SZX12 bright-field microscope.
Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) The expression levels of GUS mRNA in transgenic tobacco and Arabidopsis plants developed for the plasmids pKCaMV35SGUS and pSiM24GUS were evaluated by real-time quantitative RT-PCR [38] using GUS-specific forward (59-d-TTACGTCCTGTA-GAAACCCCA-39) and reverse (59-d-ACTGCCTGGCACAG-CAAT TGC-39) primers. The qPCR assays were performed using the iTaq SYBR Green Supermix with ROX (Bio-Rad, USA) according to the manufacturer's instructions. Tobacco tubulin (by using forward 59-d-ATGAGAGAGTGCATATCGAT-39 and reverse 59-d-TTCACTGAAGAAGGTGTTGAA-39 primers) was used as an internal control to normalize the expression of GUS. The comparative threshold cycle (Ct) method (Applied Biosystems bulletin, part No. 4376784 Rev. C, 04/2007) was used to evaluate the relative expression levels of the transcripts. The threshold cycle was automatically determined for each reaction by the system set with default parameters (Step One Real-Time PCR System, Applied Biosystems). The specificity of the PCR was determined by melting curve analysis of the amplified products using the standard method installed in the system (Step One Real-Time PCR System, Applied Biosystems).

b-Glucuronidase (GUS) assay and histochemical GUS staining
Fluorometric GUS enzymatic assays for measuring GUS activities in tobacco protoplast extracts, Arabidopsis and tobacco plant extracts were performed as described previously [9,39]. The total protein content in protoplast and plant extracts was estimated by the Bradford method using BSA as a standard [40]. Histochemical GUS staining was carried out in plants following the published protocol [9,34], and photographs were taken under a bright-field microscope (Olympus SZX12).

GFP detection
GFP fluorescence images of electroporated tobacco protoplasts, onion epidermal cells and transgenic Arabidopsis leaves expressing GFP were analyzed using a confocal laser scanning microscope (TCS SP5; Leica Microsystems CMS GmbH, D-68165 Mannheim, Germany) using LAS AF (Leica Application Suite Advanced Fluorescence) 1.8.1 build 1390 software under a PL FLUOTAR objective (10.0X/N.A.0.3 DRY) using a confocal pinhole set of 1 airy unit and a 16 zoom factor for improved 8-bit resolution, as described previously [28,38]. To excite the expressed GFP in transgenic plants, a 488-nm argon laser (30%) with an AOTF (allowing for 40% transmission) was used, and fluorescence emission spectra were collected between 501 and 580 nm with the photomultiplier tube (PMT) detector gain set to 1050 V [28].

Transient expression of GUS using pSiM24 vector through vacuum infiltration method
Suspensions of the A. tumefaciens strain GV3850 bearing pSiM24 and pSiM24-GUS constructs were prepared as previously described [35], and the infiltration procedure was conducted following a previously reported protocol [41]. Leaves of N.
benthamiana were weighed and submerged in a suspension of A. tumefaciens strain GV3850 bearing pSiM24 or pSiM24-GUS plasmids. A vacuum level of 760 mm Hg was applied and released several times until the leaves became translucent [41]. Leaves were transferred into MS-media-containing plates and incubated at room temperature for two days. GUS expression in the infiltrated leaves was evaluated by the GUS histochemical staining method and GUS assay [9,39]. ; selection marker genes (KanR, neomycin phosphotransferase II, nptII) directed by nopaline synthase promoter (Nos promoter); terminator sequences of ribulose bisphosphate carboxylase small subunits (39rbcSE9); nopaline synthase terminator (Nos terminator); multiple cloning sites (first MCS, second MCS and third MCS) with various restriction sites; replicon unit pRK2 oriV; trfA gene for agrobacterium; ColE1 origin of replication for E. coli; and 'bla' AmpR gene for resistance to ampicillin. doi:10.1371/journal.pone.0098988.g001 Analysis GFP-AtCESA3 ixr1-2 , Vip3A(a), KMP-11, IL-10 and nat-T-phyllo-GFP after transient expression in tobacco using pSiM24 vector The GFP fused Arabidopsis mutated CESA3 (GFP-AtCESA3 ixr1-2 ) fragment with Xho I and Sst I sites was obtained from pKM24KH-MD1 (GenBank accession no. JX996118) [42][43] by restriction digestions. Likewise, the native T-phylloplanin fused GFP with the apoplast targeting sequence (nat-T-phyllo-GFP) fragment with Xho I and Sst I sites was obtained from pKM24-ibm8 (GenBank accession no. KF951257) [44] by restriction digestions. Both these fragments were cloned in pSiM24 following standard protocols [28] and the resulted plasmids were named as pSiM24-GFP-AtCESA3 ixr1-2 and pSiM24-nat-T-phyllo-GFP. Suspension of A. tumefaciens strain pGV3850 harboring pSiM24, pSiM24-GFP-AtCESA3 ixr1-2 and pSiM24-nat-T-phyllo-GFP constructs were infiltrated into leaves of tobacco plants (N. tabacum cv. SamsunNN) as described earlier [35]. After two days of Agroinfiltration the transient AtCESA3 ixr1-2 expression was evaluated by RT-PCR by using gene specific primers and also by Western blotting using AtCESA3 ixr1-2 polyclonal antibody as described earlier [42]. The transient nat-T-phyllo-GFP expression was evaluated by RT-PCR by using gene specific primers and also by confocal microscopy as previously described [44].

Features of assembled binary expression vector pSiM24
The binary expression vector pSiM24 was designed to reduce the size of the vector backbone by eliminating non-essential elements of our previous vector pKM24KH (size 12,945-bp, GenBank accession no. HM036220), a derivative of pKYLX7 [4]. The pKM24KH vector is a low-copy-number plasmid. We replaced the E. coli replication unit with a high-copy-number replicon ColEI in pSiM24, making the identification and characterization of gene inserts easier. We also modified the agrobacterium replicon unit (Oriv-trfA of pRK2) by optimizing the trfA open reading frame for better expression. The overall DNA yields and transformation frequency of the new vector pSiM24 were several times greater than those of the previous vector pKM24KH in both E. coli and A. tumefaciens. The binary vector pSiM24 (Figure 1; GenBank Accession no. KF032933) has The bacteria were transformed with equal molar amounts of each of the plasmid DNA. Statistical analysis of the data was performed adopting one way ANOVA analysis (using GraphPad Prism version 5.01) and presented as the means 6 S.D. A P value of less than 0.05 was considered significant indicated by different superscript letters. doi:10.1371/journal.pone.0098988.t002 Table 3. Binary Ti vectors pSiM24 produced higher plasmid DNA yields in Escherichia coli strain TB1 over pCAMBIA. DNA yield and transformation frequencies of E. coli and A. tumefaciens with pSiM24 binary vectors DNA yield and transformation frequencies in E. coli and A. tumefaciens were evaluated and presented (Tables 1-3). Transformations were performed with equal molar amounts of each of the plasmids to normalize increasing plasmid size as previously described [50]. The transformation frequencies of pSiM24 vectors are four-to six-fold higher than pCAMBIA and pKM24KH vectors, in E. coli ( Table 1). The transformation frequency of the pSiM24 vector is 1.4-to 1.8-fold higher than conventional pCAMBIA and pKM24KH vectors, in A. tumefaciens, although the effect is not as marked as in E. coli ( Table 2). The DNA yields of pSiM24 vectors were approximately three-fold greater than those of the pCAMBIA vector and seven to eight-fold higher than those of pKM24KH in E. coli (Table 3).  promoter showed approximately 10 times higher GUS activity than the CaMV 35S promoter (Figure 2). The pBTRKM24-GUS construct was also evaluated by the biolistic bombardment of epidermal cells of onion peels, showing strong GUS expression, as detected histochemically (Figure 3).
The pSiM24-GFP (with a different reporter gene, i.e., GFP) was studied in a tobacco protoplast system, where GFP fluorescence was visualized by confocal microscopy (Figure 4). The pSiM24-GUS construct was tested in an Agrobacterium infiltration assay in N. benthamiana leaves. The A. tumefaciens (strain C58C1-GV3850) carrying pSiM24 (empty vector), pK-CaMV35S-GUS and pSiM24-GUS constructs was used for agro-infiltration. Transient GUS expression detected histochemically, showed stronger GUS expression in agro-infiltrated patches for pSiM24-GUS construct than for pK-CaMV35S-GUS ( Figure 5). The pSiM24-GUS/GFP plasmids were also bombarded in onion cells, and strong GUS or GFP expression was observed in transformed onion epidermal cells (Figure 3-4). Tobacco leaf discs were co-cultivated with A. tumefaciens for three days, and transformed leaf discs were selected in the presence of 250 mg/L kanamycin and 500 mg/L of cefotaxime for four weeks. The increase in fresh weight in transformed leaf discs was evaluated as described previously [51]. The increases in fresh weight of four-week-old leaf discs were compared and are presented in Table 4. Leaf discs treated with binary vectors showed a seven-to eight-fold increase in fresh weight over the vector-less control, remaining green with multiple regenerating shoots. In the negative vector-less control, leaf discs did not induce callus and turned yellow within two weeks of antibiotic selection. Thus, the percentage of leaf discs showing an increase in fresh  After the co-cultivation with A. tumefaciens strain GV3850 at 25uC for 3 days, leaf discs were selected on shooting medium containing 250 mg/L of kanamycin and 500 mg/L of cefotaxime for four weeks. Each treatment involved 5 plates with 10 leaf discs per plate. The same experiment was repeated two more times. Statistical analysis of the data was performed adopting one way ANOVA analysis (using GraphPad Prism version 5.01) and presented as the means 6 S.D. A P value of less than 0.05 was considered significant indicated by different superscript letters. weight over the negative vector-less control indicates the proportion of putatively transformed leaf discs, which ranged from 79 to 98%. There appeared to be no detectable difference between pSiM24 binary vectors and the positive control pCAMBIA and pKM24KH vectors. The effect of pSiM24 binary vector on transformation frequency was also studied in A. thaliana. The pSiM24 and pSiM24-GUS/GFP vectors exhibited approximately two-fold more transformation frequency in A. thaliana than pCAMBIA2300 and pKM24KH vectors (Table 5).

Expression analysis of pSiM24-GUS/GFP in transgenic plants
Agrobacterium carrying the pSiM24-GUS reporter gene was used to transform Arabidopsis and tobacco plants. GUS histochemical analysis confirmed that the pSiM24 vector successfully expressed GUS genes in transgenic Arabidopsis and tobacco (both by agrobacterium-mediated transformation as well as by gene-gun methods) plants (Figure 6-7). The Arabidopsis pSiM24-GUS and pSiM24-GFP transgenic plants successfully expressed GUS and GFP proteins, as detected by GUS histochemical staining and confocal microscopy of GFP (Figure 7-8). Furthermore, GUS analysis of second-generation plants confirmed the successful inheritance of the transgene from one generation to another generation in both transgenic tobacco and Arabidopsis plants (Figure 9-10). The GUS activity was estimated biochemically in R2-generation transgenic Arabidopsis and tobacco plants; it was observed that approximately two times higher GUS activity accumulated in leaf and stem tissues of Arabidopsis plants than in tobacco plants ( Figure 9). The expression of GUS activities in different tissues of transgenic Arabidopsis and tobacco plants containing pSiM24-GUS showed the following pattern: Root .
Leaf . Stem (Figure 9). In addition, the histological GUS staining documented that the level of GUS expression was high in the reproductive tissues of transgenic pSiM24-GUS tobacco and Arabidopsis plants ( Figure 10). Both in tobacco and Arabidopsis transgenic plants, GUS transcript levels were higher for the pSiM24 binary vector than for the pKYLX-based expression vector pKCaMV35 ( Figure 5).

Transient expression of GUS using pSiM24 vector through vacuum infiltration method
A. tumefaciens carrying pSiM24 and pSiM24-GUS constructs infiltrated N. benthamiana leaves were assayed and histochemically stained for GUS enzyme. The completely infiltrated leaves showed approximately 1800 GUS units, whereas the partially infiltrated leaves exhibited approximately 850 GUS units ( Figure 11A). One unit of GUS activity was defined as the amount of enzyme that liberated 1 p mol 4-methylumbelliferone mg 21 protein min 21 [52]. The agro-infiltrated leaves showed strong GUS expression, as detected by histochemical staining, in leaves of both N. benthamiana and Zea mays ( Figure 11B and Figure 12).
Transient expression of GFP-AtCESA3 ixr1-2 , Vip3A(a), KMP-11, IL-10 and nat-T-phyllo-GFP genes using pSiM24 vector Western blot analysis of pSiM24-GFP-AtCESA3 ixr1-2 agroinfiltrated leaf samples showed the expected bands of size 145 kD for GFP-AtCESA3 ixr1-2 as detected with AtCESA3-specific polyclonal antibodies ( Figure 13A). In addition, RT-PCR analysis of agroinfiltrated leaf samples exhibited expected 1318 bp band for a portion of GFP-AtCESA3 ixr1-2 ( Figure 13A). The transient expression of Vip3A(a) using pSiM24 expression vector in tobacco protoplast was detected by Vip3A-specific polyclonal antibodies that showed the expected bands of size 88 kD ( Figure 13B). Using pSiM24 expression vector KMP-11 and IL-10 were also expressed transiently in tobacco protoplasts and showed expression up to 0.03 mg of KMP-11 and 0.08 mg of IL-10 per mg of protoplast protein samples by indirect ELISA (Figure 13C). The RT-PCR analysis and localization analysis of apoplast targeted nat-Tphyllo-GFP by confocal laser scanning microscopy showed the successful expression of nat-T-phyllo-GFP using pSiM24 expression vector ( Figure 14).

Discussion
A binary vector, used as a standard tool in the transformation of higher plants mediated by A. tumefaciens, consists of T-DNA and  [7,53]. The vector backbone carries plasmid replication functions for E. coli and A. tumefaciens, selectable marker genes for the bacteria, optionally a function for plasmid mobilization between bacteria and other accessory components [7,8].
The binary vector pSiM24 has an overall size of 7.08 kb and carries a plant-gene expression cassette containing a highly active, constitutive promoter (M24) (GenBank Accession no. KF032933). The size of the pSiM24 vector is approximately 2000 bp shorter than the commercially available pCAMBIA vectors (www.cambia. org) and approximately 6000 bp shorter than pKYLX-based vectors [4]. In the pSiM24 binary vector, only the necessary elements were included to attain a minimum size. The right border (RB) and the left border (LB) of pSiM24 are imperfect, direct repeats of 25 bases. The RB and LB are considered to be the only essential cis-elements for T-DNA transfer [54]. The promoter carried by the expression cassettes described here has been studied in transgenic plants (present study) and is also functional in plants such as tobacco [41][42][43][44]55], Arabidopsis and corn (Sahoo and Maiti, Unpublished Data). It has been documented that the Mirabilis mosaic virus full-length transcript promoter is constitutive in nature, exhibiting 14 to 25 times stronger activity than CaMV35S in the tobacco protoplast transient system and transgenic tobacco plants, respectively [27,34,42]. The modified full-length transcript promoter (M24) of the Mirabilis mosaic virus with duplicated enhancer domains [27,29,[41][42][43][44]55] was used in the pSiM24 vector to evaluate gene expression in plants. In the pSiM24-GUS vector, the coding sequence of GUS was placed between the heterologous M24 promoter and the terminator sequence from the rbcSE9 gene ( Figure 1) [43][44]. We showed that microT-DNAs in pSiM24 containing a kanamycin resistance gene and reporter gene (GUS or GFP) were integrated stably in the nuclear chromosomal DNA of transgenic plants for successive generation.
Selectable markers need to be expressed in calli, in cells from those plants that are being regenerated or in germinating embryos to facilitate plant transformation. Therefore, promoters for constitutive expression are preferred. In pSiM24, the Nos Representative transgenic Arabidopsis plant leaves (second generation, two weeks old) generated by agrobacterium-mediated transformation were imaged to determine GFP activity. Fluorescent, bright-field and superimposed (bright-field and green fluorescent) confocal laser scanning micrographs of transgenic Arabidopsis leaves. Scale bar represents 320 mm. (B) Representative transgenic Arabidopsis plant stems (second generation, two weeks old) generated by agrobacterium-mediated transformation were imaged to determine GFP activity. Fluorescent, bright-field and superimposed (bright-field and green fluorescent) confocal laser scanning micrographs of transgenic Arabidopsis stems. Scale bar represents 220 mm. (C) Representative transgenic Arabidopsis plant stem-root junctions (second generation, two weeks old) generated by agrobacterium-mediated transformation were imaged to determine GFP activity. Fluorescent, bright-field and superimposed (bright-field and green fluorescent) confocal laser scanning micrographs of transgenic Arabidopsis stem-root junctions. Scale bar represents 220 mm. (D) Representative transgenic Arabidopsis plant roots (second generation, two weeks old) generated by agrobacterium-mediated transformation were imaged to determine GFP activity. Fluorescent, bright-field and superimposed (bright-field and green fluorescent) confocal laser scanning micrographs of transgenic Arabidopsis roots. Scale bar represents 225 mm. doi:10.1371/journal.pone.0098988.g008 pSiM24: A User Friendly Small Ti Binary Vector pSiM24: A User Friendly Small Ti Binary Vector promoter derived from nopaline synthase (Nos) of A. tumefaciens [56] was used to express the selectable marker gene (Figure 1). The choice of promoters responsible for selectable marker gene expression also plays an important role in the efficiency of transformation [57][58][59]. The use of weak promoters may not always be a bad idea because the levels of expression of marker genes and genes of interest are often linked, and the selection of transformants with weak selectable markers may cause strong expression of the gene of interest to be obtained [8]. It is generally recommended that different promoters be used for the selectable marker and expressing the gene of interest [57][58][59], as in the pSiM24 vector (Figure 1), which carries the M24 promoter for the expression of the gene of interest (here GUS in pSiM24-GUS) and Nos for the selectable marker (here, KanR). Homology-based gene silencing has been reported to occur extensively in transgenic plants [60]. Gene silencing due to promoter homology can be avoided by either using diverse promoters isolated from different plant and viral genomes or by designing synthetic promoters [27,28,34,38,[61][62][63][64][65][66][67][68].
Depending on the plant species to be transformed, the choice of selectable markers greatly affects the efficiency of transformation, and permissive concentrations of selective agents vary considerably among plant species. Genes resistant to antibiotics or herbicides, such as kanamycin, hygromycin, phosphinothricin and glyphosate, are very popular. Kanamycin resistance has been most frequently exploited in the transformation of many dicotyledonous plants such as tobacco, tomato, potato and Arabidopsis [34,42,69]. The pSiM24 binary vector contains a synthetic 'nptII' KanR gene (nos promoter-KanR cDNA-Nos terminator), the open reading frame of which is optimized for plant codon bias; hence, the nptII gene serves both as a selectable marker for the regeneration of plantlets on kanamycin-containing medium (for tobacco 250-300 mg/ml) and as a screenable marker for agrobacterium (25 mg/ml). In the present study, pSiM24-containing nptII gene was used to select the transformed Arabidopsis and tobacco plants in 30 mg/ml and 250 mg/ml kanamycin, respectively, (Figure 6-7). Choice of antibiotics is an important factor in plant transformation. For example, kanamycin may not suitable for rice and maize cells, whereas hygromycin resistance (hpt) is very good for rice transformation [10], and the phosphinothricin resistance gene (bar) is efficient for maize and other cereals [70,71]. We also developed a binary vector pKDH, which has a structure similar to that of pSiM24, but the selection marker KanR gene was replaced with a hygromycin resistance (HygR, Hygromycin B transferase, HPH) gene for the selection of transgenic monocot plants, and the sequence information of the binary vector pKDH was provided (Genebank Accession no. KF041008).
The components of the pSiM24 expression system vector are arranged in a modular configuration in which the promoter, terminator and MCS cassettes are flanked by unique restriction endonuclease cleavage sites. The pSiM24 vector provides nine unique cloning sites in the first multiple cloning site (MCS) between the left T-DNA border and the M24 promoter (BstXI, StuI, EspAI, PasI, KflI, Bstz17I, SmaI, XmaI and EcoRI), twelve unique cloning sites in the second MCS between the M24 promoter and the pea rbcSE9 terminator (HindIII, AbsI, PspXI, The data represent means 6 S.D. of four second generation individuals from one line for each tissue (n = 4). The values significantly differ between control and transgenic plants at P,0.01 based on Student's t-test. T1, T2 and T3: Representative transgenic lines generated by floral-dip plant transformation procedure. doi:10.1371/journal.pone.0098988.g009 SciI, XhoI, HpaI, MluI, Eco53kI, SacI, SbfI, PstI, and XbaI) and seven unique cloning sites in the third MCS between the Nos promoter and the right T-DNA border (BglII, BstEII, EcoNI, FseI, SwaI, NruI, and PacI). This configuration facilitates the modification or replacement of individual components in the pSiM24 vector. The MCS in pSiM24 contains more additional cleavage sites than that of pUC19. It should be noted that the orientation of the MCS in the pSiM24 plasmid, relative to the rbcS and M24 promoters, is opposite that in pUC19, relative to the lac promoter. The presence of a number of cloning sites unique to the three MCS allow for gene-stacking applications to introduce multiple gene with additional sequences, such as translational initiation, signal and transit peptide sequences and translational termination, into these plasmids. The pSiM24 vector provides a number of options for cloning, transformation and expression strategies. The M24 promoter in the pSiM24 plasmid can be easily replaced with other promoters as EcoRI-HindIII cassettes, thus making different strategies for the regulated expression of foreign genes possible.
Reporter genes, whose expression can be easily monitored, are useful in many ways in plant transformation. Strength and temporal, spatial and other types of regulation of promoters and elements may be conveniently assayed by connecting these elements to the reporter genes. Genes for b-glucuronidase (GUS) [9], luciferase [72] and GFP [73] are popular examples. In the present study, two different reporter genes, GUS and GFP, were introduced into the pSiM24 vector to monitor and analyze their expression under the M24 promoter in both stable and transient systems. Reporter genes that are connected to constitutive promoters may be used to monitor the process of transformation. The expression of the reporter genes soon after the inoculation of plant cells with A. tumefaciens, which is referred to as ''transient expression'', is a good indication of the transfer of the T-DNA from the bacteria to the nuclei of plant cells. The expression of the reporter genes later in a cluster of cells growing on selection media provides evidence of the integration of the T-DNA in plant chromosomes. A binary vector that carries a constitutive selectable marker and a constitutive reporter is very useful as a control vector both in transformation experiments and in assays of gene expression. Hence, in pSiM24, both ''nptII'' and GUS/GFP were constitutively expressed by using two different constitutive promoters, i.e., Nos for nptII and M24 for GUS/GFP, for expression in transgenic plants ( Figure 6-10).
The rbcSE9 polyadenylation signal used in the pSiM24 vector has previously been used to direct efficient mRNA39 end formation from chimeric genes in transformed tobacco [74][75][76]. These 39 ends are identical to those observed in pea, indicating that this signal is suitable for the predictable expression of foreign genes in plants. The 39 regions of the cauliflower mosaic virus 35S transcript and the nopaline synthase gene in the wild-type T-DNA of A. tumefaciens are frequently used as a 39 signal to direct selectable marker genes expression.
In pSiM24, the ''bla'' AmpR gene, which confers resistance to ampicillin, was used as the marker for bacterial selection for E. coli. The selectable marker for plants, Nos-nptII, in pSiM24 also provides fair levels of resistance to both E. coli and A. tumefaciens. Binary vectors need to be replicated both in E. coli and A. tumefaciens. Hence, the pSiM24 vector carries all of the functions necessary for replication and transfer in Escherichia coli and A. tumefaciens, which includes a ColE1-replicon and an RK2-replicon derived from pRK2013 [77]. The pSiM24 binary vector carries the origin of vegetative replication (OriV) and the transacting  replication functions (Trf) of plasmid incompatibility group P (IncP) plasmids [78]. The types of replication functions exploited determine the copy numbers and stability of the plasmids in bacterial cells. E. coli exhibited a transformation frequency up to five-to six-fold higher with pSiM24 than with conventional pCAMBIA and pKM24KH vectors, (Table 1) and the plasmid DNA yields of pSiM24 binary Ti vectors were three-fold and seven-to eight-fold higher in E. coli than those of conventional pCAMBIA 2300 and pKM24KH, respectively ( Table 3). The pSiM24 binary vector contains the ColE1 replicon without a bom (basis of mobility) sequence, which again reduces its size. The bom function is necessary for plasmid mobilization from E. coli to A. tumefaciens [79]. This function is not necessary when vectors are introduced into A. tumefaciens by electroporation or freeze-thaw methods.
Not only reporter genes, other introduced genes of size up to 4 kb were also effectively expressed using pSiM24 expression vector. In the present study, nat-T-phyllo-GFP [44] was expressed transiently using pSiM24 expression vector in tobacco leaves. Tphylloplanins have antimicrobial properties and are known to inhibit blue mold disease caused by Peronospora tabacina [41,44,80,81]. Both the native and mature tobacco phylloplanin gene fused with GFP targeted to the apoplasm increases resistance to blue mold disease in tobacco [41,44]. Here, the expression of nat-T-phyllo-GFP using pSiM24 vector was confirmed by the GFP fluorescence in apoplast region of agroinfiltrated plant leaves ( Figure 14). Another, chimeric gene (GFP-AtCESA3 ixr1-2 ) of size 4086-bp fragment was also successfully expressed transiently using pSiM24 following agro-infiltration procedure. The overexpression of GFP fused to the Arabidopsis CESA3 ixr1-2 (GFP-AtCESA3 ixr1-2 ) gene in transgenic tobacco was known for increasing cellulose digestibility and biomass saccharification [42,43]. Further genes like Vip3A(a), KMP-11 and IL-10 were also successfully expressed transiently in tobacco protoplasts using pSiM24 expression vector (Figures 13). The gene product of novel vegetative insecticidal gene, vip3A(a) shows activity against lepidopteran insects [45,46]. KMP-11, a flagellar protein is known to play an essential role in regulating cytokinesis in both amastigote and promastigote forms of leishmania [47] and is a potential stimulator of T-lymphocyte proliferation [82]. Interleukin-10 (IL-10), an anti-inflammatory cytokine secreted under different conditions of immune activation by a variety of cell types, including T cells, B cells, and monocytes/ macrophages [83,84] has been shown to suppress a broad range of inflammatory responses and as an important factor in maintaining homeostasis of overall immune responses [85,86] and thus has been used for developing novel therapies for several human diseases such as allergic responses and autoimmune diseases [87].
The effect of pSiM24 binary vector on transformation frequency studied in A. thaliana verified the pSiM24 and pSiM24-GUS/GFP vectors exhibited more transformation frequency in A. thaliana than pCAMBIA2300 and pKM24KH vectors ( Table 5) however strong GUS transgene expression in pSiM24-GUS transgenic tobacco and Arabidopsis than pK-CaMV35S-GUS transgenic plants depends upon the strong M24 promoter ( Figure 5) [41,42].
The pSiM24 vector was observed to be active in transferring the transgene both transiently (Figure 3-5, Figure 11-14) and stably ( Figure 6-10) in plant systems, making it useful for various plant biotechnological applications. This plasmid has multiple cloning sites and can act as a platform for various applications, such as gene expression studies and different promoter expressional analyses. In addition, pSiM24 offers a wide selection of cloning sites and high copy numbers in E. coli for the facile manipulation of different genetic elements. Thus, the pSiM24 binary vector system described in this study has a high degree of flexibility and may serve as a useful tool for the transformation of plants, making it apt for future use in field release experiments.