Functional Characterization of a Strong Bi-directional Constitutive Plant Promoter Isolated from Cotton Leaf Curl Burewala Virus

Cotton leaf curl Burewala virus (CLCuBuV), belonging to the genus Begomovirus, possesses single-stranded monopartite DNA genome. The bidirectional promoters representing Rep and coat protein (CP) genes of CLCuBuV were characterized and their efficacy was assayed. Rep and CP promoters of CLCuBuV and 35S promoter of Cauliflower mosaic virus (CaMV) were fused with β-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes. GUS activity in individual plant cells driven by Rep, CP and 35S promoters was estimated using real-time PCR and fluorometric GUS assay. Histochemical staining of GUS in transformed tobacco (Nicotiana tabacum cv. Xanthi) leaves showed highest expression driven by Rep promoter followed by 35S promoter and CP promoter. The expression level of GUS driven by Rep promoter in transformed tobacco plants was shown to be two to four-fold higher than that of 35S promoter, while the expression by CP promoter was slightly lower. Further, the expression of GFP was monitored in agroinfiltrated leaves of N. benthamiana, N. tabacum and cotton (Gossypium hirsutum) plants using confocal laser scanning microscopy. Rep promoter showed strong consistent transient expression in tobacco and cotton leaves as compared to 35S promoter. The strong constitutive CLCuBuV Rep promoter developed in this study could be very useful for high level expression of transgenes in a wide variety of plant cells.


Introduction
Members of the family Geminiviridae, commonly called as geminiviruses, are characterized by having single-stranded, circular DNA genome of 2.5-3.0 kb, which is either monopartite or bipartite and encapsidated within virions having geminate morphology. Different geminiviruses can infect either monocotyledonous or dicotyledonous plants, and collectively, these viruses cause a variety of major crop diseases. On the basis of genome organization, insect vector and host range, the family, Geminiviridae is differentiated into seven genera, Curtovirus, Topocuvirus, Mastrivirus, Begomovirus, Becurtovirus, Eragrovirus and Turncurtovirus [1][2][3].
Begomovirus is the largest genus among geminiviruses having more than 200 species and transmitted by whitefly (Bemisia tabaci) vector. Monopartite begomoviruses have one genomic component (DNA-A) with six open reading frames (ORFs). The coat protein gene (AV1) and AV2 are expressed from the viral sense strand while Rep gene (AC1), AC2, AC3 and AC4 are expressed from the complimentary sense strand [4]. Most monopartite begomoviruses are associated with satellite molecule called betasatellite which is often required for the development of wild type symptom in naturally-infected host plant. It requires a helper begomovirus for its replication, systemic infection and whitefly vector-mediated transmission [5][6][7][8]. Some begomovirus betasatellite disease complexes are associated with a nanovirus like component named alphasatellite [8][9][10][11].
The large intergenic region (LIR) of monopartite begomovirus and common region (CR) of bipartite begomovirus contain bidirectional promoter which includes cis-acting regulatory elements, transcription factor binding sites (TFBs) and transcription start site required for the control of gene expression and replication [12]. Functional analysis of CR of bipartite and LIR of several monopartite geminiviruses have demonstrated that this region, located between 5 0 ends of the first complimentary and virion sense ORFs, possesses promoter activity and is essential for bidirectional transcription of both complimentary as well as virion sense genes [13][14][15][16]. In the absence of transcriptional activator protein AC2, complimentary sense (Rep) promoter of African cassava mosaic virus (ACMV), Tomato golden mosaic virus (TGMV) and Cotton leaf curl Multan virus (CLCuMuV) showed much stronger activity than the virion sense (CP) promoter [17][18][19]. LIR sequence of several geminiviruses share little sequence homology to one another but there are several conserved motifs present in almost all geminiviruses, like, two TATA boxes located at either side of LIR and GC-rich box (except ACMV) [20]. The TATA box motif and G-box motif located 15 nucleotide (nt) upstream of the stem-loop motif in TGMV is essential for activation of complementary sense gene transcription [21]. In Maize streak virus, deletion of GC-rich region resulted in 70% reduction of promoter activity [22]. The LIR also contains the origin of replication of viral genome. The iterated elements (8-13 nt) and stem-loop motif have been identified in the region of the putative TATA box of the complimentary sense promoter [20,22,23].
In the present study, the bidirectional promoters of Rep and CP genes from the LIR sequence of CLCuBuV were isolated and characterized. Further, expression patterns and activities of GUS reporter gene tagged with Rep and CP promoters were monitored, quantified and compared to CaMV 35S promoter in the transformed tobacco plants. Simultaneously, the GFP reporter gene tagged with Rep and CP promoters was also monitored both in tobacco and cotton leaves by agroinfiltration method. The observations of the present study revealed a promising future of CLCuBuV Rep promoter as a potential tool for ectopic gene expression in plants. Further, it may be an alternate for the most widely used CaMV 35S promoter in plant biotechnology applications.

Materials and Methods
Cloning and sequencing of CLCuBuV genome Cotton plants (Gossypium hirsutum) showing typical leaf curling, vein thickening and enation on the abaxial side of leaf were collected from a field in Lucknow, U. P., India (26°50 0 49.2@ N, 80°56 0 49.2@ E). The leaf sample of the diseased cotton plant was collected from a private land with the verbal consent of the owner to pick the leaf sample for academic purpose (Gossypium hirsutum is not an endangered or protected species), in September of 2010. Total genomic DNA from diseased cotton leaf was isolated using DNAeasy plant mini kit (QIAGEN, Germany) following the manufacturer's instructions. Begomoviral complex was confirmed as described by Kumar et al. [30]. Full-length genome of CLCuBuV was amplified using templiPhi DNA amplification kit (GE healthcare, USA) as per manufacturer's instructions. The RCA (rolling circle amplification) product was partially digested with 1 U of HindIII restriction enzyme (Fermentas, USA) and incubated at 37°C for 25 min. The purified DNA fragment was ligated into pBluscriptSK+ vector and transformed into Escherichia coli DH5α cells. The clone was sequenced by automated sequencer (Ocimum Biosolutions Ltd., India) and the nucleotide sequence was submitted in GenBank under the accession number KF767352. Genome organization of DNA-A component of CLCuBuV is represented in Fig. 1A.

Cloning of large intergenic region
Primers viz. PR For and PR Rev were designed on the basis of the submitted nucleotide sequence (KF767352) for amplification of LIR ( Table 1). The amplification was performed in 50 μl of reaction mixture containing 100 ng of template DNA (RCA product), 50 pmol of each primer, 1x Taq DNA polymerase buffer, 200 μM dNTPs and 1.25 U AmpliTaq DNA Polymerase (Applied Biosystems, USA). The reactions were conducted at 94°C for 5 min as initial denaturation followed by 30 cycles of 94°C for 1 min, 48°C for 2 min, 72°C for 1 min and final extension at 72°C for 5 min. The amplified DNA product was run on 1.5% agarose gel, stained with EtBr and visualized in Gel documentation system (BIO-RAD, USA). The PCR product of the expected size ca. 350 bp was recovered from the agarose gel and purified by using QIAEXII Gel Extraction Kit (QIAGEN, Germany). The quantity and purity of gel eluted DNA were measured by BioPhotometer plus (Eppendorf, Germany) and cloned into pDrive PCR cloning vector (QIAGEN, Germany). The positive colonies were confirmed by PCR and restriction digestion, and nucleotide sequences were determined using an automated sequencer (ABI3730XL, Applied Biosystems, USA).

Sequence analysis
The cis-regulatory elements of the bidirectional promoter sequence of CLCuBuV were analyzed using the Plant Cis-Acting Regulatory Element (Plant CARE) database [31] and Cister: Cis-Element Cluster Finder [32]. Transcription Factor Binding sites (TFBs) were identified by using Plant cis-acting regulatory DNA elements (PLACE) database and PlantPAN: Plant Promoter Analysis Navigator [33,34]. Neural Network Promoter Prediction (NNPP) server was used for the prediction of transcription start site (TSS) [35].

Development of LIR-GUS and LIR-GFP gene fusion vectors
Four plant expression vectors viz. pRepGUS1391Z, pCPGUS1391Z, pRepGFP0029 and pCPGFP0029 representing bidirectional CLCuBuV Rep/Cp promoter were constructed (Fig. 1B). For generating pRepGUS1391Z and pCPGUS1391Z vectors, LIR of CLCuBuV, previously cloned in pDrive vector, was digested with BglII restriction enzyme (Fermentas, USA) and ligated into pCAMBIA1391Z vector (Promoterless Vector kindly provided by Dr Sunil K Mukherjee, ICGEB, New Delhi, India). The integration and orientation of the bidirectional promoter was confirmed by PCR amplification and restriction digestion. The resulting plasmid was named pRepGUS1391Z and pCPGUS1391Z. For constructing LIR-GFP fusion constructs, LIR was first amplified using primer pair LIR For and LIR Rev ( Table 1). The PCR amplicon was cloned in pGEM-T Easy Vector (Promega, USA). GFP cloned in pBI121 vector was digested with EcoRI and BamHI restriction enzymes (Fermentas, USA) and ligated into plant expression vector pGreen0029 (kindly provided by Dr Neera Bhalla Sarin, Jawaharlal Nehru University, New Delhi, India). The resulting plasmid was named pGFP0029. LIR previously cloned into pGEM-T Easy Vector was digested with XbaI restriction enzyme (Fermentas, USA) and ligated into pGFP0029 vector. The orientation of the bidirectional promoter of CLCuBuV was confirmed by PCR, restriction digestion and sequencing. The resulting gene constructs were named as pRepGFP0029 and pCPGFP0029. pCAMBIA2301 containing Table 1. List of oligonucleotide primers used for amplifying promoter fragments and genes.

Name of primer
Primer sequences in 5 0 -3 0 CaMV 35S promoter-GUS-NOS polyA cassette and pBI121 containing CaMV 35S promoter-GFP-T NOS cassette were used as positive control.

Plant transformation
The plant expression vectors were introduced into Agrobacterium tumefaciens strain LBA4404 by freeze thaw method [36]. The gene constructs, namely pRepGFP0029 and pCPGFP0029, were mobilized in A. tumefaciens strain LBA4404 along with pSoup helper plasmid [37]. Agrobacterium-mediated transformation of leaf discs of Nicotiana tabacum cv. Xanthi was carried out essentially as described by Horsch et al. [38]. Regenerated hygromycin-resistant plants were grown in glasshouse and the presence of the promoter was confirmed by PCR.

Histochemical localization of GUS activity
GUS staining was performed essentially as described [39]. Apical leaves of transformed tobacco plants (or tissue samples), one month after sub culture were incubated overnight at 37°C in a solution containing 1 mM X-Gluc (Fermentas, USA), 50 mM sodium phosphate buffer pH 7.0, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 1 mM Na 2 EDTA, 0.1% Triton X100. The chlorophyll was removed using 70% ethanol. Petioles and leaves were sectioned by hand after staining. The samples were observed and photographed under Nikon 80I microscope (Nikon, Japan).

Fluorometric GUS activity assay
Quantitative GUS fluorometric assays were conducted according to the method of Jafferson [40] by measuring the fluorescence of 4-methylumbelliferone (MU) formed as a result of the cleavage of 4-methylumbelliferyl-β-D-glucuronide (MUG, Himedia) by GUS with a spectrofluorometer FP-6300 (JASCO, Japan). GUS activity was calculated as the production of MU from MUG in pmol/min/μg of protein. Agroinfiltration experiments were performed as essentially described previously [42]. Seeds of N. benthamiana, N. tabacum and G. hirsutum were planted and grown in growth chamber at 25°C under 16 h: 8 h, light: dark photoperiod. Plants were grown for 4 to 6 weeks before infiltration. The bacterial suspension was infiltrated into the intercellular space on the abaxial side of each intact leaf by using 2 ml syringe without needle. The plants were then placed in growth chamber (25°C, photoperiod, 16 h: 8 h, light: dark). The expression of GFP was monitored 72 h post infiltration under UV light. Further, the GFP expression was monitored under fluorescence microscope ECLIPSE 80i (Nikon, Japan) in the wavelength 330-380 nm.

Promoter expression analysis (CLSM)
Leaves of tobacco and cotton were agroinfiltrated with the individual promoter constructs carrying GFP reporter gene for assaying their transient activities as described earlier. The fluorescent images were captured with CLSM (Leica Microsystems, Germany) using LAS AF (Leica Application Suit Advanced Fluorescence) software under oil-immersion objective (40.0X1.15OIL). To excite the expressed GFP in leaves the AOTF of 488 nm (at 40%) was used and the fluorescence emissions were collected between 501 and 598 nm as described earlier [43].

Sequence analysis of CLCuBuV promoter
The bidirectional promoter was isolated from the LIR monopartite genome (DNA-A) of CLCuBuV. Its size was 353 nucleotides long (Fig. 2). Analysis of the promoter sequence of CLCuBuV revealed the presence of several putative cis elements such as G-box, stem-loop motif, TATA boxes, a GC-rich region (GTGGGCCCTACC) and a conserved late element (CLE) (Fig. 2, Tables 2 and S1). The TATA boxes and the GC rich region have been shown to be essential for the activity of the geminivirus promoters. The putative CLE (TTGTGGGCCCTA) sequence at +189 was suggested to be a potential functional target for AC2 gene to trans-activate the virion sense gene transcription [20,44]. The promoter sequence also revealed several potential TFBs, the E-box motifs (CANNTG) at sites +24, +80, +223, −29, −85 and −228 [45], the maize Dof transcription factor recognition core sequence AAAG at sites −7, −35, −131 and +237 [46] and the root specific ATATT motifs at sites +88, +162 and −165 [47] (Table 3).
Other potential TFBs and cis regulatory DNA elements were also identified by searching the Plant CARE, PLACE and PlantPAN databases as listed in Tables 2, 3

and S2.
Histochemical localization of GUS expression driven by CLCuBuV Rep, CLCuBuV CP and CaMV 35S promoter constructs PCR positive transformed tobacco plants were stained for GUS activity using X-Gluc staining solution. Staining patterns of representative plant leaves are shown in Fig. 3. GUS staining of the transgenic plants containing CLCuBuV Rep was shown to be stronger than that of the plants carrying CaMV 35S promoter and CLCuBuV CP. The expression pattern of CLCuBuV CP promoter was observed to be weaker than that of CaMV 35S promoter (Fig. 3A). The transverse section of leaf (vascular tissue, lamina and midrib) of tobacco plants transformed with CLCuBuV Rep promoter construct, showed strong GUS expression in the mesophyll (spongy and palisade) cells and trichome ( Fig. 3B and C).

GUS activity of CLCuBuV Rep and CP promoter in transformed tobacco
The GUS activity in hygromycin-resistant transformed tobacco plants was assayed by a fluorometric quantification method and measured in pmol 4-methyl umbelliferone (4MU)/min/μg proteins. The transgenic tobacco plants containing promoter-GUS constructs were regenerated from hygromycin-resistant calli. Transgenic lines of tobacco plants were established for each construct. Presence of the GUS gene and promoter sequence was confirmed by PCR. The GUS activity was assayed in mature leaves, shoots and roots of 4 weeks old transgenic lines for each construct. The average level of GUS activity in mature leaves, transformed with pRe-pGUS1391Z, pCPGUS1391Z and pCAMBIA2301 gene constructs, were 5175.12 (±180.74),   Average value of GUS activity in roots, transformed with pRepGUS1391Z, pCPGUS1391Z and pCAMBIA2301 constructs, were 12835.02 (±492.25), 2882.52 (±262.83) and 4607.38 (±466.80) pmol 4MU/min/μg proteins, respectively (Fig. 4C) (p < 0.001 by student's t test). The control tobacco plants showed no expression of GUS.
The efficacy of CLCuBuV Rep promoter was found to be higher than CaMV 35S promoter in leaves, shoots and roots of all transgenic plants. However, the expression level of CLCuBuV CP promoter was lower than that of CaMV 35S promoter in the tissues of leaves, shoots and roots of the transformed plants. The GUS activity in the roots appeared to be higher than that of the shoots and leaves (Fig. 4A, B and C).

Transcriptional regulation of GUS
To confirm the expression of GUS gene at mRNA level, RT-PCR analysis was performed using total RNA samples from transgenic and non-transgenic plants of Nicotiana tabacum cv. Xanthi. The RT-PCR analysis of transgenic plants showed that the transgenes were expressed differentially in these transgenic plants (Fig. 5A). Further, no amplification was observed in non-transgenic tobacco plant employing GUS-specific primers but amplification with actinspecific primers was observed in both transgenic and non-transgenic tobacco plants (Fig. 5A). The CLCuBuV Rep promoter transformed plants had shown higher expression level than those of CLCuBuV CP and CaMV 35S promoter. The qRT-PCR was performed to study the expression of GUS gene under the control of CLCuBuV Rep, CLCuBuV CP and CaMV 35S promoter. Results revealed that GUS gene transcript level was four-fold higher in CLCuBuV Rep promoter transformed plants compared to that of CaMV 35S promoter (Fig. 5B). Transcript level of GUS gene under the control of CaMV 35S promoter was observed slightly higher than that of CLCuBuV CP promoter.

Visualization of transiently expressed GFP in tobacco and cotton leaves
The expression of GFP was detected in leaves of agroinfiltrated plants of both tobacco and cotton under ultraviolet light 3 days post-infiltration. Fluorescent image of leaves expressing GFP in tobacco and cotton were captured by the confocal microscope and fluorescence microscope as described earlier and are presented in Figs. 6 and 7, respectively. Higher fluorescent intensity of GFP was found in both tobacco and cotton leaves agroinfiltrated with CLCuBuV Rep promoter construct as compared to CaMV 35S and CLCuBuV CP promoter constructs.

Discussion
Efficient genetic modification of plants for agronomic traits requires the use of efficient regulatory sequences to drive the expression of trans-genes in specific plant tissues or at certain developmental stages. The pattern of expression provided by different promoters must be analyzed before they can be used to generate transgenic plants commercially. In this study the sequence of LIR from CLCuBuV was analyzed. We have compared the expression patterns of GUS gene driven by CLCuBuV Rep and CLCuBuV CP promoters with CaMV35S promoter in transformed transgenic tobacco plants. The highest expression of GUS gene was obtained using the CLCuBuV Rep promoter followed by the CaMV 35S promoter and the lowest activity was observed with CLCuBuV CP promoter (Figs. 4 and 5). In the transgenic tobacco plants the CLCuBuV Rep promoter showed strong GUS activity while the CLCuBuV CP promoter showed very weak activity. This may be due to the absence of AC2 gene product in CLCuBuV [24][25][26]. Role of transactivator AC2 protein for activation of virion sense promoter has already been reported [65,66]. The supremacy of Rep (AC1) promoter over the CP (AV1) promoter, in the absence of transactivtor AC2 protein was demonstrated in CLCuMuV [19], ACMV [18], Wheat dwarf virus [67] and Mungbean yellow mosaic India virus [68]. Gene expression process in geminivirus shows an early expression of complimentary sense gene, whose products are then involved in viral replication such as AC1 (Rep) or they may act as transcription activator for the virion sense gene expression, such as AC2 (TrAp). In contrast the expression of virion sense genes usually appears later and requires complimentary sense gene product/products for activation. The staining of GUS activity showed that the CLCuBuV promoter was active in all the tobacco cells observed. The strongest histochemical staining for GUS activity was observed in the mesophyll cells and vascular tissue of the leaf transformed with CLCuBuV Rep promoter construct (Fig. 3). The quantitative analysis of GUS assay clearly revealed that CLCuBuV Rep promoter had two to four-fold higher activity than the CaMV 35S promoter, while CLCuBuV CP promoter was lagging behind CLCuBuV Rep and CaMV 35S promoter, demonstrating least activity among the three (Figs. 4 and 5).
Transcription level of the GUS reporter gene driven by CLCuBuV was quantified for the first time in this study. RT-PCR and RT-qPCR based analysis of GUS-specific transcripts isolated from transgenic tobacco plants containing CLCuBuV Rep, CLCuBuV CP and CaMV 35S promoter constructs clearly showed a fair correlation between the steady-state level of mRNA (transcript level) and GUS activity (protein level) (Fig. 5). Based on the above experimental evidence, it was confirmed that the activity of CLCuBuV Rep promoter was significantly higher than that of CaMV 35S and CLCuBuV CP promoters.
For the first time transient expression of GFP reporter gene under the control of CLCuBuV promoters in different plants, such as, N. tabacum, N. benthamiana and G. hirsutum was also investigated. Simultaneously, a positive control constitutive promoter CaMV 35S associated with GFP was also used to study the degree of effectiveness between the promoters such as CLCuBuV Rep, CLCuBuV CP and CaMV 35S. In this study, the expression and localization of the GFP reporter gene in agroinfiltrated leaves was monitored by confocal laser scanning microscopy (Fig. 6). Our histochemical results clearly showed that expression pattern of GFP under the control of CLCuBuV Rep promoter were found to be more effective than CaMV 35S promoter, used in consequent experiments.
Though several groups have carried out similar studies in tobacco and cotton plants, they did not quantify the expression level driven by these promoters at transcriptional level [15,16,19]. Ashraf et al. [15] quantified the transiently expressed GUS activity driven by CLCu-BuV C1 gene promoter by fluorometric assay. Evidence was provided exhibiting 2-3 fold higher GUS activity of CLCuBuV promoter than that of 35S. In present study, GUS activity was quantified in stably transformed transgenic tobacco plants by RT-qPCR as well as fluorometric assay. RT-qPCR clearly demonstrated that the expression of GUS activity was up to four fold higher than 35S promoter. Further, for the first time, expression of GFP reporter gene Characterization of Cotton Leaf Curl Burewala Virus Promoter under the control of CLCuBuV promoters (Rep and CP) was in situ localized in three different plant species such as N. tabacum, N. benthamiana and G. hirsutum using confocal laser scanning microscopy. Not only the novelty and degree of effectiveness of these promoters in three different plants (N. tabacum, N. benthamiana and G. hirsutum) was demonstrated, but it also reflects that it may be used to develop other valuable agronomic traits. The observations of the present study reveal a promising future of CLCuBuV Rep promoter as a potential tool for ectopic gene expression in plants, particularly cotton.