Cloning of a Novel Antifungal Promoter from Phaseolus vulgaris and the Determination of its Activity in Stably Transformed Nicotiana tabacum plants

To investigate the transcriptional regulation of gene expression, chimeric fusions, between the β-glucuronidase reporter gene (GUS) and the isolated promoter regions of the pvPDF gene (pvPDF-PRO: GUS), were constructed and introduced into Nicotiana tabacum. Analysis of transgenic pvPDF-PRO:GUS tobacco plants indicated that GUS activity was observed with all the promoter constructs with the strongest being in leaf followed by stem and roots. These results clearly demonstrate that pvPDF-PRO is a strong inducible and near-constitutive promoter and emphasize the great application potential for plant genetic engineering studies. Interestingly, a search for putative cis-acting elements in the pvPDF promoter architecture revealed the presence of some important transcription regulatory elements including: CAAT Box, TATA Box, CATA Box, and light regulatory elements (CCA1, GATA, GT-1). Taken together, these results further our understanding of the regulation of the pvPDF promoter activity.


Introduction
Genetic transformation is a powerful tool for production of crop plants with increased resistance to phytopathogens.A number of transgenic cultivars with heightened tolerance to economically important pests and disease agents are in commercial production.However, in most cases the transgene is driven by a powerful constitutive promoter, such as the cauliflower mosaic virus 35S (CaMV35S) and its derivatives, and is expressed at high levels even in the absence of pathogen invasion.Continuous synthesis and high accumulation of transgene products, especially toxins, could interfere with plant metabolic pathways and the overall expression of other valuable traits.In contrast, the use of promoters of plant defense genes has distinct advantages as the majority is only activated following plant attack by pests or pathogens.The use of native plant promoters can also help to avoid transgene silencing often associated with the presence of promoters of non-plant origin in the plant genome (Yevtushenko et al., 2004;Vieweg et al., 2004;Nishiuchi et al., 2004;Ross et al., 2004;Matarasso et al., 2005;Rubio-Somoza et al., 2006).
Plants have developed a variety of physical and biochemical defense barriers against pests and pathogens.Mechanical wounding of plant tissue, mimicking pathogen invasion or insect chewing, leads to the accumulation of mRNAs that encode proteins thought to be involved in plant defense (Yevtushenko et al., 2004;Bowles et al., 1990), and provides a convenient system to isolate and study defense-related genes and their upstream regulatory regions in transgenic hosts (Clarke et al., 1994;Hollick et al., 1993).Potato plants can be engineered for broad-spectrum disease resistance by expression of antimicrobial peptides under control of a constitutive CaMV 35S promoter (Osusky et al., 2000).
In recent years, concerns over genetic modification issues have resulted in regulatory authorities requiring comprehensive analysis of transgene insertion events in the plants that are to be commercialized.Recent studies have suggested that applied and modified a genomic walking method that combines vectorette and suppression PCR walking.Some studies have suggested that stably expressed transgenes comprise relatively simple T-DNA arrangements flanked on at least one side by plant DNA and that unstably expressed loci tend to be composed of multiple T-DNA copies (Iglesias et al., 1997).Several PCR-based walking methods have been described (Ochman et al., 1988;Rosenthal and Jones, 1990;Riley et al., 1990;Lagerstrom et al., 1991;Parker et al., 1991;Trueba and Johnson, 1996;Jones and Winistorfer, 1993).However, these methods generally have not been applied in determining T-DNA insertion sites because they are too inefficient and/or complicated.
The genes which encode the various components of the pre-mRNA splicing or rRNA processing machinery provide a rich source of promoters for transgene

Phaseolus vulgaris and the Determination of its Activity in Stably Transformed Nicotiana tabacum plants
*For correspondence: helshemy@hotmail.comi56 Mahmoud et al.
expression in plant biotechnology.As most genes are organised in multigene families with great variability in expression levels and patterns, a novel approach has been developed to allow the identification and isolation of promoters with the required expression characteristics.This approach proves valuable for promoter isolation and exploitation (Sunter and Bisaro, 2003;Van et al., 2002).
This work describes the isolation and functional characterization of the plant-defensin 5′-untranscribed promoter from Phaseolus vulgaris, designated pvPDF-PRO1, pvPDF-PRO2 and pvPDF-PRO3 fragments.Their functional analyses and promoter activity in chimeric reporter constructs (pvPDF-PRO: GUS) in transgenic tobacco plants are discussed, in the light of the potential use of these promoters in plant biotechnology.

Plant material and growth conditions
Kidney Bean, Phaseolus vulgaris, seeds were grown on MS media at 25 ± 2ºC for 7 days in dark.The harvested material was immediately frozen in liquid N 2 and stored at -70ºC.For transformation studies, Nicotiana tabacum var Xanthi plants were grown at 25 ± 2ºC in 16 h light and 8 h dark.

Cloning strategy of kidney bean pvPDF promoters
The cloning of the pvPDF promoter was carried out according to Reddy et al. (1999 and2002).Purified Phaseolus vulgaris genomic DNA was restrictiondigested overnight at 37ºC, with 80 units of restriction enzymes for the concentration of 40 µg/l DNA: e.g.BamHI, BglII and Sou3A (NEB).Following heat inactivation of enzymes at 65ºC for 10 min and centrifugation, the resulting DNA pellets were then washed with 70% EtOH, allowed to air dry and subsequently resuspended in 20 µl sterile double-distilled H 2 O.The digested DNA were electrophoresed on 0.8% agarose gel and processed for cutting the region from 0.5 to 1.0 kb.Each fragment was purified using Miniprep columns (Qiagen).The digested DNA (20 µg) was partially end-filled by dGTP and dATP .The reaction mixture contained dNTPs (2mM each), 10X reaction buffer (2.5 µl), klenow enzyme (2 µl) in reaction volume of 50 µl and was incubated at 37ºC for 30 min.The DNA was purified through Miniprep purification Columns (Qiagen).The following oligonucleotide adaptors were employed: ADOP-32, 5´-AATACGACTCACTATAGGGCGGCCGCCCGGGC-3´ and ADOP-27, IDT Inc.).A volume of 20 µl of each adaptor (50 ng/l) was pipetted into a 0.5-ml Eppendorf microfuge tube and overlaid with mineral oil.The adaptors were heated at 99ºC for 4 min in a beaker of water.The heat was removed, and the solution was allowed to cool for 1 h at room temperature.The annealed adaptors were decanted from under oil and stored at -20ºC.Then, 10 µl of the genomic restriction digest was ligated to 1 µl of the annealed adaptors with 2 µl of T4 DNA ligase buffer and 2 µl of T4 DNA ligase (5 U/µl) in a 20-µl reaction.The ligation was incubated overnight at 12ºC and heat inactivated at 65ºC for 10 min.A 180-µl volume of TE (pH 8) was added to the ligation mix; this is called the adaptor library.Excess primer-adapter was removed by purification through QIAquik Columns (Qiagen).

Manipulation of plant nucleic acids
Nucleic acid manipulations were essentially carried out according to Sambrook et al. (1989).Genomic DNA was extracted and purified on a mini-prep scale as described by Murray and Thompson (1980).Total RNA from plant tissues was isolated as mentioned by Chomczynski and Sacchi (1987) with some modifications.Target PCR products were cleaned up by Gel Extraction Kit (Promega) and cloned into the pGEMT vector kit (Promega), then transformed into E. coli DH5α according to Hanahan (1985).Positive clones were confirmed by restriction analysis and sequenced using Sanger Sequencing Technology on ABI Prism 3730XL (Applied Biosystems/ Sanger) according to the dideoxy chain-termination method (Sanger et al., 1977).

Preparation of chimeric constructs
Plasmid vector pBI121 (Bevan, 1984) was used for cloning of pvPDF promoter upstream from GUS gene (Fig. 1).These constructs were then utilized for tobacco transformation.To generate pvPDF::GUS promoter construct, pvPDF promoter fragments, double-digested with BamH1/HindIII, were ligated into pBI121 binary vector digested with the same restriction enzymes.Ligation was carried out overnight at 14ºC in the presence of T 4 DNA Cloning of a Novel Antifungal Promoter from Phaseolus vulgaris i57 ligase.The ligation reaction was employed to transforme DH5α competent cells.

Plant transformation
Constructs were transferred to Agrobacterium tumefaciens strain LBA4404 by the freeze-thaw method according to An et al. (1988).To test for bona fide transformants, 3-day-old Agrobacterium colonies were further analyzed by PCR for the presence of the selectable marker gene nptII, using the following primers combination: Neo5, 5′-GAGGCTATTCGGCTATGACTG-3′ and NeoSHR, 5′-GGCCATTTTCCACCATGATA-3′.Leaf discs of sterilegrown Nicotiana tabacum were transformed essentially as described by Horsch et al. (1985), with minor modifications.Transformed plants were selected by rooting several times on kanamycin-containing medium and transferred to compost.

GUS histochemistry and quantitative assay
This method for screening the transgenic plants was done according to (Jefferson, 1987 andAllan et al., 1993) and allows for the verification of the expression of uid A gene in transgenic plants.Leaf tissue from wild type and transgenic plants was collected and rinsed in 50 mM Naphosphate buffer (pH 7.0).Then the tissue was stained with 2 mM 5-bromo-4-chloro-3-indolyl glucuronide (X-gal from Biosynth.Inc) in 50 mM of Na-phosphate buffer (pH 7.0), followed by brief vacuum infiltration and placed at 37ºC for overnight in dark.After staining, tissues were rinsed extensively in ethanol to remove chlorophyll before examination.Preparing X-GLUC Stain by Dissolve 5 mg X-gluc in 1.0 ml dimethyl formamide and adding 50 mM NaPO 4 pH 7.0 to 10 ml.
4-Methylumbelliferone (4-MU) production was measured at four time points using a fluorometer fitted with a microtiter plate reader (Millipore, Cytofluor 2350).Total protein determination was carried out according to Bradford (1976) using Bradford reagent ((Bio-Rad) and BSA as standard.Measurements were performed in triplicate and GUS activity expressed as pmol 4-MU/min/ mg protein.

Results and discussion
Identification and cloning of pvPDF promoters Plant promoter architecture is important for understanding regulation and evolution of the promoters, but our current knowledge about plant promoter structure, especially with respect to the core promoter, is insufficient.Several promoter elements including TATA box, and several types of transcriptional regulatory elements have been found to show local distribution within promoters, and this feature has been successfully utilized for extraction of promoter constituents from plant genome.
Promoters of various strengths and specificities are required for expression of foreign genes in plants for analysis of gene function or for biotechnological improvement of crop species.To meet such requirements and to have maximum precision in expression, a range of promoters with different expression levels and patterns is desired.Keeping this in mind, we carried out a program of isolation of plant promoters and functionally characterized them for suitability to express foreign genes using tobacco as a model system.One of the promoters, designated pvPDF, was identified and was shown to belong to a plant defensin gene encoding an antifungal protein from beans.
The isolation of unknown DNA sequences flanking known regions is critical for gene expression analysis.Several protocols have been developed to isolate an unknown DNA sequence (promoter) adjacent to DNA fragments of known sequence (cDNA) by PCR (Hui et al., 1998).A number of modifications were developed to isolate the unknown 5′ and 3′ flanking regions of the DNA.Usually, PCR was carried out with restriction enzyme(s)digested genomic DNA fragments after ligation (Siebert et al., 1995) or cloning into a vector (Niu and Fallon, 1999) or ligated to double-stranded, partially double-stranded (Iwahana et al., 1994;Willems, 1998), or single-stranded oligonucleotide cassettes (Kilstrup and Kristiansen, 2000).In these cases the amplifications were carried out with locus-specific primer(s) and a vector/oligonucleotide cassette specific-primer to amplify a fragment contiguous to the known sequence.In this method all the template molecules are likely to be amplified linearly leading to the generation of a lot of noise.In this study we have followed a protocol developed by Reddy et al. (2002) that involved the use of restriction digestion of genomic DNA followed by partial filling whereby preventing selfligation between fragments.In addition, the present protocol also employed biotinilated primers that enrich specific template prior to nested PCR.Also, one of the adopter strands was blocked at the 3′ end by attaching an amine group ( Reddy et al., 2002).Following this method a number of unknown 3′ and 5′ regions of pvPDF gene promoters were isolated in this paper (data not shown).Namely, a 730 bp fragment corresponding to the 5′ UTR region of pvPDF gene from bean, was isolated following this new method (Fig. 2).The major transcript was found to initiate from 270 bases upstream of the translation    (Hall, 1999).B The nucleotide sequence of the pvPDF full length gene after isolated from Kidney Beans and sequencing.
Cloning of a Novel Antifungal Promoter from Phaseolus vulgaris i59 initiation site.The minor transcript was found to initiate from 150 nucleotides upstream of the translation initiation site (data not shown).Similar result has been obtained by Reddy et al. (1999), in which they isolated genomic DNA fragments from tobacco-and pea-derived promoter regions for DNA topoisomerase I (topo I) using similar PCR based 5′ genome walking.In case of pea, they isolated 1140 bp and in tobacco a 482 bp upstream of ATG -5′ flanking region of tobacco topo I using 5′-RACE PCR-based approach (Reddy et al., 1999).Moreover, we have managed to cloned a putative 5′-UTR promoter region for the isolated pvPDF gene and have found that it possess various known plant regulatory motifs (Table 1, Fig. 2 and Fig. 3 A).Futhermore, the corresponding pvPDF gene was also successfully PCRamplified (Fig. 3 B).The pvPDF gene open reading frame was found to be composed of 195 bp and the exact sequence will be submitted to GenBank (Mahmoud et al., unpublished data).

Database-assisted pvPDF promoter sequence analyses
The sequence homology of the 5′-UTR region of the pvPDF gene isolated in the present study (Fig. 2) revealed the existence of some differences within the reported pvPDF gene sequence (data not shown).This could be due to the fact that the pvPDF exists in multiple copies and the difference could be due to isolation of promoter for the gene other than the one that was previously reported by (Siva Reddy ICGEB-India, personal communication).Analysis of regulatory sequences is greatly facilitated by database-assisted bioinformatic approaches.In this context, the TRANSFAC database contains information on transcription factors, their origin, functional properties and their sequence-specific binding activities (http://www.sphinx.rug.ac.be: 8080/PLANTCARE/) (Hobohm and Sander, 1995;Stultz et al., 1993).
By employing software tools it is possible to screen the database with a given DNA sequence for interacting transcription factors.If a regulatory function is already attributed to this sequence the database assisted identification of binding sites for proteins or protein classes and subsequent experimental verification may establish functionally relevant sites within this sequence.The binding transcription factors as well as interacting factors may already be present in the database.The putative cis-acting elements present in the isolated pvPDF promoter were identified using PLACE (Higo et al., 1999, http://www.dna.affrc.go.jp) (Table 1).A search for putative cis-acting elements in the pvPDF promoter revealed the presence of some important elements including: CAAT Box, TATA Box, CATA Box, and light regulatory elements or LREs (CCA1, GATA, GT-1).Interestingly, the pvPDF promoter has a TATA Box near the 5′ end of the pvPDF gene.The first TATA-like element detected is at −237 and the second TATA Box was at −685.The presence of CAAT Box1 motifs in the pvPDF promoter correlates with higher expression in leaves as compared to roots.Similar motifs have been reported in the promoter of the legA gene of pea (Shirsat et.al. 1989).The presence of light regulatory elements has the consensus GT1 motif binding site in many light-regulated genes (Le Gourrierec et.al. 1999).
In the pvPDF promoter one light regulatory element could been detected at the region between −251 to −258.Moreover, two GATA Box elements were identified, one at −678 to −682 and the other between −320 to −325.In this context, similar cis-acting elements have been reported in CaMV 35S promoter and in promoters from Petunia (Benfey and Chua, 1990;Gilmartin et al., 1990).The presence of light regulatory elements and tissue specific elements accounts for the tissue specific light regulation of pvPDF.Three putative Dof binding sites are present in the region between −703 to −706.Dof proteins are unique to plants and contain a highly conserved DNA binding domain that binds to a core AAAG sequence.
In maize, Dof1 is constitutively expressed in roots, leaves and stem and acts as a transcriptional activator while Dof2 is expressed in roots and stem and acts as a transcriptional repressor (Yanagisawa and Sheen, 1998) for light mediated expression of C4 photosynthetic phosphoenolpyruvate carboxylase (C4-PEPC).Thus, Dof proteins may have tissue specific effects.Dof proteins have also been implicated in regulation of hormonal responses and pathogen attack (Yanagisawa and Schmidt, 1999;Yanagisawa, 2000).The pvPDF-PRO promoter sequence also has a "Box II" box sequence at 231 to 234 sites.Similar cis-acting elements have been reported to be present in the tobacco plastid atpB gene promoter (Kapoor and Sugiura, 1999).
Responsiveness of Promoter Transgenic pvPDF: GUS Tobacco Plants Agrobacterium mediated leaf disk method was followed for transformation, pvPDF promoter sequences and the GUS expression.A frequency of 5-20 plantlets were obtained per explants, which was comparable to control pBI121 binary vector transformation frequency, indicating that the cloning of pvPDF promoter had no adverse effects on the overall transformation frequencies.Transformed plants rooted normally and no abonormalities associated with the transformation was observed (Fig. 4).Histochemical GUS staining of transgenic plants indicated that pvPDF: GUS promoter was active in almost all reproductive organs and the highest level was in the roots (Fig. 5).
We have utilized HindIII and BamHI sites present in the pBI121 vector to clone pvPDF promoters.Three different constructs were created that differed in their length.The pvPDF-PRO1 is the smallest promoter which was only 350 bp long, the pvPDF-PRO2 was 500 bp and the longest promoter, pvPDF-PRO3, contained 730 bp.All the promoters were cloned into the same HindIII and BamHI sites (Fig. 1) for a better comparison of promoter strength.As a control, pBI121 having 35S promoter was used.The CaMV 35S is a strong and constitutive promoter and used most extensively to express foreign genes in plants.Under 35S promoter, GUS expression was detected in all tissue types and at all developmental stages of tobacco plant growth.The expression was strongest in leaf tissue.The expression of GUS was observed with all three promoter constructs.The expression was strongest in the leaf followed by stem and roots (data not shown).Among the three constructs, was the highest pvPDF-PRO3 followed by pvPDF-PRO2, whereas GUS activity was low with pvPDF-PRO1 promoter construct (data not shown).However, the expression pattern for various tissues was similar in all the constructs.The activity of the pvPDF gene promoter, encoding a bean cysteine-rich antifungal peptide, was investigated in transgenic pvPDF::GUS tobacco plants.Quantitative GUS activity analysis of the transgenic plant leaves showed the average activity of the bean pvPDF-PRO was 2-to 3-fold higher than that of the CaMV35S promoter.Histochemical GUS staining of transgenic plants indicated that pvPDF::GUS promoter was active in almost all reproductive organs and the highest level was in the roots (data not shown).
Functional analysis of promoter 5′-deletion series indicated that promoter activity of a 355 nucleotide fragment (-355 to the transcription initiation site) and a 460 nucleotide fragment (-460 to the transcription initiation site) were 2-fold and 3-fold stronger than that of the pvPDF full-length promoter, respectively.These results demonstrate that the bean pvPDF promoter is a strong inducible and near-constitutive promoter in plants and has great application potential for plant genetic engineering studies.Although the CaMV35S promoter appeared to be a strong, constitutive promoter in assays involving cell extracts, detailed histological analysis of a reporter gene product that is detectable at the cell and tissue level showed a rather high degree of variability of expression of this gene product.This histological analysis revealed an unknown and unexpected variability in the expression of a gene product driven by the CaMV35S promoter.This variable level and site of expression is believed to have two primary causes, one reason is attributed to the fact that variability is an intrinsic property of the CaMV35S promoter (Rubio-Somoza et al., 2006;Matarasso et al., 2005;Vieweg et al., 2004;Nishiuchi et al., 2004;Tjaden et al., 1995;Ross et al., 2004).
Promoters of various strengths and specificities are required for expression of foreign genes in plants for functional analysis of gene expression and for biotechnological improvement of economically-important crop plants.To meet such requirements, a range of promoters with different expression levels and patterns is required.Keeping this in mind, we begun a program of isolation of plant promoters and characterize them for their suitability to express foreign genes using tobacco as a model system.In this work, we described the cloning and functional analysis of a Phaseolus vulgaris promoter (pvPDF), controlling the expression of a plant defensin gene encoding an antifungal protein.Based on the obtained results, we envisage that this promoter can be valuable for various plant biotechnological applications.

Fig. 1 .
Fig. 1.The pBI121 binary vector used in chimeric construct generation including the map positions of some restriction sites.

Fig. 3 .Fig. 4 .
Fig. 3.A Photograph of a gel showing a purified PCR product corresponding to the 730 bp full-length promoter of pvPDF from Kidney Beans prior to pGEMT cloning and sequencing.M,marker lane.B PCR-based isolation of the coding region of the pvPDF gene from Kidney Beans using the full-length primer set (See Materials and Methods).

Table 1 .
Putative cis-acting elements identified in the cloned pvPDF promoter sequence.These elements were identified using the Signal Scan Program at PLACE (http://www.dna.affrc.go.jp).