Plant Cell Tiss Organ Cult DOI 10.1007/s11240-010-9888-0
ORIGINAL PAPER
Botrytis cinerea-resistant marker-free Petunia hybrida produced using the MAT vector system Raham Sher Khan • Syed Sartaj Alam • Iqbal Munir • Pejman Azadi • Ikuo Nakamura Masahiro Mii
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Received: 21 July 2010 / Accepted: 15 November 2010 Ó Springer Science+Business Media B.V. 2010
Abstract The presence of marker genes conferring antibiotic or herbicide resistance in transgenic plants has been a controversial issue and a serious problem for their public acceptance and commercialization. The MAT (multi-autotransformation) vector system has been one of the strategies developed to excise the selection marker gene and produce marker-free transgenic plants. In an attempt to produce transgenic marker-free Petunia hybrida plants resistant to Botrytis cinerea (gray mold), we used the ipt gene as a selectable marker gene and the wasabi defensin (WD) gene, isolated from Wasabia japonica (a Japanese horseradish which has been a potential source of antimicrobial proteins), as a gene of interest. The WD gene was cloned from the binary vector, pEKH-WD, to an ipt-type MAT vector, pMAT21, by gateway cloning technology and transferred to Agrobacterium tumefaciens strain EHA105. Infected leaf explants of P. hybrida were cultured on hormone- and antibiotic-free MS medium. Extreme shooty phenotype (ESP)/ipt shoots were produced by the explants infected with the pMAT21-WD. The same antibiotic- and hormone-free MS medium was used in subcultures of the ipt shoots. Ipt shoots subsequently produced morphologically normal shoots. Molecular analyses of genomic DNA
R. S. Khan (&) S. S. Alam P. Azadi I. Nakamura M. Mii (&) Graduate School of Horticulture, Chiba University, 648 Matsudo, Matsudo, Chiba 271-8510, Japan e-mail:
[email protected] M. Mii e-mail:
[email protected] R. S. Khan I. Munir Institute of Biotechnology and Genetic Engineering, Khyber Pakhtunkhwa Agricultural University, Peshawar, Pakistan
from the transgenic plants confirmed the integration of the gene of interest and excision of the selection marker. Expression of the WD gene was confirmed by northern blot and western blot analyses. A disease resistance assay of the marker-free transgenic plants exhibited enhanced resistance against B. cinerea strain 40 isolated from P. hybrida. Keywords MAT vector ipt gene Wasabi defensin gene Agrobacterium tumefaciens Petunia hybrida
Introduction Antibiotic- and/or herbicide-resistant genes are widely used as selectable markers in plant transformation (Bevan et al. 1983; Waldron et al. 1984; Akama et al. 1995). Once the transgenic plants have been developed, however, these marker genes are redundant, although still incorporated into the genome, and this continuous existence in the genome of the transgenic plants raises issues of environmental and ecological concerns (Dale 1992; Gressel 1992; Nap et al. 1992; Hill and Sendashonga 2006). Moreover, it is difficult to pyramid transgenes (gene stacking) with the same selectable marker. In addition, the existence of marker genes in transgenic crops—and their gene products—needs to be evaluated by additional and lengthy risk assessment process for their public use (Goldstein et al. 2005). Several strategies have been tested to produce transgenic plants free from selection marker genes (Yoder and Goldsbrough 1994; Ebinuma et al. 1997a; Puchta 2000; Jaiwal et al. 2002; Hare and Chua 2002), such as the introduction of only the gene of interest and laboriously screening the regenerants using PCR analyses (Vetten et al. 2003; Ballester et al. 2010), site-specific recombination (Gleave et al. 1999; Zubko et al. 2000; Roy et al. 2008;
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Zhang et al. 2009; Khan et al. 2010a), and cotransformation (Komari et al. 1996; Lu et al. 2009) [reviewed in Hohn et al. (2001) and Darbani et al. (2007)]. In the study reported here, we employed the multi-autotransformation (MAT) vector system to develop diseaseresistant marker-free Petunia hybrida without a sexual crossing step. The MAT vector system, which was developed by Ebinuma and co-workers (Ebinuma et al. 1997a; Sugita et al. 1999; Ebinuma and Komamine 2001), is a novel transformation system in which transgenic tissues are visually selected by their morphological changes caused by oncogenes [isopentenyltransferase (ipt) gene] or rhizogenes (or the rol gene) of Agrobacterium tumefaciens. The MAT vectors are equipped with the yeast site-specific recombination R/RS system to mediate the excision of the DNA fragment and the ipt gene located between two directly oriented recombination sites (Araki et al. 1987). The ipt gene codes for isopentenyltransferase, which catalyzes the formation of isopentenyl AMP, a precursor of several cytokinins. Following transformation, overexpression of the ipt gene results in an increase in endogenous cytokinins and, consequently, the production of extreme shooty phenotype/ ipt shoots that are characterized by the loss of apical dominance, short internodes, abnormal morphological changes, and lack of rooting ability. As a result, the ipt shoots can be visually selected and subcultured. The excision of the ipt gene by site-specific recombination induced by recombinase of the R/RS system during subculturing produces morphologically normal marker-free transgenic plants. The MAT vector system has been evaluated in tobacco (Ebinuma et al. 1997a; Sugita et al. 2000), Antirrhinum majus (Cui et al. 2000, 2001; Ebinuma and Komamine 2001), hybrid aspens (Matsunaga et al. 2002), rice (Endo et al. 2002), Nierembergia (Khan et al. 2006a), white poplar (Zelasco et al. 2007), Citrus (Ballester et al. 2007), Cassava (Saelim et al. 2009), Medicago truncatula (Scaramelli et al. 2009), Kalanchoe blossfeldiana (Thirukkumaran et al. 2009) and Petunia hybrida (Khan et al. 2010a, b). Most of these studies have evaluated the applicability of the MAT vector system using mostly the bglucuronidase (GUS) gene as a model gene of interest. Petunia hybrida, an important floral plant, is susceptible to a number of pathogenic bacteria and fungi that affect both its growth and market value. As a useful strategy to confer resistance to wide range of pathogens, genes for cysteine-rich antimicrobial peptides (AMPs) have been introduced in different plant species. Antimicrobial peptides (also called host defence peptides) are an evolutionarily conserved component of the innate immune response and found among all living organisms. These AMPs have been shown to play a role in plant defense against a wide range of pathogens, including bacteria and fungi (Broekaert et al. 1995, 1997; Cammue et al. 1992, 1994).
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The antimicrobial protein gene WjAMP-1 isolated from leaves of Wasabia japonica was found to inhibit fungal and bacterial growth when expressed in Nicotiana benthamiana (Saito et al. 2001; Kiba et al. 2003). Transgenic tobacco plants overexpressing the wasabi defensin gene have also been reported to be resistant against Botrytis cinerea (gray mold; Nishihara, unpublished). Similarly, the growth of blast fungus was inhibited in transgenic rice carrying wasabi defensin (Kanzaki et al. 2002). In an earlier study, we produced transgenic potato plants carrying wasabi defensin that exhibited antifungal activity against B. cinerea (Khan et al. 2006b). Transgenic Phalaenopsis plants expressing wasabi defensin protein (WD) showing resistance to Erwinia caratovora have also been produced (Sjahril et al. 2006). Transgenic ‘Egusi’ melon (Colocynthis citrullus) was produced recently that exhibited resistance against Aletnaria leaf spot and Fusarium wilt (Ntui et al. 2010). Previous work by our group, using the GUS gene as a model gene of interest, showed that marker-free P. hybrida can be produced using the MAT vector system (Khan et al. 2010a). The aim of the study reported here was to develop transgenic marker-free P. hybrida plants resistant to B. cinerea (gray mold) through the introduction of the wasabi defensin (WD) gene using an ipt-type MAT vector, pMAT21-WD.
Materials and methods Vector construct Escherichia coli strain Top10 was used as the host for recombinant vector constructions. The ipt-type MAT vector, pMAT21 (Ebinuma et al. 1997b), which has the LacZ (located outside the ‘hit and run’ cassette), GUS, ipt, and recombinase (R) genes [located between directly oriented recombination site (RS) sequences], was used as the destination vector. The binary vector construct used in this study was constructed by Gateway LR clonase reactions (Fig. 1; Invitrogen, CA, USA). The WD gene was isolated from the binary vector pEKH by digestion with HindIII and cloned in the pCR8/GW/TOPO TA vector (Invitrogen) by TA cloning to produce the entry vector PCR8/WD. The destination vector was constructed by inserting the ccdB gene (a highly lethal gene whose protein blocks growth of E. coli wild-type gyrA ? strain) in Sma1 site of LacZ in pMAT21 (Fig. 1a). WD was integrated into the destination vector by LR clonase (Invitrogen) to produce pMAT21-WD, an ipttype MAT vector harboring the WD gene and transferred to A. tumefaciens strain EHA105 by the freeze/thaw method (Weigel and Glazebrook 2006).
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Fig. 1 Schematic representation of the T-DNA region of the isopentenyltransferase (ipt)-type multi-auto-transformation (MAT) vector, pMAT21-WD. a The MAT vector with a ‘hit and run’ cassette in which the chimeric ipt, b-glucuronidase (GUS), and recombinase (R) genes are inserted into the R/RS system between two directly oriented recombination sites (RS). The wasabi defensin (WD) gene is located outside the ‘hit and run’ cassette. RB Right border sequence of a T-DNA, LB left border sequence of a T-DNA. The cauliflower
mosaic virus 35S promoter (35SP) drives the R and WD genes. The GUS and ipt genes are driven by the nopaline synthase (NosP) and native ipt (IptP) promoters, respectively. The terminators (T) of the ipt, R, GUS, and WD genes are derived from nopaline synthase. b TDNA region after excision of the ‘hit and run’ cassette. Primer positions and length of PCR products are indicated by double arrows. Recognition sites of restriction enzymes (HindIII, Pst1, EcoRI, BamHI) are also indicated
A. tumefaciens harboring pMAT21-WD was grown overnight in a reciprocal shaker (120 cycles min-1) at 28°C in LB medium (10 g l-1 tryptone, 5 g l-1 yeast extract, 10 g l-1 NaCl, pH 7.2) containing 50 mg l-1 kanamycin (Wako Pure Chemical Industries, Osaka, Japan) and 25 mg l-1 chloramphenicol (Sigma–Aldrich Chemie, Steinheim, Germany). The bacterial culture was centrifuged (3,000 g) for 10 min and the bacterial pellet resuspended in hormone-free MS (Murashige and Skoog 1962) medium [bacterial suspension:MS medium, 1:2 (v:v)] containing 100 lM acetosyringone (3,5-dimethoxy-4hydroxy-acetophenone; Sigma-Aldrich) and 30 g l-1 sucrose to a final density of OD600 = 0.6.
Histochemical GUS assay
Transformation Leaf explants of in vitro-grown P. hybrida ‘Dainty Lady’ were infected with the overnight-grown bacterial suspension (OD600 = 0.6) for 5 min, blotted dry with sterilized filter paper to remove excess bacteria, and incubated on plant growth regulator (PGR)-free MS medium supplemented with 30 g l-1 sucrose, 0.25% gellan gum (Gelrite; Kelco, Division of Merck, San Diego, CA), and 100 lM acetosyringone, for 3 days under the dark condition for cocultivation. The infected explants were then washed with liquid PGR-free MS medium supplemented with 10 mg l-1 meropenem (Meropen; Sumitomo Pharmaceuticals, Osaka, Japan) and transferred to PGR- and antibiotic-free MS medium containing 20 mg l-1 meropenem. The infected and the uninfected control explants were kept under normal growth conditions. The regenerated shoots were subcultured on the same MS medium.
Histochemical GUS assays of leaves from ESP (extreme shooty phenotype) shoots and normal transgenic P. hybrida were carried out by soaking the tissues in X-Gluc (5bromo-4-chloro-3-indolyl-beta-D-glucuronic acid) solution (Jefferson 1987). After an overnight (15–16 h) incubation at 37°C, chlorophyll was removed by soaking the tissues for several hours in 70% ethanol. PCR analysis For the PCR analysis, genomic DNA was extracted from ipt and morphologically normal shoots and from control Petunia plants following the CTAB (cetyl trimethyl ammonium bromide) method with slight modifications (Rogers and Bendich 1988). PCR cycling was carried out using the oligonucleotide primers (Bex, Japan) for the GUS, ipt, and WD genes. The sequences of the oligonucleotide PCR primers were: GUS1, 50 -GGTGGGAAAGC GCGTTACAAG-30 ; GUS2, 50 -TTTACGCGTTGCTTCCG CCA-30 ; IPT1, 50 -CTTGCACAGGAAAGACGTCG-30 ; IPT2, 50 - AATGAAGACAGGTGTGACGC-30 ; WD1, 50 -TT TGCTTCTATCATCGCTCTTC-30 ; WD2, 50 -TTATTAGT ACAACAAACCAACA-30 . Southern blot hybridization For Southern blot hybridization, genomic DNA (10 lg) from ipt and morphologically normal shoots and nontransformed control P. hybrida plants were digested with
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HindIII. The digested samples were separated on a 0.8% (w/v) agarose gel, blotted to a positively charged nylon membrane (Hybond-N?; Amersham Pharmacia Biotech, Amersham, UK), and hybridized with a digoxigenin (DIG)labeled probe of the WD gene. The probe DNA fragment, corresponding to a part of the WD gene, was labeled by PCR using DIG-dUTP, following the supplier’s instructions (Boehringer Ingelheim, Ingelheim am Rhein, Germany). Hybridization, washing, and detection were performed using the DIG Easy Hyb (hybridization solution) and DIG Wash and Block Buffer set following the supplier’s instructions (Boehringer Ingelheim). Hybridization with the DIG-labeled probe was performed for 16 h at 41°C, and the hybridization patterns were detected with the chemiluminescent substrate CDP-Star (Roche Molecular Biochemicals, Mannheim, Germany) and anti-DIG-AP (anti-DIG antibody linked to alkaline phosphatase). The hybridized blot was exposed to Hyperfilm TM-MP X-ray film (Amersham Pharmacia Biotech) for 15–20 min at room temperature. RNA extraction and northern blot hybridization For northern blot analysis, total RNA was extracted using a one-step acid phenol–guanidine isothiocyanate–chloroform method (Sambrook and Russell 2001) to detect the expression of the WD at the mRNA level. A 20-lg sample of total RNA was fractionated by electrophoresis on a 1.5% agarose-formaldehyde gel in 19 morphpropane sulphonic acid (MOPS) and transferred overnight by capillary action to a Hybond-N? nylon membrane (Amersham Biosciences, Arlington Heights, IL) using 209 SSC (3 M NaCl, 0.3 M sodium citrate). The RNA on the membrane was fixed by UV-crosslinking for 2–3 min. Northern blot hybridization was carried out using the DIG-labeled DNA probe of the WD gene following the same procedure as that described for Southern blot analysis, with slight modification where necessary. Reverse transcription-PCR Reverse transcription (RT)–PCR was performed using a SuperScript Transcriptase III kit (Life Technologies, Carlsbad, CA). Total RNA (1.0 lg per 20 ll) with the WD-specific primers was used for RT–PCR. cDNA was synthesized at 55°C for 40 min and the reaction stopped at 70°C for 15 min. The cDNA was denatured prior to the PCR analysis at 94°C for 2 min. PCR amplification was then performed in 30 cycles of 94°C for 30 s, 59°C for 30 s, and 72°C for 1 min. The resultant PCR products were analyzed by electrophoresis on a 1.2% agarose gel.
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Western blot analysis To evaluate the expression of the integrated WD in the genomic DNA of transgenic plants, total proteins were extracted from fresh leaves of 4- to 5-week-old marker-free transgenic and non-transformed control in vitro petunia plants (containing 5–7 mature leaves). The samples were homogenized with extraction buffer [(62.5 mM Tris-HCl, pH 6.8, 2% (v/v) sodium dodecyl sulphate (SDS), 10% (v/ v) glycerol)] and 0.2% b-mercaptoethanol after being ground in liquid nitrogen. The homogenized samples were centrifuged (20,000 g) for 5 min at 4°C, and the total proteins in the supernatant from each sample were denatured by boiling for 3 min followed by incubation on ice for 2 min and separation by SDS–polyacrylamide gel electrophoresis (PAGE 15%) using a Bio-Rad Mini electrophoresis system per the manufacturer’s instructions (Bio-Rad, Hercules, CA. The fractionated proteins were electroblotted onto a polyvinylidine difluoride (PVDF) membrane (Amersham Biosciences). The PVDF membrane was blocked for 1 h in 5% bovine serum albumin (BSA) in TBS (20 mM Tris-HCl, pH 7.5, 150 mM NaCl). Detection of the WD protein was performed using polyclonal antisera raised in rabbit against the defensin protein (primary antibody, 1:1000, v/v) and subsequently goat-anti-rabbit IgG (Amersham Biosciences, USA) conjugated to horseradish peroxidase (HRP) as secondary antibody (1:100000, v/v) with an enhanced chemiluminescence (ECL) western blot detection system (Amersham Biosciences). Fungal-resistance assays of transgenic plants Antifungal activity of the WD protein in the marker-free transgenic Petunia plants was tested against B. cinerea strain 40 (isolated from P. hybrida). B. cinerea was grown on potato dextrose agar (PDA) until the surface of agar in the petri dish (20 9 90 mm) was covered with the fungal mycelia. For inoculation, a block of the agar (with cultured mycelia) was placed near the base of in vitro marker-free transgenic and nontransformed control plants (2–3 weeks old) grown in glass bottles and incubated in a growth chamber maintained at 25°C and 100% relative humidity under a 16/8-h (light/dark) photoregime. Pictures were taken 2 weeks after inoculation (Fig. 7a). Detached leaf assay For the detached leaf assay, a spore suspension was prepared by flooding the cultures of B. cinerea strain 40, grown in petri dishes on PDA at 25°C for 7–8 days, with 10 ml of sterile distilled water and then rubbing the agar surface gently with a sterilized loop to dislodge the sporangia. The spore suspension was collected in Eppendorf
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tubes with a sterile Pasteur pipette and vortexed vigorously to induce the spores. The spores were counted on a hemocytometer and the suspension concentration adjusted to 1 9 106 spores per milliliter using sterile water. The spore suspension was used immediately for inoculation. The youngest fully expanded mature leaves from the marker-free transgenic and control plants were harvested and immediately placed, adaxial side up, on wet filter paper in petri plates. The center of each leaf was wounded on both sides of midrib, and 20 ll of spore suspension (1 9 106 spores ml-1) was pipetted onto the wounded spot (Fig. 7c, d). The petri plates were placed in a growth chamber maintained at 25°C and 100% relative humidity under a 16/8-h (light/dark) photoregime. The area (mm2) of the necrotic lesions on the leaf-disks was measured 5 days after inoculation. Restriction of fungal colonization To further confirm the anti-fungal activity of WD protein in preventing the colonization of B. cinerea strain 40, we challenged in vitro transgenic and control Petunia plants with the fungal spores, as described earlier. The inoculated plants were incubated in a growth chamber maintained at 25°C and 100% relative humidity under a 16/8-h (light/dark) photoregime. After 1 week, leaves and stem sections from the inoculated plants were cultured on PDA in petri plates and incubated again under the same conditions. The cultures were photographed (Fig. 7e, f) 1 week after inoculation.
Results and discussion Production of marker-free normal plants Leaf explants of P. hybrida were infected with the A. tumefaciens harboring pMAT21-WD, an ipt-type MAT vector containing the ipt, GUS, and R (recombinase) genes in the removable cassette flanked by directly oriented recombination sites (RS) and cultured on hormone- and antibiotic-free MS medium. Uninfected control explants were also cultured under the same conditions. Approximately 2 weeks after infection, nodular compact calli appeared on the infected leaf explants (Fig. 2a); in contrast, the uninfected control leaf explants were unable to produce calli and ultimately died (Fig. 2c). Adventitious shoot lines (ASLs) were separated from the explants, approximately 1 month after infection, and transferred to PGR- and selective antibiotic-free 0.25% gelrite-solidified MS medium supplemented with 3% sucrose and 20 mg l-1 meropenem (Fig. 2b). These ASLs differentiated into normal looking shoots and ipt shoots. The ipt shoots were identified and selected from each subculture based on distinctive
morphological characteristics, including abnormal morphology, short internodes, and a lack of apical dominance and rooting ability (Fig. 2d, e). The selected ipt shoots were subcultured at 2-week intervals on the same MS medium. Approximately 6 months after infection, 23 normallooking shoots were obtained from the separation of ipt shoots of different ASLs. The recombinase of the R/RS system mediated excision of the ipt gene (selectable marker) and, consequently, the reduction in cytokinin production caused the emergence of normal-looking shoots. Morphologically normal-looking shoots and ipt shoots were subjected to the histochemical GUS assay (Fig. 3). As expected, all of the ipt shoots showed GUS expression and all of the normal shoots did not. The ipt GUS? and normal GUS- shoots were analyzed by PCR to confirm that the gene of interest, WD, was intact and the selection marker had been excised from the normal-looking plants. Molecular analyses Three oligonucleotide primers that were able to amplify the ipt, GUS, and WD genes were used in the PCR analyses. The predicted 1.2-kb GUS, 0.5-kb WD, and 0.8-kb ipt fragments were amplified in all ipt shoots (Fig. 4, lanes 3–5), and the ipt and GUS genes, as expected, were not detected in normal shoots (Fig. 4, lanes 6–8). WD was detected in four of the 23 normal shoots, verifying that the selection marker was excised and that these normal shoots were marker-free and contained only the gene of interest, WD. No transgene was detected in genomic DNA of the remaining normal-looking shoots based on the results of the PCR analysis (data not shown). The cytokinin produced in the transgenic cells may diffuse to adjacent cells, leading to the production of escape plants. Importantly, all of the transgenic normal-looking shoots were marker-free, in contrast to our findings in an earlier study (Khan et al. 2006a, 2010a) in which two of the transgenic normallooking shoots in Nierembergia caerulea and one of the eight well-rooted transgenic plants in P. hybrida contained the intact ipt gene. It can be concluded that the R/RS sitespecific recombination caused excision of the ipt gene from all cells of the normal shoots and the latter were free from chimerism. However, autoexcision of the selection marker gene by recombinase driven by the constitutively expressed promoter, CaMV35S, took a long time (6–8 months). The induction of site-specific recombination and the excision of the ipt gene by inducible promoters directing recombinase expression is an alternative approach to overcome the problems of chimerism and inefficient excision (Endo et al. 2002; Matsunaga et al. 2002). Southern blot analysis of genomic DNA from the ipt and morphologically normal marker-free transgenic plants was
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Fig. 2 Regeneration of ipt and normal shoots from leaf explants of Petunia hybrida through ipt shoot formation after infection with Agrobacterium tumefaciens harboring the ipt-type MAT vector, pMAT21-WD. a Calli and shoot formation by infected leaf explants of Petunia on hormone- and selective antibiotic-free MS medium 3 weeks after infection. b Adventitious shoot lines (ASLs) isolated from the regenerated explants and cultured on MS medium for ipt or
normal shoot formation. c Control uninfected explants showing no calli or shoot formation 5 weeks after co-cultivation. d Differentiation of ASLs into ipt-like shoots (arrows) 5 weeks after infection. e ipt shoots (arrows) that could be visually distinguished from other shoots 3 months after infection. f Morphologically normal shoot free from selection marker, produced from ipt shoots approximately 6 months after infection
Fig. 3 Histochemical GUS assay of ipt and morphologically normal shoots of P. hybrida ‘Dainty Lady’ obtained through ipt shoot formation after infection with A. tumefaciens harboring pMAT21-WD. Ipt shoots (a) and marker-free transgenic shoots (b) were tested for
histochemical GUS expression by soaking in X-Gluc solution. After overnight (15–16 h) staining, chlorophyll was removed by soaking the tissues for several hours in 70% ethanol
performed to determine the copy number of the WD gene in transgenic plants. Genomic DNA from ipt and morphologically normal transgenic and non-transformed control plants was digested with HindIII and hybridized with the
DIG-labeled probe of the WD gene. Southern blot hybridization indicated that the transgenic plants had one to three T-DNA insertions (Fig. 5). Hybridization was not detected with the non-transformed plant DNA (Fig. 5, lane 1).
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Fig. 4 PCR analysis of ipt and morphologically normal shoots of P. hybrida ‘Dainty Lady’ obtained through ipt shoot formation after infection with A. tumefaciens harboring the ipt-type MAT vector, pMAT21-WD. Lanes M Size marker (øX174/HaeIII digests), 1 negative control (DNA from non-transformed plant), 2 positive control (plasmid DNA) for the GUS (1.2 kb, upper band), ipt (0.8 kb, middle band), and WD (0.5 kb, lower band) genes, 3–5 amplification of GUS (1.2 kb, upper band), ipt (0.8 kb, middle band), and WD (0.5 kb, lower band) genes in ipt shoots, 6–8 amplification of WD gene in marker-free transgenic shoots. Ipt and GUS genes were not detected in normal-looking transgenic shoots
Fig. 5 Southern blot analysis of the WD gene in ipt and morphologically normal shoots of P. hybrida ‘Dainty Lady’ obtained through ipt shoot formation after infection with A. tumefaciens harboring pMAT21-WD. Genomic DNA from ipt shoots and morphologically normal transgenic plants was digested with HindIII and hybridized with the DIG-labeled probe of the WD gene. Lanes M DIG-labeled DNA molecular weight marker III, 1 nontransformed control, 2–4 ipt shoots, 5–7 marker-free transgenic normal shoots produced from ipt shoots
RT–PCR was carried out to confirm the expression of the WD gene at the mRNA level (Fig. 6a). Expression was confirmed in all of the transgenic marker-free lines (Fig. 6a, lanes 3–6); no PCR product was detected in the control plant (lane 2). When DNase-treated RNA samples (without reverse transcriptase) from control and transgenic plants were used as a PCR template, the absence of the product confirmed that the RNA samples were not contaminated by genomic DNA (data not shown). Northern blot analysis of total RNA from marker-free and non-transformed control plants was performed to
Fig. 6 Reverse transcription (RT)–PCR, northern blot, and western blot analyses of marker-free transgenic and control P. hybrida plants ‘Dainty Lady’ obtained through ipt shoot formation after infection with A. tumefaciens harboring the ipt-type MAT vector pMAT21-WD. a RT–PCR was performed to confirm the expression of the WD gene at the mRNA level. Lanes M Size marker (øX174/HaeIII digests), 1 positive control (plasmid cDNA) for WD gene, 2 negative control (cDNA from non-transformed plant), 3–6 amplification of WD transcripts in marker-free transgenic plants. b Northern blot analysis of marker-free petunia plants. Northern hybridization was carried out using the digoxigenin (DIG)-labeled DNA probe of the WD gene. Lanes 1 Non-transformed control, 2–5 mRNA transcription from the WD gene in marker-free transgenic petunia plants. c Western analysis for the expression of WD protein in marker-free transgenic plants of petunia. Protein extracts of leaves from in vitro-grown transgenic independent clones and non-transformed control plants were fractionated on a 15% polyacrylamide gel and subjected to immunoblot analysis using a rabbit polyclonal antiserum for WD. Lanes: 1 Nontransformed control, 2–5 marker-free transgenic plants. Arrow indicates the 5-kDa band of the WD protein
further confirm the expression of WD at the mRNA level. The findings revealed that all of the transgenic plants had detectable levels of WD gene transcripts (Fig. 6b, lanes 2–5). mRNA transcripts were not detected in the control plant (Fig. 6b, lane 1). To confirm the expression of WD gene at protein level, we carried out western blot analysis of protein extracts from marker-free and non-transformed petunia plants. In the western blot analysis, the primary antibody immunoreacted with the antigen (WD) and the secondary antibody (goat-anti-rabbit IgG). The secondary antibody, conjugated with a HRP enzyme, converted the luminol substrate (ECL) to a light-releasing substance, which was detected as a spot on film and a 5-kDa fragment was detected in the total
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Plant Cell Tiss Organ Cult b Fig. 7 Disease resistance assay of marker-free transgenic plants of P.
hybrida ‘Dainty Lady’ expressing the WD gene against B. cinerea. a, b Transgenic and non-transformed control in vitro plants were challenged with B. cinerea grown on potato dextrose agar (PDA). Two weeks after inoculation, the control plant could not stand upright and fell over (b), whereas the transgenic plants expressing the WD gene remained green and continued to grow through the fungal hyphae (a). c, d Detached leaf assay of control and marker-free transgenic petunia plants. Leaf from the control plant (d) developed expanded lesions around the inoculation spot, whereas the leaf from the transgenic plant had only a small necrotic region at the site of inoculation (c). e, f Leaves and stem sections from B. cinereainoculated control and transgenic petunia plants were cultured on PDA. Explants from the control favored the growth of the fungus (f), whereas growth of the fungus was restricted by the WD-expressing plant parts from the transgenic petunia (e). g Area (mm2) of necrotic lesion developed on detached leaves of control and transgenic petunia plants after inoculation with spores of B. cinerea. WT Wild type
protein extracts of only transgenic plants (Fig. 6c, lanes 2–5). No reaction was detected in the non-transformed control plants (Fig. 6c, lane 1). Disease resistance assay of marker-free transgenic plants Defensin, a protein belonging to the defensin family, is one of the antimicrobial proteins expressed in plants against stresses such as infection by a foreign pathogen. Low
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concentrations of WD was found to inhibit the growth of the rice blast fungus in vitro (Saito et al. 2001). Earlier studies on the expression of WD in transgenic Nicotiana benthamiana (Saito et al. 2001; Kiba et al. 2003), rice (Kanzaki et al. 2002), potato (Khan et al. 2006b), Phalaenopsis (Sjahril et al. 2006), and ‘‘Egusi’’ melon (Colocynthis citrullus) (Ntui et al. 2010) all showed the enhanced antimicrobial activities of this protein. Fungal resistance of transgenic plants expressing the WD gene was evaluated against B. cinerea, one of the main pathogenic fungus that causes severe damage to the leaves and flowers of P. hybrida. In vitro transgenic and nontransformed control plants were challenged with B. cinerea strain 40 grown on PDA to evaluate the resistance imparted by the expression of WD protein in transgenic petunia plants. Following the placement of an agar block (1 cm2) with grown mycelia at the base of 2- to 3-week-old in vitro control and transgenic plants, the fungal mycelia readily penetrated the stem of the control plants, resulting in dehydration and stem softening. Approximately 10 days after inoculation, the control plant could not stand upright and fell over (Fig. 7b). In contrast transgenic plants expressing the WD gene remained green and continued to grow through the fungal hyphae (Fig. 7a). In another set of experiments, detached leaves from control and marker-free transgenic petunia plants were placed in petri dishes with wet filter paper, wounded, and inoculated with the B. cinerea strain 40 spore suspension (two inoculation spots per leaf). Water-soaked spots on the leaf disks could be observed after 24 h. One day later, necrotic lesions appeared which expanded over time as increasingly more tissue was damaged by the pathogen. After 5 days of incubation at room temperature, most of the leaf area from the control plant turned brown and decomposed (Fig. 7d). In contrast, the leaf from the transgenic plants expressing WD protein was still green, with small necrotic spots only at the sites of inoculation (Fig. 7c). The
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necrotic area (mm2) affected by B. cinerea around the inoculation spot was threefold larger in non-transformed control leaf (Fig. 7g). In our previous study, WD protein in transgenic potato imparted partial resistance to B. cinerea, as evidence in the detached leaf assay (Khan et al. 2006b). The results of the present study further confirm the resistance potential of WD protein against B. cinerea. The antimicrobial activity of WD protein in restricting the multiplication of B. cinerea in plants was further confirmed by challenging in vitro transgenic and control Petunia plants with the fungal spores in glass bottles and incubating the challenged plants at room temperature and 100% relative humidity. After 1 week, leaf and stem sections from the inoculated plants were cultured on PDA in petri plates and incubated again under the same conditions. Following the second incubation period, fungal growth appeared to be unrestricted on all plant parts from the control plants (Fig. 7f). In comparison, the multiplication of B. cinerea in the leaves and stem sections from the transgenic petunia expressing WD was restricted (Fig. 7e). Based on the results reported here, we suggest that the WD gene was successfully integrated into the genome of transgenic petunia plants, producing the defensin protein. Expression of the antimicrobial peptide, WD, provided enhanced resistance to infections of B. cinerea in markerfree transgenic petunia. However, further research is needed to test the resistance provided by WD in transgenic petunia to other pathogenic fungi and bacteria. Acknowledgments We would like to thank the Japan Society for Promotions of Sciences (JSPS) for their financial support for this research project. Our thanks are also extended to Pulp and Paper Research group, Nippon Paper Industries, Tokyo who kindly provided the MAT vector constructs.
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