Plant Science 154 (2000) 171 – 181 www.elsevier.com/locate/plantsci
Transgenic expression of a gene encoding a synthetic antimicrobial peptide results in inhibition of fungal growth in vitro and in planta Jeffrey W. Cary a,*,1, Kanniah Rajasekarana,1, Jesse M. Jaynes b, Thomas E. Cleveland a a
USDA, ARS, Southern Regional Research Center, Food and Feed Safety Research Unit, 1100 Robert E. Lee Bl6d., New Orleans, LA 70124, USA b Demegen Inc., 1051 Brinton Rd., Pittsburgh, PA 15221, USA Received 16 August 1999; received in revised form 16 December 1999; accepted 17 December 1999
Abstract Transgenic tobacco plants producing the synthetic antimicrobial peptide D4E1, encoded by a gene under the control of an enhanced cauliflower mosaic virus 35S RNA promoter, were obtained by Agrobacterium-mediated transformation. Successful transformation was demonstrated by PCR and Southern hybridization analysis of tobacco DNAs. Expression of the synthetic D4E1 gene was shown by RT-PCR of tobacco mRNA. Crude protein extracts from leaf tissue of transformed plants significantly reduced the number of fungal colonies arising from germinating conidia of Aspergillus fla6us and Verticillium dahliae by up to 75 and 99%, respectively, compared to extracts from plants transformed with pBI121. Compared to negative controls, tobacco plants expressing the D4E1 gene showed greater levels of disease resistance in planta to the fungal pathogen, Colletotrichum destructi6um, which causes anthracnose. © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Aspergillus fla6us; Colletotrichum destructi6um; Disease resistance; Nicotiana tabacum; Synthetic peptide; Transgenic; Verticillium dahliae
1. Introduction Plants do not possess a complex immunoglobulin-based system such as that found in higher vertebrates to defend themselves against attacking microbial pathogens; however, they do have a wide variety of innate host defense mechanisms at their disposal. These include the production of antimicrobial reactive oxygen species (ROS), secondary metabolites, hydrolytic enzymes, and a wide array of antimicrobial proteins and peptides [1,2]. Recombinant DNA technologies and plant transformation procedures have been used to introduce and express genes encoding these types of * Corresponding author. Tel.: +1-504-2864264; fax: + 1-5042864419. E-mail address:
[email protected] (J.W. Cary) 1 Both JWC and KR contributed equally to this work.
antimicrobial agents in plants in an effort to increase host resistance to plant pathogens. Of particular interest has been the identification and characterization of ribosomally synthesized antimicrobial peptides. Antimicrobial peptides appear to be ubiquitous in nature being found in many organisms, from humans to bacteria [3–5]. Various plants produce, either preformed or in response to microbial invasion, cysteine-rich antimicrobial peptides such as thionins, defensins, lipid transfer proteins, and hevein- and knottin-type peptides [6]. Examples of antimicrobial peptides of mammalian and insect origin include bovine or human defensins and protegrins [7], magainins from amphibians [8], and cecropins from the giant silk moth, Hyalophora cecropia [9]. These peptides have been shown to be effective against a wide array of microorganisms including fungi [10]. Antifungal peptides act either
0168-9452/00/$ - see front matter © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 0 0 ) 0 0 1 8 9 - 8
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by lysing the fungal cell [4,5,11,12] or by interfering with cell wall synthesis [13]. Cecropin and cecropin analogs have been expressed in transgenic tobacco (Nicotiana tabacum) with mixed results regarding pathogen resistance. Huang et al. [14] and Jaynes et al. [15] demonstrated reduced disease severity in transgenic tobacco expressing cecropin analogs upon infection with the bacterial pathogens, Pseudomonas syringae pv. tabaci and P. solanacearum, respectively. However, tobacco plants expressing a native cecropin did not confer resistance to P. syringae pv. tabaci or P. solanacearum presumably due to degradation of the peptide by host proteases [16,17]. The advent of automated peptide synthesizers and combinatorial peptide chemistry has made it possible to rapidly synthesize, and screen large numbers of peptides for their ability to inhibit the growth of target microbial pathogens. These linear peptides often can be less than half the size (10–20 amino acids) of their native counterparts and many times more potent without concomitant toxicity to host tissues. DeLucca et al. [18] reported on the antifungal activity of a 17 amino acid linear, synthetic peptide designated D4E1. This peptide was shown to interact with sterols present in the conidial cell walls and resist degradation by fungal and host proteases. In vitro assays with D4E1 demonstrated a minimal lethal concentration needed to kill 100% of germinating conidia of Aspergillus fla6us, A. fumigatus, and A. niger of 12.5, 12.5, and 25 mM, respectively. In this study, we report on the successful Agrobacterium tumefaciens-mediated transformation and expression of D4E1 in tobacco and subsequently, demonstrate antifungal activity from crude extracts of the transformed leaves against A. fla6us and Verticillium dahliae. Additionally, in planta assays demonstrated reduced disease severity upon inoculation of transgenic tobacco leaves with the fungal pathogen, Colletotrichum destructi6um.
2. Materials and methods
2.1. Cloning and manipulations A synthetic gene encoding the peptide D4E1 (FKLRAKIKVRLRAKIKL) was generated by polymerase chain reaction (PCR) amplification of
a plasmid, pD4E1, containing a 66 bp fragment representing D4E1 (unpublished data). Oligonucleotide primers for PCR of pD4E1 were designed with NcoI (5% D4E1) and SacI (3% D4E1) restriction endonuclease sites for subsequent subcloning of the D4E1 coding region into the binary plasmid vector. Using plasmid pD4E1 as template, primers 5% D4E1 (5%-CCATGGGATTTAAGTTGAGAGCTAAG-3%) and 3% D4E1 (5%-GAGCTCT TACAACTTAATCTTAGCT-3%) were used to amplify the D4E1 coding region with Pfu polymerase (Stratagene, LaJolla, CA). The presence in primer 5% D4E1 of the 5% NcoI site (double underline) and an additional GA (single underline) to the D4E1 coding region would result in the addition at the 5% end of the D4E1 gene of codons for a translational initiator methionine (ATG of NcoI site) and glycine (GGA) adjacent to the phenylalanine (see D4E1 amino acid sequence above). The PCR product was subcloned into the vector pCRScript Cam (Stratagene) to produce pCR ScriptD4E1 (Fig. 1). A binary vector for the transformation and expression of D4E1 was constructed as depicted in Fig. 1. The PCR generated D4E1 gene was placed under the control of a double cauliflower mosaic virus 35S RNA (CaMV 35S) promoter that contained a tobacco mosaic virus (TMV) 5% untranslated RNA leader (omega) sequence [19]. The promoter was released from the plasmid vector pAGUS1-TN2 by SphI-SacI double digestion and ligated into SphI-SacI digested plasmid pBI221 (Clontech, Palo Alto, CA) to produce plasmid pUC-d35S-GUS-nos (nos refers to the nopaline synthase transcriptional terminator from pBI221). This plasmid was then digested with NcoI-SacI as was pCR Script-D4E1 allowing the 67 bp D4E1 coding region to be ligated downstream of the double CaMV35S-TMV omega leader (d35S) promoter region. This construct was designated pUC-d35S-D4E1-nos. The d35S-D4E1 fragment from pUC-d35S-D4E1-nos was isolated and purified following HindIII-SacI digestion as were the T-DNA, nos terminator, and nptII gene regions of plasmid pBI121 (Clontech) after separation from the CaMV 35S promoter-uidA gene region by HindIII-SacI digestion. These two fragments were ligated together to produce the binary vector pBI-d35S-D4E1-nos. All plasmid constructs were transformed into and isolated from competent E. coli DH5a cells (Gibco-BRL, Bethesda, MD). The pBI-d35S-D4E1-nos binary vector was
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transformed into electrocompetent A. tumefaciens LBA 4404 (Gibco-BRL) via electroporation using a Cell-Porator (Bio-Rad, Hercules, CA) according to the manufacturer’s procedure. Total genomic DNA was isolated from tobacco leaf tissue using the method of Paterson et al. [20]. Total RNA was prepared from tobacco leaf tissue by the method of Bugos et al. [21].
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ant, adventitious shoot buds were transferred again to MS medium containing kanamycin (50 mg l − 1) and only the shoots that formed healthy root systems were subsequently transferred to pots containing a commercial soil mix for further evaluation in an environmentally-controlled growth chamber (28°C, 16 h day). The potted plants were also assayed for the presence of NPT II protein by ELISA [24].
2.2. Plant transformation 2.3. PCR and Southern blot analysis Transformation of tobacco (Nicotiana tabacum L. cv. SR-1) was accomplished using the A. tumefaciens-mediated leaf disk transformation system [22]. Thirty leaf disks were cultured on Murashige and Skoog (MS) nutrient medium [23] supplemented with 0.75 mg l − 1 6-benzylaminopurine, 200 mg l − 1kanamycin, and 200 mg l − 1 each of cefotaxime and carbenicillin. The antibiotic-toler-
To determine if the d35S-D4E1-nos T-DNA region had successfully integrated into the plant genome, a 465 bp region spanning from within the d35S promoter to the 3% end of the nos terminator was PCR amplified from total plant genomic DNA. The d35S promoter primer, 5%-ATGACGCACAATCCCACTATCCT-3% and the 3% nos ter-
Fig. 1. Diagram depicting the plasmids and enzymes used in the construction of the binary vector pBI-d35S-D4E1-nos. The DNA regions and methodology for the generation of pBI-d35S-D4E1-nos are described in Section 2. Abbreviations: Sp, SphI; N, NcoI; S, SacI; E, EcoRI; H, HindIII. RB and LB denote the right and left T-DNA border sequences.
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minator primer, 5%-CTAGTAACATAGATGTCTCCGCGC-3% were used to amplify plant genomic DNA using AmpliTaq Gold polymerase (Stratagene). Thermocycler (MJ Research, Watertown, MA) parameters were as follows: 1 cycle of 95°C, 10 min; 60°C, 1 min; 72°C, 30 s; 34 cycles of 95°C, 1 min; 65°C, 1 min; 72°C, 30 s; followed by a final extension of 72°C for 2 min. PCR products were analyzed by electrophoresis in a 1.2% agarose gel followed by ethidium bromide staining. For Southern blot analysis, tobacco genomic DNA (20 mg) was digested to completion with EcoRI and electrophoresed in a 1% agarose gel. DNA fragments were transferred to nylon membranes (Schleicher & Schuell, Keene, NH) by vacuum and hybridized with an approximately 1 kb random-primed (High Prime Kit; Roche, Indianapolis, IN), 32P-labelled, d35S promoter-D4E1nos terminator HindIII-EcoRI fragment from the pBI-d35S-D4E1-nos binary vector.
2.4. RT-PCR of transgenic tobacco RNA Due to the small size of the expected D4E1 transcript (approximately 90 bp), standard northern blotting procedures would not be feasible as a means of detecting these transcripts. Reverse transcription-polymerase chain reaction (RTPCR) amplification of RNA provided a means to easily detect the presence of these small transcripts. Poly(A) mRNA was isolated from total RNA using the PolyATract mRNA Isolation System (Promega, Madison, WI). Reverse transcription and first strand cDNA synthesis from the purified tobacco mRNA was performed using the Advantage RT-for-PCR Kit (Clontech, Palo Alto, CA). The antisense oligonucleotide primer used for PCR was designed to the 5% end of the nos transcriptional terminator sequence including the SacI site (underlined) that linked it with the 3% end of the D4E1 coding sequence (5%-TTTGAACGATCGGGGAAATTCGAGCTC - 3%). The sense PCR primer represented the 5% end of the D4E1 coding region starting with the NcoI site (underlined) used to link it with the d35S promoter (5%-CCATGGGATTTAAGTTGAGAGCTAAGATTA-3%). Using AmpliTaq polymerase (Stratagene), first strand cDNA representing the D4E1 transcript was amplified using the
following thermocycler parameters: 95°C, 5 min; 60°C, 1 min; 72°C, 1 min; 95°C, 1 min; 34 cycles at 65°C, 1 min; 72°C, 1 min; 72°C, 2 min. PCR products were separated by electrophoresis in a 10% polyacrylamide gel. To confirm that there was no DNA contaminating the RNA samples, all cDNAs from the reverse transcription of tobacco mRNA were amplified with two primers based on the tobacco ribulose 1,5-bisphosphate carboxylase small sunbunit (GenBank accession number X02353). Due to the presence of three introns in the small subunit coding region, these two primers, 5%RubSS (5%-ATGGCTTCCTCAGTTCTTTCCTCTGC-3%) and 3%RubSS (5%-GCTTGTAGGCAATGAAACTGATGCAC3%) were expected to generate a 525 bp product from mRNA, whereas a product of 922 bp would be expected upon amplification of genomic DNA.
2.5. DNA sequencing The validity of the nucleotide sequence for the synthetic D4E1 NcoI-SacI coding region, its proper ligation to the d35S promoter and nos terminator, as well as the nucleotide sequence of the tobacco D4E1 transcript RT-PCR products were determined by non-radioactive sequencing using a LI-COR 4000 automated sequencer (LICOR, Lincoln, NE) and standard sequencing primers.
2.6. In 6itro analysis of antifungal acti6ity of plant extracts to A. fla6us and V. dahliae The inhibitory activity of extracts from tobacco plants transformed with D4E1 was assessed in vitro following the method of DeLucca et al. [25]. Briefly, conidial suspensions were prepared from cultures grown on potato dextrose agar (PDA) slants for seven days at 30°C (A. fla6us) or 22°C (V. dahliae). Conidial suspensions in 1% (w/v) potato dextrose broth (PDB, pH 6.0) were adjusted to a density of 105 conidia/ml and were germinated in PDB for 8 h at 30°C (A. fla6us) or overnight at 22°C (V. dahliae) prior to assay. Plant homogenates were prepared by directly grinding tobacco leaves into a fine powder in liquid nitrogen with no buffer added. Ground tissues were then centrifuged at 8200 ×g for 10
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min at room temperature and extract collected from each sample. Negative control samples consisted of extract from tobacco leaves transformed with pBI121. Conidial suspensions (25 ml) were then added to 225 ml of plant extract, mixed, and incubated for 1 h at 30°C (A. fla6us) or 22°C (V. dahliae). Three 50 ml aliquots from each sample were then spread onto PDA plates and incubated at 30°C (A. fla6us) or 22°C (V. dahliae) for 24– 48 h and fungal colonies enumerated. One-way ANOVA was used to determine the significance of the effect of transgenic plant extracts on germinating conidia. Mean separations were performed using the method of Tukey [26].
2.7. In planta assay for anthracnose resistance Seven days prior to plant inoculation, Czapek yeast autolysate agar (CYA) plates were inoculated with mycelium of a virulent isolate of Colletotrichum destructi6um (ATCC 42492) and incubated at 24°C. A single CYA plate was flooded with 9 ml of sterile distilled water and spores were aseptically removed to yield a final inoculum density of approximately 1× 106 spores/ml. Tobacco plants transformed with D4E1 were inoculated by placing 10 drops of 10 ml each onto the adaxial surface of three young, but fully expanded tobacco leaves. Three leaves each of three non-transformed as well as pBI121transformed tobacco plants were also inoculated to serve as controls. Inoculated leaves were then covered with a plastic bag for 4 days to allow for lesion development in a growth chamber (27°C, 95 – 100% RH, 60 mE m − 2 s − 1, 16 h day:8 h dark). Disease severity was scored for each inoculation site of each leaf separately, 7 days after inoculation using the following scale: 0 = no visible symptoms; 1= slight chlorosis, 0–5 necrotic flecks in area of inoculation; 2 = chlorosis, 6 – 10 necrotic flecks in area of inoculation; 3 = necrotic flecks coalescing, overall lesion area less than total area inoculation (B 5 mm); 4= total inoculation site covered with necrotic lesion (necrotic area ca. B 5 mm in diameter); 5=necrotic area expanding beyond original inoculation site (necrotic lesion \ 5 mm in diameter). One-way ANOVA was used to detect significant differences in anthracnose severity. Mean separation was performed using the method of Tukey [26].
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3. Results
3.1. Transformation of tobacco Each leaf disk produced at least five transformed shoots as evidenced by the adventitious shoot formation in the presence of toxic levels of kanamycin (200 mg l − 1) and the ability of shoots to successfully root in growth media containing inhibitory levels of kanamycin (50 mg l − 1). Furthermore, the putative antibiotic resistant plantlets were assayed for the presence of NPT II. To obtain different individual transformants only one NPT II-positive plantlet from each leaf disk was transferred to the greenhouse. All of the transgenic tobacco plants carrying the D4E1 gene were morphologically similar to non-transformed or pBI121-transformed controls with respect to flowering and seed set. Six R0 plants, labeled T302, T303, T304, T306, T307, and T310, were assayed for disease resistance. The transformation and the disease resistance assays were duplicated with a second set of ten R0 transformants with similar results (data not shown). Seed collected from selfed R0 plants were germinated on MS medium containing 100 mg l − 1 kanamycin and the seedlings showed a 3:1 Mendelian segregation for antibiotic resistance, as expected.
3.2. PCR and Southern blot analysis PCR products from the six putative transgenic plant genomic DNA samples had the expected size of 465 bp representing the region spanning from 140 bp within the d35S promoter to the 3% end of the nos terminator. The DNA from pBI121-transformed control plants did not amplify any detectable PCR product (Fig. 2, panel A), as expected. These products hybridized with a 32 P-labeled 1 kb d35S promoter-D4E1-nos terminator HindIII-EcoRI fragment confirming that the PCR products were amplified from the d35S promoter-D4E1-nos region (Fig. 2, panel B). No hybridization signal was observed for the pBI121-transformed plant DNA control lane or the no DNA lane. In addition, EcoRI digested genomic DNA from these transformed tobacco plants also showed hybridization signals with the same probe (Fig. 3). Plants T302, T303, T304, and T310 demonstrated multiple hybridization signals. However, extremely faint hybridization signals
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Fig. 2. Southern hybridization of PCR products amplified from genomic DNA of six tobacco R0 plants transformed with pBI-d35S-D4E1-nos. Primers were designed to amplify an approximately 465-bp region spanning from within the d35S promoter to the 3% end of the nos terminator. Lane ‘no DNA’ represents PCR amplification reaction without any template DNA (negative control); Lane ‘plasmid’ denotes PCR amplification of the pBI-d35S-D4E1-nos binary vector (positive control). (A) Ethidium bromide stained PCR products from amplification of tobacco genomic and plasmid DNA. (B) Autoradiograph of Southern blot of the gel in Panel A hybridized with the 32P-labeled d35S-D4E1-nos HindIII-EcoRI fragment from the pBI-d35S-D4E1-nos plasmid.
sized from putative transgenic tobacco mRNA. As shown in Fig. 4, PCR of the pBI-d35S-D4E1-nos binary vector positive control DNA produced a fragment of approximately 90 bp as did the first strand cDNA of transgenic tobacco samples T302, T303, T304, T306, T307, and T310. The tobacco SR-1 (pBI121) negative control sample did not produce this product, as expected. These cDNAs were also amplified with primers specific for the small subunit of tobacco ribulose 1,5-bisphosphate carboxylase. Primers 5%RubSS and 3%RubSS were designed to anneal to DNA sequences 922 bp apart in the gene and they spanned three introns thus allowing for the determination of DNA contamination of the mRNA samples. All samples except the no DNA control gave one product of 525 bp indicating that there was no DNA contamination and therefore the PCR products observed with both primer sets were the result of amplification of their respective gene transcripts. It also demonstrated that the SR-1 (pBI121) mRNA was of sufficient quality for amplification. DNA sequence analysis showed that the sequence of the
corresponding to the two lower bands (approximately 6.6 and 8.0 kb) observed for T302 and T304 and the two upper bands of sample T303 were also detected in the SR-1 (pBI121-transformed) and T306 and T307 samples. These bands were believed to represent non-specific hybridization of the probe to regions of genomic DNA common to all of the samples. Therefore, it appears that samples T302, T303, T304, T306, and T307 have one copy of the T-DNA integrated into their genome. However, the possibility of multiple integration events in T302, T303, and T304 cannot be ruled out. Sample T310 gave as many as 4 or 5 unique hybridization signals indicating that multiple integration events occurred in this plant genome.
3.3. Analysis of D4E1 transcripts Due to the expected small size of D4E1 transcripts, RT-PCR of purified mRNA from putative transgenic tobacco plants was performed as a means of accurately determining if the D4E1 gene was being expressed. The primers used in the PCR reaction were expected to give a product of 89 bp upon amplification of first strand cDNA synthe-
Fig. 3. Southern hybridization of tobacco genomic DNA with a 32P-labeled d35S-D4E1-nos HindIII-EcoRI probe. Tobacco genomic DNA (20 mg) was digested to completion with EcoRI, blotted to nylon membrane and hybridized with 32Plabelled probe as described in Section 2. HindIII-digested lambda DNA (in kb) was used as a molecular size standard.
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3.4. In 6itro assay of antifungal acti6ity
Fig. 4. RT-PCR analysis of expression of the D4E1 gene in transgenic tobacco plants. RT-PCR was performed and products analyzed as described in Section 2. (A) Ethidium bromide stained RT-PCR products amplified from transgenic tobacco cDNA samples following reverse transcription of purified mRNA. cDNA from tobacco transformed with plasmid pBI121 served as a negative control while plasmid pBI-d35SD4E1-nos DNA was used as a positive control. (B) Ethidium bromide stained RT-PCR PCR products amplified from ribulose bisphosphate carboxylase small subunit cDNA.
Plant extracts from each transformed tobacco plant significantly reduced (PB0.05) the number of fungal colonies arising from germinating conidia of both A. fla6us and V. dahliae compared to the extracts from pBI121-transformed negative controls (Figs. 5 and 6). Germinating conidia of A. fla6us were susceptible to the extracts from transformed plants resulting in a greater than 60% reduction in the number of colonies. Extracts from all the transformants significantly reduced (PB 0.05) the number of colonies compared to the control (Fig. 5). Germinating conidia of V. dahliae were more susceptible to extracts from the transformed plants than A. fla6us. Extracts from the transformed plants reduced the number of germinating conidia of V. dahliae by 61–99% compared to extracts from tobacco transformed with pBI121 (Fig. 6). More than 90% control was observed with all the transgenic plants tested except T303 (61% control). Results from transgenic plants were similar to the results from in vitro antifungal assays using purified D4E1, which demonstrated IC50 values of 7.75 and 0.60 mM for A. fla6us and V. dahliae, respectively (unpublished). However, results from in vitro antifungal assays using control plant extract spiked with different concentrations of D4E1 (0 to 900 mM) were inconclusive. Antifungal effects were observed only at levels
Fig. 5. Inhibition of germinated conidia of A. fla6us by exposure to leaf extracts from tobacco plants expressing the antifungal peptide D4E1 for 1 h. (*) denotes a significant reduction (F=14.16, P B0.00001) in the number of A. fla6us colonies compared to extracts of pBI121-transformed control. Error bars indicate standard error of means. Mean separation was performed using the method of Tukey.
PCR insert from T302 and T307 exactly matched the expected sequence from amplification of the region spanning the d35S-D4E1-nos DNA region (data not shown).
Fig. 6. Inhibition of germinated conidia of V. dahliae by exposure to leaf extracts from tobacco plants expressing the antifungal peptide D4E1 for 1 h. (*) denotes a significant reduction (F =286.9; PB 0.0001) in the number of V. dahliae colonies compared to extracts of pBI121-transformed control. Error bars indicate standard error of means. Mean separation was performed using the method of Tukey.
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after inoculation. The one-way ANOVA indicated that anthracnose severity was significantly less (PB0.05) on transformants T304, T307, and T310 than on control tobacco leaves (Figs. 7 and 8). However, anthracnose severity on transformant T302 was not significantly different from the pBI121-transformed control. Similar inhibitory effect in vitro was also demonstrated in assays with the purified D4E1 against C. destructi6um (IC50 =13.02 mM; data not shown). 4. Discussion Fig. 7. Anthracnose severity among tobacco plants transformed with the antifungal gene coding for D4E1. (*) indicates a significant reduction (F =18.55, PB 0.0001) in anthracnose severity from pBI121-transformed control. Error bars indicate standard error of means. Mean separation was performed using the method of Tukey. Details of anthracnose severity assessment are given in Section 2.
greater than 50 mM and this did not correlate with the in vitro antifungal effects of purified D4E1.
3.5. In planta anthracnose resistance Leaves inoculated with C. destructi6um developed anthracnose lesions within 48–72 h
In recent years, there has been a large body of information generated on potential applications of antimicrobial peptides in disease resistance in plants. Many of these studies have focused on the in vitro efficacy of antimicrobial peptides and their related synthetic analogs or totally synthetic peptides against bacterial and, to a lesser extent, fungal pathogens [6,10,27]. Antimicrobial peptides are excellent candidates to augment disease resistance mechanisms in plants due to their i) rapid biocidal or biostatic ability against target cells; ii) activity against a wide spectrum of organisms at low concentrations; and iii) nontoxic nature with respect to mammalian cells [28–31]. Hemolytic
Fig. 8. Anthracnose symptoms on leaves of a pBI121-transformed negative control plant and a transgenic plant (T304) expressing the antifungal peptide D4E1. Leaves were detached and photographed 7 days after inoculation.
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activity against mammalian red blood cells was found to be extremely low for the synthetic peptide D4E1 at levels toxic to phytopathogens (J.M. Jaynes, unpublished). In many cases, synthetic analogs of natural antimicrobial peptides or totally synthetic antimicrobial peptides offer even more target specificity, increased efficacy at lower concentrations, and reduced degradation by plant proteases than their natural counterparts [15,32]. The synthetic peptide D4E1 has been shown to be inhibitory to growth of about twenty bacterial and fungal phytopathogens in our laboratory (Rajasekaran et al., unpublished). However, the high cost of producing large quantities of synthetic antimicrobial peptides prohibits their direct application to crop plants for the purpose of pathogen control. Instead, transgenic approaches are being undertaken as a means to provide the amount of antimicrobial peptides necessary to impart improved resistance to plant pathogens. Transgenic studies utilizing antimicrobial peptides to obtain resistance to bacterial pathogens have been reported from several laboratories [27,33,34]. However, only a few recent reports dealt with antifungal effects of transgenic plants expressing natural peptides. For example, Terras et al. [35] reported on the antifungal activity in transgenic tobacco expressing a radish plantdefensin peptide gene, Rs-AFP2. In their study, in vitro assays using crude protein fractions of transgenic leaves inhibited growth of Alternaria longipes and conferred enhanced resistance in planta to the same foliar pathogen. Expression of onion AceAMP1 in scented geranium resulted in increased resistance to Botrytis cineria leaf infection [36]. This study represents the first report of the successful generation of transgenic tobacco plants expressing a gene encoding a synthetic peptide with antifungal activity. Transgenic plants demonstrated a significant reduction in anthracnose severity caused by the tobacco pathogen, C. destructi6um (Figs. 7 and 8). Additionally, in vitro assays using crude protein extracts from transgenic plants expressing D4E1 demonstrated significant growth inhibition of the aflatoxigenic fungus A. fla6us and the wilt fungus V. dahliae (Figs. 5 and 6). A. fla6us, which is not a tobacco pathogen, produces aflatoxins in several economically important crop plants such as cotton, corn, peanut and treenuts compromising food and feed safety. Taken together, the antifungal effects of transgenic plants (present study) along with the effects
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of purified D4E1 on several phytopathogens (Rajasekaran et al., unpublished), offer promise of broad-spectrum resistance to bacterial as well as fungal phytopathogens. Southern hybridization analysis indicated that the transgenic plants contained from one to five copies of the gene encoding D4E1. The highest levels of resistance to pathogens both in vitro and in planta were observed for the plant T310 (Figs. 5 and 6) that had up to five copies of the D4E1 gene integrated into its genome (Fig. 3). This gene dosage effect may have contributed to higher levels of D4E1 peptide being produced. The transgenic plant T302 did not show a significant control in planta of anthracnose (Fig. 7), in a marked contrast to its significant activity in vitro against A. fla6us and V. dahliae, the reasons for which are not clearly understood. The modes of action of antifungal peptides have been studied extensively [10,37]. Those that interact specifically with the lipid components of cell membranes often cause formation of pores or ion channels that result in leakage of essential cellular minerals or metabolites or dissipate ion gradients in cell membranes. Other peptides have been shown to inhibit chitin synthase or b-D-glucan synthase. The synthetic peptide D4E1 complexes with ergosterol, a sterol present in germinating conidia of a number of fungal species, suggesting that its mode of action is lytic [18]. D4E1 has been shown to take on a b-sheet conformation in solution or during interaction with cell membranes, which is in contrast to cecropin A, and magainins which assume a-helical structures upon binding to acidic phospholipid bilayers. In addition, D4E1 was shown to be more resistant to fungal and cotton leaf proteases than cecropin A [18]. However, preliminary results in our laboratory on the effect of non-transformed control tobacco plant extracts, spiked with different levels of D4E1, did not correlate with in vitro antifungal effects of purified D4E1. Moreover, addition of plant protease inhibitors to tobacco plant extracts prior to spiking with D4E1 did not enhance the antifungal effect of the peptide (data not shown). The interaction of amphipathic peptides among themselves and with biological membranes is a complex phenomenon that is not well understood. Further research is needed to elucidate the mode of action in planta of this antimicrobial peptide.
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Acknowledgements The authors would like to thank Demegen, Mycogen for the D4E1 peptide and its sequence, Tony DeLucca for his advice on antifungal assays, and Maren Klich for the V. dahliae culture. We are grateful to Pamela Harris, Kurt Stromberg, and Neel Barnaby for their excellent technical assistance.
[16]
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