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Applied and Environmental Microbiology, October 2005, p. 6360-6367, Vol. 71, No. 10
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.10.6360-6367.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Production of an Engineered Killer Peptide in Nicotiana benthamiana by Using a Potato virus X Expression System

Marcello Donini,1 Chiara Lico,1 Selene Baschieri,1 Stefania Conti,2 Walter Magliani,2 Luciano Polonelli,2 and Eugenio Benvenuto1*

ENEA, UTS Biotecnologie, Sezione Genetica e Genomica Vegetale, C.R. Casaccia, P.O. Box 2400, I-00100 Rome, Italy,1 Universita' di Parma, Sezione di Microbiologia-Dipartimento di Patologia e Medicina di Laboratorio, Parma, Italy2

Received 2 March 2005/ Accepted 2 June 2005


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ABSTRACT
 
The decapeptide killer peptide (KP) derived from the sequence of a single-chain, anti-idiotypic antibody acting as a functional internal image of a microbicidal, broad-spectrum yeast killer toxin (KT) was shown to exert a strong microbicidal activity against human pathogens. With the aim to exploit this peptide to confer resistance to plant pathogens, we assayed its antimicrobial activity against a broad spectrum of phytopathogenic bacteria and fungi. Synthetic KP exhibited antimicrobial activity in vitro towards Pseudomonas syringae, Erwinia carotovora, Botrytis cinerea, and Fusarium oxysporum. KP was also expressed in plants by using a Potato virus X (PVX)-derived vector as a fusion to the viral coat protein, yielding chimeric virus particles (CVPs) displaying the heterologous peptide. Purified CVPs showed enhanced antimicrobial activity against the above-mentioned plant pathogens and human pathogens such as Staphylococcus aureus and Candida albicans. Moreover, in vivo assays designed to challenge KP-expressing plants (as CVPs) with Pseudomonas syringae pv. tabaci showed enhanced resistance to bacterial attack. The results indicate that the PVX-based display system is a high-yield, rapid, and efficient method to produce and evaluate antimicrobial peptides in plants, representing a milestone for the large-scale production of high-added-value peptides through molecular farming. Moreover, KP is a promising molecule to be stably engineered in plants to confer broad-spectrum resistance to phytopathogens.


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INTRODUCTION
 
The search for novel molecules with a wide spectrum of antimicrobial activity against bacterial and fungal pathogens is of challenging interest in human and plant pathology. Phytopathogens are the major cause of crop loss, preventing effective food distribution in the world. With the increased public concern about the use of chemicals for food crops, there is an urgent need to search for alternatives means to protect plants from pathogens. Recent advances in plant biotechnology have driven the development of new strategies for plant protection (29, 43). In particular, molecular strategies have been adopted which involve the expression in plants of antibacterial proteins of both plant (14, 17, 20, 28) and nonplant (9, 13, 31, 36) origins.

In the attempt to isolate novel potent antimicrobial molecules for therapeutic use, a killer toxin (KT) produced by the yeast Pichia anomala was isolated and characterized for its ability to kill other microorganisms presenting specific cell wall receptors (KT receptors) (24). Although therapeutically attractive for its wide spectrum of microbicidal activity in vitro against diverse pathogens (Candida albicans, Pneumocystis carinii, and Mycobacterium tuberculosis), KT was found to have no practical use because of its instability in the physiological milieu of mammalian cells as well as its antigenicity and toxicity (32). In the attempt to find a novel strategy to exploit KT's microbicidal properties, a KT-neutralizing monoclonal antibody (KT4) was used to raise anti-idiotypic antibodies representing the internal image of the KT active domain. These anti-idiotypic antibodies showed effective binding to KT receptors, KT-like microbicidal activity in vitro, and pronounced protective effects in vivo (33, 34). A single-chain antibody (KT-scFvH6) was also obtained from a phage display library derived from animals vaccinated with monoclonal antibody KT4 (25). KT-scFvH6 showed in vitro and in vivo microbicidal activity against a broad range of pathogens (10, 11, 12, 25). In the attempt to identify minimal active peptides within the antigen binding region of the scFv antibody for possible therapeutic use, a decapeptide consisting of seven amino acids of framework region 1 and the first three amino acids (Ser, Ala, and Ser) of complementarity-determining region 1 of the immunoglobulin light chain was selected and optimized by single-alanine replacement, yielding the killer peptide (KP) (35). The selected KP exerted strong microbicidal activity against C. albicans, Cryptococcus neoformans, and Paracoccidioides brasiliensis both in vitro and in vivo. Competition assays suggested that it interacts with a ß1-3 glucan KT receptor on target microbial cells (8, 35, 47).

In our work we assayed the in vitro antimicrobial potential of the chemically synthesized KP against important bacterial and fungal phytopathogens. Moreover, to address the problems associated with the efficiency of expression of peptides due to their toxicity and to evaluate the antimicrobial potential of KP against phytopathogens prior to performing plant engineering experiments, we exploited a transient-expression system based on the Potato virus X (PVX) vector (42). Viral expression vectors based on PVX have been extensively used to transiently express a wide array of different proteins in plants (3, 5, 19, 22, 23, 38, 39, 40), including an antimicrobial defensin (21, 41). In particular, the wasabi (Wasabia japonica) defensin was expressed in Nicotiana benthamiana by adopting the so-called "gene insertion" strategy, in which the gene encoding the heterologous protein is cloned as an additional open reading frame into a viral vector.

In the present study we adopted a "peptide display" strategy based on a modified PVX expression vector (26, 44) to obtain high expression levels of the KP decapeptide. To this end, the sequence coding for KP was fused in a PVX expression vector to the 5' end of the viral coat protein (CP) gene, generating chimeric virus particles (CVPs) that display the heterologous peptide fused to CP. Purified CVPs were characterized and assayed for antimicrobial activity against different phytopathogenic bacteria and fungi in vitro, showing a higher activity than the chemically synthesized KP. Moreover, in vivo assays designed to challenge KP-expressing plants (as CVPs) with Pseudomonas syringae pv. tabaci showed enhanced resistance to bacterial attack. Our results indicate that the PVX-based display system is a high-yield, rapid, and efficient method to produce and evaluate the activity of antimicrobial peptides in plants and that KP is a promising molecule of potential use for engineering plants with broad-spectrum resistance to pathogens.


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MATERIALS AND METHODS
 
Fungal and bacterial strains.
The bacterial and fungal strains used in this study were obtained from the National Collection of Plant Pathogenic Bacteria (NCPPB) (United Kingdom), the German Collection of Microorganisms and Cell Cultures (DMSZ) (Braunschweig, Germany), and the University of Perugia, Italy (DAPP-PG) and included Pseudomonas corrugata (NCPPB 2445), Pseudomonas syringae pv. tomato (NCPPB 2563 and DAPP-PG214), Erwinia carotovora subsp. carotovora (DSMZ 30170), Botrytis cinerea (DSMZ 877), and Fusarium oxysporum (DSMZ 62306). Pseudomonas syringae pv. tabaci (Pt11528) was a kind gift of P. Tavladoraki, University of "Roma Tre," Italy. Candida albicans (UP10) and Staphylococcus aureus (a38) were supplied by L. Polonelli, University of Parma, Italy.

KP and SP decapeptides.
The synthesis of KP (AKVTMTCSAS) on the basis of the sequence of scFv H6 and its optimization through alanine scanning have been described in detail elsewhere (35). A scrambled peptide (SP) (MSTAVSKCAT), containing the same amino acids as KP in a different order, was included as a negative control (35).

In vitro evaluation of the synthetic peptides and chimeric virus particles.
Antibacterial activity was assayed essentially as described previously (37). Bacterial strains were grown in LB medium to an optical density at 590 nm of approximately 1 and then diluted in phosphate-buffered saline to about 2 x 104 CFU/ml. KP or control peptide SP dissolved in PBS was added to the diluted cultures to final concentrations ranging from 5 to 100 µg/ml. After 6 h of incubation at 24°C, 50-µl aliquots were withdrawn, diluted in LB, and spread on LB agar plates. After overnight incubation at 28°C, the number of surviving bacteria, expressed as CFU, was estimated. Each experiment was performed in triplicate. The 50% inhibitory concentration (IC50) of the peptide was calculated by nonlinear regression analysis of curves obtained by plotting the number of CFU versus log of peptide concentration. Growth inhibition at 50 µg/ml peptide was calculated by the following equation: percentage of inhibition = (1 – number of CFU for KP treated/number of CFU for SP treated) x 100.

Assays with the CVPs were performed as described above. The PVX KP or control PVX SP was added to diluted cultures to final concentrations ranging from 10 to 50 µg/ml. Growth inhibition at 50 µg/ml concentration was calculated as described above.

Botrytis cinerea and Fusarium oxysporum were grown and maintained on potato dextrose agar (PDA) plates at 26°C. Inhibition of Fusarium spore germination was performed essentially as described previously (9). Briefly, spores were collected from PDA plates with a sterile loop and resuspended in ultrapure water. The spore suspension was pelleted at 8,100 x g in an Eppendorf microcentrifuge for 10 min at room temperature. Subsequently, spores were washed twice with sterile water and counted using a hemocytometer. The spore suspension was diluted to 400 spores/µl, and 1 µl of the suspension was added to 100 µl of peptide solutions at concentrations ranging from 10 to 100 µg/ml and incubated at 26°C. The number of germinating spores was counted using a hemocytometer, and inhibition of germination was calculated as follows: percentage of inhibition = 1 – (percentage of germination for KP treated/percentage of germination for SP treated) x 100. Alternatively, 40-µl aliquots of the spore suspension incubated with the peptide were spread on PDA plates, and after 48 h of incubation at 26°C, germinated spores were counted as CFU.

Inhibition of spore germination in Botrytis was performed as described before (48). Spore suspensions (100 µl) containing 103 conidia and increasing concentrations of peptides (10 to 100 µM) were incubated at 26°C. After 24 h, the number of germinating spores was evaluated and inhibition of germination was calculated using the above equation.

Antifungal assays with the CVPs were performed essentially as described above. The spore suspension was diluted to 400 spores/µl, and a 1-µl aliquot of this suspension was added to 100 µl of virus solution at different concentrations ranging from 10 to 150 µg/ml and incubated at 26°C. The number of germinating spores was counted, and inhibition of germination was calculated as described above.

Alternatively, 40-µl aliquots of the above-described spore suspension incubated with the virus were spread on PDA plates, and after 48 h of incubation at 26°C, germinated spores were counted as CFU. To visualize the growth inhibition, 30-µl aliquots of the suspension containing 103 conidia treated with the virus (10 to 150 µg/ml) were dropped at the centers of PDA plates. After incubation at 26°C, growth inhibition was observed as reduced colony radius.

The in vitro candidacidal activity was determined essentially as reported previously (35). In brief, approximately 150 viable germinating C. albicans UP10 cells, suspended in 10 µl of water, were added to 90 µl of a solution containing the CVPs at a final concentration of 100 µg/ml. After incubation for 6 h at 37°C, the fungal cells were spread on the surface of Sabouraud dextrose agar plates and incubated at 30°C; colony counting was performed after 48 h. In vitro antimicrobial activity against Staphylococcus aureus (a38) was assayed in a bacterial suspension in 1:1 Mueller-Hinton medium-physiological solution. Ten microliters of bacterial suspension was added to 90 µl of a solution containing the CVPs at a final concentration of 100 µg/ml. After incubation for 4 h at 37°C, the suspensions were dispensed, spread on Müller-Hinton agar plates, and incubated at 37°C. Colony counting was performed after 24 h.

Construction of PVX CP-peptide fusions.
The KP sequence was inserted into the coat protein of the variant PVX-Sma virus as an N-terminal in-frame fusion. PVX-Sma is a variant of the PVX-201 virus (5) carrying an N-terminal deletion of 22 amino acids in the CP. The PVX-Sma plasmid, essentially derived from pPVX-201 (5), was used as a vector. Briefly, a double-stranded DNA fragment encoding the KP peptide, with SmaI and NheI ends appended, was obtained by in vitro annealing of two oligonucleotides, 5'-CTAGCATGGCTAAAGTCACAATGACATGTTCAGCTTCA-3' and 5'-TGAAGCTGAACATGTCATTGTGACTTTAGCCATG-3'. The double-stranded oligonucleotide was inserted between the SmaI and NheI sites of pPVX-Sma, yielding plasmid pPVX-KP. The SP sequence corresponding to the scramble peptide used as a control was also cloned between the SmaI and NheI sites of pPVX-Sma, yielding plasmid pPVX-SP. DNA sequencing analysis of all constructs was performed on an automated DNA sequencer (MWG Biotech), confirming the predicted sequences.

Nicotiana benthamiana plant inoculation and virus analysis.
Plants were inoculated with virus carrying plasmid pPVX-KP, pPVX-SP, pPVX-Sma, or pPVX-201 by gently rubbing the surfaces of two leaves per plant with carborundum and distributing on each leaf 20 µl of water containing 20 µg of plasmid DNA. Upon systemic infection (10 to 12 days after inoculation), the correct expression of the foreign sequence was verified on systemic leaves by reverse transcription-PCR (RT-PCR) and DNA sequencing. Briefly, total RNA was extracted using the RNeasy plant minikit (QIAGEN), and the RT-PCRs were performed using the GeneAmp PCR kit (Perkin-Elmer). The cDNA was synthesized using oligo(dT)16 as a primer, and PCR was performed with two primers mapping on the region of the PVX genome containing the CP (PVX-back, 5'-CTGGGGAATCAATCACAGTGTTGGCTTG-3'; PVX-for, 5'-TCAGATCGAGACGACTACGGCAA-3'). Sequencing of the RT-PCR products was performed using the PVX-back primer. Stability and infectivity of the recombinant viruses were assayed by performing cycles of reinfection using the sap extracted from systemic leaves of the plants inoculated with plasmid vectors. Briefly, infected tissue (0.2 g) was finely ground in liquid nitrogen, and 100 µl of phosphate buffer (PBS, 0.1 M, pH 7) was added. After a 10 min of centrifugation in an Eppendorf microcentrifuge at 20,000 x g and 4°C, the supernatant was recovered and used to inoculate N. benthamiana plants as described above.

The presence of the CVPs in the sap of infected plants was assayed by Western blot analysis. Briefly, the infected plant tissue was harvested and homogenized in 1x PBS containing protease inhibitors ("Complete Mini"; Boehringer Mannheim). After two centrifugations at 20,000 x g for 30 min at 4°C, the supernatant was analyzed by sodium dodecyl sulfate-12.5% polyacrylamide gel electrophoresis. The total soluble protein concentration in the supernatants was quantified using the Bradford colorimetric assay (Bio-Rad). An amount of 5 µg of total leaf proteins were analyzed in each lane. Apparent molecular masses were estimated with prestained standards (Rainbow Markers; Amersham). Blotting was performed on a polyvinylidene difluoride membrane (Hybond-P; Amersham), and immunodetection was carried out using the anti-PVX-CP rabbit polyclonal antibody (Adgen Limited, United Kingdom) specific for the coat protein at 5 µg/ml, followed by biotinylated anti-rabbit polyclonal antibody and streptavidin-horseradish peroxidase conjugate (Amersham). Signal development was obtained by enhanced chemiluminescence (ECL Plus; Amersham).

Virus purification.
The chimeric PVX-KP, PVX-SP, and PVX-Sma virus particles were purified from plant tissues with the typical symptoms of systemic viral infection (upper leaves showing chlorotic mosaic with rugose areas) 10 to 12 days after inoculation, as described previously (1). Briefly, leaf tissues were ground, and the sap was separated from cellular debris by centrifugation. Virus particles were purified on a cesium chloride density gradient (CsCl concentration of 0.43 g/ml) using a Beckman TL100 ultracentrifuge with a TLA 100.4 rotor at 86,000 x g and 4°C for 12 h. The virus suspension was collected, dialyzed against 1x PBS, and stored at –20°C. The yield of CVPs in the cases of PVX-KP and PVX-SP was in the range of 0.5 to 1.5 mg per 20 g (fresh weight) of leaf tissue. Purified viruses were analyzed by sodium dodecyl sulfate-12.5% (wt/vol) polyacrylamide gel electrophoresis followed by AgNO3 staining (27), and quantification was performed spectrophotometrically as described previously (1). The amount of 20 µg of purified virus corresponds to approximately 0.8 µg of fused recombinant peptide.

In vivo resistance assays on PVX-infected plants.
N. benthamiana plants were inoculated with the pPVX constructs. After 5 days, upper leaves not yet showing systemic viral symptoms were challenged with 50 µl of a Pseudomonas syringae pv. tabaci (Pt11528) suspension (105 CFU/ml LB medium) by infiltrating bacteria into the lamina with a needleless 5-ml syringe. The syringe was placed on the abaxial side of the leaf in the laminar area between two lateral veins, and the suspension was slowly injected in the intercellular space of the leaf. Inoculum concentration was determined by optical density at 600 nm and confirmed by plating serial dilutions on LB plates. Plants were then grown at 25°C with 16 h of light. After about 8 days, the necrotic zone around the inoculation site was observed and pictures were taken. All necrotic lesions showed an irregular outline, and it was difficult to exactly quantify the area of necrosis. In a separate round of experiments, the bacterial growth in the infiltrated region was quantified, and leaf disks (1-cm diameter) excised within a single infiltrated area from each plant were collected. A total of nine disks were collected from each treatment. The disks were then divided into three sets of three and quickly ground in 500 µl LB. Homogenates were plated on LB agar at three different dilutions (10–1, 10–2, and 10–3). P. syringae colonies were counted after incubation at 28°C for 24 to 48 h. Values are means ± standard errors of the means from three independent experiments.


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RESULTS
 
In vitro assay of the synthetic KP decapeptide.
The synthetic KP decapeptide was tested in vitro for its inhibitory activity against several bacterial and fungal phytopathogens. The growth of P. corrugata, P. syringae pv. tomato (NCPPB 1106, NCPPB 2563, and DAPP-PG 214), P. syringae pv. tabaci (Pt11528), and E. carotovora was measured in the presence of KP or its irrelevant control scramble peptide (SP) at different concentrations. In Fig. 1A a comparison of the growth inhibition percentages for all bacterial strains at the concentration of 50 µg/ml is shown. The IC50s, calculated from the dose-response curves for P. syringae pv. tomato (NCPPB 1106) and P. syringae pv. tabaci (Pt11528), were 32 µg/ml and 44 µg/ml, respectively, whereas it was 50 µg/ml in E. carotovora (Table 1).



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  		FIG. 1. In vitro inhibitory activity of the KP synthetic decapeptide against bacterial and fungal pathogens. (A) The growth inhibition activity of synthetic KP (50-µg/ml concentration) against P. corrugata, P. syringae pv. tomato (NCPPB 1106, NCPPB 2563, and DAPP-PG 214), P. syringae pv. tabaci (Pt11528), and E. carotovora was measured by the reduction in the number of CFU compared to that with the irrelevant SP scramble peptide used as a control. Growth inhibition was calculated with the following equation: percentage of inhibition = (1 – number of CFU for KP treated/number of CFU for SP treated) x 100. (B) Effect of KP on spore germination of F. oxysporum and B. cinerea. Spore germination assay at a KP concentration of 50 µg/ml shows a 50% inhibition of B. cinerea and a 62% inhibition of F. oxysporum. Values are means of triplicate determinations ± standard errors of the means.


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  		TABLE 1. Antimicrobial activities of KP decapeptide and PVX-KP recombinant virus against phytopathogenic bacteria and fungi

Similar studies were performed in vitro against the fungal pathogens F. oxysporum and B. cinerea. Spore germination in the presence of 50 µg/ml of KP showed 50% inhibition for B. cinerea and 62% for F. oxysporum, whereas fungal growth was totally inhibited at 120 µg/ml and 100 µg/ml, respectively (Fig. 1B; Table 1).

Fusion of KP and SP sequences to the coat protein of PVX and production of chimeric virus particles in plants.
We fused the sequences encoding the KP decapeptide and the SP irrelevant peptide to be used as a control in the antimicrobial assays to the PVX CP-encoding gene (Fig. 2A). We used the pPVX-Sma vector, derived from a PVX mutant which was previously isolated in our lab. This stable PVX mutant bears a 5' deletion of 66 nucleotides (22 amino acids) in the CP-coding gene compared to the PVX-201 (5) and a SmaI cloning site used for the N-terminal fusion of recombinant sequences to the viral coat protein. Recombinant pPVX-KP and pPVX-SP plasmids containing the full-length cDNA of the mutant PVX-Sma virus with the modified CP sequences were used to inoculate N. benthamiana plants. Inoculation with the plasmid constructs was performed by gently rubbing the surfaces of two leaves per plant with carborundum and distributing on each leaf 20 µl of water containing 20 µg of plasmid DNA. The expression of the foreign sequence was confirmed 12 days after inoculation (Fig. 2B) by extracting total RNA and performing RT-PCR using PVX-specific oligonucleotides followed by DNA sequence analysis (data not shown). Modified CPs were highly stable in the format of choice (KP and SP sequences fused at the 5' end of the CP) and retained their ability to form CVPs after three cycles of reinfection (data not shown).



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  		FIG. 2. Schematic representation of PVX-derived vectors. The diagrams show the locations of the viral genes coding for the viral replicase (166K), the movement proteins (25K, 8K, and 12K), and the coat protein (CP) of the PVX-Sma variant. The PVX-Sma virus bears an N-terminal deletion of 22 amino acids compared to the wild-type PVX-201 (5) and was used for the antimicrobial peptide fusions. The viral genome is inserted between the constitutive promoter 35S, derived from cauliflower mosaic virus, and the transcription terminator of the nopaline synthase gene (NOS), which if important for the regulation of viral genome expression upon plant infection with plasmid DNA. (A) pPVX-KP is the plasmid carrying the virus genome engineered to express the KP sequence fused to the CP and was obtained by inserting a double-stranded oligonucleotide between the SmaI and NheI sites of the pPVX-Sma plasmid, derived from pPVX-201 (5). The SP sequence corresponding to the scramble peptide used as a negative control was also cloned between the SmaI and NheI sites of pPVX-Sma, yielding plasmid pPVX-SP (see Materials and Methods). (B) Phenotypes at 12 days postinoculation of leaves of Nicotiana benthamiana that were systemically infected with pPVX-KP and pPVX-SP. No difference was observed in the viral symptoms compared to those after infection with the virus without the insert (PVX-Sma) and the wild-type PVX-201. After the mechanical inoculation on a basal leaf, the virus moves from cell to cell in the infected tissue through the plasmodesmata and spreads systemically in the upper leaves through the phloem vein network. As shown, PVX produces typical mosaic symptoms on systemic leaves. Further experiments demonstrated that the modified CPs were highly stable and retained their ability to form CVPs after three cycles of reinfection.

Analysis and yield of CVPs.
To study the expression of the recombinant CPs bearing the KP and SP decapeptide fusions, leaves showing systemic infection were harvested 10 days after inoculation with pPVX-KP, pPVX-SP, and the pPVX-Sma displaying no peptide. Total protein extracts from leaves were analyzed by Western blotting using the anti-PVX-CP rabbit polyclonal antibody (Adgen Limited, United Kingdom) followed by biotinylated anti-rabbit polyclonal antibody and streptavidin-horseradish peroxidase conjugate (Fig. 3). The results indicate the presence of single bands with the expected molecular weight (approximately 23,900) corresponding to KP-CP and SP-CP fusion proteins (lanes 2 and 3, respectively). The band corresponding to the coat protein of the PVX-Sma virus fusion is lower than those of KP-CP and SP-CP, as expected (lane 1). A protein extract from a noninoculated plant (lane 4) was used as a negative control.



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  		FIG. 3. Analysis of PVX-infected plants. Western blot analysis of total leaf protein from Nicotiana benthamiana leaves is shown. Gels were blotted to polyvinylidene difluoride membranes, and immunodetection was performed using anti-PVX CP mouse polyclonal antibodies (Adgen Limited, United Kingdom). An amount of 5 µg of total leaf protein was analyzed in each lane. Lane 1, proteins from upper leaves of pPVX-Sma-infected plants; lane 2, proteins from upper leaves of pPVX-KP-inoculated plants; lane 3, proteins from leaves of pPVX-SP-infected plants; lane 4, mock-inoculated plants used as negative controls. The results indicate the presence of single bands with the expected molecular weight (approximately 23,900) corresponding to KP-CP and SP-CP fusion proteins (lanes 2 and 3, respectively). The coat protein of the PVX-Sma virus bearing no peptide fusion is also shown as a control (lane 1), showing a lower band as expected (molecular weight, 23,000). The arrow indicates the position of the 25-kDa band of the molecular mass marker (Rainbow Markers; Amersham).

Recombinant PVX-KP and PVX-SP and PVX-Sma viruses were extracted from infected leaf tissues and purified by CsCl density gradients, giving yields in the range of 0.5 to 1.5 mg per 20 g of fresh weight.

In vitro inhibition of bacterial and fungal pathogens by purified chimeric virus particles.
Purified PVX-KP virus particles were tested in vitro for inhibitory activity against the bacterial phytopathogens P. corrugata, P. syringae (pv. tomato NCPPB 1106, NCPPB 2563, and DAPP-PG 214), and E. carotovora. In Fig. 4A the growth inhibition percentage at the CVP concentration of 50 µg/ml, corresponding to a concentration of approximately 2 µg/ml of fused recombinant peptide, is shown. The IC50s for P. syringae pv. tomato (NCPPB 1106) and E. carotovora were 24 µg/ml and 42 µg/ml, respectively, while the IC50 for P. syringae pv. tabaci was 28 µg/ml (Table 1).



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  		FIG. 4. In vitro inhibitory activity of the PVX-KP chimeric virus particles against bacterial and fungal pathogens. (A) Growth inhibition activity of PVX-KP (50 µg/ml) against P. corrugata, P. syringae (pv. tomato NCPPB 1106, NCPPB 2563, and DAPP-PG 214), and E. carotovora was measured by the reduction in the number of CFU compared to the controls treated with the irrelevant PVX-SP. Growth inhibition was calculated by the following equation: percentage of inhibition = (1 – number of CFU for PVX-KP treated/number of CFU for PVX-SP treated) x 100. The in vitro activity against the human pathogens Candida albicans and Staphylococcus aureus at a concentration of 100 µg/ml is also reported (shaded bars). (B) Effect of PVX-KP on spore germination of Fusarium oxysporum and Botrytis cinerea. The reduction in the number of germinated spores was compared to that for controls treated with the irrelevant PVX-SP. Spore germination assay at 50-µg/ml PVX-KP shows a 90% inhibition of B. cinerea and a 95% inhibition of F. oxysporum. Values are means of triplicate determinations ± standard errors of the means.

The inhibitory activity of the peptide-displaying virus particles was also tested against the two human pathogens Candida albicans (UP10) and Staphylococcus aureus (a38), causing growth inhibition of 86% and 80%, respectively, at the concentration of 100 µg/ml (Fig. 4A).

In vitro inhibition studies were also performed against the phytopathogenic fungi F. oxysporum and B. cinerea. Spore germination assays at 50 µg/ml of PVX-KP showed 90% inhibition of B. cinerea and 95% inhibition of F. oxysporum (Fig. 4B). Moreover, the growth of both fungi was totally inhibited at 80 µg/ml PVX-KP, corresponding to approximately 3.2 µg/ml of fused recombinant peptide (Table 1).

In vitro activity of the recombinant PVX-KP virus against P. syringae pv. tomato (DAPP-PG 214) was compared to that of chemically synthesized KP. Figure 5 is representative of a CFU assay after incubation with 50 µg/ml of reagent. The rate of bacterial killing after 1 h was approximately 99% with PVX-KP, whereas it was 21% with simple KP peptide.



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  		FIG. 5. In vitro activity of the recombinant PVX-KP virus against P. syringae pv. tomato DAPP-PG 214 compared to the chemically synthesized KP decapeptide. Growth inhibition was monitored counting the number of surviving bacteria (CFU) at different incubation times. After incubation for 1 h with 50 µg/ml of PVX-KP, the rate of bacterial killing was 99%, whereas with KP decapeptide (50 µg/ml), the killing rate was 21% at 1 h and reached 92% after 4 h. It should be noted that the concentration of 50 µg/ml of PVX-KP corresponds to a theoretical concentration of fused peptide of 2 µg/ml. As negative controls, bacteria were incubated either with PBS, with the chemically synthesized SP scramble peptide, or with the PVX-SP virus displaying the scramble peptide. Values for each time point are means of triplicate determinations ± standard errors of the means.

In vivo inhibition of Pseudomonas syringae by plants producing chimeric virus particles.
To evaluate the potential resistance to bacterial pathogens, plants infected with PVX-KP and PVX-SP were challenge inoculated with suspensions (105 CFU/ml LB medium) of P. syringae pv. tabaci (Pt11528) by infiltrating bacteria into the abaxial side of the leaf with a needleless 5-ml syringe. Chlorotic and necrotic symptoms at 5 days postinoculation are shown in Fig. 6A. Symptoms developed markedly on plants infected with PVX-SP, while PVX-KP-infected plants exhibited reduced areas of necrosis surrounding the point of inoculation. To assess whether the reduced symptoms reflected a reduction in bacterial growth, bacteria in the infiltrated region were quantified. Leaf disks (1 cm in diameter) were collected within a single infiltrated area from each treated plant and were quickly ground in 500 µl LB. Homogenates were plated on LB agar at three dilutions (10–1, 10–2, and 10–3). P. syringae colonies were counted after incubation at 28°C for 24 to 48 h. The results, reported in Fig. 6B, show a 90% reduction in bacterial growth in PVX-KP-infected plants compared to the controls (PVX-SP and PVX-Sma, bearing no peptide fusion). Data from plants infiltrated with just H2O show no bacterial growth, excluding any possible bacterial contamination from the leaves.



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  		FIG. 6. In planta assays. N. benthamiana plants were inoculated with the viral constructs pPVX-KP, bearing the killer peptide, and pPVX-SP, bearing the irrelevant scramble peptide as a control. After 5 days, the upper leaves of these plants were challenge infiltrated with diluted cultures of P. syringae pv. tabaci (Pst) (105 CFU/ml LB medium) by infiltrating bacteria into the lamina with a needleless 5-ml syringe. (A) Representative pictures of the chlorotic and necrotic symptoms observed after 8 days from the infiltration experiments conducted with P. syringae pv. tabaci (top). The symptoms appear weaker with pPVX-KP-inoculated plants than with pPVX-SP-inoculated plants, used as negative controls. Mock-infiltrated plants (H2O), used as negative controls (bottom), show no necrotic area, as expected. (B) The number of surviving bacteria following the leaf infiltration with P. syringae was determined. Leaf disks (1-cm diameter) were collected within a single infiltrated area from each treated plant and were quickly ground in 500 µl LB. Homogenates were plated on LB agar at three dilutions (10–1, 10–2, and 10–3). P. syringae colonies were counted after incubation at 28°C for 24 to 48 h. A reduced number of surviving bacteria is observed in the PVX-KP-infected plants compared to plants infected with PVX-SP and PVX-Sma, lacking the peptide, used as controls. A plant not infected with virus (N.I.) but infiltrated with P. syringae was also used as a control. Data for plants infiltrated with distilled water are also reported (H2O). Values are means ± standard errors of the means from three independent experiments.


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DISCUSSION
 
The KP peptide derived from an anti-idiotypic scFv has potent antimicrobial activity against different fungal animal pathogens, such as C. albicans, C. neoformans, and P. brasiliensis (8, 35, 47). In this work we assayed the efficacy of this peptide in inhibiting the growth and spore germination of important fungal and bacterial phytopathogens. The synthetic decapeptide showed strong activity against different isolates of Pseudomonas syringae and Erwinia carotovora, as well as a weaker but still significant inhibitory effect against the phytopathogenic fungi B. cinerea and F. oxysporum. Such a broad-spectrum activity has been reported for only a few classes of antimicrobial peptides. In fact, the expression in plants of natural antimicrobial peptides, such as barley {alpha}- or ß-hordothionin (15), barley {alpha}-thionin (7), horseshoe crab tachyplesin (2), or cecropin B (16), conferred only weak resistance to a limited range of bacteria and fungi. More encouraging results were obtained with the synthetic chimeric cecropin-melittin cationic peptide (31), which exhibited a broader spectrum of antimicrobial activity. In our work the synthetic KP decapeptide was demonstrated to posses an antibacterial activity similar to that reported for other plant defensins (30, 45, 46) but lower those of the cecropin-melittin chimera (13) or the synthetic analogue of magainin, MSI 99 (31). Furthermore, in spore germination assays against the phytopathogenic fungus F. oxysporum, KP showed inhibition levels similar to those reported for MSI 99 (9).

Therefore, the KP peptide has efficacy against both animal and plant pathogenic bacteria and fungi. This broad-spectrum activity could be explained by taking into account the molecular target of KP. In fact, it has been demonstrated that the decapeptide interacts with specific cell wall constituents common both in bacteria and fungi, such as the ß-1,3-glucan laminarin (35).

Many attempts to overexpress antimicrobial peptides in transgenic plants failed as a consequence of their toxicity or low expression yields, proving in many cases to be ineffective to enhance resistance against pathogens (18). Viral vector-based transient-expression systems have proved to be a useful alternative to stable genetic transformation. Therefore, we used an epichromosomal expression system to produce large quantities of the antimicrobial KP peptide and rapidly evaluate possible unintended effects in plant cells.

It was previously demonstrated that the N terminus of the PVX coat protein is exposed to the surface of the virion with the first 33 amino acid residues of CP, forming a ß-strand (4). Consequently, PVX represented a good candidate carrier for foreign peptides, as it self-assembles in an ordered fashion and accumulates to high levels in infected tissues. In addition, it does not reveal size and packaging constraints usually found in icosahedral viruses. Hence, the decapeptide was transiently expressed as an N-terminal fusion with the coat protein, engendering the formation of CVPs that likely display KP in an accessible and functional format. The main advantages of this transient-expression system are in the rapidity of the procedure and yields such as those reported here (0.5 to 1.5 mg per 20 g of infected tissue). These findings are basically different from what was reported for the antimicrobial wasabi defensin expressed via a PVX-derived vector in an additional open reading frame configuration (21), which exhibited low expression and purification levels (40 µg per 100 g of infected tissue). Notably, our system can be further refined for the production of free peptides by simple digestion of the purified virus particle with the appropriate proteolytic enzyme.

The main constraints to this whole approach are the general low stability and infectivity of the chimeric virus particles carrying exogenous sequences fused at the N terminus of CP, traits that depend mainly on the fused sequence length and amino acid composition (6). However, in this work we demonstrate that the two PVX constructs (pPVX-KP and pPVX-SP) retain both viral stability and infectivity even after three rounds of reinfection. Therefore, the additional fusion sequences seem not to interfere with virus assembly or with cell-to-cell movement. Moreover, analysis of the infected plant extracts shows that the CP of the recombinant virus is present as a single band on Western blots, indicating its stability to protease degradation.

The in vitro antimicrobial activity of the KP-displaying PVX chimeric virus assayed against both bacteria and fungi was surprisingly high compared to that of the synthetic KP at equimolar concentrations. The significantly higher efficacy observed for the virus-fused peptides could be explained taking into account a higher stability of the fused KP in solution (less aggregation or protease digestion and highly ordered display of peptides by the viral carrier) with respect to the chemically synthesized peptide.

The in vivo antimicrobial activity of PVX-KP chimeric virus particles assayed against P. syringae pv. tabaci in N. benthamiana plants showed a strong protection against bacterial disease. Whatever the mechanism, it can be hypothesized that the PVX-engineered KP functionally exerts the antimicrobial activity in the intercellular space, inhibiting the early stages of P. syringae growth.

On the basis of the results presented here, we provide a promising field of evidence to widen the spectrum of antimicrobial activity of the KP decapeptide from animal to plant pathogens. We also introduce the concept of biological synthesis of antimicrobial peptides through the PVX-based display system for both mass production and evaluation of protective effects in plants. Of particular interest is the potential use of the KP decapeptide to engineer plants with broad-spectrum resistance to pathogens.


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ACKNOWLEDGMENTS
 
We thank A. Desiderio, M. E. Villani, U. De Corato, and D. De Martinis for useful discussions during the preparation of this work.


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FOOTNOTES
 
* Corresponding author. Mailing address: ENEA, UTS Biotecnologie, Sezione Genetica e Genomica Vegetale, C.R. Casaccia, P.O. Box 2400, I-00100 Rome, Italy. Phone: (39) 06 3048-6558 Fax: (39) 06 3048-4171. E-mail: benvenutoe{at}casaccia.enea.it. Back


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REFERENCES
 
    1
  1. AbouHaidar, M. G., H. Xu, and K. L. Hefferon. 1998. Potexvirus isolation and RNA extraction. Methods Mol. Biol. 81:131-143.[Medline]
    2. 2
  2. Allefs, S. J. H. M., E. R. DeJong, D. E. A. Florack, C. Hoogendoorn, and W. J. Stiekema. 1996. Erwinia soft rot resistance of potato cultivars expressing antimicrobial peptide tachyplesin I. Mol. Breeding 2:97-105.
    3. 3
  3. Avesani, L., A. Falorni, G. B. Tornelli, C. Marusic, A. Porceddu, A. Polverari, C. Faleri, F. Calcinaro, and M. Pezzetti. 2003. Improved in planta expression of the human islet autoantigen glutamic acid decarboxylase (GAD65). Transgenic Res. 12:203-212.[CrossRef][Medline]
    4. 4
  4. Baratova, L. A., N. I. Grebenshchikov, E. N. Dobrov, A. V. Gedrovich, I. A. Kashirin, A. V. Shishkov, A. V. Efimov, L. Jarvekulg, Y. L. Radavsky, and M. Saarma. 1992. The organization of potato virus X coat proteins in virus particles studied by tritium planigraphy and model building. Virology 188:175-180.[CrossRef][Medline]
    5. 5
  5. Baulcombe, D. C., S. Chapman, and S. Santa Cruz. 1995. Jellyfish green fluorescent protein as a reporter for virus infections. Plant J. 7:1045-1053.[CrossRef][Medline]
    6. 6
  6. Bendahmane, M., M. Koo, E. Karrer, and R. N. Beachy. 1999. Display of epitopes on the surface of tobacco mosaic virus: impact of charge and isoelectric point of the epitope on virus-host interactions. J. Mol. Biol. 290:9-20.[CrossRef][Medline]
    7. 7
  7. Carmona, M. J., A. Molina, J. A. Fernandez, J. J. Lopez-Fando, and F. Garcia-Olmedo. 1993. Expression of the alpha-thionin gene from barley in tobacco confers enhanced resistance to bacterial pathogens. Plant J. 3:457-462.[CrossRef][Medline]
    8. 8
  8. Cenci, E., F. Bistoni, A. Mencacci, S. Perito, W. Magliani, S. Conti, L. Polonelli, and A. Vecchierelli. 2004. A synthetic peptide as a novel anticryptococcal agent. Cell Microbiol. 6:953-961.[CrossRef][Medline]
    9. 9
  9. Chakrabarti, A., T. R. Ganapathi, P. K. Mukherjee, and V. A. Bapat. 2003. MSI-99, a magainin analogue, imparts enhanced disease resistance in transgenic tobacco and banana. Planta 216:587-596.[CrossRef][Medline]
    10. 10
  10. Conti, S., F. Fanti, W. Magliani, M. Gerloni, D. Bertolotti, A. Salati, A. Cassone, and L. Polonelli. 1998. Mycobactericidal activity of human natural, monoclonal, and recombinant yeast killer toxin-like antibodies. J. Infect. Dis. 177:807-811.[Medline]
    11. 11
  11. Conti, S., W. Magliani, S. Arseni, E. Dieci, R. Frazzi, A. Salati, P. E. Varaldo, and L. Polonelli. 2000. In vitro activity of monoclonal and recombinant yeast killer toxin-like antibodies against antibiotic-resistant gram-positive cocci. Mol. Med. 6:613-619.[Medline]
    12. 12
  12. Conti, S., W. Magliani, S. Arseni, R. Frazzi, A. Salati, L. Ravaneti, and L. Polonelli. 2002. Inhibition by yeast killer toxin-like antibodies of oral streptococci adhesion to tooth surfaces in an ex vivo model. Mol. Med. 8:313-317.[Medline]
    13. 13
  13. DeGray, G., K. Rajasekaran, F. Smith, J. Sanford, and H. Daniell. 2001. Expression of an antimicrobial peptide via the chloroplast genome to control phytopathogenic bacteria and fungi. Plant Physiol. 127:852-862.[Abstract/Free Full Text]
    14. 14
  14. Epple, P., K. Apel, and H. Bohlmann. 1997. Overexpression of an endogenous thionin enhances resistance of Arabidopsis against Fusarium oxysporum. Plant Cell 9:509-520.[Abstract]
    15. 15
  15. Florack, D. E., W. G. Dirkse, B. Visser, F. Heidekamp, and W. J. Stiekema. 1994. Expression of biologically active hordothionins in tobacco. Effects of pre- and pro-sequences at the amino and carboxyl termini of the hordothionin precursor on mature protein expression and sorting. Plant Mol. Biol. 24:83-96.[CrossRef][Medline]
    16. 16
  16. Florack, D., S. Allefs, R. Bollen, D. Bosch, B. Visser, and W. Stiekema. 1995. Expression of giant silkmoth cecropin B genes in tobacco. Transgenic Res. 4:132-141.[CrossRef][Medline]
    17. 17
  17. Gao, A. G., S. M. Hakimi, C. A. Mittanck, Y. Wu, B. M. Woerner, D. M. Stark, D. M. Shah, J. Liang, and C. M. Rommens. 2000. Fungal pathogen protection in potato by expression of a plant defensin peptide. Nat. Biotechnol. 18:1307-1310.[CrossRef][Medline]
    18. 18
  18. Gura, T. 2001. Innate immunity. Engineering protection for plants. Science 291:2070.[Free Full Text]
    19. 19
  19. Hendy, S., Z. C. Chen, H. Barker, S. Santa Cruz, S. Chapman, L. Torrance, W. Cockburn, and G. C. Whitelam. 1999. Rapid production of single-chain Fv fragments in plants using a potato virus X episomal vector. J. Immunol. Methods 231:137-146.[CrossRef][Medline]
    20. 20
  20. Holtorf, S., J. Ludwig-Muller, K. Apel, and H. Bohlmann. 1998. High-level expression of a viscotoxin in Arabidopsis thaliana gives enhanced resistance against Plasmodiophora brassicae. Plant Mol. Biol. 36:673-680.[CrossRef][Medline]
    21. 21
  21. Kiba, A., H. Saitoh, M. Nishihara, K. Omiya, and S. Yamamura. 2003. C-terminal domain of a hevein-like protein from Wasabia japonica has potent antimicrobial activity. Plant Cell Physiol. 44:296-303.[Abstract/Free Full Text]
    22. 22
  22. Kooman-Gersmann, M., R. Vogelsang, E. C. Hoogendijk, and P. J. De Wit. 1997. Assignment of amino acid residues of the AVR9 peptide of Cladosporium fulvum that determine elicitor activity. Mol. Plant-Microbe Interact. 10:821-829.[Medline]
    23. 23
  23. Li, Y., Y. Geng, H. Song, G. Zheng, L. Huan, and B. Qiu. 2004. Expression of a human lactoferrin N-lobe in Nicotiana benthamiana with potato virus X-based agroinfection. Biotechnol. Lett. 26:953-957.[CrossRef][Medline]
    24. 24
  24. Magliani, W., S. Conti, M. Gerloni, D. Bertolotti, and L. Polonelli. 1997. Yeast killer systems. Clin. Microbiol. Rev. 10:369-400.[Abstract]
    25. 25
  25. Magliani, W., S. Conti, F. de Bernardis, M. Gerloni, D. Bertolotti, P. Mozzoni, A. Cassone, and L. Polonelli. 1997. Therapeutic potential of anti-idiotypic single chain antibodies with yeast killer toxin activity. Nat. Biotechnol. 15:155-158.[CrossRef][Medline]
    26. 26
  26. Marusic, C., P. Rizza, L. Lattanzi, C. Mancini, M. Spada, F. Belardelli, E. Benvenuto, and I. Capone. 2001. Chimeric plant virus particles as immunogens for inducing murine and human immune responses against human immunodeficiency virus type 1. J. Virol. 75:8434-8439.[Abstract/Free Full Text]
    27. 27
  27. Merril, C. R., D. Goldman, S. A. Sedman, and M. H. Ebert. 1981. Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science 211:1437-1438.[Abstract/Free Full Text]
    28. 28
  28. Molina, A., and F. Garcia-Olmedo. 1997. Enhanced tolerance to bacterial pathogens caused by the transgenic expression of barley lipid transfer protein LTP2. Plant J. 12:669-675.[CrossRef][Medline]
    29. 29
  29. Mourgues, F., M. N. Brisset, and E. Chevreau. 1998. Strategies to improve plant resistance to bacterial diseases through genetic engineering. Trends Biotechnol. 16:203-210.[CrossRef][Medline]
    30. 30
  30. Osborn, R. W., G. W. De Samblanx, K. Thevissen, I. Goderis, S. Torrekens, F. Van Leuven, S. Attenborough, S. B. Rees, and W. F. Broekaert. 1995. Isolation and characterisation of plant defensins from seeds of Asteraceae, Fabaceae, Hippocastanaceae and Saxifragaceae. FEBS Lett. 368:257-262.[CrossRef][Medline]
    31. 31
  31. Osusky, M., G. Zhou, L. Osuska, R. E. Hancock, W. W. Kay, and S. Misra. 2000. Transgenic plants expressing cationic peptide chimeras exhibit broad-spectrum resistance to phytopathogens. Nat. Biotechnol. 18:1162-1166.[CrossRef][Medline]
    32. 32
  32. Pettoello-Mantovani, M., A. Nocerino, L. Polonelli, G. Morace, S. Conti, L. Di Martino, G. De Ritis, M. Iafusco, and S. Guandalini. 1995. Hansenula anomala killer toxin induces secretion and severe acute injury in the rat intestine. Gastroenterology 109:1900-1906.[CrossRef][Medline]
    33. 33
  33. Polonelli, L., F. De Bernardis, S. Conti, M. Boccanera, M. Gerloni, G. Morace, W. Magliani, C. Chezzi, and A. Cassone. 1994. Idiotypic intravaginal vaccination to protect against candidal vaginitis by secretory, yeast killer toxin-like anti-idiotypic antibodies. J. Immunol. 152:3175-3182.[Abstract]
    34. 34
  34. Polonelli, L., F. Fanti, S. Conti, L. Campani, M. Gerloni, M. Castagnola, G. Morace, and C. Chezzi. 1990. Detection by immunofluorescent anti-idiotypic antibodies of yeast killer toxin cell wall receptors of Candida albicans. J. Immunol. Methods 132:205-209.[CrossRef][Medline]
    35. 35
  35. Polonelli, L., W. Magliani, S. Conti, L. Bracci, L. Lozzi, P. Neri, D. Adriani, F. De Bernardis, and A. Cassone. 2003. Therapeutic activity of an engineered synthetic killer anti-idiotypic antibody fragment against experimental mucosal and systemic candidiasis. Infect. Immun. 71:6205-6212.[Abstract/Free Full Text]
    36. 36
  36. Ponti, D., M. L. Mangoni, G. Mignogna, M. Simmaco, and D. Barra. 2003. An amphibian antimicrobial peptide variant expressed in Nicotiana tabacum confers resistance to phytopathogens. Biochem. J. 370:121-127.[CrossRef][Medline]
    37. 37
  37. Ponti, D., G. Mignogna, M. L. Mangoni, D. De Biase, M. Simmaco, and D. Barra. 1999. Expression and activity of cyclic and linear analogues of esculentin-1, an anti-microbial peptide from amphibian skin. Eur. J. Biochem. 263:921-927.[Medline]
    38. 38
  38. Roggero, P., M. Ciuffo, E. Benvenuto, and R. Franconi. 2001. The expression of a single-chain Fv antibody fragment in different plant hosts and tissues by using Potato virus X as a vector. Protein Expr. Purif. 22:70-74.[CrossRef][Medline]
    39. 39
  39. Rommens, C. M., J. M. Salmeron, D. C. Baulcombe, and B. J. Staskawicz. 1995. Use of a gene expression system based on potato virus X to rapidly identify and characterize a tomato Pto homolog that controls fenthion sensitivity. Plant Cell. 7:249-257.[Abstract]
    40. 40
  40. Sablowski, R. W., D. C. Baulcombe, and M. Bevan. 1995. Expression of a flower-specific Myb protein in leaf cells using a viral vector causes ectopic activation of a target promoter. Proc. Natl. Acad. Sci. USA 2:6901-6905.
    41. 41
  41. Saitoh, H., A. Kiba, M. Nishihara, S. Yamamura, K. Suzuki, and R. Terauchi. 2001. Production of antimicrobial defensin in Nicotiana benthamiana with a potato virus X vector. Mol. Plant-Microbe Interact. 14:111-115.[Medline]
    42. 42
  42. Scholthof, H. B., K. B. Scholthof, and A. O. Jackson. 1996. Plant virus gene vectors for transient expression of foreign proteins in plants. Annu. Rev. Phytopathol. 34:299-323.[CrossRef][Medline]
    43. 43
  43. Shah, D. M. 1997. Genetic engineering for fungal and bacterial diseases. Curr. Opin. Biotechnol. 8:208-214.[CrossRef][Medline]
    44. 44
  44. Smolenska, L., I. M. Roberts, D. Learmonth, A. J. Porter, W. J. Harris, T. M. Wilson, and S. Santa Cruz. 1998. Production of a functional single chain antibody attached to the surface of a plant virus. FEBS Lett. 441:379-382.[CrossRef][Medline]
    45. 45
  45. Terras, F. R., H. M. Schoofs, M. F. De Bolle, F. Van Leuven, S. B. Rees, J. Vanderleyden, B. P. Cammue, and W. F. Broekaert. 1992. Analysis of two novel classes of plant antifungal proteins from radish (Raphanus sativus L.) seeds. J. Biol. Chem. 267:15301-15309.[Abstract/Free Full Text]
    46. 46
  46. Terras, F. R., S. Torrekens, F. Van Leuven, R. W. Osborn, J. Vanderleyden, B. P. Cammue, and W. F. Broekaert. 1993. A new family of basic cysteine-rich plant antifungal proteins from Brassicaceae species. FEBS Lett. 316:233-240.[CrossRef][Medline]
    47. 47
  47. Travassos, L. R., L. S. Silva, E. G. Rodrigues, S. Conti, A. Salati, W. Magliani, and L. Polonelli. 2004. Therapeutic activity of a killer peptide against experimental paracoccidioidomycosis. J. Antimicrob. Chemother. 54:956-958.[Abstract/Free Full Text]
    48. 48
  48. Viterbo, A., S. Haran, D. Friesem, O. Ramot, and I. Chet. 2001. Antifungal activity of a novel endochitinase gene (chit36) from Trichoderma harzianum Rifai TM. FEMS Microbiol. Lett. 200:169-174.[CrossRef][Medline]

Applied and Environmental Microbiology, October 2005, p. 6360-6367, Vol. 71, No. 10
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.10.6360-6367.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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