2013 Volume 38 Issue 1 Pages 33-38
We describe the purification, cDNA cloning, and characterization of a defensin, AFP1, in Brassica juncea, which shares 100% amino acid sequence identity with Raphanus sativus defensin (Rs-AFP1) and has a high antifungal activity against Magnaporthe oryzae. The recombinant AFP1 synthesized in Escherichia coli showed thermostability and antifungal activity against a broad spectrum of rice pathogenic fungi. The changes of a negative to positive charge at the surface of AFP1 derived by amino acid substitutions showed more enhanced antifungal activities than the wild-type AFP1.
Rice blast caused by the fungal pathogen Magnaporthe oryzae (Pyricularia oryzae) is the most devastating disease in rice-producing countries worldwide. Rice blast causes a loss 10 to 30% of the annual rice harvest. Control of this disease has predominantly relied on breeding for rice cultivars with durable resistance and applying agrochemicals. However, the durable resistance obtained is frequently reduced by the rapid adaptation of the pathogen to resistant cultivars in the field.
Several plants are known to produce antimicrobial peptides, which defend themselves from pathogenic fungi. Some of them are basic small peptides, which have been grouped into several families in accordance with their primary structures and the mechanisms of action. These peptides when isolated from plants are thionins,1) non-specific lipid-transfer proteins,2) cyclotides3) and defensins.4)
Plant defensins are small, basic, and cysteine-rich proteins of 45 to 54 amino acids that possess antifungal and in some cases also antibacterial activities4) and are widely distributed in a vast majority of plant families, including Brassicaceae,5) Fabaceae,4) Hippocastanaceae,4) Liliaceae,4) and Solanaceae.4) The Brassicaceae family has been shown to contain very potent antifungal peptides, and several of typical antimicrobial peptides have been isolated from various species in this family.5) Plant defensins are largely isolated from seeds.5) All plant defensins share a characteristic three-dimensional folding pattern, stabilized by four disulfide bridges,5) which form a cysteine-stabilized alpha-helix beta-strand motif.6)
Mustard Brassica juncea is known to be a traditional food and a type of medicine which has been reported to be heat- and drought-tolerant and resistant to fungal diseases.7) Plant seed proteins play important roles in protecting seeds from microorganisms.8) These characteristics suggest that seeds of B. juncea might be a good source for the isolation of antifungal proteins against M. oryzae.
In this study, we searched for the B. juncea antifungal peptide and determined a defensin (AFP1) that is identical with a defensin (Rs-AFP1) from Raphanus sativus.9) We isolated a cDNA encoding AFP1 from B. juncea seeds. In addition, we expressed AFP1 cDNA in Escherichia coli and assessed the stability and antimicrobial activities against rice pathogenic fungi and bacteria. To determine the amino acids that are important for the antifungal activity of AFP1, we undertook a mutational analysis of AFP1.
The extraction and purification of antimicrobial peptides from Brassica juncea seeds (Sakata Seed Corporation, Yokohama, Japan) were performed as described by Dos Santos et al.10) with some modifications. B. juncea seed flour (100 g) was soaked for 1 hr with a 400 mL extraction buffer (10 mM Na2HPO4, 15 mM NaH2PO4, 100 mM KCl, and 2 mM EDTA) added with 4 protease-inhibitor cocktail tablets (Roche Diagnostics, Tokyo, Japan) at 4°C. The precipitate obtained with 70% ammonium sulfate saturation was solubilized in distilled water and heated at 80°C for 15 min. The resulting suspension was clarified by centrifugation, and the supernatants were extensively dialyzed against distilled water using dialysis tubing with a molecular mass cut-off of 3,500 Da. The dialyzed solutions were adjusted to 50 mM Tris–HCl (pH 9.0) with 1 M Tris–HCl (pH 9.0) and subsequently passed over a 5 mL HiTrap Q FF column (GE Healthcare, Piscataway, NJ, USA) in equilibrium with 50 mM Tris–HCI (pH 9.0), and the unbound proteins were then collected. The flow-through fraction was further purified by a cation-exchange chromatography and reverse-phase chromatography. The flow-through fraction was passed through a 5 mL HiTrap CM FF column (GE Healthcare) equilibrated with 50 mM MES (pH 6.0). The column was washed with 50 mM MES (pH 6.0) and eluted with a linear gradient from 0–0.5 M NaCl in 50 mM MES (pH 6.0). The flow rate was 2 mL/min, and the absorbances of all fractions were monitored at 280 nm. The retained peak was purified by reverse-phase HPLC on a COSMOSIL/COSMOGEL column (Nacalai Tesque, Kyoto, Japan) at a flow rate of 0.5 mL/min with buffer A (2% acetonitrile and 0.1% TFA) for 24 min and 10–60% buffer B (80% acetonitrile and 0.1% TFA) over 150 min. All these purification steps were used to check all fractions of antifungal activity against M. oryzae Guy11 using a microplate assay.
2. Assay of antimicrobial activityMagnaporthe oryzae Guy11 was obtained from Dr. M. Nishimura (National Institute of Agrobiological Sciences). Rhizoctonia solani MAFF 305003, Gibberella fujikuroi MAFF 235968, Cochliobolus miyabeanus MAFF 305425, Xhathomonas oryzae pv. oryzae MAFF 210559, Burkholderia plantarii MAFF 301723, Burkholderia glumae MAFF 301094, and Acidovorax avenae subsp. avenae MAFF 106618 were from the collection of the National Institute of Agrobiological Sciences Genebank.
Antifungal activity was measured by a method similar to that of Broekaert et al.11) using half-strength potato dextrose broth (1/2 PDB, Difco, Detroit, MI, USA) containing 2×104 fungal conidia/mL or hyphal shorten fragments/mL. The measurement of antibacterial activity was conducted in the same way of the assay of antifungal activity. A quarter-strength polypeptone medium containing, per liter, 2.5 g of polypeptone (Difco), 0.5 g of yeast extract (Difco), and 0.25 g of MgSO4–7H2O (Wako, Tokyo, Japan) was inoculated with the bacterial cells (2.0×107/mL).
A peptide solution (20 µL) and the sterilized culture solution (80 µL) were successively put into a microplate well and incubated for 96 hr at 25°C. The absorption at 595 nm served as a measure for microbial growth. IC50 values were calculated from dose-response curves.
3. Identification of antifungal proteinFor reductive alkylation, the protein sample (1 µg) was reduced with dithiothreitol and subsequently alkylated with iodoacetamide. The protein solution was digested using a Protease Profiler Kit (Sigma, St. Louis, MO, USA). The peptide digests were analyzed directly by MALDI-TOF MS. For protein identification, the peptide masses obtained by MALDI-TOF MS were analyzed using the Mascot search.
4. cDNA cloningTotal RNA was extracted from B. juncea seeds using an RNeasy Plant Mini Kit (Qiagen, Tokyo, Japan). First-strand cDNA was synthesized with 5 µg of total RNA and an Oligo (dT) primer with a Ready-To-Go You-Prime First-Strand Beads (GE Healthcare). The full-length cDNA for AFP1 was cloned with the 5′-ATG GCT AAG TYT GCT TCY ATC-3′ (Y=C or T) as a 5′-primer and a NotI-(dT)18 primer as a 3′-primer by polymerase chain reaction (PCR). The 5′-primer was referred to as previously described.12) Amplified PCR products were resolved by agarose gel electrophoresis in a 1×Tris–acetate–EDTA (TAE) buffer and visualized by staining with 0.5 µg/mL ethidium bromide. PCR amplicons were purified from 2.0% agarose gel slices using SUPREC-EZ (Takara Bio, Kyoto, Japan) following the manufacturer’s protocol. Purified DNA was cloned into a pT7Blue vector (Novagen, Madison, WI, USA) following manufacturer’s instructions.
5. Construction, expression and purification of recombinant wild or mutant AFP1A DNA fragment encoding the mature AFP1 was amplified by PCR using the AFP1 cDNA of B. juncea as a template and oligonucleotide primers AFP1Fw (5′-CGC GGA TCC -CAG AAG TTG TGC GAG AGG CC-3′) and AFP1Rv (5′-CCG GAA TTC -TTA ACA AGG GAA GTA GCA AAT ACA C-3′), which incorporated a BamHI and EcoRI restriction site, respectively. The AFP1 expression vector, pGEX-6P-1/AFP1, was constructed with a mature region of AFP1 gene digested from the amplified PCR using BamHI and EcoRI and then cloned into the pGEX-6P-1 glutathione-S-transferase (GST)-fusion expression vector (GE Healthcare).
Site-directed mutagenesis was carried out according to instructions (LA PCR in vitro mutagenesis kit, Takara Bio). In brief, the mature region of the AFP1 gene was inserted into the multicloning sites between the PstI and EcoRI sites of pBluescript KS+. The plasmid pBluescript/AFP1, carrying the mature region of the AFP1 gene, was used as a template for site-directed mutagenesis (E5M, G9R, V39R, and E5M/G9R). The single-mutation DNA fragment (V39R) was used as a template for the construction of mutant DNA fragments E5M/V39R or G9R/V39R. The triple mutational DNA fragment (E5M/G9R/V39R) was generated by PCR using as a template the double mutational DNA fragment (E5M/G9R). The four mutagenesis primers used were as follows: MUT-1, 5′-TGG CCT CAT GCA CAA CTT CTG-3′ (E5M); MUT-2, 5′-GAC CAT GTC CTA CTT GGC CTC-3′ (G9R); MUT-3, 5′-GGG AAG CGA TAG TTG CA-3′ (V39R); and MUT-4, 5′-GTC CTA CTT GGC CTC ATG CAC AA-3′ (E5M/G9R). Two PCRs were performed separately with two combinations of the primers: with the mutagenesis primer and M13 primer M4 or with MUT and M13 primer RV. These two PCR products were combined and amplified by subsequent PCR with M13 primers M4 and RV. The sequence of all PCR products was confirmed by DNA sequencing. The obtained mutant AFP1 fragment was subcloned into pGEX-6P-1 for the construction of the expression plasmids.
Escherichia coli Rosetta-gami B (DE3) pLysS cells (Novagen) harboring pGEX-6P-1/AFP1 or a mutant plasmid was grown in a modified Davis medium containing, per liter, 7 g of K2HPO4, 2 g of KH2PO4, 0.2 g of MgSO4 · 7H2O, 0.5 g of (NH4)2SO4, 83 g of yeast extract (Difco), 30 g of glucose, and 100 µg/mL ampicillin at 37°C to an OD600 of 0.6. At that time, the expression of the GST fusion AFP1 was induced by adding isopropyl-β-d(−)-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. After 18 hr of cultivation at 23°C, the cells were harvested and disrupted by addition with Triton X-100 after freeze/thawing in 1×PBS. The total soluble fraction was separated by centrifugation and filtration using a 0.45 µm syringe filter, and the supernatant was subjected to a 5 mL GSTrap FF column (GE Healthcare) for affinity purification. Mature-form AFP1 was released by digestion for 18 hr at 4°C with PreScission Protease (GE Healthcare) from the bound GST-AFP1. The eluted fraction was further purified by cation-exchange chromatography using a 5 mL HiTrap CM FF column (GE Healthcare) in equilibrium with 50 mM MES (pH 6.0). The eluted recombinant mature AFP1 (rAFP1) was concentrated and substituted with ultra pure water using Microcon YM-3 spin column (Amicon, Tokyo, Japan).
6. Thermal and pH stability assessmentThe stability of rAFP1 was assessed by an antifungal assay as described above. The thermal stability of the peptide was assessed with the IC50 values against M. oryzae conidia with the peptide pretreated at 50°C, 80°C, and 100°C for 30 min. The activity of AFP1 was scored against the control reaction performed at 25°C. The AFP1 peptide was also subjected to a pH stability assay. AFP1 was pretreated with a 50 mM Glycine–HCl buffer (pH 3.0, 5.0) and a 40 mM Glycine–NaOH buffer (pH 9.0, 11.0) at 25°C for 1 hr. The assessment procedure of pH stability of AFP1 was the same as that described for the thermal stability of AFP1. Values represent the average of at least nine independent measurements.
7. The effect of cations on the AFP1 antifungal activityThe effects of the AFP1 antifungal activity on different cations were assessed with the IC50 values against M. oryzae conidia with the addition of various concentrations of KCl, NaCl, MgCl2, and CaCl2. The control examined it in the condition that did not add cations. The assessment procedure for the coexistence stability of cations of AFP1 was the same as that described for the thermal and pH stability of AFP1. Values represent the average of at least three independent measurements.
The plant antifungal peptide was purified from B. juncea seeds as described in Materials and Methods (Supplemental Figs. S1A, B). After establishing the highest antifungal activity fraction against M. oryzae, MALDI-TOF mass spectrometry showed that a polypeptide of 5670 Da was present in the active fraction (Supplemental Fig. S1C). The antifungal peptide identification was performed by peptide mass fingerprinting using Arg-N, Asp-N, Glu-C, Lys-C, or trypsin digestion and a MALDI mass spectrometer. The Mascot search of the Swiss-Prot database revealed that the amino acid sequence of the antifungal peptide (designated AFP1) purified from B. juncea is identical to that of Rs-AFP1, which is found in the defensin from Raphanus sativus.9)
The antifungal activity of purified AFP1 was tested against M. oryzae. The IC50 value against M. oryzae was calculated to be 2.00 µg/mL. This value was equivalent to that of kasugamycin, which is distributed as an agricultural chemical (Table 1).
| Rice pathogens | IC50 (µg/mL) |
|---|---|
| Magnaporthe oryzae Guy11 | 1.90 (±0.29) |
| Rhizoctonia solani MAFF 305003 | 3.24 (±1.10) |
| Gibberella fujikuroi MAFF 235968 | 2.24 (±0.15) |
| Cochliobolus miyabeanus MAFF 305425 | 1.14 (±0.20) |
| Xanthomonas oryzae pv. oryzae MAFF 210559 | >25 |
| Burkholderia plantarii MAFF 301723 | >25 |
| Burkholderia glumae MAFF 301094 | >25 |
| Acidovorax avenae subsp. avenae MAFF 106618 | >25 |
Standard deviations of three replicates (n=3) are indicated in the brackets. The concentrations of rAFP1 used in these studies were in the ranges of 0–50 µg/mL. MAFF numbers denote the accession number of microorganisms in the National Institute of Agrobiological Sciences (NIAS) Genebank. The IC50 (µg/mL) of native AFP1 and kasgamycin were 2.00 (±0.63) and 2.53 (±0.25), respectively.
A cDNA clone for AFP1 of B. juncea was isolated and named AFP1. Sequence analysis of AFP1 indicated that the cDNA fragment contains an open reading frame (ORF) coding for a mature peptide of 51 amino acids with a calculated molecular mass of 5694 Da without pyroglutamylation and disulfide bridge formation. The nucleotide sequence of the AFP1 (deposited in Genbank with the accession No. AB537492) is very similar to the Rs-AFP1 nucleotide sequence (accession No. U18557), showing 95% sequence identity.
2. Escherichia coli expression and purification of recombinant AFP1To obtain purified AFP1 recombinant protein (rAFP1) and to determine its antimicrobial activity against phytopathogens, we constructed the expression cassette pGEX-6P-1/AFP1, which was transformed into Escherichia coli Rosetta-gami B (DE3) pLysS cells for protein expression. The expressed GST-AFP1 fusion protein had a molecular mass of 28 kDa and was purified by GST affinity chromatography. The fusion protein was cleaved with PreScission Protease to release recombinant AFP1 (rAFP1) with a molecular mass of ca. 6.2 kDa from the GST binding domain. The eluted rAFP1 was purified further on an ion-exchange chromatography. The molecular mass measured by MALDI-TOF MS for the purified rAFP1 with N-terminal tetra peptides from the pGEX-6P-1 vector was 6098 Da. The difference between the calculated mass (6106 Da) and the measured mass (6098 Da) may have been caused by the loss of eight hydrogens, as the eight cysteines of the rAFP1 molecule formed four disulfide bridges.
3. Effect of AFP1 on several rice pathogenic fungi and bacteria growthThe antifungal potency of rAFP1 relative to its native AFP1 was assessed on M. oryzae by measuring the percentage of growth inhibition due to serial dilutions of the peptide samples. The rAFP1 provided nearly the same antifungal potency (IC50=1.90 µg/mL) as its native AFP1 (IC50=2.00 µg/mL) (Table 1). To further evaluate the antimicrobial activity of rAFP1, the IC50 values obtained with rAFP1 are summarized in Table 1. The rAFP1 was active against fungal strains with an IC50 of 1.1–3.2 µg/mL but less active against bacterial strains.
4. Influences of pH, temperature, and external cation concentration on AFP1 antifungal activityThe rAFP1 was functionally active against M. oryzae following treatments with various pH buffers (pH 3–11) (Supplemental Fig. S2A). AFP1 showed 90% higher stability in the pH 3–11 conditions compared with the pH 7 conditions.
In the thermostability investigation of rAFP1, the peptide showed 95% activity following heat treatment at 80°C for 30 min. Furthermore, heating rRs-AFP1 at 100°C for 30 min modestly reduced the antifungal activity to 85% (Supplemental Fig. S2B). Thus, it was determined that rAFP1 is a highly thermostable peptide.
As a cation-sensitive action has been reported for antimicrobial and antifungal peptides,13) we tested the influence of monovalent cations (Na+, K+) and divalent cations (Ca2+, Mg2+) on the antifungal activity of rAFP1. We found that the antifungal activity progressively decreased with increasing the mono and divalent cation concentrations in the medium (Supplemental Figs. S2C–F). The growth inhibition of M. oryzae on rAFP1 decreased approximately 90% at the concentration of 25 mM KCl or 2.5 mM NaCl. Furthermore, the presence of 25 µM MgCl2 or 100 µM CaCl2 led to a 90% decrease of the growth inhibition of M. oryzae. These results show that the antifungal activity of rAFP1 is strongly sensitive to cations.
5. Antifungal activity of AFP1 mutantsTo identify the amino acids of the AFP1 molecule that are important for antifungal activity, we performed site-directed mutagenesis on AFP1. We selected the positions 5, 9, and 39 in AFP1 as a candidate for two reasons: first, the positions 5 and 9 of N-terminal region in Medicago sativa defensin, MsDef1, which is structurally similar to AFP1, contribute to the overall antifungal activity of Msdef1,14) and, second, the substitution of valine at position 39 by arginine in Rs-AFP2 in R. sativus, which shares 94% amino acid sequence identity with AFP1, exhibits the highest antifungal activity against Fusarium culmorum.15) The functional analysis of plant defensins with single amino acid substitutions has been reported,15) while the analysis of combination of mutations in the plant defensins is scarcely known. Therefore, we investigated the effect of antifungal activity of not only single mutants but also double and triple mutants.
Table 2 lists the antifungal activity obtained for recombinant wild-type AFP1 and AFP1 mutants. All single mutants (E5M, G9R, and V39M) had a moderate effect on antifungal activity to M. oryzae. Double mutants (E5M/V39R and G9R/V39R) also affected antifungal activity more than rAFP1. In particular, E5M/V39R showed a 3.65-fold increase in antifungal activity. A double mutant, E5M/G9R, was found as inactive and insoluble aggregates. Contrary to expectations, the triple mutant E5M/G9R/V39R was less active than the double mutants.
| Peptide variant | IC50 (µg/mL) | Relative activity (%) |
|---|---|---|
| rAFP1 | 1.90 (±0.29) | 100 |
| E5M | 1.66 (±0.23) | 114 |
| G9R | 1.59 (±0.24) | 119 |
| V39R | 1.62 (±0.22) | 117 |
| E5M/G9R | ND | ND |
| E5M/V39R | 0.52 (±0.05)* | 365 |
| G9R/V39R | 1.01 (±0.25)* | 188 |
| E5M/G9R/V39R | 1.46 (±0.15) | 130 |
Standard deviations of three replicates (n=3) are indicated in the brackets. The concentrations of mutant rAFP1s used in these studies were in the ranges of 0–20 µg/mL. Asterisk indicate statistically significant differences from rAFP1 IC50 at p<0.05. ND, not determined.
In this study, we isolated an antifungal protein (AFP1) from Brassica juncea seeds, and its cDNA was identified by RT-PCR based on information from the previously identified defensin gene in Brassicaceae family.9,12) We also established an expression system for the production of the recombinant AFP1, which exhibited strong antifungal activity at a micromolar concentration against rice pathogenic fungal strains.
The heterologous production of plant defensins has already been reported, especially in the yeast Pichia pastoris. However the yeast P. pastoris is often sensitive to plant defensins,16) and oxidized cysteines may appear in cells or in culture medium. We chose the Escherichia coli expression system previously reported for human defensins to express AFP1,17) which provides an economical and fast procedure for producing a folded defensin identical to the native defensin. The recombinant AFP1 was successfully expressed in the E. coli Rosetta-gami B (DE3) pLysS strain, which is designed to enhance the expression of eukaryotic proteins that contain rare codons. This host strain further facilitates cytoplasmic disulfide bond formation, which is helpful for the folding of proteins in vivo. As AFP1 is an antifungal peptide with four disulfide bonds, it seemed difficult to effectively express AFP1 in the soluble form in E. coli. To avoid protein aggregation, AFP1 was expressed as a fusion protein containing GST-tag, which may help maintain the solubility of expressed fusion protein. GST-AFP1 was cleaved with PreScission Protease to remove the GST-tag. After the GST-tag was removed, the recombinant AFP1 was finally purified by cation-exchange chromatography. As shown in Table 1, the recombinant AFP1 exhibited nearly the same antifungal activity as its native AFP1.
AFP1 was highly heat- and pH-stable. These properties were exploited in the purification process that involved heating at 80°C for 15 min and exposure to organic solvent during the RP-HPLC purification. This inherent stability arises from the property for defensin structure known as the cystein-stabilized alpha-helix beta-strand motif.6)
The antifungal activity of AFP1 on M. oryzae described in this study exhibited a cation modulator effect (Supplemental Figs. S2C–F). The cation-dependent decrease of AFP1 antifungal activity was observed in the presence of Na+, K+, Mg2+, and Ca2+. The reduction of antimicrobial activity in the presence of mono or divalent cations has been reported for several plant,18) insect19) and mammalian defensins.20) In insect and mammalian defensins, high cationic strength disturbs positively charged defensin to interact with the negative charged microbial membrane surface.19) However, plant defensins are considered to bind the specific receptors on the target cell surface. It seems likely that the antagonistic effect of cations is the result of weakening of the electrostatic interactions between AFP1 and its putative membrane receptor.
Several previous studies have reported that the significance of both basic residues and their asymmetric distribution over the molecular surface causes the amphiphilic properties of defensin. The importance of basic residues for the antimicrobial activity of defensins has been reported previously.21) The site-directed mutational analysis of Rs-AFP2, a defensin isolated from radish seeds, was reported and showed that basic amino acid residues contribute to the antifungal potency of this peptide. The substitutions of neutral residues Gly9 or Val39 by arginine increased the Rs-AFP2 antifungal activity. The replacement of acidic residue Gln5 by a neutral methionine also showed the increase of Rs-AFP2 antifungal potency.15) Increases in net charge (E5M, G9R, and V39R) showed a moderate effect on antifungal activity. Furthermore, the combinations of point mutations (E5M or G9R with V39R) exerted higher antifungal activity against M. oryzae. The mutated residues at positions 5, 9, and 39 are located over the surface of the AFP1 molecule.6) Hence, it is expected that the interaction between AFP1 and its target site is based, in part, on electrostatic interactions. The net charge of the surface of AFP1 may be one of the significant determining factors on the antifungal activity of AFP1.
The formation of dimers and/or oligomers in antimicrobial proteins has been proposed as a factor to disrupt biological membranes.22) The recent plant defensin structural studies suggest that the oligomerization of plant defensin may provide insights into the membrane permeabilization or disruption caused by plant defensins.23,24) These structural studies also report the locations of intermolecular hydrogen bonds to form dimer formation of plant defensin.23,24) Using SDS-PAGE analysis, it has been reported that AFP1 is capable of forming an oligomeric configuration.18) In AFP1, the location of residue E5 is corresponds with the position involving the intermolecular hydrogen bonds for dimer formation. Thus, mutation of E5 to methionine would be expected to promote the oligomerization of AFP1 rather than the change of the net charge of surface of AFP1.
The exact mechanisms underlying the antifungal activity exerted by plant defensins are not known, but there is evidence that plant defensins bind to a specific receptor in the fungal membrane. Plant defensins could interact selectively with phosphorylinositol containing sphingolipids or glycosylceramides and then trigger the death of fungal cells through binding to the target cell membrane. Rs-AFP2, sharing 94% amino acid identity with AFP1, interacts with a sphingolipid glucosylceramide in the plasma membrane of the yeasts Candida albicans and P. pastoris.16) This raises the possibility that two highly similar defensins might share a common receptor.
Transgenic approaches have shown that defensin genes expressed in plants can confer protection against pathogens. In fact, transgenic plants carrying the Rs-AFP2 gene from R. sativus displayed resistance to the fungus Alternaria longipes.9) More recently, the antifungal activity against the fungus M. oryzae was reported when the defensin Rs-AFP2 from radish was expressed in transgenic rice.25) Since AFP1 has a highly similar amino acid sequence to Rs-AFP2 and a potent antifungal activity against M. oryzae in vitro, transgenic plants carrying the AFP1 gene may display resistance to a wide spectrum of fungi.
Part of this work was supported by a Grant-in-Aid for Research and Development Program for New Bio-industry Initiatives, a grant of Strategic Research Foundation Grant-aided Project for Private Universities from Ministry of Education, Culture, Sport, Science, and Technology, Japan (MEXT) 2010–2014 (S1001030), Fusion Research Project of National Agriculture and Food Research Organization (NARO) and Cooperative System for Supporting Priority Research of Japan Science and Technology Agency (JST). We thank Dr. Marie Nishimura for kindly providing us with the M. oryzae strain and Mayumi Kimizu for her technical help on mutant DNA construction. M. oryzae Guy11 was imported and used under special permission of Ministry of Agriculture, and Forestry and Fisheries of Japan.
