Identification and Characterization of an Antifungal Protein, AfAFP_R9, Produced by Marine-Derived Aspergillus fumigatus R9

Abstract

A fungal strain, R9, was isolated from the South Atlantic sediment sample and identified as Aspergillus fumigatus. An antifungal protein, AfAFP_R9, was purified from the culture supernatant of Aspergillus fumigatus R9. AfAFP_R9 was identified to be restrictocin, which is a member of the ribosome-inactivating proteins (RIPs), by MALDI-TOF-TOF-MS. AfAFP_R9 displayed antifungal activity against plant pathogenic Fusarium oxysporum, Alternaria longipes, Colletotrichum gloeosporioides, Paecilomyces variotii, and Trichoderma viride at minimum inhibitory concentrations of 0.6, 0.6, 1.2, 1.2, and 2.4 μg/disc, respectively. Moreover, AfAFP_R9 exhibited a certain extent of thermostability, and metal ion and denaturant tolerance. The iodoacetamide assay showed that the disulfide bridge in AfAFP_R9 was indispensable for its antifungal action. The cDNA encoding for AfAFP_R9 was cloned from A. fumigatus R9 by RT-PCR and heterologously expressed in E. coli. The recombinant AfAFP_R9 protein exhibited obvious antifungal activity against C. gloeosporioides, T. viride, and A. longipes. These results reveal the antifungal properties of a RIP member (AfAFP_R9) from marine-derived Aspergillus fumigatus and indicated its potential application in controlling plant pathogenic fungi.

Introduction

There are a vast number and diversity of microorganisms living in the oceans. The interaction between the marine microorganisms and their unique environments causes the development of special metabolic pathways in these microorganisms. The antifungal substances from marine microorganisms are becoming an important part of discovering new antifungal antibiotics and developing marine drugs in recent years. Many antimicrobial fungi have been isolated by culture-dependent methods from various marine organisms such as sponges and algae [35]. Rateb and Ebel [24] gave an overview of new natural products from marine fungi and their biological activities during 2006 to mid-2010, and 690 structures were presented.

Now, a great variety of antifungal proteins with different antifungal characteristics have been identified from various species of fungus. Aspergillus giganteus was the first filamentous fungus to potently produce an antifungal peptide (AFP) [18,31]. Subsequently, a number of different antifungal proteins have been derived from ascomycetes, such as PAF from Penicillium chrysogenum [6,15,19,25], NAF from Penicillium nalgiovense [7], AcAFP from Aspergillus clavatus [27,28], AnAFP from Aspergillus niger [9], NFAP from Neosartorya fisheri [5,8], and Pc-Actin from Penicillium chrysogenum A096 [3]. According to their structure, molecular mass, and antifungal mechanism, the antifungal proteins are classified into ribosome-inactivating proteins (RIPs), pathogenesis-related proteins (PR), defensins, glycine/histidine-rich proteins, lipid transfer proteins (LTPs), protease inhibitors, and other proteins [26].

RIPs have been found in bacteria, fungi, mushrooms, and plants, and have a broad spectrum of biological activities, including antitumor, antivirus, antifungus, and anti-insect activities. The RIPs have a glycosidase or phosphatase activity, resulting in the arrest of protein synthesis due to the ribosome damage caused by these two enzymes [23,30]. RIPs have been classified into three types. Type 1 RIPs are single-chain N-glycosidases with a molecular mass of 11 to 30 kDa. Type 2 RIPs contain two chains, a cell-binding lectin (B chain) and an N-glycosidase (A chain), with a molecular mass of 60 kDa [34]. Type 3 RIPs contain only one chain, which covers both the cell-binding lectin and N-glycosidase [22]. Type 1 RIPs are much less toxic, as they lack the B-chain, and thus they do not bind and enter cells [29].

In this study, an antifungal strain, Aspergillus fumigatus R9, was isolated from a South Atlantic sediment sample. Its antifungal protein AfAFPR9 was purified and identified as a member of RIPs. The antifungal properties of the natural and recombinant AfAFPR9 proteins were characterized. The results obtained suggest that AfAFPR9 may represent a potential candidate of fungicide controlling plant pathogenic fungi.

 

Materials and Methods

Tested Strains

The tested fungi, including Colletotrichum gloeosporioides (ACCC 31200, Agricultural Cultural Collection of China), Fusarium oxysporum (ACCC 31352), Trichoderma viride (ACCC 30902), Rhizoctonia solani (ACCC 36316), Alternaria longipes (ACCC 30002), and Sclerotinia sclerotiorum (ACCC 36081), were provided by Agricultural Cultural Collection of China. Paecilomyces variotii (CGMCC 3.776, China General Microbiological Culture Collection Center) was obtained from CGMCC, Institute of Microbiological Chinese Academy of Sciences. These seven tested fungi are important plant pathogenic fungi in agriculture.

Isolation and Identification of an Antifungal Strain

The sediment samples used for strain isolation were collected from the South Atlantic (W 14.87°, S 12.12°; depth of water: 2,647 m). Isolation and identification of the strains as well as analysis of their antifungal activity were carried out as previously described [3]. Briefly, the sediment samples were diluted with sterilized seawater and approximately 200 µl of the diluted sample was spread on plates containing different types of medium, such as GPY (glucose 1%, peptone 0.2%, and yeast extract 0.05%) and YTM (0.5% yeast extract, 0.3% tryptone, and 2.5% mannitol). Plates were incubated at 28℃ for growth. The strains were selected based on their morphological features and inoculated into the corresponding liquid media for further growth to evaluate their antifungal potential. For the identification of the antifungal strain, the ribosomal internal transcribed spacer (ITS) DNA sequence was amplified using the primers ITS5 (5’-GGAAGTAAAAGTCGTAACAAGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’), sequenced at Sangon Biotech (Shanghai, China) and analyzed for similarity using BLAST (http://blast.ncbi. nlm.nih.gov/Blast.cgi).

Purification and Identification of Antifungal Protein from A. fumigatus R9

A. fumigatus R9 was cultured in GPY medium at 28℃ for 7 days. The purification of the antifungal protein was performed as previously presented [3]. Briefly, the culture supernatant of A. fumigatus strain R9 was obtained by vacuum filtration through qualitative filter paper. The culture supernatant was fully saturated with ammonium sulfate and then centrifugated at 12,000 ×g for 30 min at 4℃. The precipitate containing crude proteins was dissolved in distilled water and dialyzed at 4℃ for 24 h, and finally lyophilized.

The crude proteins and the fractions separated by ion exchange chromatography were tested for antifungal activity against the tested fungi. Ion-exchange chromatography was performed with an AKTA FPLC system (GE Healthcare, USA). The crude protein solution was loaded onto a DEAE Sepharose Fast Flow column (GE Healthcare), which was pre-equilibrated with starting buffer A (pH 8.1, 20 mM Tris–HCl) for the primary purification step. The elution program was as follows: 0% elution buffer B (pH 8.1, 20 mM Tris–HCl, 1 M NaCl), 3 CV (coloum volume); 0~40% elution buffer B, 10 CV; 100% elution buffer B, 2 CV; 0% elution buffer B, 2 CV. The bioactive fraction was concentrated by ultrafiltration in a Vivaspin 15R (molecular weight cutoff 5,000; Sartorius, Germany) and further purified on a CM Sepharose Fast Flow column (GE Healthcare, USA) under the same program. The resulting active component was concentrated by ultrafiltration in a Vivaspin 15R and its purity was assessed by 15% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and silver nitrate staining. The purified antifungal protein, named AfAFPR9; was identified by MALDI-TOF-TOF-MS at Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. The database search was performed on the Mascot server (http://www.matrixscience.com/search_from_select.html) with MALDI-TOF-TOF-MS data.

Assay of Antifungal Activity

The assay for antifungal activity toward the seven phytopathogenic fungal species was carried out in PDA plates. One 0.6 cm diameter piece of tested phytopathogenic fungal strains’ cylinder agar with mycelial growth was placed on the center of a PDA plate. After the mycelial colony had developed, sterile blank paper discs of 0.65 cm diameter were placed at a distance of 0.8 cm away from the rim of the growing mycelial colony. Fifty microliter aliquots of the supernatant of A. fumigatus R9 and the fractions of ionexchange chromatography were added to each paper disc. Fifty microliters of GPY medium or starting buffer A, which dissolved the antifungal protein, was used as blank controls. The plates were incubated at 28℃ until mycelial growth enveloped discs containing the control disc, or formed crescents of inhibition around discs containing samples with antifungal activity.

MIC Determination of Antifungal Protein AfAFPR9

The minimum inhibitory concentration (MIC) of AfAFPR9 against different pathogenic fungi was determined by the paper disc dilution method [32]. Two-fold serial dilutions of AfAFPR9 solution ranging from 0.4 to 0.00625 µg/µl were prepared, and 50 µl of each diluted solution was added onto paper discs placed 0.8 cm from the edge of growing mold on a PDA plate. The plates were placed at 28℃ for several days, depending on the tested pathogen. The MIC was determined as the lowest concentration of active protein that could inhibit visible mold growth and calculated as the total protein added on each paper disc (microgram per disc). Starting buffer A without active protein was used as the blank control.

Physiochemical Properties of Antifungal Protein

To examine the effects of metal ions on AfAFPR9 activity, several metal ions, such as Na+, K+, Mg2+, Cu2+, and Ag+, were dissolved in starting buffer A to a final concentration of 10 mM. AfAFPR9 (0.4 µg/µl) was treated with the different ion solutions at room temperature for 1 h before being tested for antifungal activity. Fifty microliters of the treated AfAFPR9 was used for the antifungal tests. The AfAFPR9 protein without metal ion treatment was used as the positive control, and starting buffer A and ion solutions were used as blank and negative controls.

To evaluate the thermostability of AfAFPR9, AfAFPR9 solution (0.4 µg/µl) was treated at 100℃ for 20, 40, 60, and 80 min, respectively. After cooling to room temperature, the residual antifungal activity of AfAFPR9 was tested against A. longipes. Fifty microliters of the heat-treated AfAFPR9 was used for the antifungal test. AfAFPR9 solution without heat treatment and starting buffer A were used as the positive and blank controls, respectively.

For denaturant-resistant test, AfAFPR9 (0.4 µg/µl) was treated with 0.1% SDS, 0.1% carbamide, and 0.1% guanidine hydrochloride at 28℃ for 24 h. The treatment effect was analyzed by observing the residual antifungal activity of AfAFPR9. Fifty microliters of the treated antifungal protein was used for the antifungal tests. The AfAFPR9 without denaturant treatment was used as the positive control. Only denaturants and starting buffer A were used as blank controls. All experiments, including metal-ion-resistant, thermostability, and denaturant-resistant tests, were done in triplicate and the results are shown as mean ± standard deviation (SD) of three experiments.

Cloning of AfAFPR9 cDNA

Total RNA was extracted from strain R9 by using TRIzol reagent (Invitrogen, USA), according to the manufacturer’s instructions. First-strand cDNA was synthesized from 1 µg of total RNA using oligo-dT-adaptor primer (TaKaRa, Dalian, China) and used as the template for PCR amplification of AfAFPR9 cDNA. Based on the restrictocin gene sequence (GenBank Accession No. AAA32707.1), two primers were designed: forward primer 5’-GCGACCTGGACATGCATCAACCAA-3’ and reverse primer 5’-CTAATGAGAACACAGTCTCAAGTC-3’. PCR was performed with an initial denaturation step of 3 min at 94℃ and then 35 cycles were run as follows: 30 sec denaturation at 94℃, 30 sec annealing at 55℃, and 30 sec extension at 72℃, followed by a cycle of 72℃ for 10 min. The amplified product was sequenced at Sangon Biotech. Sequence homology search was performed using the BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple sequence alignment was carried out using the DNAMAN tool (Lynnon Biosoft).

Heterologous Expression of AfAFPR9 in Escherichia coli and Antifungal Activity Analysis of Its Recombinant Protein

AfAFPR9 was expressed as a fusion protein with 6× His-tag and thioredoxin (TRX) using a pET-32a vector in the E. coli Rosetta strain (Amersham Pharmacia Biotech). The AfAFPR9 cDNA was amplified using the following primers: forward primer 5’-CCGGAATTCGCGACCTGGACATGC-3’ with an EcoRI digestion site (underlined), and reverse primer 5’-CCGCTCGAGCTAATGAGAACACAGTC-3’ with an XhoI digestion site (underlined). The PCR product was cloned into the EcoRI/XhoI-digested pET-32a. The resulting plasmid pET-32a-AfAFPR9 was transformed into the competent cells of E. coli Rosetta using pET-32a as a vector control. The positive colonies were identified by PCR and DNA sequencing. AfAFPR9 fusion protein was expressed by 1 mM isopropy l-β-D-thiogalactopyranoside (IPTG) induction at 16℃ for 48 h. The recombinant proteins were then purified using Ni2+ IDA affinity chromatography (Novagen, USA) as described in the supplier’s instructions. SDS-PAGE was performed for analysis of recombinant protein expression and purification. The antifungal activity of the purified recombinant protein was determined using a microtiter plate assay as previously described [10].

Effect of the Disulfide Bridge on the Antifungal Activity of AfAFPR9

AfAFPR9 (0.4 µg/µl) was reduced by β-mercaptoethanol, and the partially reduced AfAFPR9 was alkylated by iodoacetamide [2,13]. Freshly prepared β-mercaptoethanol was added to the AfAFPR9 solution to a final concentration of 1 mM. The mixture was incubated at 37℃ for 10 min followed by dialyzing against distilled water at 4℃ for 24 h with several changes of water. After that, 100 µl of the partially reduced AfAFPR9 was placed on a paper disc and tested for its antifungal activity against A. longipes. Another 400 µl of the AfAFPR9 was diluted into the same volume of iodoacetamide solution and then kept in the dark for 3 min. Five micrograms of iodoacetamide was dissolved in 400 µl of Tris-HCl buffer (50 mM, pH 8.5). After alkylation, the mixture was dialyzed against distilled water at 4℃ for 24 h with several changes of water. Finally, 200 µl of the alkylated AfAFPR9 was placed on a paper disc for antifungal test. The antifungal experiments were done in triplicate and the results are shown as the mean± SD.

 

Results

Strain Identification and Antifungal Activity Detection

A total of 24 strains were purified based on their morphological features and inoculated into corresponding liquid media for further growth to evaluate their antifungal potential. The supernatant of the strain R9 culture showed obvious inhibitory activity against several plant pathogenic fungi, including C. gloeosporioides, F. oxysporum, T. viride, R. solani, A. longipes, S. sclerotiorum, and P. variotii. The R9 strain produced a large number of green spores. Its ITS gene sequence showed the highest identity of 99% with that of A. fumigatus KARVS04 (GenBank Accession No. KC119200.1) and has been submitted to the NCBI GenBank with an accession number of KF985037. Based on the morphological characteristics and molecular information, strain R9 was identified as A. fumigatus. The A. fumigatus R9 strain has now been deposited at China Center for Type Culture Collection (CCTCC, Wuhan, China) with a preservation number of M2013206.

Purification of Antifungal Protein AfAFPR9

Further analysis showed that the precipitate from A. fumigatus R9 culture supernatant by saturation with ammonium sulfate and some fractions of ion-exchange chromatography also displayed antifungal activity against several tested fungi, including F. oxysporum, A. longipes, C. gloeosporioides, P. variotii, and T. viride. The precipitate was dissolved in water and first separated into 12 different fractions on a DEAE Sepharose Fast Flow column (Fig. 1A). Evaluation the of antifungal activity of the fractions revealed that only the second fraction AP2 had obvious antifungal activity. The AP2 fraction was then collected and further separated on a CM Sepharose Fast Flow column, and two main fractions (BP1, BP2) were obtained (Fig. 1B). Fraction BP2 showed antifungal activity against the tested fungi above (Fig. 2). SDS-PAGE analyses showed that only one protein band corresponding to about 18 kDa was observed in fraction BP2 (Fig. 3). This purified antifungal protein produced by A. fumigatus R9 was named AfAFPR9.

Fig. 1.Isolation of the antifungal protein by ion-exchange chromatography. (A) The crude protein was loaded on a DEAE Sepharose Fast Flow column. (B) Antifungal fraction AP2 was loaded on a CM Sepharose Fast Flow column.

Fig. 2.Antifungal activity of fraction BP2 (disc 2) against the sensitive tested fungi with starting buffer A (disc 1) as blank control. (A) A. longipes. (B) T. viride. (C) C. gloeosporioides. (D) P. variotii. (E) F. oxysporum.

Fig. 3.15% SDS-PAGE of the purified antifungal protein. Lane 1. Standard protein marker; Lane 2. Antifungal fraction BP2 collected from CM Sepharose Fast Flow column.

Identification and cDNA Cloning of AfAFPR9

After being treated with trypsin, four peptide sequences of AfAFPR9 were identified with MALDI-TOF-TOF. All of them were contained in a protein named restrictocin (GenBank Accession No. AAA32707.1) (Table 1), which was a member of the RIPs. The AfAFPR9 cDNA was then cloned using RT-PCR. It is 450 bp long, encoding a protein of 149 amino acids. It has been submitted to NCBI GenBank with an accession number of KJ081439. The deduced AfAFPR9 protein contained all the four peptides identified by MALDI-TOF-TOF (Fig. 4), indicating that the cDNA cloned here encodes the antifungal protein AfAFPR9. Multiple sequence alignment showed that AfAFPR9 shared 84.66% amino acid sequence identity with restrictocin from Aspergillus restricts (AAA32707.1), 84.09% with mitogillin from Aspergillus restrictus (P67876.1), 72.32% with α-sarcin from Aspergillus giganteus (CAA43180.1), 71.75% with clavin from Aspergillus clavatus (ACC49407.1), 5.66% with RIP from Momordica charantia L (AAS17014.1), and 8.15% with antiviral protein 1 from Bougainvillea xbuttiana (AAY34283.2). These results indicate that the antifungal protein AfAFPR9 is a member of the RIP family from marine fungi, belonging to type 1 RIP.

Table 1.Mass spectrum identification of antifungal protein AfAFPR9.

Fig. 4.Nucleotide sequence of AfAFPR9 cDNA and deduced amino acid sequence. The regions underlined show the four matched peptides identified by MALDI-TOF-TOF. ▲: indicates the first and last amino acid residues of the characteristic microbial RNases superfamily sequence (21-148 aa); △: indicates the five active sites, Tyr47, His49, Glu95, Arg120, and His136; ☆: indicates the two cysteine residues in AfAFPR9.

Physiochemical Properties of AfAFPR9

In order to investigate the MICs of AfAFPR9 against the five tested strains, the paper disc dilution method was performed. The MICs of AfAFPR9 were 0.6, 0.6, 1.2, 1.2, and 2.4 µg/disc against F. oxysporum, A. longipes, C. gloeosporioides, P. variotii, and T. viride, respectively. In addition, metal ions, such as Na+, K+, Mg2+, Cu2+, and Ag+ were tested for their effects on the antifungal activity of AfAFPR9. The antifungal activity of AfAFPR9 against all five tested strains was not reduced when it was treated with Na+ (Table 2). When AfAFPR9 was treated with K+, its antifungal activity against F. oxysporum was 87.16 ± 13.31% retained but was largely impaired against the other four strains (Table 2). AfAFPR9 was sensitive to metal ions Mg2+, Cu2+, and Ag+, since its antifungal activity was largely or even completely lost after being treated with 10 mM Mg2+, Cu2+, or Ag+ (Table 2). However, the antifungal activity of AfAFPR9 against P. variotii was almost not reduced when it was treated with Mg2+ (Table 2). Different metal ions thus have different effects on the antifungal activity of AfAFPR9. Finally, the denaturant-resistant test was also carried out. Relatively, the antifungal activity of AfAFPR9 against all five tested strains was slightly or not affected by 0.1% SDS, 0.1% carbamide, and 0.1% guanidine hydrochloride (Table 3).

Table 2.Antifungal activity of AfAFPR9 against the sensitive tested fungi after treatment with metal ions (10 mM).

Table 3.Antifungal activity of AfAFPR9 against the sensitive tested fungi after treatment with several denaturants.

AfAFPR9 maintained undamaged activity against A. longipes when it was heated at 100℃ for 20 min (Fig. 5). However, with the extension of heating time, the activity of AfAFPR9 was gradually lost. When heated at 100℃ for 80 min, its antifungal activity was completely lost (Fig. 5). These results indicated that AfAFPR9 exhibited a certain extent of thermostability.

Fig. 5.Antifungal activity of AfAFPR9 against A. longipes after heat treatment at 100℃ for 20, 40, 60, and 80 min. The antifungal activity was indicated as the average radius, with standard deviation, of the inhibition zone forming on the plates by AfAFPR9 against A. longipes.

The Disulfide Bridge in AfAFPR9 Contributing to Its Antifungal Activity

In order to investigate whether the two cysteine residues in AfAFPR9 form a disulfide bridge that contributes to its antifungal activity, the iodoacetamide assay was carried out. When AfAFPR9 was partially reduced by β-mercaptoethanol, its antifungal activity against A. longipes was reduced by 25.83 ± 10.10% (Fig. 6, middle colomn). When the mercapto groups of the two cysteine residues were fully alkylated by iodoacetamide, the antifungal activity of AfAFPR9 against A. longipes was almost lost completely (Fig. 6, right colomn). These results indicated that the disulfide bridge in AfAFPR9 contributed to its antifungal activity.

Fig. 6.Effect of the disulfide bridge on the antifungal activity of AfAFPR9 against A. longipes. The antifungal activity was indicated as the average radius, with standard deviation, of the inhibition zone forming in the plates by AfAFPR9 against A. longipes.

Expression of AfAFPR9 in Escherichia coli

The AfAFPR9 cDNA was cloned into the pET-32a vector and expressed in E. coli Rosetta after IPTG induction. Both induced and non-induced E. coli Rosetta/pET-32a-AfAFPR9 and E. coli Rosetta/pET-32a (the vector control) were analyzed by SDS-PAGE (Fig. 7). A 35 kDa band (Fig. 7, lane 5) corresponding to the size of recombinant AfAFPR9 protein with 6× His-tag and thioredoxin (TRX) was only observed in the induced E. coli Rosetta/pET-32a-AfAFPR9, indicating that the recombinant AfAFPR9 was specifically expressed in E. coli Rosetta/pET-32a-AfAFPR9. The recombinant AfAFPR9 protein was then purified using Ni2+ IDA affinity chromatography (Fig. 7, lane 6).

Fig. 7.SDS-PAGE analysis of recombinant AfAFPR9. Lane 1: protein marker; Lane 2: non-induced E. coli Rosetta/pET-32a; Lane 3: induced E. coli Rosetta/pET-32a; Lane 4: non-induced E. coli Rosetta/pET-32a-AfAFPR9; Lane 5: induced E. coli Rosetta/pET-32a-AfAFPR9; Lane 6: purified recombinant AfAFPR9 protein.

Antifungal Activity of the Recombinant AfAFPR9

The antifungal activity of the recombinant AfAFPR9 was measured against the tested fungi in comparison with the natural AfAFPR9, using culture medium and recombinant TRX protein as control. In all of the experiments, five replicates were prepared for each treatment and the concentrations of proteins were all at 400 ng/µl. The antifungal activity was observed by measuring the optical density at 600 nm (OD600) of the tested fungi growing on microplates at 24 h after treatment with AfAFPR9. The recombinant AfAFPR9 had obvious antifungal activity against C. gloeosporioides, T. viride, and A. longipes, and almost no effect on the growth of F. oxysporum (Fig. 8). This method is not appropriate for P. variotii, whose hyphae entwined together, thereby resulting in an unchanged OD600 (Fig. 8). These results indicate that the recombinant AfAFPR9 produced in E. coli exhibits good antifungal activity.

Fig. 8.Analysis of antifungal activity of the recombinant AfAFPR9 protein. The antifungal activity was expressed as the growth inhibition of tested fungi by measuring the OD600. of the tested fungi at 24 h after treatment with AfAFPR9.

 

Discussion

The marine-derived RIP AfAFPR9 had a broad antifungal spectrum in that it could inhibit some plant pathogenic fungi, including F. oxysporum, A. longipes, C. gloeosporioides, P. variotii, and T. viride. AfAFPR9 was different from other RIPs in origin, function, and antimicrobial spectrum. Lyophyllin was isolated from mushroom Lyophyllum shimeji and exhibited good antifungal activity against Physalospora piricola and Coprinus comatus [11]. Luffacylin from sponge gourd seeds exerted antifungal activity against Mycosphaerella arachidicola and Fusarium oxysporum [21]. Hispin from seeds of the hairy melon was also found to have antifungal activity against C. comatus [17]. Bougainvillea xbuttiana antiviral protein 1 from E. coli exhibited antiviral activity against sunnhemp rosette virus [4]. As with some other RIPs expressed in E. coli, recombinant AfAFPR9 also exhibited antifugal activity. Recombinant Momordica charantia L RIP can inhibit the growth of the Sphaerotheca fuliginea in vitro [33]. Clavin from the filamentous fungus A. clavatus IFO 8605 was expressed in E. coli and its recombinant protein showed antitumor activity [20].

The deduced AfAFPR9 protein of the cDNA has a characteristic microbial RNases superfamily sequence at 21-148 aa and five active sites, Tyr47, His49, Glu95, Arg120, and His136, as found in restrictocin from A. restrictus (Fig. 4) [12]. This provided further evidence that AfAFPR9 is a member of the RIP family.

The MIC test results showed the difference in antifungal activities of AfAFPR9 against five tested strains, which could be attributed to the different sensitivity of these five strains to AfAFPR9. In the metal ion tests, the antifungal activity of AfAFPR9 was maintained by treatment of monovalent ions, and partially or completely lost by treatment by divalent ions such as Mg2+ and Cu2+. It was presumed that the divalent ions Mg2+ and Cu2+ could change the structure of AfAFPR9, thus leading to the loss of activity. Although the divalent ions such as Mg2+ and Cu2+ are rich in soil, they have no restriction in practical field application since AfAFPR9 will be sprayed on the plants.

It was shown that AfAFPR9 exhibited a certain extent of thermostability. Two cysteine residues in AfAFPR9 may form a disulfide bridge, which would contribute to its thermostability (Fig. 4). Previous reports showed that antifungal proteins AFP and PAF had eight and six cysteine residues, respectively, which might form disulfide bridges contributing to their heat stability [14,16]. Further study also revealed that the disulfide bridge in AfAFPR9 also contributed to its antifungal activity. This was consistent with the previous results that the disulfide bridges in antifungal PAF was indispensable for its antifungal action [1].

The antifungal protein AfAFPR9 was purified from marinederived A. fumigatus and identified as a member of the RIP family. The broad antifungal spectrum, good antifungal activity, thermostability, and resistance to several metal ions and denaturants of AfAFPR9 suggest that it may represent a potential candidate of fungicide controlling plant pathogenic fungi.