Save

Cloning of a serine protease gene from the nematophagous fungus Esteya vermicola and expressed activity of the recombinant enzyme against Bursaphelenchus xylophilus

In: Nematology
View More View Less
  • 1 Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Department of Plant Pathology, Nanjing Agricultural University, Nanjing 210095, P.R. China
Full Access

A serine protease gene Evsp was cloned from the nematophagous fungus Esteya vermicola with strong virulence against Bursaphelenchus xylophilus. The full-length cDNA of Evsp contains 2280 nucleotides with a 1656 bp ORF encoding a protein with 551 amino acids. The genomic Evsp includes two exons (396 bp and 1260 bp) separated by an intron (207 bp). There is only one copy of Evsp gene in the fungal genome. The deduced amino acids sequences of Evsp showed highly homology with the catalytic domains in subtilisin serine proteases. Phylogenetic analyses based on the protein sequences revealed that E. vermicola is separated from nematode-trapping fungi but close to other nematophagous and entomopathogenic fungi. The recombinant serine protease rEvsp was induced in Escherichia coli with expression vector pET28a(+). The tests of protease and nematicidal activities for the purified and refolded rEvsp indicated it is possibly involved in the fungal infection process against B. xylophilus.

Abstract

A serine protease gene Evsp was cloned from the nematophagous fungus Esteya vermicola with strong virulence against Bursaphelenchus xylophilus. The full-length cDNA of Evsp contains 2280 nucleotides with a 1656 bp ORF encoding a protein with 551 amino acids. The genomic Evsp includes two exons (396 bp and 1260 bp) separated by an intron (207 bp). There is only one copy of Evsp gene in the fungal genome. The deduced amino acids sequences of Evsp showed highly homology with the catalytic domains in subtilisin serine proteases. Phylogenetic analyses based on the protein sequences revealed that E. vermicola is separated from nematode-trapping fungi but close to other nematophagous and entomopathogenic fungi. The recombinant serine protease rEvsp was induced in Escherichia coli with expression vector pET28a(+). The tests of protease and nematicidal activities for the purified and refolded rEvsp indicated it is possibly involved in the fungal infection process against B. xylophilus.

The pine wood nematode (PWN), Bursaphelenchus xylophilus, causes pine wilt disease (PWD) and seriously damages the coniferous forests in East Asian countries, such as Japan, Korea and China (Mota & Vieira, 2008). The spread to European countries, Portugal (Mota et al., 1999) and Spain (Robertson et al., 2011), makes PWN a great threat to pine trees worldwide (Futai, 2013). Management programmes for the control of PWD are based on strict plant quarantine, felling, de-barking and burning of damaged pine trees, as well as replanting resistant Pinus species (Suzuki, 2002). Alternative measures have also been developed, including biocontrol of PWN and its Monochamus vectors (Shimazu, 2004).

Nematophagous fungi are proposed as biological agents to control plant-parasitic nematodes due to their unique infection abilities, as well as their compatibility and security to human health and the environment. More than 700 nematophagous fungi have been reported (Zhang et al., 2011) but most of them are parasites of root-knot nematodes (Meloidogyne spp.) and cyst nematodes (Heterodera spp. and Globodera spp.). Esteya vermicola is the first recorded endoparasite of B. xylophilus (Liou et al., 1999), although some associated nematode-trapping fungi have been described (Mamiya, 1983). The strains of E. vermicola can kill B. xylophilus populations completely in a week in vitro and are considered as potential biocontrol agents with high infectivity (Liou et al., 1999; Wang et al., 2008, 2014).

The infection process of nematophagous fungi includes critical events such as cuticle penetration, nematode immobilisation and fungal invasion (Tunlid et al., 1991). Extracellular hydrolytic enzymes secreted by fungi, including serine proteases, chitinases and collagenases, may be involved in these events to aid penetration and compromise nematode defences (Morton et al., 2004). Research has demonstrated that serine proteases secreted by nematophagous fungi play essential roles during parasitism. For example, the Ver112 from Lecanicillium psalliotae degrades the cuticle of the saprophytic nematode Panagrellus redivivus (Yang et al., 2005), the Hnsp from Hirsutella rhossiliensis kills the soybean cyst nematode Heterodera glycines and degrades its cuticle (Wang et al., 2007) and the SprT from Trichoderma pseudokoningii SMF2 kills juveniles of M. incognita and inhibits hatching (Chen et al., 2009). Moreover, the overexpression of serine proteases in Purpureocillium lilacinum (= Paecilomyces lilacinus) enhances the fungal virulence against nematodes (Wang et al., 2010; Yang et al., 2011a).

In our pervious study, the strain NKF 13222 of E. vermicola was demonstrated to have high infectivity against PWN and potential biocontrol capabilities against plant-parasitic nematodes (Wang et al., 2014). Little is known about the infection process of E. vermicola strains in relation to extracellular enzymes. In this paper we described the cloning of a cuticle-degrading serine protease gene Evsp from E. vermicola. The coding sequences and phylogenetic relationship of the protease were analysed. The heterologous expression of Evsp in Escherichia coli was carried out and the nematicidal activity against PWN was evaluated.

Materials and methods

Organisms and growth conditions

The strain NKF 13222 of E. vermicola with the deposition code CCTCC M2013394 from the China Center for Type Culture Collection, was purified from an isolate of B. rainulfi that was intercepted at Tianjin port, P.R. China, from wood packaging material originating from Brazil (Wang et al., 2014) and maintained on potato dextrose agar (PDA) plates in our laboratory. One small agar piece with the strain was inoculated into 50 ml YEB liquid medium (0.5% beef extract, 0.5% peptone, 0.1% yeast extract, 0.5% sucrose and 0.04% MgSO4 ⋅ 7H2O) in a 250 ml flask, kept still in a 25°C incubator and cultured for 8 days. The mycelia of E. vermicola for nucleotide extractions were collected by filtering the medium with a sterilised tissue paper in a funnel and removing the agar piece and liquid.

The population BxLYG of B. xylophilus collected from dead tree of Pinus thunbergii from Lianyungang of Jiangsu Province, P.R. China, was inoculated on the lawn of Botryotinia fuckeliana grown on PDA media and cultured for 7 days at 25°C (Li et al., 2009). The nematodes were harvested from PDA media using the Baermann funnel at 25°C for 6 h, and washed three times with M9 buffer to remove the remaining mycelia.

cDNA and genomic cloning of gene Evsp and sequences analysis

All primers used in the study are listed in Table 1. The total RNA was isolated from mycelia of E. vermicola by the Trizol Total RNA Isolation Kit (Invitrogen). Reverse transcription was performed using the oligo(dT) primer and the PrimeScript RT-PCR Kit (TaKaRa). The degenerate primers SPDPF and SPDPR were designed using online ICODEHOP and CODEHOP software (http://blocks.fhcrc.org/codehop.html) on the basis of the highly conserved amino acid sequences within other fungal serine proteases from NCBI and were used to amplify the partial fragment of Evsp from strain NKF 13222. The full-length cDNA was amplified using the SMARTER RACE cDNA Amplification Kit (Clontech) with the primer pairs designed according to the obtained partial fragment of Evsp. The external primer GSP1 and the internal primer NGSP1 were used for 5′-RACE PCR, the external GSP2 and the internal NGSP2 were used for 3′-RACE PCR. The sequences of putative full-length cDNA were deduced from the overlapping sequences of both amplification products using BioEdit Version 7.0.1 (Hall, North Carolina State University, Raleigh, NC, USA). The genomic DNA was extracted from E. vermicola mycelia using the Fungal DNA Kit (Omega). The Evsp gene from genomic DNA was amplified with primers EvspGF/EvspGR. After purification, all PCR products were cloned into the pMD19-T vector (TaKaRa) and sequenced by Invitrogen.

Table 1.

Primers used in this study.

Table 1.

The DNA sequences were aligned using ClustalW in BioEdit Version 7.0.1. The amino acid sequences alignment and isoelectric points (pI) determination were performed with program NCBIBLAST, DNAMAN Version 6.0.3.99 and ProtParam (http://web.expasy.org/protparam/). The signal peptide prediction was determined using the SignalP program (http://www.cbs.dtu.dk/services/SignalP). The phylogenetic analysis of the fungal serine proteases was conducted by the neighbour-joining method using PAUP 4.0b10 program (Swofford, 2002). The bootstrap tests were performed with 1000 replicates to assess the confidence intervals of the branch points.

Southern blot analysis of fungal genomic DNA

The genomic DNA of E. vermicola was digested with EcoRI and HindIII at 37°C overnight. The fragments were separated on 0.8% agarose gel by electrophoresis and transferred to a Hybond N+ nylon membrane (Roche). The fragment used for probe was amplified from genomic DNA of E. vermicola with the primers GSP1/GSP2 and purified using DNA Fragment Purification Kits (Omega). The probes were synthesised with digoxigenin-labelled probes. The procedures of prehybridisation, hybridisation and signal detection were performed according to the instructions of the DIG High Prime DNA Labeling and Detection Starter Kit (Roche).

Plasmid construction and expression of Evsp in E. coli

In order to construct pET28a(+) expressing the Evsp gene, the fragment of encoding sequences without signal peptides was amplified from the full-length cDNA of Evsp using a forward primer EvspPEF and reverse primer EvspPER. The amplified fragment was cloned into pMD19-T vector and sequencing performed. The pMD19-T with the fragment was first digested with BamHI and HindIII and then ligated into BamHI/HindIII-linearised pET28a(+) using T4 DNA ligase (TaKaRa) at 4°C overnight. The gfp gene encoding green fluorescent protein (GFP) was amplified from pLGFP-C1 vector (Clontech) with the primers GFPF/R, then subcloned into pET28a(+), as described above, and used as a negative control. Both recombinant plasmid pET28a(+)-Evsp and control pET28a(+)-gfp were transformed into E. coli BL21 (DE3) for protein induction.

The transformed E. coli BL21 (DE3) strain was inoculated into 3 ml LB medium with 50 μg ml−1 Kanamycin and shaken at 37°C for 12 h. A 30 μl culture were further inoculated into 100 ml Kanamycin LB medium and shaken to 0.5 OD600. The expressions of rEvsp and rGFP were induced by adding 1 mM isopropyl-β-d-1-thiogalactopyranoside (IPTG) at 37°C for 6 h (Li & Li, 2009). The bacteria expressing rEvsp and rGFP were harvested by centrifugation and washed three times with phosphate-buffered saline (PBS).

Purification and refolding of recombinant fractions

The bacteria pellets were resuspended in lysis buffer (pH 8.0) containing 50 mM Tris-HCl, 5 mM EDTA and 1 mM PMSF, and then subjected to 10 min sonication of 5 s on-off cycle with a Digital Sonifier® (Branson). Part of the lysate was centrifuged at 10 000 g for 5 min and then both the supernatant and the pellet were used for sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12% polyacrylamide gels with 0.1% SDS using a Mini-PROTEAN II electrophoresis cell (Bio-Rad) (Laemmli, 1970). Gels were stained with Coomassie brilliant blue R-250. The remaining lysate was used for isolation of rEvsp and rGFP inclusion bodies as indicated by SDS-PAGE.

The lysate was first centrifuged at 10 000 g for 30 min, then the inclusion body pellet was washed with rinsing buffer (pH 8.0) containing 50 mM Tris-HCl, 5 mM EDTA and 2% deoxycholate, and continuously washed with distilled water to remove the contaminated salts and detergents and centrifuged at 10 000 g for 30 min (Sheng et al., 2006). Finally, the inclusion bodies were solubilised for 6 h at room temperature in solution buffer (pH 8.0) containing 50 mM Tris, 8 M urea, 100 mM NaCl, 1 mM EDTA and 5 mM DTT. This solution sample was applied to a Ni2+-chelating Sepharose column (25 ml) (Qiagen), which was equilibrated with buffer (10 mM Tris-HCl and 30 mM imidazole) and was loaded at a concentration of 1.5 mg ml−1. The column was washed with 100 ml equilibration buffer, and the proteins were eluted using elution buffer (10 mM Tris-HCl and 500 mM imidazole) (Hainfeld et al., 1999). Four ml of fractions was collected and part of the fractions was analysed by SDS-PAGE.

The recombinant fractions were then refolded at 4°C during nine dialysis steps with refolding buffer containing 0.7 M Tris-HCl (pH 8.0), 5 mM DTT, 1 M NaCl, 1.33 mM GSH, 1 mM GSSG, 10% glycerin and decreased concentrations of urea (7, 6, 5, 4, 3, 2, 1 and 0 M). At the end, the refolding buffer was replaced by the PBS buffer and incubated for 2 days at 4°C (fresh PBS every 8 h). The precipitate was removed by centrifugation at 12 000 g for 20 min and the supernatant was collected as the refolded rEvsp and rGFP (Singh & Panda, 2005).

Protease and nematicidal activity analysis

The rEvsp activity was qualitatively assayed by the Folin method according to the measurement of protease activity (Lowry et al., 1951). One ml supernatant of refolded rEvsp was added to 1 ml 2% casein and incubated at 40°C for 10 min. The rGFP and PBS buffer were used as controls. The reactions were terminated by adding 2 ml 0.4 M TCA and incubated for 20 min at 40°C. The colour reactions were carried out by adding 5 ml 0.4 M Na2CO3 and 1 ml 50% Folin to the solution and were observed by incubating the glass tubes at 40°C for 20 min.

The effect of rEvsp on B. xylophilus was evaluated by adding about 100 mixed-stage nematodes to the refolded rEvsp solution in a 5 ml Petri dish. The rGFP and PBS buffer were used as the controls. Each treatment was in triplicate. All dishes were incubated at 30°C and examined at 12, 24 and 36 h. The changes in nematode cuticle were observed under a BX51 microscope (Olympus) and numbers of dead and living nematodes were determined using a binocular microscope (Leica). Nematodes that show no reaction to continuous physical stimulation using a needle were scored as dead. The significant differences of mean mortality were determined by the Student’s t-test (P<0.005) using SAS software (SAS Institute).

Fig. 1.
Fig. 1.

Complete nucleotide and deduced amino acid sequences of the Evsp cDNA (GenBank accession No. KP698922). The 5′- and 3′-UTR are shown in lowercase letters, the open reading frame (ORF) is shown in uppercase. The predicted secretion signal sequence is boxed, and the amino acids used for degenerate primer design are shaded, the gene-specific primers used for PCR are underlined. The intron splice sites are shown with vertical arrows. The stop codon (TAA) is marked with an asterisk and the putative polyadenylation signal sequence (aataac) is marked with double line above, and three catalytic triad (Asp203, His235 and Ser401) are marked with black triangle.

Citation: Nematology 17, 9 (2015) ; 10.1163/15685411-00002924

Results

Characterisation of the Evsp gene and phylogenetic analysis

An approx. 500 bp cDNA fragment was first amplified from E. vermicola by RT-PCR using degenerate primers. Sequencing and blast searching revealed the fragment was part of the serine protease gene. The full-length cDNA of a subtilase-like serine protease gene designated as Evsp were generated by RACE-PCR. The Evsp gene contains 2280 nucleotides with a 1656 bp open reading frame (ORF), which encodes a polypeptide of 551 amino acids (aa) with a 21 aa signal peptide (MKHVLALSLAACAYAAPAVNT) (Fig. 1). There is a 5′ end noncoding sequence of 263 bp and a 3′-end noncoding sequence of 361 bp containing a putative polyadenylation signal located 12 bp upstream of the polyA tail. The theoretical molecular mass of polypeptide is predicted to be 58.8059 kDa and the pI 5.97. The deduced amino acids of Evsp share conserved motifs with peptidase S8, which are characteristic of the subtilisin-like serine proteases. The genomic clone Evsp is 2458 bp long with two exons (396 bp and 1260 bp) and one intron (207 bp).

The southern blot revealed that one band was detected both in the genomic DNA of E. vermicola NKF 13222 digested with restriction enzymes EcoRI and HindIII (Fig. 2). Both EcoRI and HindIII sites were not present either in cDNA or in the genomic clone of Evsp, which indicates a single copy of Evsp gene presented in the genome of NKF 13222.

Phylogenic analyses based on amino acid sequences of Evsp and representative serine proteases from other nematophagous fungi and entomopathogenic fungi were performed. All these proteases appeared to be divided into two groups in the tree (Fig. 3). Serine proteases from nematode-trapping fungi clearly formed one group, Esvp and those from three other endoparasitic-, opportunistic-, toxin-producing nematophagous fungi and entomopathogenic fungi formed another group.

Purification of rEvsp and detection of protease activity

After induction at 37°C for 6 h, SDS-PAGE analysis showed that the induction cells of E. coli BL21 (DE3) transformed with plasmid pET28a-Evsp and negative control pET28a-gfp both produced a large amount of new protein in the form of inclusion bodies. A small amount of soluble protein was also produced in supernatant by pET28a-gfp. A single band of each recombinant fraction was obtained after the inclusion bodies were dissolved in high concentration urea, then, following the purification by Ni2+-chelating Sepharose column, the molecular mass of the homogeneous protein rEvsp and rGFP was determined as approx. 60 kDa and 30 kDa, respectively, similar to the theoretical molecular mass (62.02828 kDa and 31.87388 kDa) of the deduced amino acid flanked by His-tag (Fig. 4A).

Fig. 2.
Fig. 2.

Southern blot of genomic DNA of strain Esteya vermicola NKF 13222 digested with EcoRI and HindIII.

Citation: Nematology 17, 9 (2015) ; 10.1163/15685411-00002924

The activity of the refolded rEvsp dialysed in decreased concentrations of urea was confirmed by the Folin method showing the colour changed into blue (Fig. 4B). This indicated that rEvsp has protease activity by which the medium casein is hydrolysed into amino acid and the reaction of free tyrosine with Folin phenol is triggered to produce blue-coloured chromophores. In the control with rGFP and PBS buffer, the colour was unchanged.

Nematicidal ability of rEvsp against B. xylophilus

The nematicidal activity of the purified rEvsp on PWN was evaluated by observation of changes in nematode cuticle and the mortality. After incubation in rEvsp solution for 12 h, the cuticle of nematodes became slightly shrunken. The cuticle became markedly shrunken after being incubated for 24 h and apparently wrinkled after 36 h (Fig. 5A-F). However, the nematodes in both rGFP solution and PBS control had smooth and bright cuticle surface and a clear body content (Fig. 5G-H). The result indicated some of the cuticle components may be degraded by the rEvsp.

After incubating in the refolded rEvsp solution for 12 and 24 h, the mortality of PWN was 41.3 and 58.9%, respectively, which is significantly different from those in refolded rGFP solution (P<0.01) (Fig. 6), with a mortality of 15.2% and 18.1%, respectively. After 36 h, the mortality of nematodes increased to 76.8%, which is significantly different from those in refolded rGFP solution (P<0.005), with a mortality of 22.3%. There were no significant differences in the nematode mortality between incubations with refolded rGFP solution and the PBS control.

Fig. 3.
Fig. 3.

Phylogenetic analysis based on the deduced peptide sequences of serine protease from different nematophagous and entomopathogenic fungi. The Aspergillus niger protease is used as outgroup. The phylogenetic tree was obtained by the neighbour-joining method using the PAUP program and the numbers below the branches indicate the percentages at which given branches were supported in 1000 bootstrap replications.

Citation: Nematology 17, 9 (2015) ; 10.1163/15685411-00002924

Fig. 4.
Fig. 4.

Purification and enzyme activity assay of Esteya vermicola rEvsp. A: The purification of recombinant proteins expressed in BL21 (DE3) were separated by SDS-PAGE (12%) and visualised by Coomassie brilliant blue G-250 staining. Lane M: Protein marker; 1: Purified rEvsp protein; 2: Purified rGFP protein. B: colour changes in Folin phenol test. 1: Refolded rEvsp protein; 2: Refolded rGFP protein; 3: PBS buffer. This figure is published in colour in the online edition of this journal, which can be accessed via http://booksandjournals.brillonline.com/content/journals/15685411.

Citation: Nematology 17, 9 (2015) ; 10.1163/15685411-00002924

Fig. 5.
Fig. 5.

Effects on Bursaphelenchus xylophilus cuticle incubated in the refolded Esteya vermicola rEvsp solution. A, B: Nematodes at 12 h; C, D: Nematodes at 24 h; E, F: Nematodes at 36 h; G, H: Nematodes incubated in refolded rGFP solution and PBS for 36 h. Arrows indicate the cuticle wrinkles. (Scale bars = 10 μm.)

Citation: Nematology 17, 9 (2015) ; 10.1163/15685411-00002924

Discussion

Esteya vermicola is the first reported endoparasitic nematophagous fungus of B. xylophilus and is considered as a potential biocontrol agent with high infectivity (Liou et al., 1999). In the glasshouse test by Wang et al. (2011), the survival index of 4-year-old pine seedlings sprayed with fungus 1 month before PWN inoculation was 0.670 compared with only 0.067 for control seedlings without fungus. Although E. vermicola has strong nematicidal efficacy in vitro (Wang et al., 2014), the information of its nematicidal virulence factors was lacking.

In this study, we designed degenerate primers based on the conserved domain of other identified serine proteases and successfully generated the full-length cDNA of Evsp using RACE amplification. Sequence analysis of the deduced amino acid of Evsp revealed high homology with the subtilisin serine proteases from other nematophagous fungi. The active site of Evsp consists of the catalytic triad aspartate (Asp203)-histidine (His235)-serine (Ser401), which are almost completely conserved among serine proteases from nematophagous fungi (Yang et al., 2007). The presence of a signal peptide suggested that Evsp might be secreted to the nematode cuticle by E. vermicola during parasitism, which indicated that the function of Evsp might have an essential role during infection and penetration.

The nematophagous fungi are divided into four main groups as nematode-trapping fungi, endoparasitic fungi, opportunistic fungi and toxin-producing fungi (Yang et al., 2013). Our constructed phylogenetic tree of the serine proteases revealed two independent clusters. The serine proteases from nematode-trapping fungi were all grouped into one cluster, the Esvp was grouped with serine proteases from other groups of nematophagous fungi and entomophagus fungi. The pathogenic mechanisms of nematophagous fungi and entomophagous fungi against nematodes and insects may overlap (Li et al., 2010). Up to now it is unclear whether E. vermicola can parasitise insects. However, one E. vermicola strain, CBS 115803, has been isolated from the surface of larvae and adult beetles of Scolytus intricatus and their galleries under the bark of oak branches in the Czech Republic (Kubátová et al., 2000). Esteya vermicola is confirmed to have strong infection ability to PWN, whose transmission and distribution in pine forest mainly relies on the vector insect Monochamus alternatus. Therefore, the possible genetic selection for Evsp of E. vermicola in phylogenetic evolution still needs further study.

The nematophagous fungus could produce several different proteases, whose function in the infection of nematodes can be examined by treating the fungus with various protease inhibitors. The serine protease inhibitor significantly decreased the nematicidal activity of nematophagous fungus, which may imply the serine protease is involved in the penetration of nematode cuticle and host-cell digestion (Tunlid & Jansson, 1991). The fungal and bacterial serine proteases can degrade diverse protein-containing substrates, such as casein, BSA, gelatin, denatured collagen and nematode cuticle (Yang et al., 2013). The rEvsp also showed similar characters. After purification and renaturation, the rEvsp could hydrolyse casein, which induced the blue colour reaction in the Folin test. It also could degrade the cuticle of B. xylophilus, which finally resulted in the death of nematodes. These attributes indicate that rEvsp has protease activities that function in helping E. vermicola to degenerate the nematode cuticle wall and penetrate into the body. Moreover, the Evsp maybe not the only serine protease existing in E. vermicola, because 11 subtilisins are expressed by Metarhizium anisopliae during its growth on insect cuticle (Bagga et al., 2004), and 24 genes encoding putative subtilases have been identified in the complete genome of Arthrobotrys oligospora (Yang et al., 2011b).

Fig. 6.
Fig. 6.

Nematicidal activity of the refolded Esteya vermicola rEvsp on Bursaphelenchus xylophilus. Each bar value represents the means ± SD of three replicates. Asterisks indicate statistically significant differences using Student’s t-test, ∗∗P<0.01, ∗∗∗P<0.005.

Citation: Nematology 17, 9 (2015) ; 10.1163/15685411-00002924

Molecular techniques are well developed and widely used and have been applied in genetic engineering to improve the virulence of nematophagous fungi. The overexpression of the cuticle-degrading protease PII in A. oligospora mutants constructed by restriction enzyme-mediated integration (REMI) transformation, produced a greater number of traps and increased the speed of capturing nematodes compared to the wild-type strain (Åhman et al., 2002). The overexpression of the serine protease Psp-3-encoding gene in P. lilacinum through Agrobacterium tumefaciens-mediated transformation, resulted in an increase of about 20% in the parasitism of M. incognita eggs by the transformants in both conidial suspension and mycelium bioassays (Wang et al., 2010). Moreover, the serine protease gene ver112 from L. psalliotae was introduced into the commercialised P. lilacinum by REMI transformation, and this significantly increased the protease activity and nematicidal activities against P. redivivus and Caenorhabditis elegans when compared to the wild strain (Yang et al., 2011a). As a nematophagous fungus with high pathogenic ability against B. xylophilus, E. vermicola also can be subjected to genetic engineering to enhance virulence factors and improve the biocontrol ability of the strain. Further studies are needed to ascertain the roles of extracellular enzymes and other attributes of E. vermicola involved in the infection of nematodes.

Acknowledgements

Xuan Wang and Tinglong Guan contributed equally to the work. The work was supported by the National Key Basic Research Program of China (973 Program, 2013CB127501), the Natural Science Foundation of China (Grant No. 31471751) and the Special Fund for Agro-scientific Research in the Public Interest (Grant No. 201103018).

References

  • Åhman J., Johansson T., Olsson M., Punt P.J., van den Hondel C.A., Tunlid A. (2002). Improving the pathogenicity of a nematode-trapping fungus by genetic engineering of a subtilisin with nematotoxic activity. Applied and Environmental Microbiology 68, 3408-3415.

    • Search Google Scholar
    • Export Citation
  • Bagga S., Hu G., Screen S.E., Leger R.J. St (2004). Reconstructing the diversification of subtilisins in the pathogenic fungus Metarhizium anisopliae. Gene 324, 159-169.

    • Search Google Scholar
    • Export Citation
  • Chen L.L., Liu L.J., Shi M., Song X.Y., Zheng C.Y., Chen X.L., Zhang Y.Z. (2009). Characterization and gene cloning of a novel serine protease with nematicidal activity from Trichoderma pseudokoningii SMF2. FEMS Microbiology Letters 299, 135-142.

    • Search Google Scholar
    • Export Citation
  • Futai K. (2013). Pine wood nematode, Bursaphelenchus xylophilus. Annual Review of Phytopathology 51, 61-83.

  • Hainfeld J.F., Liu W., Halsey C.M., Freimuth P., Powell R.D. (1999). Ni-NTA-gold clusters target His-tagged proteins. Journal of Structural Biology 127, 185-198.

    • Search Google Scholar
    • Export Citation
  • Kubátová A., Novotný D., Prášil K., Mráček Z. (2000). The nematophagous hyphomycete Esteya vermicola found in the Czech Republic. Czech Mycology 52, 227-235.

    • Search Google Scholar
    • Export Citation
  • Laemmli U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.

  • Li A.N., Li D.C. (2009). Cloning, expression and characterization of the serine protease gene from Chaetomium thermophilum. Journal of Applied Microbiology 106, 369-380.

    • Search Google Scholar
    • Export Citation
  • Li H., Trinh P.Q., Waeyenberge L., Moens M. (2009). Characterization of Bursaphelenchus spp. isolated from packaging wood imported at Nanjing, China. Nematology 11, 375-408.

    • Search Google Scholar
    • Export Citation
  • Li J., Yu L., Yang J.K., Dong L.Q., Tian B.Y., Yu Z.F., Liang L.M., Zhang Y., Wang X., Zhang K.Q. (2010). New insights into the evolution of subtilisin-like serine protease genes in Pezizomycotina. BMC Evolutionary Biology 10, 68.

    • Search Google Scholar
    • Export Citation
  • Liou J.Y., Shih J.Y., Tzean S.S. (1999). Esteya, a new nematophagous genus from Taiwan, attacking the pinewood nematode (Bursaphelenchus xylophilus). Mycological Research 103, 242-248.

    • Search Google Scholar
    • Export Citation
  • Lowry O.H., Rosebrough N.J., Farr A.L., Randall R.J. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 265-275.

    • Search Google Scholar
    • Export Citation
  • Mamiya Y. (1983). Pathology of the pine wilt disease caused by Bursaphelenchus xylophilus. Annual Review of Phytopathology 21, 201-220.

    • Search Google Scholar
    • Export Citation
  • Morton C.O., Hirsch P.R., Kerry B.R. (2004). Infection of plant-parasitic nematodes by nematophagous fungi – a review of the application of molecular biology to understand infection processes and to improve biological control. Nematology 6, 161-170.

    • Search Google Scholar
    • Export Citation
  • Mota M.M., Vieira P. (2008). Pine wilt disease: a worldwide threat to forest ecosystems. Berlin, Germany, Springer.

  • Mota M.M., Braasch H., Bravo M.A., Penas A.C., Burgermeister W., Metge K., Sousa E. (1999). First report of Bursaphelenchus xylophilus in Portugal and in Europe. Nematology 1, 727-734.

    • Search Google Scholar
    • Export Citation
  • Robertson L., Arcos S.C., Escuer M., Merino R.S., Esparrago G., Abelleira A., Navas A. (2011). Incidence of the pinewood nematode Bursaphelenchus xylophilus Steiner & Buhrer, 1934 (Nickle, 1970) in Spain. Nematology 13, 755-757.

    • Search Google Scholar
    • Export Citation
  • Sheng J., An K., Deng C., Li W., Bao X., Qiu D. (2006). Cloning a cuticle-degrading serine protease gene with biologic control function from Beauveria brongniartii and its expression in Escherichia coli. Current Microbiology 53, 124-128.

    • Search Google Scholar
    • Export Citation
  • Shimazu M. (2004). Effects of temperature on growth of Beauveria bassiana F-263, a strain highly virulent to the Japanese pine sawyer, Monochamus alternatus, especially tolerance to high temperatures. Applied Entomology and Zoology 39, 469-475.

    • Search Google Scholar
    • Export Citation
  • Singh S.M., Panda A.K. (2005). Solubilization and refolding of bacterial inclusion body proteins. Journal of Bioscience and Bioengineering 99, 303-310.

    • Search Google Scholar
    • Export Citation
  • Suzuki K. (2002). Pine wilt disease – a threat to pine forest in Europe. Dendrobiology 48, 71-74.

  • Swofford D. (2002). PAUP version 4.0. Phylogenetic analysis using parsimony (and other methods). Sunderland, MA, USA, Sinauer Associates.

    • Search Google Scholar
    • Export Citation
  • Tunlid A., Jansson S. (1991). Proteases and their involvement in the infection and immobilization of nematodes by the nematophagous fungus Arthrobotrys oligospora. Applied and Environmental Microbiology 57, 2868-2872.

    • Search Google Scholar
    • Export Citation
  • Wang B., Wu W., Liu X. (2007). Purification and characterization of a neutral serine protease with nematicidal activity from Hirsutella rhossiliensis. Mycopathologia 163, 169-176.

    • Search Google Scholar
    • Export Citation
  • Wang C.Y., Fang Z.M., Sun B.S., Gu L.J., Zhang K.Q., Sung C.K. (2008). High infectivity of an endoparasitic fungus strain, Esteya vermicola, against nematodes. Journal of Microbiology 46, 380-389.

    • Search Google Scholar
    • Export Citation
  • Wang C.Y., Fang Z.M., Wang Z., Zhang D.L., Gu L.J., Lee M.R., Sung C.K. (2011). Biological control of the pinewood nematode Bursaphelenchus xylophilus by application of the endoparasitic fungus Esteya vermicola. BioControl 56, 91-100.

    • Search Google Scholar
    • Export Citation
  • Wang J., Wang J., Liu F., Pan C. (2010). Enhancing the virulence of Paecilomyces lilacinus against Meloidogyne incognita eggs by overexpression of a serine protease. Biotechnology Letters 32, 1159-1166.

    • Search Google Scholar
    • Export Citation
  • Wang X., Wang T., Wang J., Guan T., Li H. (2014). Morphological, molecular and biological characterization of Esteya vermicola, a nematophagous fungus isolated from intercepted wood packing materials exported from Brazil. Mycoscience 55, 367-377.

    • Search Google Scholar
    • Export Citation
  • Yang J.K., Huang X.W., Tian B.Y., Wang M., Niu Q.H., Zhang K.Q. (2005). Isolation and characterization of a serine protease from the nematophagous fungus, Lecanicillium psalliotae, displaying nematicidal activity. Biotechnology Letters 27, 1123-1128.

    • Search Google Scholar
    • Export Citation
  • Yang J.K., Tian B.Y., Liang L.M., Zhang K.Q. (2007). Extracellular enzymes and the pathogenesis of nematophagous fungi. Applied Microbiology and Biotechnology 75, 21-31.

    • Search Google Scholar
    • Export Citation
  • Yang J.K., Zhao X.N., Liang L.M., Xia Z.Y., Lei L.P., Niu X.M., Zou C.G., Zhang K.Q. (2011a). Overexpression of a cuticle-degrading protease Ver112 increases the nematicidal activity of Paecilomyces lilacinus. Applied Microbiology and Biotechnology 89, 1895-1903.

    • Search Google Scholar
    • Export Citation
  • Yang J.K., Wang L., Ji X.L., Feng Y., Li X.M., Zou C.G., Xu J.P., Ren Y., Mi Q.L., Wu J.L. et al. (2011b). Genomic and proteomic analyses of the fungus Arthrobotrys oligospora provide insights into nematode-trap formation. PLoS Pathogen 7, e1002179.

    • Search Google Scholar
    • Export Citation
  • Yang J.K., Liang L.M., Li J., Zhang K.Q. (2013). Nematicidal enzymes from microorganisms and their applications. Applied Microbiology and Biotechnology 97, 7081-7095.

    • Search Google Scholar
    • Export Citation
  • Zhang Y., Li G.H., Zhang K.Q. (2011). A review on the research of nematophagous fungal species. Mycosystema 30, 836-845.

Content Metrics

All Time Past Year Past 30 Days
Abstract Views 302 56 0
Full Text Views 162 2 0
PDF Views & Downloads 9 1 0