The Aphelenchus avenae genome highlights evolutionary adaptation to desiccation.

Some organisms can withstand complete body water loss (losing up to 99% of body water) and stay in ametabolic state for decades until rehydration, which is known as anhydrobiosis. Few multicellular eukaryotes on their adult stage can withstand life without water. We still have an incomplete understanding of the mechanism for metazoan survival of anhydrobiosis. Here we report the 255-Mb genome of Aphelenchus avenae, which can endure relative zero humidity for years. Gene duplications arose genome-wide and contributed to the expansion and diversification of 763 kinases, which represents the second largest metazoan kinome to date. Transcriptome analyses of ametabolic state of A. avenae indicate the elevation of ATP level for global recycling of macromolecules and enhancement of autophagy in the early stage of anhydrobiosis. We catalogue 74 species-specific intrinsically disordered proteins, which may facilitate A. avenae to survive through desiccation stress. Our findings refine a molecular basis evolving for survival in extreme water loss and open the way for discovering new anti-desiccation strategies.

Cathepsin L-like Cysteine Proteinase Genes Are Associated with the Development and Pathogenicity of Pine Wood Nematode, Bursaphelenchus xylophilus.

The pine wood nematode (PWN), Bursaphelenchus xylophilus, is the pathogen of pine wilt disease (PWD), resulting in huge losses in pine forests. However, its pathogenic mechanism remains unclear. The cathepsin L-like cysteine proteinase (CPL) genes are multifunctional genes related to the parasitic abilities of plant-parasitic nematodes, but their functions in PWN remain unclear. We cloned three cpl genes of PWN (Bx-cpls) by rapid amplification of cDNA ends (RACE) and analyzed their characteristics using bioinformatic methods. The tissue specificity of cpl gene of PWN (Bx-cpl) was studied using in situ mRNA hybridization (ISH). The functions of Bx-cpls in development and pathogenicity were investigated using real-time quantitative PCR (qPCR) and RNA interference (RNAi). The results showed that the full-length cDNAs of Bx-cpl-1, Bx-cpl-2, and Bx-cpl-3 were 1163 bp, 1305 bp, and 1302 bp, respectively. Bx-cpls could accumulate specifically in the egg, intestine, and genital system of PWN. During different developmental stages of PWN, the expression of Bx-cpls in the egg stage was highest. After infection, the expression levels of Bx-cpls increased and reached their highest at the initial stage of PWD, then declined gradually. The silencing of Bx-cpl could reduce the feeding, reproduction, and pathogenicity of PWN. These results revealed that Bx-cpls play multiple roles in the development and pathogenic processes of PWN.

A nematicidal tannin from Punica granatum L. rind and its physiological effect on pine wood nematode (Bursaphelenchus xylophilus).

The ethanol extract of Punica granatum L. rind was tested to show significant nematicidal activity against pine wood nematode. Three nematicidal compounds were obtained from the ethanol extract by bioassay-guided fractionation and identified as punicalagin 1, punicalin 2, and corilagin 3 by mass and nuclear magnetic resonance spectral data analysis. Punicalagin 1 was most active against PWN among the purified compounds with the LC50 value of 307.08µM in 72h. According to the enzyme assays in vitro, punicalagin 1 could inhibit the activity of acetylcholinesterase, amylase and cellulase from PWN with IC50 value of 0.60mM, 0.96mM and 1.24mM, respectively. The morphological structures of PWNs treated by punicalagin 1 were greatly changed. These physiological effects of punicalagin 1 on PWN may helpful to elucidate its nematicidal mechanism.

In silico comparative analysis of tylenchid nematode pectate lyases.

Phytoparasitic nematodes can infect a wide range of crop plants, and cause billions of dollars of agricultural losses each year. These parasites represent the largest source of biotic stress experienced by plants. The order Tylenchida comprises the most important parasitic nematodes, particularly the root-knot and cyst nematodes. These parasitic organisms obtain nutrients to support their development through complex interactions with their hosts. Plant-parasitic nematodes secrete a mixture of cell-wall degrading enzymes to facilitate migration through the plant root. Enzymes are secreted that degrade the principal cell-wall components, cellulose, hemi-cellulose, or pectin. Pectate lyases are important parasitism factors in plant-parasitic nematodes. These enzymes degrade polygalacturonic acid, which is a fundamental constituent of pectin of host cell walls. Thus, pectate lyases permit the penetration and colonization of plant host cells by parasites. Here, we analyzed 22 pectate lyase protein sequences from tylenchid nematode species. Our results revealed great variation in the isoelectric points of pectate lyases, and groups of acidic and alkaline proteins that may have distinct enzymatic activities were identified. Phylogenetic analysis also revealed the presence of two main groups of pectate lyases with distinct chemical properties. Seven conserved motifs were identified, but only five were present in all sequences. Results of the molecular docking analysis revealed differences in the predicted interaction sites in the pectate lyases from the two groups. These results may provide a theoretical basis for future studies of host plant resistance to nematode infection.

Versatile glycoside hydrolase family 18 chitinases for fungi ingestion and reproduction in the pinewood nematode Bursaphelenchus xylophilus.

The glycoside hydrolase family 18 (GH18) of chitinases is a gene family widely expressed in archaes, prokaryotes and eukaryotes, and hydrolyzes the ß-1,4-linkages in chitin. The pinewood nematode Bursaphelenchus xylophilus is one of the organisms that produces GH18 chitinases. Notably, B. xylophilus has a higher number of GH18 chitinases compared with the obligate plant-parasitic nematodes Meloidogyne incognita and Meloidogyne hapla. In this study, seven GH18 chitinases were identified and cloned from B. xylophilus based on genomic analyses. The deduced amino acid sequences of all these genes contained an N-terminal signal peptide and a GH18 catalytic domain. Phylogenetic analysis showed that the origin of B. xylophilus GH18 chitinases was independent of those from fungi and bacteria. Real-time quantitative reverse transcription PCR analysis indicated that GH18 chitinase genes had discrete expression patterns, representing almost all the life stages of B. xylophilus. In situ hybridisation showed that the mRNA of GH18 chitinase genes of B. xylophilus were detected mainly in the spermatheca, esophageal gland cells, seminal vesicle and eggs. RNA interference (RNAi) results revealed different roles of GH18 chitinase genes in B. xylophilus. Bx-chi-1, Bx-chi-2 and Bx-chi-7 were associated with reproduction, fungal cell-wall degradation and egg hatching, respectively. Bx-chi-5 and Bx-chi-6 may be involved in sperm metabolism. In conclusion, this study demonstrates that GH18 chitinases have multiple functions in the life cycle of B. xylophilus.

Glycoside Hydrolase (GH) 45 and 5 Candidate Cellulases in Aphelenchoides besseyi Isolated from Bird's-Nest Fern.

Five Aphelenchoides besseyi isolates collected from bird's-nest ferns or rice possess different parasitic capacities in bird's-nest fern. Two different glycoside hydrolase (GH) 45 genes were identified in the fern isolates, and only one was found in the rice isolates. A Abe GH5-1 gene containing an SCP-like family domain was found only in the fern isolates. Abe GH5-1 gene has five introns suggesting a eukaryotic origin. A maximum likelihood phylogeny revealed that Abe GH5-1 is part of the nematode monophyletic group that can be clearly distinguished from those of other eukaryotic and bacterial GH5 sequences with high bootstrap support values. The fern A. besseyi isolates were the first parasitic plant nematode found to possess both GH5 and GH45 genes. Surveying the genome of the five A. besseyi isolates by Southern blotting using an 834 bp probe targeting the GH5 domain suggests the presence of at least two copies in the fern-origin isolates but none in the rice-origin isolates. The in situ hybridization shows that the Abe GH5-1 gene is expressed in the nematode ovary and testis. Our study provides insights into the diversity of GH in isolates of plant parasitic nematodes of different host origins.

Influence of Bxpel1 Gene Silencing by dsRNA Interference on the Development and Pathogenicity of the Pine Wood Nematode, Bursaphelenchus xylophilus.

As the causal agent of pine wilt disease (PWD), the pine wood nematode (PWN), Bursaphelenchus xylophilus, causes huge economic losses by devastating pine forests worldwide. The pectate lyase gene is essential for successful invasion of their host plants by plant-parasitic nematodes. To demonstrate the role of pectate lyase gene in the PWD process, RNA interference (RNAi) is used to analyze the function of the pectate lyase 1 gene in B. xylophilus (Bxpel1). The efficiency of RNAi was detected by real-time PCR. The result demonstrated that the quantity of B. xylophilus propagated with control solution treatment was 62 times greater than that soaking in double-stranded RNA (dsRNA) after B. xylophilus inoculation in Botrytis cinerea for the first generation (F1). The number of B. xylophilus soaking in control solution was doubled compared to that soaking in Bxpel1 dsRNA four days after inoculation in Pinus thunbergii. The quantity of B. xylophilus was reduced significantly (p < 0.001) after treatment with dsRNAi compared with that using a control solution treatment. Bxpel1 dsRNAi reduced the migration speed and reproduction of B. xylophilus in pine trees. The pathogenicity to P. thunbergii seedling of B. xylophilus was weaker after soaking in dsRNA solution compared with that after soaking in the control solution. Our results suggest that Bxpel1 gene is a significant pathogenic factor in the PWD process and this basic information may facilitate a better understanding of the molecular mechanism of PWD.

Genome wide comprehensive analysis and web resource development on cell wall degrading enzymes from phyto-parasitic nematodes.

BACKGROUND: The plant cell wall serves as a primary barrier against pathogen invasion. The success of a plant pathogen largely depends on its ability to overcome this barrier. During the infection process, plant parasitic nematodes secrete cell wall degrading enzymes (CWDEs) apart from piercing with their stylet, a sharp and hard mouthpart used for successful infection. CWDEs typically consist of cellulases, hemicellulases, and pectinases, which help the nematode to infect and establish the feeding structure or form a cyst. The study of nematode cell wall degrading enzymes not only enhance our understanding of the interaction between nematodes and their host, but also provides information on a novel source of enzymes for their potential use in biomass based biofuel/bioproduct industries. Although there is comprehensive information available on genome wide analysis of CWDEs for bacteria, fungi, termites and plants, but no comprehensive information available for plant pathogenic nematodes. Herein we have performed a genome wide analysis of CWDEs from the genome sequenced phyto pathogenic nematode species and developed a comprehensive publicly available database. RESULTS: In the present study, we have performed a genome wide analysis for the presence of CWDEs from five plant parasitic nematode species with fully sequenced genomes covering three genera viz. Bursaphelenchus, Glorodera and Meloidogyne. Using the Hidden Markov Models (HMM) conserved domain profiles of the respective gene families, we have identified 530 genes encoding CWDEs that are distributed among 24 gene families of glycoside hydrolases (412) and polysaccharide lyases (118). Furthermore, expression profiles of these genes were analyzed across the life cycle of a potato cyst nematode. Most genes were found to have moderate to high expression from early to late infectious stages, while some clusters were invasion stage specific, indicating the role of these enzymes in the nematode's infection and establishment process. Additionally, we have also developed a Nematode's Plant Cell Wall Degrading Enzyme (NCWDE) database as a platform to provide a comprehensive outcome of the present study. CONCLUSIONS: Our study provides collective information about different families of CWDEs from five different sequenced plant pathogenic nematode species. The outcomes of this study will help in developing better strategies to curtail the nematode infection, as well as help in identification of novel cell wall degrading enzymes for biofuel/bioproduct industries.

Inhibition of acetylcholinesterases of the pinewood nematode, Bursaphelenchus xylophilus, by phytochemicals from plant essential oils.

To understand the nematicidal mode of action of phytochemicals derived from plant essential oils against the pinewood nematode (Bursaphelenchus xylophilus), we evaluated 97 compounds (49 monoterpenes, 17 phenylpropenes, 16 sesquiterpenes, and 15 sulfides) for their inhibitory effects on B. xylophilus acetylcholinesterases (BxACEs). In the primary inhibition assay using B. xylophilus crude protein, more than 50% BxACE inhibition activity was observed with 3 monoterpenes, (+)-α-pinene, (-)-α-pinene, and 3-carene; 2 phenylpropenes, ο-anisaldehyde, and coniferyl alcohol; and 1 sesquiterpene, cis-nerolidol. Other compounds showed moderate or weak inhibitory activity. The inhibitory activities against 3 recombinant BxACEs were subsequently estimated using the identified active compounds in a primary inhibition assay. (+)-α-Pinene showed the strongest inhibition of BxACE-1 followed by 3-carene, coniferyl alcohol, (-)-α-pinene, o-anisaldehyde, and cis-nerolidol. The half maximal inhibitory concentration (IC50) values of (+)-α-pinene, 3-carene, o-anisaldehyde, cis-nerolidol, and (-)-α-pinene against BxACE-2 were found to be 0.64, 1.41, 8.18, 8.53, 15.28, and 18.03mM, respectively. Coniferyl alcohol showed the strongest inhibition of BxACE-3 followed by (+)-α-pinene and cis-nerolidol.

Pine wood nematode, Bursaphelenchus xylophilus.

After devastating vast areas of pine forests in Asian countries, the pine wilt disease spread into European forests in 1999 and is causing worldwide concern. This disease involves very complicated interactions between a pathogenic nematode, its vector beetle, host pine species, and fungi in dead hosts. Pathogenicity of the pine wood nematode is determined not only by its physical and chemical traits but also by its behavioral traits. Most life history traits of the pine wood nematode, such as its phoretic relationship with vector beetles, seem to be more effective in virulent than in avirulent isolates or species. As the pathogenicity determinants, secreted enzymes, and surface coat proteins are very important, they have therefore been studied intensively. The mechanism of quick death of a large pine tree as a result of infection by a tiny nematode could be ascribed to the dysfunction of the water-conducting system caused by the death of parenchyma cells, which must have originally evolved as an inherent resistant system.

Rather than by direct acquisition via lateral gene transfer, GHF5 cellulases were passed on from early Pratylenchidae to root-knot and cyst nematodes.

BACKGROUND: Plant parasitic nematodes are unusual Metazoans as they are equipped with genes that allow for symbiont-independent degradation of plant cell walls. Among the cell wall-degrading enzymes, glycoside hydrolase family 5 (GHF5) cellulases are relatively well characterized, especially for high impact parasites such as root-knot and cyst nematodes. Interestingly, ancestors of extant nematodes most likely acquired these GHF5 cellulases from a prokaryote donor by one or multiple lateral gene transfer events. To obtain insight into the origin of GHF5 cellulases among evolutionary advanced members of the order Tylenchida, cellulase biodiversity data from less distal family members were collected and analyzed. RESULTS: Single nematodes were used to obtain (partial) genomic sequences of cellulases from representatives of the genera Meloidogyne, Pratylenchus, Hirschmanniella and Globodera. Combined Bayesian analysis of ≈ 100 cellulase sequences revealed three types of catalytic domains (A, B, and C). Represented by 84 sequences, type B is numerically dominant, and the overall topology of the catalytic domain type shows remarkable resemblance with trees based on neutral (= pathogenicity-unrelated) small subunit ribosomal DNA sequences. Bayesian analysis further suggested a sister relationship between the lesion nematode Pratylenchus thornei and all type B cellulases from root-knot nematodes. Yet, the relationship between the three catalytic domain types remained unclear. Superposition of intron data onto the cellulase tree suggests that types B and C are related, and together distinct from type A that is characterized by two unique introns. CONCLUSIONS: All Tylenchida members investigated here harbored one or multiple GHF5 cellulases. Three types of catalytic domains are distinguished, and the presence of at least two types is relatively common among plant parasitic Tylenchida. Analysis of coding sequences of cellulases suggests that root-knot and cyst nematodes did not acquire this gene directly by lateral genes transfer. More likely, these genes were passed on by ancestors of a family nowadays known as the Pratylenchidae.

In vitro proteolysis of nematode FMRFamide-like peptides (FLPs) by preparations from a free-living nematode (Panagrellus redivivus) and two plant-parasitic nematodes (Heterodera glycines and Meloidogyne incognita).

Proteolytic activities in extracts from three nematodes, the plant parasites Heterodera glycines and Meloidogyne incognita, and the free-living Panagrellus redivivus, were surveyed for substrate preferences using a battery of seven FRET-modified peptide substrates, all derived from members of the large FMRF-amide like peptide (FLP) family in nematodes. Overall protease activity in P. redivivus was four- to fivefold greater than in either of the parasites, a result that might reflect developmental differences. Digestion of the M. incognita FLP KHEFVRFa (substrate Abz-KHEFVRF-Y(3-NO2)a) by M. incognita extract was sevenfold greater than with H. glycines extract and twofold greater than P. redivivus, suggesting species-specific preferences. Additional species differences were revealed upon screening 12 different protease inhibitors. Two substrates were used in the screen, Abz-KHEFVRF-Y(3-NO2)a and Abz-KPSFVRF-Y(3-NO2)a), which was digested equally by all three species. The effects of various inhibitor, substrate and extract source combinations on substrate digestion suggest that M. incognita differs significantly from P. redivivus and H. glycines in its complement of cysteine proteases, particularly cathepsin L-type protease.

Functional analysis of the cellulose gene of the pine wood nematode, Bursaphelenchus xylophilus, using RNA interference.

Cellulases are pathogenic substances suspected to be responsible for the development of the early symptoms of nematode disease. The pine wood nematode, Bursaphelenchus xylophilus (Parasitaphelenchidae), is the causal agent of pine wilt disease, which kills millions of pine trees. We used RNA interference (RNAi), a reverse genetic tool, to analyze the function of the endo-ß-1,4-glucanase gene of B. xylophilus, which causes the most serious forest tree disease in China and the rest of eastern Asia. Silencing of this gene was detected through real-time PCR and cellulase activity assays after soaking for 24 h in dsRNA. The cellulase gene silencing effects differed among various siRNAs. The propagation and dispersal ability of these nematodes decreased when the endo-ß-1,4-glucanase gene was silenced. It is important to select an effective siRNA before performing an RNAi test.

Cloning and characterization of a 2-cys peroxiredoxin in the pine wood nematode, Bursaphelenchus xylophilus, a putative genetic factor facilitating the infestation.

The pine wood nematode, Bursaphelenchus xylophilus, is an invasive plant parasitic nematode and a worldwide quarantine pest. An indigenous species in North America and the causal agent of pine wilt disease, B. xylophilus has devastated pine production in Southeastern Asia including Japan, China, and Korea since its initial introduction in the early 1900s. The reactive oxygen species (ROS) is the first line of defense utilized by host plants against parasites, while nematodes, counteractively, employ antioxidants to facilitate their infection. Peroxiredoxins (Prxs) are a large class of antioxidants recently found in a wide variety of organisms. In this report, a gene encoding a novel 2-cysteine peroxiredoxin protein in B. xylophilus was cloned and characterized. The 2-cysteine peroxiredoxin in B. xylophilus (herein refers to as "BxPrx") is highly conserved in comparison to 2-cysteine peroxiredoxins (Prx2s) in other nematodes, which have two conserved cysteine amino acids (Cp and Cr), a threonine-cysteine-arginine catalytic triad, and two signature motifs (GGLG and YF) sensitive to hydrogen peroxide. In silico assembly of BxPrx tertiary structure reveals the spatial configuration of these conserved domains and the simulated BxPrx 3-dimensional structure is congruent with its presumed redox functions. Although no signal peptide was identified, BxPrx was abundantly expressed and secreted under the B. xylophilus cuticle. Upon further analysis of this leader-less peptide, a single transmembrane α-helix composed of 23 consecutive hydrophobic amino acids was found in the primary structure of BxPrx. This transmembrane region and/or readily available ATP binding cassette transporters may facilitate the transport of non-classical BxPrx across the cell membrane. Recombinant BxPrx showed peroxidase activity in vitro reducing hydrogen peroxide using glutathione as the electron donor. The combined results from gene discovery, protein expression and distribution profiling (especially the "surprising" presence under the nematode cuticle), and recombinant antioxidant activity suggest that BxPrx plays a key role in combating the oxidative burst engineered by the ROS defense system in host plants during the infection process. In summary, BxPrx is a genetic factor potentially facilitating B. xylophilus infestation.

Three acetylcholinesterases of the pinewood nematode, Bursaphelenchus xylophilus: insights into distinct physiological functions.

Acetylcholinesterase (AChE) plays a key role in postsynaptic transmission in most animals. Nematodes encode multiple AChEs, implying its functional diversity. To explore physiological functions of multiple AChEs, three distinct AChEs (BxACE-1, BxACE-2, and BxACE-3) were identified and characterized from the pinewood nematode. Sequencing comparison with Torpedo AChE and Caenorhabditis elegans ACEs identified choline-binding site, catalytic triad functional site, three internal disulfide bonds and aromatic residues for the catalytic gorge. Transcriptional profiling by quantitative real-time PCR revealed that BxACE-3 is more actively transcribed than BxACE-1 (2-3 times) and BxACE-2 (9-18 times) in both propagative and dispersal stages. The three BxACEs were functionally expressed using baculovirus system. Kinetic analysis of in vitro-expressed BxACEs revealed that the substrate specificity was highest in BxACE-1 whereas the catalytic efficiency was highest in BxACE-2. In inhibition assay, BxACE-3 showed the lowest inhibition rate. Taken together, it appears that both BxACE-1 and BxACE-2 play common but non-overlapping roles in synaptic transmission, whereas BxACE-3 may have non-neuronal functions. The current findings should provide valuable insights into the evolutionary process and various physiological roles of AChE.

The role of pseudo-endoglucanases in the evolution of nematode cell wall-modifying proteins.

In this article, the characterization and evolution of pseudo-endoglucanases and a putative expansin-like gene in the migratory nematode Ditylenchus africanus are described. Four genes were cloned with a very high similarity to the endoglucanase Da-eng1, which, however, lack a part of the catalytic domain most probably due to homologous recombination. Owing to this deletion, at least one of the catalytic residues of the corresponding protein is missing, and hence these genes are possibly pseudogenes. In two of the pseudo-endoglucanase genes, the deletions cause a frameshift (Da-engdel2, Da-engdel4), while two others (Da-engdel1, Da-engdel3) code for protein sequences with an intact carbohydrate-binding module (CBM). Recombinant proteins for Da-ENG1, Da-ENGDEL1, and Da-ENGDEL3 were demonstrated to bind to cellulose, while only Da-ENG1 showed cellulose-degrading activity. This indicates that Da-ENGDEL1 and Da-ENGDEL3 which lack cellulase activity, could still exert a function similar to cellulose-binding proteins (CBPs). Next to the pseudo-endoglucanases, a putative expansin-like gene (Da-exp1) was identified, consisting of a signal peptide, an expansin-like domain, and a CBM. This domain structure was never found before in nematode expansin-like proteins. Interestingly, the CBM of the expansin-like gene is very similar to the endoglucanase CBMs, and a conserved intron position in the CBM of nematode endoglucanases, expansin-like genes, and CBPs indicates a common origin for these domains. This suggests that domain shuffling is an important mechanism in the evolution of cell wall-modifying enzymes in nematodes.

Analysis of expressed sequence tags and identification of genes encoding cell-wall-degrading enzymes from the fungivorous nematode Aphelenchus avenae.

BACKGROUND: The fungivorus nematode, Aphelenchus avenae is widespread in soil and is found in association with decaying plant material. This nematode is also found in association with plants but its ability to cause plant disease remains largely undetermined. The taxonomic position and intermediate lifestyle of A. avenae make it an important model for studying the evolution of plant parasitism within the Nematoda. In addition, the exceptional capacity of this nematode to survive desiccation makes it an important system for study of anhydrobiosis. Expressed sequence tag (EST) analysis may therefore be useful in providing an initial insight into the poorly understood genetic background of A. avenae. RESULTS: We present the generation, analysis and annotation of over 5,000 ESTs from a mixed-stage A. avenae cDNA library. Clustering of 5,076 high-quality ESTs resulted in a set of 2,700 non-redundant sequences comprising 695 contigs and 2,005 singletons. Comparative analyses indicated that 1,567 (58.0%) of the cluster sequences had homologues in Caenorhabditis elegans, 1,750 (64.8%) in other nematodes, 1,321(48.9%) in organisms other than nematodes, and 862 (31.9%) had no significant match to any sequence in current protein or nucleotide databases. In addition, 1,100 (40.7%) of the sequences were functionally classified using Gene Ontology (GO) hierarchy. Similarity searches of the cluster sequences identified a set of genes with significant homology to genes encoding enzymes that degrade plant or fungal cell walls. The full length sequences of two genes encoding glycosyl hydrolase family 5 (GHF5) cellulases and two pectate lyase genes encoding polysaccharide lyase family 3 (PL3) proteins were identified and characterized. CONCLUSION: We have described at least 2,214 putative genes from A. avenae and identified a set of genes encoding a range of cell-wall-degrading enzymes. This EST dataset represents a starting point for studies in a number of different fundamental and applied areas. The presence of genes encoding a battery of cell-wall-degrading enzymes in A. avenae and their similarities with genes from other plant parasitic nematodes suggest that this nematode can act not only as a fungal feeder but also a plant parasite. Further studies on genes encoding cell-wall-degrading enzymes in A. avenae will accelerate our understanding of the complex evolutionary histories of plant parasitism and the use of genes obtained by horizontal gene transfer from prokaryotes.

Small subunit rDNA-based phylogeny of the Tylenchida sheds light on relationships among some high-impact plant-parasitic nematodes and the evolution of plant feeding.

Cyst (Heteroderidae), root knot (Meloidogyne spp.), and lesion (Pratylenchus spp.) nematodes all belong to a single nematode order, Tylenchida. However, the relationships between and within these economically highly relevant groups, and their relatedness to other parasitic Tylenchida is unclear. We constructed a phylogeny of 116 Tylenchida taxa based on full length small subunit ribosomal DNA (small subunit [SSU] rDNA) sequences. Ancestral state reconstruction points at a gradual development of simple to more complex forms of plant parasitism. Good resolution was observed in distal clades that include cyst, root knot, and lesion nematodes, and monophyly of most families was confirmed. Our data suggest that root knot nematodes have evolved from an ancestral member of the genus Pratylenchus, but it remains unclear which species is closest to this branching point. Contrary to the notoriously polyphagous distal representatives, basal members of the genus Meloidogyne (and probably, their common ancestor) have narrow host ranges. Our analysis also shows that mitotic parthenogeny has arisen at least two times independently among root knot nematodes. In many cases resolution till species was observed, suggesting that SSU rDNA sequences have a potential for DNA barcode-based species identification with, due to the overall conserved nature of this gene, limited intra-species variation.

Purification and characterization of recombinant endoglucanases from the pine wood nematode Bursaphelenchus xylophilus.

A family of endoglucanases belonging to glycoside hydrolase family (GHF) 45 have been isolated from the pine wood nematode Bursaphelenchus xylophilus. Here we describe the purification and characterization of the recombinant enzymes, named Bx-ENG-1, 2, and 3, expressed in Pichia pastoris. The respective molecular masses of purified Bx-ENG-1, 2, and 3 were estimated to be 18, 33-39, and 100-140 kDa by SDS-PAGE, and 18, 67, and 252 kDa by gel filtration, suggesting that Bx-ENG-1 existed in an unglycosylated monomeric form and Bx-ENG-2 and Bx-ENG-3 in a glycosylated dimeric form. The enzymatic properties of the recombinant enzymes were similar to each other: optimal activity at 60 degrees C at about pH 6.0, like other endoglucanases of GHF45. The recombinant enzymes displayed the highest activity toward lichenan, and lower activities were observed on carboxymethyl cellulose and amorphous cellulose. Nematode enzymes also hydrolyzed glucomannan, the most abundant hemicellulose in the cell walls of softwood. These substrate specificities suggest that B. xylophilus endoglucanases acted on the cellulose-hemicellulose complex in the cell walls, resulting in a weakening of the mechanical strength of the cell walls to facilitate the nematode's feeding on plant cells.

Pathogenic cellulase assay of pine wilt disease and immunological localization.

The pine wilt disease caused by Bursaphelenchus xylophilus (BX), also known as the pine wood nematode (PWN), is the most devastating disease of pine trees. In this work, a high molecular weight B. xylophilus cellulase antigen (BXCa) was purified from total homogenates of nematodes. BXCa was found to be able to hydrolyze carboxymethyl cellulose (CMC) efficiently (155.65 U/mg) and to have an approximate molecular mass of 58.9 kDa. We harvested anti-BXCa antibodies and performed immunocytochemical assays, which revealed the localization of cellulase pools in the esophageal gland cells of the PWN. It was also discovered that cellulase was secreted from the stylet and was used to hydrolyze cellulose to facilitate the PWN entering host cells. These results are consistent with other plant parasitical nematodes. Interestingly, strong fluorescence signals from cellulase staining were observed in tracheid cells in naturally infected pine wood, in addition to ray cells and the resin canal zone. These results strongly suggest that the cellulase released by the PWN is one of the pathogenic substances of pine wilt disease and is responsible for the development of the early symptoms of the disease.