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

Abstract

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.

Figure 1

Simplified tree showing relationships of Aphelenchus avenae, Bursaphelenchus and cyst/root-knot nematodes. Recently published phylogenetic tree based on SSU of ribosomal DNA [38] has been adapted for drawing this simplified tree. Taxonomic positions are indicated based on superfamily [3].

Figure 2

Histogram showing the distribution of ESTs from A. avenae by cluster size. For example, there were five clusters of size 23 containing a sum total of 115 ESTs. Distribution of contig sizes is not shown.

Figure 3

Comparison of A. avenae cluster sequences with C. elegans, other nematodes and non-nematode protein sequence databases using SimiTri. The numbers at each vertex indicate the number of cluster sequences matching only that specific database. The numbers on the edges indicate the number of cluster sequences matching the two databases linked by that edge. The number within the triangle indicates the number of A. avenae genes with matches to sequences in all three databases.

Figure 4

Summary of the Gene Ontology annotation as assigned by BLAST2GO. (A) Most represented GO terms (based on number of represented sequences) of the main category "biological process"; (B) Most represented GO terms of the main category "molecular function (C) Most represented GO terms of the main category "cellular component". Multi-level pie charts were generated using the sequence cut-offs 140, 50 and 40 for "biological process", "molecular function" and "cellular component", respectively.

Figure 5

Aa-eng-1cDNA sequence and predicted amino acid sequences. The predicted signal peptide for secretion and polyadenylation signal sequence are underlined. Predicted positions of the five intron sequences identified are indicated by darkened triangles. Primers used for obtaining the full length cDNA sequence and genomic amplification are indicated by arrows. The amino acids within the boxes represent the predicted active site residues.

Figure 6

Unrooted phylogenetic tree of GHF5 catalytic domains based on the protein sequences using the maximum likelihood method. The GHF5 proteins from A. avenae (AA-ENG-1 and AA-ENG-2) are labeled in bold. GenBank accession numbers of GHF5 proteins from Meloidogyne incognita (MI-ENG-1, MI-ENG-2, MI-ENG-3 and MI-ENG-4), Pratylenchus penetrans (PP-ENG-1 and PP-ENG-2), Pratylenchus coffeae (PC-ENG-1), Radopholus similis (RS-ENG-1A, RS-ENG-1B, RS-ENG-2 and RS-ENG-3), Globodera rostochiensis (GR-ENG-1, GR-ENG-2 and GR-ENG-4), Heterodera glycines (HG-ENG-2, HG-ENG-4, HG-ENG-5 and HG-ENG-6), Ditylenchus africanus (DA-ENG-1), beetles, bacteria and protists are indicated in brackets. The bootstrap values are calculated from 1000 replicates. The scale bar represents 10 substitutions per 100 amino acid positions.

Figure 7

Multiple sequence alignment of A. avenae pectate lyase protein sequences (AA-PEL-1 and AA-PEL-2) with the sequences of pectate lyases from plant parasitic nematodes, bacterium, and a fungus. BX-PEL-1 [BAE48369], BX-PEL-2 [BAE48370] from Bursaphelenchus xylophilus; BM-PEL-1 [BAE48373], and BM-PEL-2 [BAE48375] from Bursaphelenchus mucronatus; MI-PEL-1 [AAQ09004] and MI-PEL-2 [AAQ97032] from Meloidogyne incognita; MJ-PEL-1 [AAL66022] from Meloidogyne javanica; GR-PEL-1 [AAF80747] from Globodera rostochiensis; SC-PEL-1 [NP625403] from Streptomyces coelicolor and FS-PEL-B [AAA8738] from Fusarium solani. Identical residues are highlighted in black and functionally conserved are in gray. Black bars (I to IV) indicate the conserved regions characteristic of PL3 pectate lyases. Very highly conserved charged residues are indicated beneath the alignment by a number symbol (#), while an asterisk (*) indicates conserved cysteine residues. The positions of the intron in AA-PEL-1 and AA-PEL-2 are indicated by A1 and A2 respectively, that in BX-PEL-1/2 and BM-PEL-1/2 by B, and those in MI-ENG-1 by Mi1. Triangles and diamonds represent phase 0 and 1 introns, respectively.

Figure 8

Unrooted phylogenetic tree of selected polysaccharide lyase family 3 proteins generated using maximum likelihood analysis. The PL3 proteins from A. avenae (AA-PEL-1 and AA-PEL-2) are labeled in bold. GenBank accession numbers of PL3 proteins from Bursaphelenchus mucronatus (BM-PEL-1 and BM-PEL-2), Bursaphelenchus xylophilus (BX-PEL-1 and BX-PEL-2), Meloidogyne incognita (MI-PEL-1, MI-PEL-2 and MI-PEL-3), Meloidogyne javanica (MJ-PEL), Globodera rostochiensis (GR-PEL-1 and GR-PEL-2), Heterodera schachtii (HS-PEL-1 and HS-PEL-2), bacteria and fungi are indicated in brackets. The bootstrap values are calculated from 1000 replicates. The scale bar represents 20 substitutions per 100 amino acid positions.

Figure 9

Localization of Aa-eng-1 and Aa-pel-1 transcripts in the esophageal gland cells of A. avenae by in situ hybridisation. Nematode sections were hybridized with antisense (A) and sense (B) Aa-eng-1 digoxigenin-labeled cDNA probes. Hybridisation was also carried out with digoxigenin-labeled antisense (C) and sense (D) Aa-pel-1 cDNA probes. G, esophageal glands; S, stylet; M, metacorpus. The bar indicates a length of 20 μm.