Analysis of the Transcriptome of the Infective Stage of the Beet Cyst Nematode, H. schachtii.

The beet cyst nematode, Heterodera schachtii, is a major root pest that significantly impacts the yield of sugar beet, brassicas and related species. There has been limited molecular characterisation of this important plant pathogen: to identify target genes for its control the transcriptome of the pre-parasitic J2 stage of H. schachtii was sequenced using Roche GS FLX. Ninety seven percent of reads (i.e., 387,668) with an average PHRED score > 22 were assembled with CAP3 and CLC Genomics Workbench into 37,345 and 47,263 contigs, respectively. The transcripts were annotated by comparing with gene and genomic sequences of other nematodes and annotated proteins on public databases. The annotated transcripts were much more similar to sequences of Heterodera glycines than to those of Globodera pallida and root knot nematodes (Meloidogyne spp.). Analysis of these transcripts showed that a subset of 2,918 transcripts was common to free-living and plant parasitic nematodes suggesting that this subset is involved in general nematode metabolism and development. A set of 148 contigs and 183 singletons encoding putative homologues of effectors previously characterised for plant parasitic nematodes were also identified: these are known to be important for parasitism of host plants during migration through tissues or feeding from cells or are thought to be involved in evasion or modulation of host defences. In addition, the presence of sequences from a nematode virus is suggested. The sequencing and annotation of this transcriptome significantly adds to the genetic data available for H. schachtii, and identifies genes primed to undertake required roles in the critical pre-parasitic and early post-parasitic J2 stages. These data provide new information for identifying potential gene targets for future protection of susceptible crops against H. schachtii.

De novo analysis of the transcriptome of Pratylenchus zeae to identify transcripts for proteins required for structural integrity, sensation, locomotion and parasitism.

The root lesion nematode Pratylenchus zeae, a migratory endoparasite, is an economically important pest of major crop plants (e.g. cereals, sugarcane). It enters host roots, migrates through root tissues and feeds from cortical cells, and defends itself against biotic and abiotic stresses in the soil and in host tissues. We report de novo sequencing of the P. zeae transcriptome using 454 FLX, and the identification of putative transcripts encoding proteins required for movement, response to stimuli, feeding and parasitism. Sequencing generated 347,443 good quality reads which were assembled into 10,163 contigs and 139,104 singletons: 65% of contigs and 28% of singletons matched sequences of free-living and parasitic nematodes. Three-quarters of the annotated transcripts were common to reference nematodes, mainly representing genes encoding proteins for structural integrity and fundamental biochemical processes. Over 15,000 transcripts were similar to Caenorhabditis elegans genes encoding proteins with roles in mechanical and neural control of movement, responses to chemicals, mechanical and thermal stresses. Notably, 766 transcripts matched parasitism genes employed by both migratory and sedentary endoparasites in host interactions, three of which hybridized to the gland cell region, suggesting that they might be secreted. Conversely, transcripts for effectors reported to be involved in feeding site formation by sedentary endoparasites were conspicuously absent. Transcripts similar to those encoding some secretory-excretory products at the host interface of Brugia malayi, the secretome of Meloidogyne incognita and products of gland cells of Heterodera glycines were also identified. This P. zeae transcriptome provides new information for genome annotation and functional analysis of possible targets for control of pratylenchid nematodes.

de novo analysis and functional classification of the transcriptome of the root lesion nematode, Pratylenchus thornei, after 454 GS FLX sequencing.

The migratory endoparasitic root lesion nematode Pratylenchus thornei is a major pest of the cereals wheat and barley. In what we believe to be the first global transcriptome analysis for P. thornei, using Roche GS FLX sequencing, 787,275 reads were assembled into 34,312 contigs using two assembly programs, to yield 6,989 contigs common to both. These contigs were annotated, resulting in functional assignments for 3,048. Specific transcripts studied in more detail included carbohydrate active enzymes potentially involved in cell wall degradation, neuropeptides, putative plant nematode parasitism genes, and transcripts that could be secreted by the nematode. Transcripts for cell wall degrading enzymes were similar to bacterial genes, suggesting that they were acquired by horizontal gene transfer. Contigs matching 14 parasitism genes found in sedentary endoparasitic nematodes were identified. These genes are thought to function in suppression of host defenses and in feeding site development, but their function in P. thornei may differ. Comparison of the common contigs from P. thornei with other nematodes showed that 2,039 were common to sequences of the Heteroderidae, 1,947 to the Meloidogynidae, 1,218 to Radopholus similis, 1,209 matched expressed sequence tags (ESTs) of Pratylenchus penetrans and Pratylenchus vulnus, and 2,940 to contigs of Pratylenchus coffeae. There were 2,014 contigs common to Caenarhabditis elegans, with 15.9% being common to all three groups. Twelve percent of contigs with matches to the Heteroderidae and the Meloidogynidae had no homology to any C. elegans protein. Fifty-seven percent of the contigs did not match known sequences and some could be unique to P. thornei. These data provide substantial new information on the transcriptome of P. thornei, those genes common to migratory and sedentary endoparasitic nematodes, and provide additional understanding of genes required for different forms of parasitism. The data can also be used to identify potential genes to study host interactions and for crop protection.