BMC Genomics 2009, 10:58 http://www.biomedcentral.com/1471-2164/10/58 (data not shown). These results indicate that the cross- hybridizing probesets in all likelihood originated from soybean plants and P. sojae and were not H. glycines con- taminations in cDNA libraries of these two organisms because we did not find a single hit with a bona fide H. gly- cines database entry, which otherwise would have to have occurred. The possibility of falsely annotated H. glycines ESTs can be ruled out based on the same results. To deter- mine whether in fact there are highly conserved cyst nem- atode sequences that could cross-hybridize with gene sequences of soybean or P. sojae, we conducted BLASTN searches of the cross-hybridizing soybean and P. sojae probesets against an in-house database of all cyst nema- tode (Heterodera spp. and Globodera spp.) sequences. Of 576 soybean probesets, 119 (20.7%) indeed matched known cyst nematode genes and of 134 P. sojae probesets 12 (9.0%) in fact had matches with known Heterodera spp. or Globodera spp. genes (Additional files 13, 14). In other words, in all likelihood, sequence conservation was responsible for the observed cross-hybridization results. To complement the BLAST searches of the cross-hybridiz- ing soybean and P. sojae probesets, we used InterProScan [44] to identify known protein domains. We found 336 soybean probesets that aligned to 207 unique InterPro domains and 83 P. sojae probesets that aligned to 70 unique InterPro domains, respectively. A summary of the 25 most abundant InterPro domains for both organisms can be viewed in Table 2. Taken together, the soybean and P. sojae probesets that surprisingly cross-hybridized with H. glycines probes potentially provide a lead to an additional set of nema- tode genes from which novel parasitism-associated genes can be isolated and confirmed. Ultimately, this aspect can be advanced further only with a future release of a com- plete H. glycines genome sequence, because the recent dep- osition of a large number of genome fragments did not allow an exhaustive and conclusive analysis (data not shown). Discussion Here we have presented the results of an extensive in silico study aiming at the identification of a group of H. glycines genes that is enriched in genes with potential functions during host-parasite interactions. This is the first study combining microarray data of all major life stages (except adult males) and exhaustive sequence analyses for the identification of parasitism-associated proteins in any parasitic nematode. Our findings now enable more detailed studies to identify true parasitism genes among the individual candidate genes identified here, which would not have been feasible for larger data sets. It has been widely assumed that cyst nematode parasitism genes have well-defined roles in early and late stages of the infection process, and our findings of two distinct expression clusters representing early or late upregulation for all known parasitism genes support this. When com- paring the temporal expression patterns of H. glycines gene groups identified here, it became evident that certain expression clusters showed profiles that were similar to those of known parasitism genes. Specifically, Cluster 1 of the currently known parasitome (Figure 1) (i.e., early upregulation) was strikingly similar to Cluster 3 of the secretome identified by us here (Figure 3), while Cluster 2 of the parasitome (i.e., late upregulation) was mirrored by Cluster 1 of the secretome, Cluster 1 of plant-like H. gly- cines genes (Additional file 8) and Cluster 5 of soilmi- crobe-like H. glycines genes (Additional file 10). The H. glycines genes represented in these clusters might be partic- ularly promising candidates for future studies aiming at the verification of their roles in parasitism. Alternatively, Clusters 2 and 4 of the secretome might be of interest because the genes in these groups were strongly upregu- lated in very late parasitic stages (post-J3). These clusters might harbor novel parasitism-associated genes that are very different from the currently known parasitome, which is biased towards early stages of parasitism. It has been suggested that nematode proteins that enter the secretory pathway evolve more rapidly than those that do not and hence are less likely to match sequences of other organisms [33]. Even though the difference we found in the number of genes with BLASTX hits between all H. glycines genes represented on the Affymetrix micro- array and those with a predicted signal peptide was rela- tively low, we identified a significant difference in the relative distribution of BLASTX scores. This means that our results support previous findings and a model in which nematode secretory proteins evolve more rapidly than other gene products. None of the libraries used here was based on splice leader 1 (SL1) sequences, which tend to be skewed towards shorter sequences [24], such that no bias towards complete 5'-ends (and hence signal pep- tides) was possible in the data analyzed here. Some of the known parasitism proteins showed similari- ties to plant or microbe sequences [10-12,14,16], and in a closely related nematode species additional genes with similarity to nitrogen-fixing soil bacteria, collectively called rhizobia, could be identified [38]. It has been sug- gested that microbial genes might have been acquired by horizontal gene transfer [10,38] and that plant-like genes might have evolved in the nematode for mimicry of plant proteins and interference with plant signaling pathways [7,40,41]. For all of these genes to have any effect in a par- asitic relationship between host and parasite, they need to be secreted. Hence, we were interested in analyzing whether H. glycines genes represented on the Affymetrix Soybean Genome Array GeneChip with similarity to microbes or plants might encode signal peptide-bearing Page 8 of 17 (page number not for citation purposes)