BMC Genomics 2009, 10:58 http://www.biomedcentral.com/1471-2164/10/58 pFTreigomubperoseer1tasl expression patterns of H. glycines parasitism gene Temporal expression patterns of H. glycines parasit- ism gene probesets. All 62 H. glycines parasitism gene probesets were differentially expressed (FDR 5%) over the entire life cycle and were grouped into two clusters. The average expression pattern of each cluster is represented by a bold line. teins) or for which no function during parasitism can be ascribed yet (e.g., chitinase, venom allergen proteins). The expression profiles of these genes showed a clear down- regulation after the parasitic J2 stage, which is the begin- ning of the sedentary phase of the nematode and of feeding site development. Furthermore, those parasitism genes that were upregulated in later life stages did not match any other known sequences, apart from two excep- tions: annexin and a cellulose-binding protein (Addi- tional file 1). Annexins bind to calcium-dependent phospholipid membranes, and while their role during parasitism remains unknown, the cellulose-binding pro- tein binds to a plant pectin methylesterase and functions in cell wall modifications during syncytium development [39]. In addition to analyses of gene expression changes throughout the whole life cycle, in which each life stage was compared to all others, we also performed statistical analyses to identify gene expression changes in consecu- tive life stages to isolate specific shifts in gene expression. The results of these studies showed that most parasitism genes were differentially expressed when eggs were com- pared with infective J2 as well as when infective J2 were compared with parasitic J2. All of these differentially expressed genes were upregulated (Table 1). This pattern drastically changed from parasitic J2 to J3, where only a smaller subset of genes was differentially expressed and, interestingly, these were all down-rather than upregu- lated. H. glycines genes for secretory proteins frequently are novel sequences and change expression with the onset of parasitism To identify new H. glycines genes that are potentially involved in host-parasite interactions we identified all genes that encode secretory proteins. Specifically, we searched for signal peptide-coding regions in the consen- sus sequences of all 7,530 H. glycines probesets on the Affymetrix Soybean Genome Array GeneChip as described in Methods. This analysis identified 633 unique H. glycines genes that encode proteins with a putative sig- nal peptide but lack a transmembrane helix and have an open reading frame (ORF) of at least thirty amino acids after the predicted signal peptide cleavage site (Additional file 2). However, it can be assumed that H. glycines pos- sesses more gene products with signal peptides than we could detect here because a significant portion of ESTs lacks a complete 5'-end so that no signal peptide-coding region could possibly be identified. All known parasitism genes are included in these 7,530 probesets, and our detection protocol for secretory protein coding genes re- identified all but seven of the known sixty-six parasitism genes analyzed here, giving a re-discovery rate of 89%. The probesets of genes that were missing did not meet all of our stringent selection criteria. Because most parasitism proteins have no database hits to known proteins, we determined whether the signal pep- tide-encoding genes identified here differ in their likeli- hood of having database matches compared to other genes. For this purpose, we conducted BLASTX searches of all 6,860 unique H. glycines genes underlying the 7,530 probesets of the Affymetrix GeneChip against the non- redundant GenBank database. We found that 52.8% (334/633) of the gene products predicted to be secreted had matches when using a cutoff value of 1e-05 (Addi- tional file 2). Similarly, 58.5% (3,644/6,227) of the gene products not thought to be secreted had matches. Even though this difference of 5.7% is relatively small, we found a significant difference when the relative BLASTX scores were compared. Out of all 6,860 unique genes, 3,978 had matches, and resulted in a median BLASTX score of 310 (min 41, max 5,249). Using only the 334 gene products of secreted candidates, the median BLASTX score was reduced to 129 (min 27, max 3,299), which sug- gests that as was the case for previously reported parasit- ism genes, our newly identified cohort of secretory proteins containing potential parasitism proteins evolved more rapidly than non-secreted sequences. To analyze with which organisms H. glycines signal pep- tide-bearing proteins share conservation, we sorted the top BLASTX hits meeting a cutoff of 1e-05 by organism Page 4 of 17 (page number not for citation purposes)