BMC Genomics 2009, 10:58 http://www.biomedcentral.com/1471-2164/10/58 (Figure 2). Approximately 26% (166 genes) of the 633 H. glycines genes with putative signal peptide-coding regions matched Caenorhabditis elegans or Caenorhabditis briggsae sequences, followed by 12% that matched other H. gly- cines genes and 7% that aligned best with sequences from animals other than nematodes. However, 147 out of the total 166 Caenorhabditis matches represented unknown or hypothetical proteins and, similarly, 47 out of 78 H. gly- cines sequences were novel, i.e., they did not share similar- ity with other known sequences. Using statistical analyses, all Affymetrix probeset expres- sion profiles obtained for the 633 signal peptide-encoding genes separated into nine expression clusters (Figure 3) as described in Methods. When all experimental data, i.e., expression data throughout all life cycle stages, were taken into consideration, 94% of these probesets were differen- tially expressed (FDR 5%) during the life cycle. When con- ducting statistical analyses of expression data for consecutive life stages in pairwise comparisons, we deter- mined that 49% of the Affymetrix probesets representing the 633 secretory protein genes were differentially expressed (FDR 5%) during the transition from eggs to infective J2. Similarly, 48% of these Affymetrix probesets were differentially expressed from infective J2 to parasitic J2 and 33% from parasitic J2 to J3. The fewest significant changes (10%) occurred between J3 and J4, followed by the comparison between J4 and adult females (20%) (Table 1). Taken together, the clusters for overall differen- tially expressed probesets as well as the stage by stage comparisons showed that the strongest expression changes in genes that code for secretory proteins took place at the transitions into and out of the infective J2 stage, which marks the preparations for host invasion and a parasitic lifestyle. Identification and expression profiling of H. glycines genes that are conserved in microbes or plants Previous studies showed that a substantial proportion of the known cyst nematode parasitism proteins share a high degree of similarity with bacterial [10,11,13] and fungal [11-13] proteins and not to the non-pathogenic Caenorhabditis spp. These observations led to the sugges- tion that certain nematode genes that are important for a parasitic relationship with the host might have been acquired by horizontal gene transfer from microbes [7,10,38]. Further studies demonstrated that other cyst nematode secretory proteins with potential involvement in parasitism have a striking similarity to plant proteins [15,16,40,41], which could hint at mimicry of plant regu- latory proteins by cyst nematodes to alter the physiology of the host. Therefore, we identified H. glycines proteins that showed significant sequence similarity with microbe or plant proteins by manually sorting the results of the BLASTX searches of all 6,860 H. glycines genes against the non-redundant GenBank database mentioned above. We isolated all matches to proteins from plants, microbial phytopathogens/phytosymbionts (termed phytomi- crobes here), soil-living microbes and other microbes. A counter-selection protocol ensured that highly conserved sequences that are present in diverse organisms (including Caenorhabditis spp.) were removed in order to discard sequences that are unlikely to be involved in parasitic rela- tionships (see Methods). These analyses revealed that, using our criteria, 29 H. glycines protein sequences were conserved in plants, 41 in microbial phytomicrobes, 33 in soil-living microbes, and 53 in other microbes, resulting in a total of 156 such proteins (Additional files 3, 4, 5, 6). For example, we identified a plant-like H. glycines gene whose translated product had similarity to beta-amylase from Arabidopsis (HgAffx.12554.1), which is interesting because genes encoding beta-amylases have so far only been found in microbe and plant genomes, but not in ani- mals. Furthermore, we found H. glycines genes whose products were similar to a potato protein induced in the feeding site (giant-cells) of the root-knot nematode (Mel- oidogyne) (HgAffx.13422.1), or were involved in RNA interference (RNAi) like a Zwille/Pinhead-like protein from rice (HgAffx.13330.1). Phytomicrobe-like H. glycines sequences matched sugar metabolizing enzymes from Agrobacterium tumefaciens, e.g., mannitol-2-dehydroge- nase (HgAffx.18502.1) and sucrose hydrolase (HgAffx.9663.1, HgAffx.12954.1), as well as enzymes like glutamine synthetase (HgAffx.18955.1) and phosphori- bosyltransferase (HgAffx.23512.1) or aldose-1-epimerase (HgAffx.18360.1) from Mesorhizobium to name just a few. Table 1: Summary of parasitome, signal peptide-encoding and plant or microbe-like H. glycines genes. H. glycines contigs Contigs Probesets Differentially expressed probesets total (up/downregulated)** overall life cycle egg/infJ2 infJ2/parJ2 parJ2/J3 J3/J4 J4/F Secretory protein coding* H. glycines parasitome Plant-like Phytomicrobe-like Soil microbe-like Other microbe-like *As defined in Methods **FDR 5% 633 46 29 41 33 53 686 62 30 43 40 63 646 62 18 28 25 39 337 (213/124) 335 (186/149) 224 (55/169) 69 (41/28) 136 (41/95) 39 (34/5) 2 (0/2) 16 (9/7) 11 (4/7) 11 (5/6) 34 (30/4) 4 (4/0) 7 (5/2) 7 (4/3) 14 (11/3) 34 (0/34) 1 (1/0) 3 (1/2) 3 (1/2) 5 (2/3) 2 (0/2) 0 5 (5/0) 0 0 3 (0/3) 2 (0/2) 3 (1/2) 4 (4/0) 5 (0/5) Page 5 of 17 (page number not for citation purposes)