BMC Genomics 2009, 10:58 http://www.biomedcentral.com/1471-2164/10/58 iBFnLiggAugSerTneXe2shsietspaorfaHte.dglbyycinoersgapnuitsamtivgeroseucpretory protein encod- BLASTX hits of H. glycines putative secretory protein encoding genes separated by organism group. It is particularly interesting to discern if these H. glycines genes potentially are secreted from the nematode. There- fore, we examined how many of these H. glycines genes were part of the secretome identified in this study. Of the 29 plant-conserved H. glycines genes, three encoded a putative signal peptide. Similarly, four of those conserved in phytomicrobes, four of those conserved in soilmi- crobes and four in other microbes (Additional file 7) con- tained signal peptide sequences. However, these predicted proteins did not necessarily meet all our other, more strin- gent criteria. E.g., some proteins did have a putative trans- membrane helix or did have less than 30 amino acids after the predicted signal peptide cleavage site (see Methods). Also in these analyses, one needs to remember that a sig- nificant portion of the ESTs lacks a complete 5'-end so that no signal peptide-coding region could possibly be detected. Additional file 7 shows the overlap between H. glycines contigs identified as parasitism genes, secretory protein-encoding or plant/microbe-like. Interestingly, some of those H. glycines genes that did fulfill all our cri- teria for secreted plant-like proteins matched for example plant genes encoding histone deacetylase 2 from Arabi- dopsis (HgAffx.19783.1) or the Meloidogyne-induced giant-cell protein-like protein from potato (HgAffx.13422.1). H. glycines proteins with similarity to microbial gene products that passed our signal peptide selection process matched among others a flavin adenine dinucleotide (FAD)-linked oxidase from Arthrobacter (HgAffx.18477.1), histidine triad nucleotide-binding family protein 1 (HIT1) from Chaetomium globosum (HgAffx.11878.1) or a hypothetical protein from Gib- berella zeae (HgAffx.17226.1). To our knowledge, none of these genes encode proteins with signal peptides in the respective plant or microbe species to which the H. gly- cines sequences matched. We further grouped all identified plant- and microbe-like H. glycines genes represented by their respective probesets into distinct expression clusters (Additional files 8, 9, 10, 11). In order to identify specific shifts in gene expression, we performed pairwise comparisons of consecutive life stages of H. glycines for genes that were conserved in plants, phytopathogens/phytosymbionts, soilmicrobes and other microbes regardless of whether we could find a signal peptide-encoding sequence or not. The results of these analyses are summarized in Table 1. It is evident that the vast majority of these probesets was differentially expressed when eggs were compared with infective J2 and infective J2 with parasitic J2 and that up- and downregu- lated probesets were represented in about equal propor- tions in these two stage-wise comparisons. This means that these plant and microbe-like H. glycines genes are strongly regulated at the onset of parasitism. H. glycines encodes signal peptide-bearing gene products with similarity to plant histone deacetylase Histone modifications are an important regulatory ele- ment in gene expression [42]. We found here a H. glycines histone deacetylase-2 (HDA2) probeset (HgAffx.19783.1.S1_at) with an Arabidopsis HDA2 (AAM34784.1) as best BLASTX match for its consensus sequence, rather than a homologous gene in nematode species, including the fully sequenced C. elegans or C. briggsae genomes. Interestingly, this H. glycines HDA2 has a putative signal peptide and lacks a transmembrane helix, which makes it a putatively secreted protein. To our knowledge, there are no prior reports of signal-peptide- containing histone deacetylases. Cyst nematodes like H. glycines are known to possess plant-like proteins with sig- nal peptides that are normally not secreted in plants or other organisms, e.g., SKP1 or chorismate mutases [7]. Hence, a secreted Arabidopsis-like histone deacetylase would be an exciting new example of how cyst nematodes could modify gene expression of plants by altering the epigenome of their host cells. To analyze this H. glycines gene product further, we con- structed a multiple alignment and a phylogenetic tree for the translated Affymetrix consensus sequence of this probeset (HgAffx.19783.1.S1_at) and HDA2 homologs of selected plant and nematode species using CLUSTAL W [43]. It can be clearly seen that the H. glycines HDA2 is not only more similar to Arabidopsis HDA2, but to other plant HDA2s as well, and that the respective homologs of other nematode species are very different from plant HDA2s (Figure 4, Additional file 12). H. glycines probes cross-hybridize with Phytophthora sojae or Glycine max probesets In addition to the 7,530 probesets corresponding to H. glycines mRNAs, the Affymetrix Soybean Genome Array GeneChip contains 37,500 probesets for soybean (Glycine max) mRNAs and 15,800 probesets for mRNAs of Phytoph- thora sojae, an oomycete pathogen of soybean plants. We Page 6 of 17 (page number not for citation purposes)