BMC Genomics 2009, 10:58 http://www.biomedcentral.com/1471-2164/10/58 of genes that code for secretory proteins [13,33,34], and none combined expression analyses of all genes with detailed sequence mining approaches. summary, this project represents a functional genomic analysis of available cyst nematode data targeting phy- tonematode parasitism. We previously reported the expression profiling of all 7,530 H. glycines probesets on the Affymetrix Soybean Genome Array GeneChip representing up to 6,860 unique cyst nematode genes throughout the major life stages from embryonated eggs to the adult female stage [35]. The same Affymetrix GeneChip was used recently to study the expression patterns of a small subset of parasitism genes in a few juvenile life stages of H. glycines as well as of soy- bean genes during the infection process [36] and to survey the development of feeding cells in soybean plants [37]. We generated the necessary cDNA libraries and ESTs that allowed the design of these 7,530 H. glycines probesets from stage-specific soybean cyst nematode libraries including not only eggs and infective J2, but also the hard- to-isolate parasitic stages (J3, J4, adult females) that only form inside the soybean root. This latter aspect is particu- larly important for the research reported here because sed- entary endoparasitic nematodes like the cyst nematodes do not produce and/or secrete the vast number of parasit- ism proteins until they have invaded their host plants. Consequently, previous approaches that are based on pre- parasitic juvenile worms and that do not include the par- asitic stages are liable to miss the truly interesting proteins that the nematode only releases when in contact with its host cell deep inside the plant root. In this current paper, we have analyzed our previously deposited microarray data set [35] with a distinct focus on parasitism and host- parasite interactions. The primary goal of the research pre- sented here was to use critical high throughput criteria dis- cernable from gene sequence and expression characteristics to identify additional proteins that can rea- sonably be expected to function during parasitism. This pre-selection is an enabling discovery for further, more in- depth functional work to unravel cyst nematode parasit- ism. To this end, a rigorous examination of all 7,530 H. glycines probesets represented on the Affymetrix Soybean Genome Array GeneChip allowed us to identify a pool of 633 H. glycines genes that encode putative secretory pro- teins and of 156 H. glycines genes that are conserved in microbes or plants but that have significantly less similar- ity to sequences from the non-parasitic nematodes Caenorhabditis elegans and Caenorhabditis briggsae. We pro- vide here for the first time an analysis of the expression profiles of these genes, as well as of all previously described parasitism genes, in all major life stages of H. glycines excluding adult males. Together, the gene pool identified here represents a promising starting point to search for previously uncharacterized genes with func- tions in parasitism and for H. glycines genes that poten- tially were acquired by horizontal gene transfer, a mechanism by which parasitic nematodes are believed to have obtained a subset of parasitism genes [10,38]. In Results Known H. glycines parasitism genes show two different developmental expression patterns During the generation of the Affymetrix Soybean Genome Array GeneChip the previously identified 66 H. glycines parasitism genes [10,12,16,21,22] were grouped by the manufacturer into 46 contigs, which are represented by 62 probesets on the GeneChip (some contigs have multiple probesets) (Additional file 1). Using our previously described Affymetrix microarray data set, which encom- passes all life stages of H. glycines except adult males [35], we determined the developmental expression patterns of all 62 probesets representing these 46 contigs. All of the parasitism gene probesets displayed significant expression changes during the H. glycines life cycle at a false discovery rate (FDR) of 5%. We performed statistical analyses as described in Methods for all parasitism gene probesets, which identified two clusters based on gene expression patterns (Figure 1). H. glycines parasitism genes either reached a maximum mRNA abundance in infective J2, remained steady until parasitic J2 and dropped off steeply thereafter (Cluster 1) or they reached a maximum in their mRNA expression levels in parasitic J2 and then fell stead- ily after the J3 stage (Cluster 2). The gland-specific expres- sion patterns of these genes have been determined in previous studies [16,21,22]. Apart from very few excep- tions, parasitism genes expressed in the subventral glands of the nematode fell into Cluster 1, whereas parasitism genes from the dorsal gland could be grouped into Cluster 1 or Cluster 2 (Additional file 1). The majority of H. gly- cines parasitism genes, particularly those expressed in the dorsal esophageal gland, were novel and did not share similarities with any known sequences. Therefore, the expression profiles obtained here are important tools to infer putative protein functions judging from the time points at which these genes are upregulated. Genes that are important during host invasion and early sedentary phases, which includes the early events of syncytium induction (i.e., infective J2/parasitic J2), should be repre- sented in Cluster 1, because this cluster showed an expres- sion maximum in these two stages (Figure 1). Genes that are relevant during later stages of parasitism like the later stages of syncytial development, syncytium maintenance and feeding, are expected in Cluster 2, whose members were expressed at a relatively high level also in late stages (J3, J4, adult females). However, it was remarkable that such diverse proteins as secreted cellulases and pectate lyases with an obvious role in plant cell wall degradation (i.e., during early phases of parasitism) had a strikingly similar expression pattern to genes encoding secretory proteins that are believed to influence the host cell's phys- iology (e.g., chorismate mutases, ubiquitin extension pro- Page 3 of 17 (page number not for citation purposes)