Identification of putative parasitism genes expressed in the esophageal gland cells of the soybean cyst nematode Heterodera glycines

Cloning parasitism genes encoding secretory proteins expressed in the esoph ageal gland cells is the key to understanding the molecular basis of nemato de parasitism of plants. Suppression subtractive hybridization (SSH) with t he microaspirated contents from Heterodera glycines esophageal gland cells and intestinal region was used to isolate genes expressed preferentially in the gland cells of parasitic stages. Twenty-three unique cDNA sequences fr om a SSH cDNA library were identified and hybridized to the genomic DNA of H. glycines in Southern blots. Full-length cDNAs of 21 clones were obtained by screening a gland-cell long-distance polymerase chain reaction cDNA lib rary. Deduced proteins of 10 clones were preceded by a signal peptide for s ecretion, and PSORT II computer analysis predicted eight proteins as extrac ellular, one as nuclear, and one as plasmalemma localized. In situ hybridiz ation showed that four of the predicted extracellular clones were expressed specifically in the dorsal gland cell, one in the subventral gland cells, and three in the intestine In H. glycines. The predicted nuclear clone and the plasmalemma-localized clone were expressed in the subventral gland cell s and the dorsal gland cell, respectively. SSH is an efficient method for c loning putative parasitism genes encoding esophageal gland cell secretory p roteins that may have a role in H. glycines parasitism of soybean.