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Signal Peptide-Selection of cDNA Cloned Directly from the Esophageal Gland Cells of the Soybean Cyst Nematode Heterodera glycines

¹Department of Plant Pathology, North Carolina State University, Box 7616, Raleigh 27695-7616, U.S.A.; ²Department of Plant Pathology, University of Georgia, Athens 30602-7274, U.S.A.; ³Department of Plant Pathology, Iowa State University, 351 Bessey Hall, Ames 50011, U.S.A.

Secretions from the esophageal gland cells of plantparasitic nematodes play critical roles in the nematodeparasitic cycle. A novel method to isolate cDNA encoding putative nematode secretory proteins was developed that utilizes mRNA for reverse transcription-polymerase chain reaction derived from microaspiration of the esophageal gland cell contents of parasitic stages of the soybean cyst nematode Heterodera glycines. The resulting H. glycines gland cell cDNA was cloned into the pRK18 vector, and plasmid DNA was transformed into a mutated yeast host for specific selection of cDNA inserts that encode proteins with functional signal peptides. Of the 223 cDNA clones recovered from selection in yeast, 97% of the clones encoded a predicted signal peptide. Fourteen unique cDNA clones hybridized to genomic DNA of H. glycines on Southern blots and, among them, nine cDNA clones encoded putative extracellular proteins, as predicted by PSORT II computer analysis. Four cDNA clones hybridized to transcripts within the dorsal esophageal gland cell of parasitic stages of H. glycines, and in situ hybridization within H. glycines was not detected for eight cDNA clones. The protocol provides a direct means to isolate potential plant-parasitic nematode esophageal gland secretory protein genes.

Additional keywords: expressed sequence tags, functional genomics, host-parasite interactions, nematode feeding sites, parasitism genes.