Signal peptide-selection of cDNA cloned directly from the esophageal glandcells of the soybean cyst nematode Heterodera glycines

Secretions from the esophageal gland cells of plant-parasitic nematodes pla y critical roles in the nematode-parasitic Cycle. A novel method to isolate cDNA encoding putative nematode secretary proteins was developed that util izes:mRNA for reverse transcription-polymerase chain reaction derived from microaspiration of the esophageal gland cell contents of parasitic stages o f the soybean cyst nematode Heterodera glycines, The resulting H, glycines gland cell cDNA was cloned into the pRK18 vector, and plasmid DNA was trans formed into a mutated yeast host for specific selection of cDNA inserts tha t encode proteins with functional signal peptides, Of the 223 cDNA clones r ecovered from selection in yeast, 97% of the clones encoded-a predicted sig nal peptide. Fourteen unique cDNA clones hybridized to genomic DNA of H, gl ycines on Southern blots and, among them, nine cDNA clones encoded putative extracellular proteins, as predicted by PSORT Il computer analysis. Four c DNA clones hybridized to transcripts within the dorsal esophageal gland cel l of parasitic stages of H. glycines, and in situ hybridization within H. g lycines was not detected for eight cDNA clones. The protocol provides a dir ect means to isolate potential plant-parasitic nematode esophageal gland se cretory protein genes.