Signal peptide-selection of cDNA cloned directly from the esophageal gland cells of the soybean cyst nematode Heterodera glycines.
Secretions from the esophageal gland cells of plant-parasitic nematodes play critical roles in the nematode-parasitic 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.
Wang
X
X
Department of Plant Pathology, North Carolina State University, Raleigh 27695-7616, USA.
Allen
R
R
Ding
X
X
Goellner
M
M
Maier
T
T
de Boer
J M
JM
Baum
T J
TJ
Hussey
R S
RS
Davis
E L
EL
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D018076:DNA, Complementary; D004947:Esophagus / Q000166:cytology / Q000378:metabolism*; D005786:Gene Expression Regulation; D017403:In Situ Hybridization; D009348:Nematoda / Q000235:genetics / Q000502:physiology*; D021382:Protein Sorting Signals / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013025:Glycine max / Q000469:parasitology*; D013347:Subcellular Fractions / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D021382:Protein Sorting Signals