In-situ Hybridization to Messenger RNA in Heterodera glycines

A method is presented for in-situ hybridization to mRNA in second-stage juveniles (J2) of the soybean cyst nematode Heterodera glycines. The protocol was developed using a digoxigenin-labeled RNA probe transcribed from cDNA of a cellulase gene that was known to be expressed in the subventral esophageal glands of H. glycines. Formaldehyde-fixed J2 were cut into sections with a vibrating razor blade to make the inside of the nematodes accessible for probing. These nematode fragments then were hybridized in suspension with riboprobe, and labeled with an alkaline phosphatase-conjugated antibody to digoxigenin. Staining with nitroblue tetrazolium and bromo-chloro-indolyl phosphate revealed a highly specific hybridization signal to mRNA within the cytoplasm of the subventral gland cells, using this specific antisense probe. This in-situ hybridization protocol will be useful for the characterization and identification of esophageal gland secretion genes in plant-parasitic nematodes, among other applications.