In-situ hybridization to messenger RNA in Heterodera glycines

A method is presented for in-situ hybridization to mRNA in second-stage juv eniles (J2) of the soybean cyst nematode Heterodera glycines. The protocol was developed using a digoxigenin-labeled RNA probe transcribed from cDNA o f a cellulase gene that was known to be expressed in the subventral esophag eal glands of H. glycines. Formaldehyde-fixed J2 were cut into sections wit h a vibrating razor blade to make the inside of the nematodes accessible fo r 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 th e cytoplasm of the subventral gland cells, using this specific antisense pr obe. This insitu hybridization protocol will be useful for the characteriza tion and identification of esophageal gland secretion genes in plant-parasi tic nematodes, among other applications.