IN-SITU HYBRIDIZATION TO MESSENGER RNA IN HETERODERA GLYCINES.
J. M. de Boer1, Y. Yan2,
G. Smant3, E. L. Davis2, and
T. J. Baum1. 1Department of
Plant Pathology, Iowa State University, Ames, IA 50011-1020, 2Department
of Plant Pathology, North Carolina State University, Raleigh,
NC 27695-7616, and 3Nematology, Wageningen Agricultural
University, Binnenhaven 10, 6709 PD Wageningen, The Netherlands
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. The in-situ hybridization protocol presented here will be useful for the characterization and identification of esophageal gland secretion genes in plant-parasitic nematodes, among other applications.