1.14.2S
MOLECULAR ANALYSIS OF NEMATODE SECRETORY PROTEINS

RS HUSSEY and X DING

Department of Plant Pathology, University of Georgia, Athens, GA 30602-7274, USA

Background and objectives
Secretory proteins synthesized in the esophageal gland cells and secreted through the stylet of plant parasitic nematodes have critical roles in plant-nematode interactions [1]. Secretions injected by Meloidogyne spp. induce profound modifications in root cell phenotype and function in susceptible plants to form permanent feeding sites [2]. These nematodes are among nature's most successful parasites and elicit one of the most elaborate responses in plant tissue of any parasite. An integral part of understanding the mechanism of root-knot nematode parasitism of plants is the molecular characterization of the structures and functions of the secretory proteins. Molecular technologies are being used to identify and characterize nematode esophageal gland secretions that are involved in pathogenesis.

Results and conclusions
RNA fingerprinting was used to identify RNAs that were expressed in parasitic (up to 48 h post-inoculation of soybean) second-stage juveniles of M. incognita, but absent from or reduced in preparasitic second-stage juveniles. Two genes encoding secretory proteins were cloned from a M. incognita second-stage juvenile cDNA library by probing with fragments derived from fingerprinting. The full-length cDNA of one gene encoded a novel cellulose binding protein (CBP) with 203 amino acid residues including a putative N-terminal signal peptide. lmmunolocalization by CBP-raised polyclonal antibody labeled subventral gland cells of both pre- and parasitic second-stage juveniles. Labelling of secretions by the polyclonal antibody indicated the CBP could be secreted through the stylet. The C-terminal sequence of the CBP predicted protein was homologous to a bacterial type cellulose binding domain, whereas the N-terminal sequence did not show significant homology to any proteins in databases. The protein lacked cellulose activity, but did bind to carboxymethyl cellulose. In Northern blots, a 1 kb transcript was detected in both pre- and parasitic second-stage juveniles, but not in adult females. The CDNA of the second gene contained an open reading frame encoding 231 amino acids, with the first 21 amino acids being a putative secretory signal. In Northern blot analysis a 1 kb transcript was detected in both pre- and parasitic J2, but not in adult females. The predicted amino acid sequence had similarity to ASP-2, a secretory protein from the infective third stage of Ancylostoma caninum and venom allergen antigen 5 family of proteins from Hymenoptera. In Southern blot analysis, both genes were detected in M. incognita, and not in Heterodera glycines or Caenorhabditis elegans.

Cloning and characterizing genes encoding secretory proteins will enable the biologically important components of nematode stylet secretions to be identified and their role in plant pathogenesis to be determined.

References
1. Williamson VM, Hussey RS, 1996. Plant Cell 8,1735-1745.
2. Hussey RS, Davis EL, Ray C, 1994. In Advances in Molecular Plant Nematology. Plenum Press, New York, pp. 233-249.