Poster: Plant Pathogen/Symbiont Interactions

Abs # 768: The Medicago Enod8 nodulin gene encodes an esterase

Presenter:	Coque, Laurent F, Laurent@unt.edu
Authors	Coque, Laurent F (A) (B)  Pringle, David  (B)   Hu, Xuejun  (B)   Dickstein, Rebecca  (A) (B)
Affiliations:	(A): Department of Biological Sciences, University of North Texas (B): Department of Bioscience and Biotechnology, Drexel University
Web Site:	http://www.biol.unt.edu/~beccad/lab.htm

Enod8 is a nodule-specific gene from Medicago. Enod8 is expressed in empty nodules and expression persists when nitrate is added to nitrogen fixing plants, suggesting that Enod8 has a role in nodule organogenesis. Sequence comparisons show Enod8 to be a member of the GDSL esterase gene family. In alfalfa, Western blot analysis using anti-Enod8 antisera reveals the presence of 5 Enod8 proteins. These were purified from alfalfa nodule extracts using ammonium sulfate precipitation, concanavalin A chromatography, and ion-exchange chromatography. The purified proteins demonstrated esterase activity using p-nitrophenyl acetate and á-naphthyl acetate as substrates. Kinetic studies revealed that Enod8 esterase displays Michaelis-Menten kinetics. To eliminate the possibility that an undetectable contaminant was giving rise to the esterase activity, native gels were run. The esterase activity comigrated with the Enod8 protein, detected with anti-Enod8 antisera. In order to more fully characterize the Enod8 esterase activity, we tried several expression systems with E.coli and Pichia pastoris. Dectectable expression only occurred with E.coli recombinant systems. Enod8 could be only expressed in a soluble form when in fusion with the Maltose-Binding-Protein as an N-terminal tag, replacing the Enod8 putative secretion signal. We plan to perform SDS-PAGE esterase assays with various naphthyl substrates for testing the esterase activity of the recombinant Enod8 and nodule protein extracts. Supported by USDA NRICGP #96-35305-3413, #99-35305-8574, and #99-35305-08693.