Doctoral Dissertations

Date of Award

12-1999

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

Gary Stacey

Committee Members

Jeff Becker, Neil Quigley, Beth C. Mullin

Abstract

The signal exchange mechanism mediating the onset of nodulation is a complex process involving the synthesis and perception of host-specific molecules bearing structurally distinct modifications. Central to the establishment of a symbiotic association between legumes and their rhizobial symbionts is the perception of rhizobial lipo-chitin Nod signals by the host plant. These signal molecules consist of a β-l,4-linked N-acetylglucosamine backbone of 3 to 5 residues modified in a species-specific manner at both the reducing and non-reducing termini.

This study focused on the Bradyrhizobium-soybean symbiosis. In an effort to define both the structural elements required for Nod signal perception, as well as to identify putative Nod signal binding proteins, a two-fold approach was used. Previous work (Minami et al, 1996a) showed that an unsubstituted chitin oligomer could induce the expression of the early nodulin ENOD40. Therefore, a biochemical approach was used to identify and characterize a high-affinity binding site for N-acetylchitooligosaccharides localized in the plasma membrane of soybean. A 85 kDa binding protein was localized to the plasma membrane of both suspension-cultured soybean cells and root tissue from 5 day old seedlings. Inhibition analysis suggested a binding preference for larger (i.e., d.p.=8) N-acetylated chitin molecules, with a IC50 value of approximately 50 nM. N-acetyl-glucosamine and chitobiose showed no inhibitory effects at concentrations as high as 250 µM. Chitooligosaccharide specificity for induction of medium alkalinization and the generation of reactive oxygen in suspension-cultured cells paralleled the binding activity of this protein. Taken together, the presence of the chitin binding protein in the plasma membrane, and the specificity and induction of a biological response upon ligand binding, suggest a role for the protein as an initial response mechanism for chitin perception in soybean, and not as a host-specific binding site for lipo-chitin Nod signals.

Recently, Etzler et al. (1999) reported the identification of a Nod signal binding protein present in the roots of the legume Dolichos biflorus. Therefore, we sought to identify orthologs of this protein in soybean. Two soybean cDNAs, termed GS50 and GS52, predicted to encode apyrase proteins of 50 kDa and 52 kDa, respectively, were isolated. Northern blot analysis indicate that GS52 mRNA is expressed in roots, hypocotyls, and flowers, whereas GS50 mRNA was expressed weakly in roots, yet abundantly in stems and flowers. Western blot analysis using antigen-purified antibodies to both GS50 and GS52 showed similar patterns of tissue localization. Furthermore, western blot analysis of sucrose gradient-fi-actionated membranes fi-om root tissue revealed a differential cellular localization, with GS50 localizing to the Golgi, and GS52 to the plasma membrane.

GS50 was expressed constitutively in roots at a relatively low level under all conditions tested. However, GS52 mRNA levels in roots were significantly increased within six hours after inoculation with Bradyrhizobium japonicum. Therefore, GS52 can be classified as an early nodulin. Additionally, mRNA expression data also showed that GS52 was repressed in the presence of 20 mM nitrate, whereas expression of GS50 was not affected.

The differential expression pattern of GS50 and GS52 suggests that they likely have distinct cellular functions. The rapid induction of GS52 expression upon bacterial inoculation and the repression by nitrate suggest that this protein may have a function in the nodulation process.

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