A nod factor binding lectin with apyrase activity from legume roots

Abstract

A lectin isolated from the roots of the legume, Dolichos biflorus, binds to Nod factors produced by rhizobial strains that nodulate this plant and has a deduced amino acid sequence with no significant homology to any lectin reported to date. This lectin also is an enzyme that catalyzes the hydrolysis of phosphoanhydride bonds of nucleoside di- and triphosphates; the enzyme activity is increased in the presence of carbohydrate ligands. This lectin-nucleotide phosphohydrolase (LNP) has a substrate specificity characteristic of the apyrase category of phosphohydrolases, and its sequence contains four motifs characteristic of this category of enzymes. LNP is present on the surface of the root hairs, and treatment of roots with antiserum to LNP inhibits their ability to undergo root hair deformation and to form nodules on exposure to rhizobia. These properties suggest that this protein may play a role in the rhizobium-legume symbiosis and/or in a related carbohydrate recognition event endogenous to the plant.

Figure 1

Inhibition of binding of ¹²⁵I-LNP to chitin. Various concentrations of mono- and oligosaccharides were combined with 109 ng of ¹²⁵I-LNP and 250 μg of chitin in a total volume of 100 μl. B. japonicum USDA110 Nod factor (▪); R. sp. NGR234(NGR_A) Nod factor (▴); R. sp. NGR234(NGR_B) Nod factor (▿); R. meliloti Nod factor (●); N-acetylglucosamine (□); chitin disaccharide (▾); chitin tetrasaccharide (▵); chitin pentasaccharide (♦); chitin hexasaccharide (○). Most points represent single determinations. A sufficient amount of R. meliloti Nod factor was available only for the above assay; all other ligands showed similar results in assays with other chitin preparations.

Figure 2

Deduced amino acid sequence of LNP. Position 1 designates the NH₂ terminus of the native protein. Those regions of the deduced sequence verified by amino acid sequences of portions of the protein are underlined. The hydrophobic putative signal sequence is in italics and a ∗ is above the position of each consensus N-glycosylation site. The four apyrase conserved sequence motifs (25) are shown in boxes.

Figure 3

Effect of carbohydrate ligands on phosphatase activity of LNP isolated from D. biflorus roots. LNP (402 ng/ml) was preincubated for 1 hour in 10 mM MOPS buffer, pH 7.2, containing various concentrations of B. japonicum USDA110 Nod factor (▪), Rhizobium sp. NGR234(NGR_A) Nod factor (▴), Rhizobium sp. NGR234(NGR_B) Nod factor (▾), R. meliloti Nod factor (●), or cis-vaccenic acid (♦) and then assayed for phosphatase activity by using a final concentration of 3 mM Mg-ADP. Points are averages of duplicate determinations.

Figure 4

Localization of LNP on D. biflorus roots. Immunofluorescence confocal microscopy of fixed whole mounts of the nodulation zone of 7-day-old D. biflorus roots treated with antiserum prepared against recombinant LNP (A) or preimmunization serum (B). Each image is a three-dimensional composite of 26–29 optical sections. (Bars = 100 μm.)

Figure 5

Inhibition of root hair deformation by treatment of roots with anti-LNP-serum. A comparison of root hairs 62 hours after inoculation of 3-day-old D. biflorus roots with the symbiont, Bradyrhizobium sp. 24A10. Before exposure to rhizobia, the roots of the plants were incubated for 1 hour in 1:100 dilutions of anti-LNP-serum (A) or preimmunization serum (B). Whole mounts of the roots were examined by phase-contrast microscopy. (Bar = 50 μm.)