The nodulation-signaling protein NodO from Rhizobium leguminosarum biovar viciae forms ion channels in membranes.

Abstract

The secreted nodulation-signaling protein NodO was purified from the supernatant of cultures of Rhizobium kguminosarum blovar viciae. The native protein has a Mr of 467,000, suggestng that it exists as a dimer since the DNA sequence predicts a Mr of 30,002. Pure NodO protein had no protease, pectinase, or cellulase activity, and no binding was observed to lipooligosaccharide modulation factors. Although NodO R. leguminosarum biovar viciae has a nodulation gene (nodO) that encodes a secreted protein (9) that is involved in pea and vetch nodulation. Although mutation of nodO does not block nodulation, it is evident that in the absence of the nodE gene, nodO is required for the nodulation of peas or vetch (10). The nodE gene product is a key determinant of host specificity and introduces functional specificity into the Nod factor by causing the production of the C18:4 acyl group MATERIALS AND METHODSBacterial Strains and Growth. The exopolysaccharidedeficient strains of R. leguminosarum, A168 and A165, with and without the symbiotic plasmid pRL1JI, have been described (14) as has been pIJ1815, which carries the cloned nodO gene (14). Typically, 25 ml of an overnight culture (OD6w00 0.4) in TY medium (15) containing streptomycin (20 Mg/ml) was inoculated into each of five 2-liter flasks containing 600 ml ofthe same medium containing 1 ,M hesperitin to induce nodO expression. The cultures were shaken at 280C for 60 h and harvested at an OD600 of 0.9-1.0, when the pH of the growth medium remained below pH 7.8. Normally, strain A168/pIJ1815 was used for purification of NodO, but for measurements of possible interactions between NodO and nodulation factors, NodO was purified using A165/ pIJ1815. NodO isolated from either strain induced channels with similar characteristics.Purification of NodO. All procedures were carried out at 40C. Cells were harvested by centrifugation (9000 rpm in a Sorvall GS3 rotor for 45 min), and the culture supernatant was adjusted to pH 5.0 using glacial acetic acid and loaded at 2 ml/min onto a column (16 mm diameter) containing a 10-ml bed volume of DEAE-Sepharose Fast Flow (Pharmacia) that had been equilibrated with 0.1 M sodium acetate buffer at pH 5.0. Proteins were eluted with 0.25 M NaCl in the acetate buffer and then precipitated by adding (NH4)2SO4 to 701% saturation. The precipitate was resuspended in 2.5 ml of 20 mM sodium acetate buffer at pH 5.0 and desalted on a PD-10 column (Pharmacia); the protein was eluted with 20 mM sodium acetate buffer at pH 5.0. Aliquots of 1.0 ml were then loaded onto a Pharmacia FPLC Mono Q column that had been equilibrated with 20 mM sodium acetate buffer at pH 5.0, and proteins were eluted at a flow rate of 1.0 ml/min using a linear gradient of0-1.0 M NaH2PO4 in 20 mM sodium acetate at pH 5.0. Peak samples (fractions 23-28, Fig. 1) were 9990 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate t...

Identification of genes in Rhizobium leguminosarum bv. trifolii whose products are homologues to a family of ATP-binding proteins

“…viciae nodulation protein NodO has been described as partially homologous to haemolysin and is secreted from E. coli into the growth medium by HlyBD/TolC and PrtDEF protein secretion systems. This is strong evidence that an ABC exporter is required for NodO secretion (Scheu et al, 1992;Sutton et al, 1994). The Rhizobium genes encoding the proteins required for NodO secretion are unlinked to the n o d 0 gene which is located on the symbiotic plasmid.…”

Nod factor‐induced host responses and mechanisms of Nod factor perception

“…When added to lipid bilayers, it forms channels that allow the movement of monovalent cations. Therefore it has been suggested that NodO integrates into the plant plasmamembrane and amplifles a Nod factor-induced signal transduction pathway (Economou et al, 1994;Sutton et aL, 1994). We postulate that NodO does not compensate for a Nod factor structural deficiency by affecting Nod factor structure but either by amplifying the signal transduction cascade leading to infection or by partly inhibiting the negative control of the particular Sym2 receptor (Fig.…”

“…NodO is a secreted protein that is not involved in Nod factor biosynthesis (Sutton, Lea & Downie, 1994). When added to lipid bilayers, it forms channels that allow the movement of monovalent cations.…”

Signal Transduction in Rhizobium-Induced Nodule Formation

“…A phytohormone effect of ENOD40 is detected in protoplasts of the nonlegume tobacco. Tobacco protoplasts that express a soybean ENOD40 gene divide efficiently at high auxin concentrations, whereas in the control protoplasts these levels of auxin suppress the ability to divide ( Van de Sande et al, 1996). Since detailed cytological studies have shown that the expression of ENOD40 in the pericycle precedes the first cortical cell divisions, it is hypothesized that ENOD40 may encode a regulator of cell division whose induction upon Xkizobium infection starts the process of root cortical cell division leading to nodule formation.…”

“…These peptides appear to be plant growth regulators. In tobacco protoplasts END040 peptides change the response to auxin at concentrations as low as 10K1' to 10-l6 M (Van de Sande et al, 1996).…”