Nod factors stimulate plasma membrane delimited phospholipase C activity in vitro

Abstract

Nod factors are lipo‐chito‐oligosaccharides secreted by Rhizobium to initiate deformation of root hairs and other changes in host plants. Since Nod factor‐induced changes in intracellular calcium occur in responsive root hairs, we tested if phospholipase C (PLC) activity is stimulated by Nod factors. Plasma membranes were isolated from the nodulation‐competent zone of roots of Vigna unguiculata to assay PLC activity in vitro. Nod factors isolated from Rhizobium sp. NGR234, NodNGR[S] and NodNGR[Ac] significantly increased PLC activity and this increase in activity was inhibited in the presence of the PLC inhibitors, neomycin and U‐73122. The response appears specific as PLC activity was not significantly induced neither by the 4‐sugar, N,N′,N′′,N′′′‐tetracetylchitotetraose (TACT), or the five‐sugar, penta‐N‐acetylchitopentaose (PACT), backbone of Nod factors. The G‐protein activators, GTPγS and the aluminium fluoride complex, had no effect on PLC activity in the presence or absence of NodNGR[S], suggesting that Nod factors act independently of G‐proteins in vitro. However, the combination of oleic acid and TACT mimicked the effect of Nod factors on PLC activity indicating that the presence of the lipid tail may be critical. Also this combination of compounds acted synergistically together to evoke root hair deformation in vivo. Our results indicate that Nod factors can modulate membrane delimited PLC activity and indicate that PLC is likely to be a component of the Nod factor‐signalling pathway.

Nod factors activate both heterotrimeric and monomeric G-proteins in Vigna unguiculata (L.) Walp

“…NGR234 responded similarly to mastoparan, butanol and neomycin as Vicia sativa used in previous studies by den Hartog et al (2001). Pingret et al 1998) to inhibit phospholipase C and we have shown that they reduce NodNGR[S]-induced phospholipase C activity in Vigna unguiculata root plasma membranes (Kelly and Irving 2001). Both neomycin and U-73122 are widely used in animal and plant studies (e.g.…”

“…Red Caloona were obtained from PRIMAC (Murwillumbah, NSW 2484) and grown as previously reported (Kelly and Irving 2001). var.…”

“…Den Hartog et al (2001) showed that mastoparan and Nod factors can activate both phospholipase C and phospholipase D in vivo, thereby further implicating heterotrimeric G-proteins in the Nod factor signalling cascade. Recently, we showed that Nod factors can activate phospholipase C independently of heterotrimeric G-proteins in vitro (Kelly and Irving 2001). These later findings do not preclude heterotrimeric G-protein regulation of phospholipase C or phospholipase D but do raise questions about the role of heterotrimeric G-proteins in the Nod factor-signalling cascade.…”

“…The root sections were ground in extraction buffer (Irving 1998) at 6-7 ml g )1 fresh weight, filtered through four layers of gauze, then centrifuged at 9,000 g for 10 min at 4°C. In some experiments, microsomal fractions were used to prepare plasma membrane vesicles using an aqueous two-phase purification system (5.7% Dextran T500 and 5.7% PEG 3350) as previously described (Kelly and Irving 2001). In some experiments, microsomal fractions were used to prepare plasma membrane vesicles using an aqueous two-phase purification system (5.7% Dextran T500 and 5.7% PEG 3350) as previously described (Kelly and Irving 2001).…”

The non-specific lipid transfer protein N5 of Medicago truncatulais implicated in epidermal stages of rhizobium-host interaction

“…Several pieces of research have demonstrated that the signalling cascade activated by NFs implicates, at an early stage, the intermediation of heterotrimeric G-proteins and small GTPases [51-54], which, in turn, stimulate the functionality of phospholipase C (PLC) and D (PLD) [51,53,55,56]. According to the current model, the products of hydrolysis generated by PLC (inositol trisphosphate and diacyl glycerol) stimulate the activity of ligand-gated calcium pumps causing the increase in the cytosolic Ca 2+ concentration, whereas the PLD product (phosphatidic acid, PA) seems to be required for the onset of a kinase/phosphatase signalling cascade, that eventually leads to the activation of nodulin genes and to the reorganization of the cytoskeleton in preparation for the root hair inward growth [57].…”