No 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 are lipo-chito-oligosaccharides secreted by rhizobia that initiate many responses in the root hairs of the legume hosts, culminating in deformed hairs. The heterotrimeric G-protein agonists mastoparan, Mas7, melittin, compound 48/80 and cholera toxin provoke root hair deformation, whereas the heterotrimeric G-protein antagonist pertussis toxin inhibits mastoparan and Nod factor NodNGR[S]-(from Rhizobium sp. NGR234) induced root hair deformation. Another heterotrimeric G-protein antagonist, isotetrandrine, only inhibited root hair deformation provoked by mastoparan and melittin. These results support the notion that G-proteins are implicated in Nod factor signalling. To study the role of G-proteins at a biochemical level, we examined the GTP-binding profiles of root microsomal membrane fractions isolated from the nodulation competent zone of Vigna unguiculata (L.) Walp. GTP competitively bound to the microsomal membrane fractions labelled with [ 35 S]GTPcS, yielding a two-site displacement curve with displacement constants (K i ) of 0.58 lM and 0.16 mM. Competition with either ATP or GDP revealed a one-site displacement curve with K i of 4.4 and 29 lM, respectively, whereas ADP and UTP were ineffective competitors. The GTP-binding profiles of microsomal membrane fractions isolated from roots pretreated with either NodNGR[S] or the four-sugar, N-N'-N''-N'''-tetracetylchitotetraose (TACT) backbone of Nod factors were significantly altered compared with control microsomal fractions. To identify candidate proteins, membrane proteins were separated by SDS-PAGE and electrotransferred to nitrocellulose. GTP overlay experiments revealed that membrane fractions isolated from roots pretreated with NodNGR[S] or TACT contained two proteins (28 kDa and 25 kDa) with a higher affinity for GTPcS than control membrane fractions. Western analysis demonstrated that membranes from the pretreated roots contained more of another protein ($55 kDa) recognised by Ga common antisera. These results provide pharmacological and biochemical evidence supporting the contention that G-proteins are involved in Nod factor signalling and, importantly, implicate monomeric G-proteins in this process.

No abstract