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

Abstract.

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 [³⁵S]GTPγS, yielding a two-site displacement curve with displacement constants (K _i) of 0.58 µM and 0.16 mM. Competition with either ATP or GDP revealed a one-site displacement curve with K _i of 4.4 and 29 µM, 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 GTPγS than control membrane fractions. Western analysis demonstrated that membranes from the pretreated roots contained more of another protein (~55 kDa) recognised by Gα_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.