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Planta ; 216(4): 674-85, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12569410

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 Rhizobiumsp. 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]GTPgammaS, yielding a two-site displacement curve with displacement constants ( K(i)) of 0.58 micro M and 0.16 mM. Competition with either ATP or GDP revealed a one-site displacement curve with K(i) of 4.4 and 29 micro M, 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 GTPgammaS than control membrane fractions. Western analysis demonstrated that membranes from the pretreated roots contained more of another protein (~55 kDa) recognised by Galpha(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.


Subject(s)
Fabaceae/metabolism , Heterotrimeric GTP-Binding Proteins/metabolism , Lipopolysaccharides/pharmacology , Monomeric GTP-Binding Proteins/metabolism , Rhizobium/growth & development , Binding, Competitive/drug effects , Cholera Toxin/pharmacology , Fabaceae/microbiology , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Heterotrimeric GTP-Binding Proteins/agonists , Heterotrimeric GTP-Binding Proteins/antagonists & inhibitors , Intercellular Signaling Peptides and Proteins , Lipopolysaccharides/metabolism , Melitten/pharmacology , Monomeric GTP-Binding Proteins/drug effects , Oligosaccharides/metabolism , Oligosaccharides/pharmacology , Peptides , Pertussis Toxin/pharmacology , Plant Roots/drug effects , Plant Roots/growth & development , Plant Roots/metabolism , Rhizobium/chemistry , Signal Transduction/drug effects , Sulfur Radioisotopes , Symbiosis , Wasp Venoms/pharmacology
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