Nod factors activate both heterotrimeric and monomeric G-proteins in Vigna unguiculata (L.) Walp.
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.
Kelly
Marilyn N
MN
Department of Pharmaceutical Biology and Pharmacology, Victorian College of Pharmacy, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia.
Irving
Helen R
HR
D001667:Binding, Competitive / Q000187:drug effects; D002772:Cholera Toxin / Q000494:pharmacology; D007887:Fabaceae / Q000378:metabolism* / Q000382:microbiology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins; D008070:Lipopolysaccharides / Q000378:metabolism / Q000494:pharmacology*; D008555:Melitten / Q000494:pharmacology; D020559:Monomeric GTP-Binding Proteins / Q000187:drug effects / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism / Q000494:pharmacology; D010455:Peptides; D037342:Pertussis Toxin / Q000494:pharmacology; D018517:Plant Roots / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism; D012231:Rhizobium / Q000737:chemistry / Q000254:growth & development*; D015398:Signal Transduction / Q000187:drug effects; D013462:Sulfur Radioisotopes; D013559:Symbiosis; D014862:Wasp Venoms / Q000494:pharmacology
D036341:Intercellular Signaling Peptides and Proteins; D008070:Lipopolysaccharides; C092736:Nod factor, Rhizobium leguminosarum; D009844:Oligosaccharides; D010455:Peptides; D013462:Sulfur Radioisotopes; D014862:Wasp Venoms; D008555:Melitten; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C056529:N-acetylchitohexaose; C037500:mastoparan; D002772:Cholera Toxin; D037342:Pertussis Toxin; D020962:Heterotrimeric GTP-Binding Proteins; D020559:Monomeric GTP-Binding Proteins