Evidence for structurally specific negative feedback in the Nod factor signal transduction pathway.

SummaryNod factor is a critical signalling molecule in the establishment of the legume/rhizobial symbiosis. TheNod factor of Sinorhizobium meliloti carries O-sulphate, O-acetate and C 16:2 N-acyl attachments thatdeﬁne its activity and host speciﬁcity. Here we assess the relative importance of these modiﬁcations forthe induction of calcium spiking in Medicago truncatula. We ﬁnd that Nod factor structures lacking theO-sulphate, structures lacking the O-acetate and N-acyl groups, and structures lacking the O-acetatecombined with a C 18:1 N-acyl group all show calcium spiking when applied at high concentrations. Thesecalcium responses are blocked in dmi1 and dmi2 mutants, suggesting that they function through theNod factor signal transduction pathway. The dmi3 mutant, which is proposed to function in the Nodfactor signal transduction pathway downstream of calcium spiking, shows increased sensitivity to Nodfactor. This increased sensitivity is only active with wild-type Nod factor and was not present when theplants were treated with mutant Nod factor structures. We propose that the Nod factor signaltransduction pathway is under negative feedback regulation that is activated at or downstream of DMI3and requires structural components of the Nod factor molecule for activity.Keywords: Nod factor; nodulation; calcium spiking; feedback regulation; signal transduction.IntroductionLegumes have the capacity to form a symbiotic interactionwith rhizobial bacteria. This interaction results in theformation of bacterially infected nodules on the roots ofthe host plant. Bacterially derived Nod factor is a criticalsignalling molecule for the establishment of the legume/rhizobial symbiosis (De´narie´ etal., 1996; Long, 1996;Spaink, 2000). Isolated Nod factor applied to the appropri-ate plant host induces many of the responses elicited bythe bacterial symbiont, including root hair deformation,cortical cell division and gene expression (Downie andWalker, 1999). Nod factors also activate a number of veryearly responses in the plant, notably the induction of ionicﬂuctuations across the plasma membrane and calciumspiking in the cytosol (Cardenas etal., 2000; Ehrhardt etal.,1992; Ehrhardt etal., 1996). Nod factor is necessary butinsufﬁcient for the induction of the infection threadformation involved in the invasion of rhizobia into theplant.Speciﬁcity in the legume/rhizobial symbiosis is encodedin the structure of the Nod factor signal. All Nod factorsconsist of an oligomeric chitin backbone (3–5 N-acetyl-glucosamine residues) with an N-acyl group attached tothe non-reducing terminal sugar (Figure 1a,c). A numberof additional substituents are attached to the chitin back-bone, and these substituents vary widely between Nodfactors generated by different Rhizobium species (De´narie´etal., 1996; Downie and Walker, 1999; Long, 1996). Thespeciﬁcity of the plant/bacterial interaction is deﬁned in anumber of cases by the structure of the N-acyl group andthe substituents attached to the Nod factor molecule. TheNod factor of Sinorhizobium meliloti, the symbiont ofMedicago sativa and Medicago truncatula, carries anO-sulphate group on the reducing terminal sugar and anO-acetyl group and a speciﬁc C