Nod factors (NFs) are rhizobial lipo-chitooligosaccharide signals that trigger root nodule development in legumes. Modifications of NF structures influence their biological activity and affect their degradation by plant chitinases. Nodulation of certain pea cultivars by Rhizobium leguminosarum bv. viciae requires modification of NFs at the reducing end by either an O-acetyl or a fucosyl group. Fucosylated NFs were produced by an in vitro reaction with NodZ fucosyltransferase and purified. Their biological activity on pea was tested by measuring their capacity to stimulate the activity of a hydrolase that cleaves NFs. Nonmodified and fucosylated NFs displayed this activity at nano- to picomolar concentrations, while a sulfated NF from Sinorhizobium meliloti was inactive. In an additional series of experiments, the stability of non-modified and fucosylated NFs in the presence of purified tobacco chitinases was compared. The presence of the fucosyl group affected the degradation rates and the accessibility of specific cleavage sites on the chitooligosaccharide backbone. These results suggest that the fucosyl group in NFs also weakens the interaction of NFs with certain chitinases or chitinase-related proteins in pea roots.

Establishment of symbiosis between legumes and rhizobia requires bacterial Nod factors (NFs). The concentration of these lipochitooligosaccharides in the rhizosphere is influenced by plant enzymes. NFs induce on pea (Pisum sativum) a particular extracellular NF hydrolase that releases lipodisaccharides from NFs from Sinorhizobium meliloti. Here, we investigated the ability of non-nodulating pea mutants to respond to NodRlv factors (NFs from Rhizobium leguminosarum bv viciae) with enhanced NF hydrolase activity. Mutants defective in the symbiotic genes sym10, sym8, sym19, and sym9/sym30 did not exhibit any stimulation of the NF hydrolase, indicating that the enzyme is induced via an NF signal transduction pathway that includes calcium spiking (transient increases in intracellular Ca 21 levels). Interestingly, the NF hydrolase activity in these sym mutants was even lower than in wild-type peas, which were not pretreated with NodRlv factors. Activation of the NF hydrolase in wild-type plants was a specific response to NodRlv factors. The induction of the NF hydrolase was blocked by a-amanitin, cycloheximide, tunicamycin, EGTA, U73122, and calyculin A. Inhibitory effects, albeit weaker, were also found for brefeldin A, BHQ and ethephon. In addition to this NF hydrolase, NFs and stress-related signals (ethylene and salicylic acid) stimulated a pea chitinase that released lipotrisaccharides from pentameric NFs from S. meliloti. NodRlv factors failed to stimulate the chitinase in mutants defective in the sym10 and sym8 genes, whereas other mutants (e.g. mutated in the sym19 gene) retained their ability to increase the chitinase activity. These findings indicate that calcium spiking is not implicated in stimulation of the chitinase. We suggest that downstream of Sym8, a stress-related signal transduction pathway branches off from the NF signal transduction pathway.Establishment of symbiosis between legumes and nitrogen-fixing rhizobia results in the formation of a new plant organ, the root nodule. Rhizobia enter the host plant usually through infection of root hairs. Bacteria within root hairs induce the formation of an infection thread that grows toward the dividing cortical cells. In a later symbiotic stage, rhizobia are released from branched infection threads into the developing nodule tissue and differentiate into nitrogen-fixing bacteroids. Nodule formation is controlled by perception of rhizobial nodulation signals: the Nod factors (NFs). Flavonoids from the host plant, in conjugation with the rhizobial activator protein NodD, induce rhizobial nodulation genes (nod, nol, and noe) that are required for NF synthesis. NFs are modified lipochitooligosaccharides, i.e. chitin oligomers linked with a fatty acid replacing the N-acetyl group on their nonreducing end (Perret et al., 2000;Ovtsyna and Staehelin, 2003). On host plants, NFs induce various responses at picomolar to nanomolar concentrations. These rapid changes include root hair curling (Hac; e.g. Heidstra et al., 1994), depolarization of plasma membranes (Ehrha...

We have analyzed the nucleotide sequences of the nodX genes from two strains of Rhizobium leguminosarum bv. viciae able to nodulate Afghan peas (strains A1 and Himalaya) and from two strains of R. leguminosarum bv. trifolii (ANU843 and CSF). The nodX genes of strains A1 and ANU843 were shown to be functional for the induction of nodules on Afghan peas. To analyze the cause of phenotypic differences of strain A1 and strain TOM we have studied the composition of the lipochitin-oligosaccharides (LCOs) produced by strain A1 after induction by the flavonoid naringenin or various pea root exudates. The structural analysis of the LCOs by mass spectrometry revealed that strain A1 synthesizes a family of at least 23 different LCOs. The use of exudates instead of naringenin resulted only in quantitative differences in the ratios of various LCOs produced.

Rhizobium leguminosarum bv. viciae strains producing lipo-chitin oligosaccharides (LCOs) that are O-acetylated at the reducing terminus are required for nodulation of wild pea cultivars originating from Afghanistan that possess the recessive sym2A allele. The O-acetylation of the reducing sugar of LCOs is mediated by the bacterial nodX gene, which presumably encodes an acetyltransferase. We found that for nodulation on Afghan pea cultivars and sym2A introgression lines the nodX gene can be functionally replaced by the nodZ gene of Bradyrhizobium japonicum, which encodes a fucosyltransferase that fucosylates the reducing terminus of LCOs. The structure of the nodules, which were induced with normal frequency, was typical for effective pea nodules, and they fixed nitrogen with the same efficiency as nodules induced by nodX-carrying strains.

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