Interaction Between Leguminous Plants and Rhizobia Mediated by Nod Factors
	Legume plants form nitrogen-fixing nodules in the symbiosis with soil bacteria of the genera Rhizobium, Bradyrhizobium, Azorhizobium, Mesorhizobium and Sinorhizobium. Interactions of rhizobium bacteria with legume plants are controlled by strict host specificity. For instance, S. meliloti forms nodules on alfalfa and B. japonicum forms nodules on soybean. The host specificity is determined by at least two steps of the mutual signal exchange between the plants and microsymbionts (Fig. 1). First, bacterial nodulation (nod) genes are activated in response to plant-secreted signal molecules such as flavonoids, resulting in biosynthesis and secretion of lipo-chitooligosaccharides (LCOs) by rhizobium bacteria. In the second step, LCOs elicit nodule formation on the host plant roots and trigger the infection process. LCOs, which induce the formation of the root nodules on the host plants, are termed Nod factors.
Fig. 1. Interaction between rhizobia and their host plants The host specificity is determined by at least two steps in the mutual signal exchange between the plants and microsymbionts. First, bacterial nodulation (nod) genes are activated in response to plant-secreted signal molecules such as flavonoids, resulting in biosynthesis and secretion of Nod factors by rhizobium bacteria. In the second step, Nod factors elicit nodule formation on the host plant roots and trigger the infection process.
	Nod factors act as signal molecules to initiate the nodule formation process programmed in the host plant as well as to trigger the infection process. Nod factors purified from rhizobium cultures or chemically synthesized factors are able to induce, at concentrations of 10-9〜10-12M, root hair deformation and/or curling, preinfection-thread formation, and cortical cell division on the host legume plants. In plants such as alfalfa and wild soybean (Glycine soja), genuine nodule structures are shown to be induced by the application of Nod factors alone without bacteria. Nod-factors consist of a backbone of 3〜6 mer of beta-1,4 linked N-acetyl-D-glucosamine (Fig. 2). Non-reducing terminal sugar residues are modified by the N-acyl (fatty acid) moiety instead of the N-acetyl group at the C-2 position. The number of carbons and double bonds in the fatty acids varies from 16 to 20 and 0 to 4, respectively. The backbone of Nod factors is synthesized by bacterial NodA, NodB and NodC. NodC (chitine synthase) synthesizes a backbone of acetylglucosamine oligomer. NodB (N-deacetylase) eliminates an acetyl residue from the non-reducing end of the chitin oligomer. Then NodA (acyl transferase) adds an acyl moiety to the non-reducing terminal sugar. Genes encoding these enzymes responsible for forming lipochitin oligomer backbones nodA, B, and C are conserved through all rhizobium species, as is nodD, a regulatory gene for other nod genes. These genes are termed "common" nod genes.
Rhizobia The host plants R1 R2 R3 R4 R5 n S.melioti M.sativa(alfalfa) Ｈ C16:2 Ac(O-6) Sulfate H 1,2,3 M.truncatula C16:3 H R.leg.bv viciae Pisum(pea) Ｈ C18:1 Ac(O-6) H H 2,3 Vicia(vetch) C18:4 Ac(O-6) R.leg.bv trifolii Trifolium(clover) Ｈ C18:2 Ac(O-6) H H 2,3 C18:3 M.loti Lotus Me C18:1 Cb AcMeFuc H 3 C18:0 R.fredii G.max(soybean) Ｈ C18:1 H MeFuc H 1,2,3 G.soja Fuc B.japonicum G.max H C18:1 Ac(O-6) MeFuc H 3 C16:0 H C16:1 B.elkanii G.max H C18:1 Ac(O-6) MeFuc H 2,3 Me H Fuc Gro Cb Me:Methyl, Ac:Acetyl, Fuc:Fucosyl, Cb:Carbamyl, MeFuc:2-O-Methylfucosyl, Gro:Glycerol Fig. 2. The structure of Nod factors (reference 2 , modified) Nod factors are named, for example, like NodBj-V(C18:1, MeFuc). Bj represents B. japonicum, V shows the number of N-acetylglucosamine residues in a chitin backbone. The length of the acyl chain and degree of unsaturation, and substitutions on the reducing terminal sugar residue are indicated in parentheses.
	N-acetylglucosamine at the reducing ends of the chitin oligomer is modified by species-specific molecules such as sulfate, acetyl group, fucose and arabinose. In some rhizobium species, non-reducing end is also modified by carbamoylation or acetylation other than N-acylation. The host specificity is determined by the number of acetylglucosamine residules and by substitutions on the terminal sugar residues. In particular, modifications of the reducing terminal sugar are of critical importance. The genes involved in the substitutions on N-acetylglucosamine backbones are termed "host-specific" nod genes. For example, S. meliloti, whose host is alfalfa, produces a Nod factor that carries a sulfate group on the reducing end. Host-specific nod genes, nodH and Q are responsible for this sulfation. Mutation of these genes results in the formation of the non-sulfated Nod factor. Consequently, such bacterial mutants fail to infect on alfalfa, but are able to infect on vetch. B. japonicum produces a Nod factor methyl-fucosylated at the reducing end by a host-specific nodZ gene. It is shown that the methyl-fucosylation is essential to interact with the host legume, soybean. The presence of an acyl moiety at non-reducing ends is essential for the biological activity of Nod factors, but in most cases, their structural differences are not critical for determining host specificity. Mechanisms of recognition and perception of Nod factors by host legume plants are largely unknown. A number of studies on plant responses to bacterial nod gene mutants or to chemically-synthesized Nod factors with various structures strongly suggest that more than two receptors with different structural specificity are involved in Nod factor perception. In addition, the possible involvement of plant lectins in the early steps of Nod factor perception has very recently been indicated. As for the signal transduction pathways triggered by Nod factors, although the involvement of Ca-channels and G-proteins is shown, their basic characteristics remain to be elucidated. Studies on the Nod factor perception mechanisms in legume plants have just started. Extensive biochemical studies, as well as molecular genetic studies using model plants such as Lotus japonicus and Medicago truncatula, are expected to provide better understanding of plant-side mechanisms involved in nitrogen-fixing symbiosis.
	Yosuke Umehara, Hiroshi Kouchi(National Institute of Agrobiological Resources, Laboratory of Nitrogen Fixation)
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Sep. 15, 1999