Symbiosis between leguminous plants and rhizobia, under conditions of nitrogen limitation, leads to the development of new plant organs, the [N.sub.2]-fixing nodules, that are usually formed on roots but also on stems in a few plants. Inside the nodule the differentiated form of rhizobia, the bacteroids, fix molecular nitrogen, which is then used by the plant partner. The symbiotic interaction starts when the bacteria colonize the root surface and induce curling of the root hair tips (102, 141) [ILLUSTRATION FOR FIGURE 1 OMITTED]. This is followed by cell wall invagination and the formation of an infection thread that grows within the root hair. The infection thread traverses the outer cell layers to reach the nodule primordium, which is initiated by the reactivation of differentiated cells of the root cortex for division. Within the infection thread the rhizobia multiply but remain confined by the plant cell wall. As the primordium develops to a nodule, bacteria are released from the tip of the infection thread by endocytosis and differentiate into bacteroids surrounded by the peribacteroid membrane. Depending on the plant species, two major types of nodules are formed. The indeterminate nodules maintain an apical meristem and are therefore elongated, unlike the determinate nodules, which do not have a persistent meristem and are round-shaped. Both types of nodule have a peripheral vascular tissue.
The symbiotic interaction shows a high degree of host specificity, i.e. only certain combinations of plant and Rhizobium are compatible for establishing the nitrogen-fixing symbiosis. (In this review, for simplicity, the bacteria capable for symbiotic nitrogen fixation with legumes are called collectively Rhizobium or rhizobia.) Growth and development of bacterial and plant cells must be thoroughly coordinated, involving several communication steps between the partners. At each stage of nodule development, specific genetic programs are reciprocally induced in both the bacterium and its host plant.
In this review, we discuss some recent advances in elucidating the early events leading to root nodule development.
SIGNAL EXCHANGES BETWEEN RHIZOBIUM AND HOST PLANTS
Rhizobia approaching the root of compatible host plants respond to plant-derived inducing compounds (usually flavonoids) by expressing their nodulation (nod) genes. Induction of these genes leads to the production and secretion of return signals, the nodulation factors (Nod signals), which are lipochitooligosaccharides of variable structure (100) [ILLUSTRATION FOR FIGURE 1 OMITTED]. Nod factors are essential for the rhizobia to trigger root hair curling, to induce the formation of nodule primordia, and to enter the root via infection threads. Purified Nod factors are sufficient to induce root hair deformations, cortical cell divisions, and on some host plants, fully grown nodule-like structures (38). Moreover, on vetch plants they trigger the anticlinal alignment of cytoplasmic bridges through several cortical cell layers (164) and provoke a partial degradation of the cell wall opposite these bridges. This step was proposed to prepare the cells for the passage of the infection threads (167).
Under conditions of nitrogen starvation, certain alfalfa plants form nodule-like structures spontaneously in the absence of Rhizobium ([Nar.sup.+] phenotype) (161). [Nar.sup.+] plants appear to be more responsive than [Nar.sup.-] plants to Nod factors in that they form a higher number of nodule primordia and nodules containing a persistent meristem upon application of the Nod factor (72). These observations suggest that Nod factors act as external growth factors triggering an endogenous nodulation program in the host plant. However, they are not sufficient to allow the invasion of the nodule by rhizobia and differentiation of the bacteria to bacteroids. For nodule invasion, cell surface components like exopolysaccharides (EPS), lipopolysaccharides (LPS), and capsular polysaccharides (KPS) are needed (see 11, 88, 99). The polysaccharides likely have several functions, e.g. in cell-to-cell communication, shielding of the bacteria from plant defense mechanisms, or in signaling to suppress plant defense reactions.
Various nod gene inducers, Nod factors, and polysaccharides are all involved in determining host specificity. Thus, there appears to be a true communication between the symbiotic partners leading to each recognizing the other.
Control of nod Gene Expression
In rhizobia, nod genes are organized in several operons, located either on the chromosome or on large (Sym) plasmids. The complete sequence of the Sym plasmid has been determined for Rhizobium sp. NGR234 (56). The expression of nod genes is positively regulated by the LysR-type trans-activator protein NodD (138). The plant signal-inducible nod operons are preceded by a conserved cis-regulatory element, the nod box. NodD binds to the nod box sequence and upon interaction with the inducing flavonoid, activates transcription of these operons. Thus, nod genes are coordinately regulated, constituting a nod regulon. The interaction of NodD with the specific flavonoids of the host plant represents the first level of host-specific recognition. Superimposed onto this basic activation mechanism are a variety of additional control circuits. Many species of rhizobia contain several generally highly homologous nodD genes that result in the production of NodD proteins capable of interacting with a range of different inducer molecules. A particular case is represented by the NodD homologue SyrM (a symbiotic regulator) that is involved in both the activation of nod genes and the induction of exopolysaccharide synthesis (113). In some species, nodD genes control their own expression via positive or negative autoregulation. Moreover, different NodD proteins can influence the expression of each other. There may be additional nod gene activators, e.g. a two-component regulatory system with NodV as the flavonoid sensor and NodW as the regulator has been identified in Bradyrhizobium japonicum (70). NodV has a different inducer spectrum than NodD and this appears to widen the host range (see 138).
Positive regulation of nod gene expression has been extensively studied (138), but it is, in fact, also under negative control, as first shown for Rhizobium meliloti (92), and recently for a number of other rhizobia (39, 49, 66, 90). In most cases, negative regulation is important for efficient nodulation and/or influences the host plant spectrum (49, 61, 66, 92, 133). The repressor NolR has been identified in several different Rhizobium species (90, 92); it downregulates Nod factor production and influences Nod factor quality by impairing the expression of only a subset of the nodulation genes (29). Interestingly, negative control can also be exerted by certain NodD proteins. This was shown for different Bradyrhizobium species in which negatively acting nodD genes were positively regulated by NolA (61, 66). The nodulation protein NolA therefore has an overall repressing effect on nod gene expression (39). In Rhizobium sp. NGR234, nodD2 also acts as a negative regulator. A nodD2 mutant produced fivefold more Nod factor, and its production appeared not to be switched off in the infected nodules (49). The mutant was severely impaired in bacterial release and bacteroid differentiation, similarly to what was reported for the nolA mutants in B. japonicum (61).
The discovery of additional control elements adds to the complexity of nod gene regulation. For example, in R. meliloti, syrB negatively affects the expression of the positively acting nodD homologue syrM (4). There is accumulating evidence that nod gene expression, hence Nod signal abundance and quality, are tightly regulated, affecting nodulation efficiency and host specificity. The production of excess amounts of Nod factors appears to be deleterious, possibly because plant defense reactions are elicited (136), which may be a reason why downregulation of nod gene expression is needed during bacteroid differentiation. The signal that actually triggers this downregulation has not yet been determined (138). However, a possible role of dicarboxylic acids is suggested, based on their ability to inhibit nod gene expression in B. japonicum (175).
Nod Signal Structure and Host Specificity
Nod factors are derivatives of chitin-oligosaccharides in which the N-acetyl moiety at the nonreducing sugar residue is replaced by a fatty acyl chain. The basic lipochitooligosaccharide (LCO) structure is common to all rhizobia [ILLUSTRATION FOR FIGURE 1 OMITTED] and is determined by the "common" nod genes nodABC (38). The protein NodC is a chitooligosaccharide synthase (63, 149), NodB is a deacetylase that specifically removes the acetyl group at the nonreducing end (84), while NodA transfers the acyl chain to this position (3, 131). Depending on the bacterial species, the LCOs carry a number of different modifications. The fatty acid chain of Nod factors varies in length and degree of unsaturation, and both termini of the oligosaccharide chain may carry various substituents [ILLUSTRATION FOR FIGURE 1 OMITTED]. Each Rhizobium strain produces a characteristic spectrum of Nod factors that is usually unique for a given isolate. The Nod factor profile seems to reflect adaptation to the host plant. Thus rhizobia belonging to different taxonomic groups produce LCOs of similar structure when isolated from the same host plant (104).
Many different nod genes are involved in modifying the basic LCO structure specifically for different rhizobia. For instance, nodH encodes a sulfotransferase that transfers a sulfate group to the reducing end of Nod factors of R. meliloti [ILLUSTRATION FOR FIGURE 1 OMITTED] (18, 41, 129, 143). The sulfate group is indispensable for nodulation on the host plant alfalfa. nodH mutants produce nonsulfated Nod factors that are inactive on alfalfa but become active on the nonhost plant vetch (129). Sulfated Nod factors are also produced by several other rhizobia (103, 125, 172), for example, some of those that nodulate common bean. However, the sulfate group appears to be much less stringently required for Nod signal activity and nodulation on bean than is the case in the R. meliloti-alfalfa interaction. Depending on the bean cultivar, nodulation may be enhanced or impaired by the presence of the sulfate group (98).
Nod factors of numerous rhizobial species carry a fucose group at the reducing end. Fucosylation is mediated by nodZ (see 26, 38). In Rhizobium sp. NGR234, a fraction of the Nod factors is sulfated at the C3 position of the fucose residue, and this depends on the presence of another putative sulfotransferase encoded by the nodulation gene noeE (74). Interestingly, noeE shows specificity for the sulfation of the fucose, whereas nodH acts on both the fucose and the reducing end GlcNAc residue. O-acetyl groups have been found at three different positions on Nod factors, each involving a specialized O-acetyltransferase. NodL provides O-acetylation at the C6 of the nonreducing GlcNAc residue (15), nodX at the C6 of the reducing end …