2.1. Polysaccharides/surface components. In R. meliloti Rml021, a class of exopolysaccharides (succinoglycans) is necessary for the invasion of Medicago sativa nodules. Although mutants incapable of synthesizing succinoglycan (SG) produce normal root-hair curling, the nodules are devoid of bacteria and bacteroids, and thus ineffective. Fluorescence-microscope analyses show that nodule invasion is aborted at various stages in different exo-mutants (Cheng, Walker 1998). Cells which lack ExoR, a negative regulator of exo-gene expression (Reed el al. 1991), vastly overproduce succinoglycan and are unable to colonize curled root-hairs or form infection threads. In contrast, a mutant in exoY that is incapable of synthesizing succinoglycan since it lacks the first enzyme in the biosynthetic pathway (Reuber, Walker 1993) colonizes curled root-hairs, but forms few, very short infection threads. Although the exoH mutant that produces symbiotically dysfunctional succinoglycan forms infection threads longer than those of the exoY mutant, they never extend as far as the base of the root-hair cell. Thus in R. meliloti, succinoglycan is a symbiotic signal. Mutants that fail to produce the symbiotically active forms of these exopolysaccharides in adequate quantities, are incapable of penetrating the adjacent cell and thus remain blocked or trapped in the infected root-hair.
A number of mutations giving similar phenotypes, but in a number of other genes have been found (see Perret et al. 2000). Where production of cyclic-P-(1^2)glucans, rhamnose-rich SPS, LPS, SG, P"(l—>2)glucans, and KPS is impaired, Nod+ but Fix "phenotypes are observed. Different regulators appear to control the expression of these diverse genes, and the resultant phenotype depends on the plant. Quite often an Inf1" Fix phenotype is observed, suggesting that the genes play similar roles. One possibility is that such surface components are necessary for or form part of the developing infection thread. In this scenario a plant that could not itself supply the missing carbohydrate would give a Fix phenotype, while the rhizobial mutant in a plant that normally synthesizes the compound, would have no effect.
2.2. Secreted proteins. Several strains of Rhizobium have been shown to secrete symbiotically active proteins. Amongst these, nodO is required for nodulation of Vicia hirsuta by mutants of R. leguminosarum bv. viciae deleted of nodFELMNT (Downie, Surin 1990), as well as to extend the host range of a R. trifolii nodE mutant to include V. sativa (Economou et al. 1994). nodO encodes a Ca2+-binding protein that is thought to form cation-specific channels in membranes of leguminous plants (Economou et al. 1990; Sutton et al. 1994). Secretion of NodO is dependent on a C-terminal signal of about 24 residues (Sutton et al. 1996), and the prsDE genes which encode two type-I-like inner-membrane proteins (Finnie et al. 1997). In addition to NodO, at least three other proteins are secreted via this system, two of which (PlyA and PlyB) are glycanases involved in the processing of bacterial exopolysaccharides (Finnie et al. 1998). Although NodO and NodS have distinct biochemical functions (the nodS gene encodes an A^-methyl transferase; Jabbouri et al. 1995), a nodO homolog of Rhizobium sp. BR816 was shown to complement a nodS mutant of NGR234 for nodulation of L. leucocephala (van RMjn et al. 1996). This suggests that secreted proteins may complement or supplement some Nod factor deficiencies.
Sequencing the symbiotic plasmid of NGR234 revealed flavonoid-inducible genes encoding components of a type III secretion system (T3SS) (Freiberg et al. 1997). In a number of bacterial pathogens, T3SSs are induced upon contact with host cells (of plants or animals) and deliver virulence proteins directly into the eukaryotic cytosol (Lee 1997). NGR234, as well as several strains of R. fredii, was shown to excrete at least three to five proteins in a NodDl-, and T3SS-dependent manner (Krishnan et al. 1995; Viprey et al. 1998; Marie et al. 2001; M. Gottfert et al. this volume). In USDA257, the nolXWBTUV cluster (corresponding to the nopX, rhcCl, nolB, rhej, nolU and nolV genes of NGR234) (Viprey et al. 1998) regulates the nodulation of Glycine max in a cultivar-specific manner (Meinhardt et al. 1993), whereas the T3SS of NGR234 profoundly affects nodulation of various legumes such as Crotalaria juncea, Pachyrhizus tuberosus and Tephrosia vogelii (see below). The absence of conserved nod-box regulatory elements in the promoter regions of the nol, nop and rhc operons indicate that NodDl dependent transcriptional regulation of the T3SS genes is mediated by another factor. y4xl, an HrpG homolog which is under the control of a functional nod-box, is thought to be the key intermediary in the regulatory cascade between flavonoids and activation of the T3SS machinery in NGR234 (Viprey et al. 1998; Marie et al. 2001). Delayed induction of this secretion pathway in comparison with loci involved in the elaboration of the Nod factors (see Perret et al. 1999), suggests that the T3SS machinery is assembled after Nod factors have been elaborated and protein export begins when intimate contact between bacteria and root-hairs has been established. Thus, the secreted proteins (some of which have direct effects on plants and are thus "effector" proteins) such as NopJ, NopX and y4xL of NGR234 would function after the bacteria have entered the plant, possibly during development of infection threads.
It has been suggested that bacterial invasion of plant cells triggers non-specific defense reactions, and that successful pathogens overcome these defences (Gabriel, Rolfe 1990). Similarly, invading symbionts have probably evolved different strategies to lower host-plant defences. T3SS proteins and polysaccharides may contribute to this phenomenon. Some plants would perceive these compounds as part of the infection pathway, and react to their presence by increased nodulation as in the cases of M. sativa inoculated with Exo+ R. meliloti and Tephrosia vogelii which favors strains of NGR234 with a functional T3SS. In contrast, Crotalaria juncea and Pachyrhizus tuberosus, which are poorly nodulated by wild-type NGR234, produce many effective nodules when inoculated with T3SS mutants, suggesting that secreted proteins have detrimental effects on certain hosts (Viprey et al. 1998; Marie et al. 2001). It is thus not unreasonable to suggest that this last group of plants exhibit a sort of hyper-sensitive response towards NGR234 and the proteins that it secretes.
2.3. Fine-tuning of infection-thread development. Although correlations between Nod-factor structure and rhizobial specificity in nodulation are hard to draw, it is generally agreed that Nod factors are required for deformation and curling of root-hairs as well as entry of rhizobia into the plant (see Broughton et al. 2000). Once within the infection threads however, other rhizobial components are necessary for nodule development. In symbioses between Medicago sativa and Rhizobium meliloti, these include the extracellular-polysaccharides called succinoglycans, EPSII, and K antigens (Pellock et al. 2000). Succinoglycan is highly efficient in mediating both infection thread initiation and extension. EPSII is significantly less efficient than succinoglycan in mediating both steps in the invasion process while K antigen is less efficient than succinoglycan in mediating extension of infection threads (Pellock et al. 2000; Walker et al. this volume). Moreover, the mere fact that one class of compounds may substitute for another suggests that initiation and development of infection threads probably involves redundant mechanisms. If redundancy in rhizobial control of infection thread development is a general feature of nodulated legumes, then other legume-Rhizobium systems should also require extracellular-polysaccharides for infection thread formation. As discussed under Polysaccharides/surface components, the available evidence suggests that extracellular-polysaccharides probably play similar roles in many symbioses.
Redundancy in the type of extracellular-polysaccharides that support growth of infection threads coupled with preliminary observations suggesting that proteins secreted by the T3SS are important in infection-thread development (N.M. Boukli, M. Saad, P. Skorpil, unpublished) raises the question of whether polysaccharides and proteins are complementary. Another way of phrasing this question is to ask if extracellular-polysaccharides could replace secreted proteins (and vice versa) in nodule development of specific plants? If the answer is yes, it would explain the various phenotypes found when different plants are inoculated with, e.g. a rhcN deletion mutant (rhcN encodes the ATPase that provides the energy to drive protein export). Perhaps the effector proteins play irreplaceable roles in plants such as Tephrosia vogelii, while in hosts like Lotus japonicus that are indifferent to the presence or absence of a functional T3SS (Marie et al. 2001), extracellular-polysaccharides functionally replace the effector proteins.
To generalize these speculations, it is necessary to find an explanation for the absence of T3SSs in certain rhizobia. Although most rhizobia probably contain a T3SS, and this includes the genome of the only Mesorhizobium loti strain that has been completely sequenced, that of the "symbiotic" island of a related M. loti strain does not (see Marie et al. 2001). Similarly, the sequence of R. meliloti lacks this type of secretion system (see Long et al. this volume). In both cases, components of a type four-secretion system (T4SS) including most of the v/r-gencs (from virBl to virB 11) have been found however. Although it remains to be seen if these v;>-loci function in protein secretion into the plant, a unifying hypothesis would be that extracellular-polysaccharides and proteins are able to complement or supplement one another in infection-thread development. Either T3SS or T4SS machines could secrete the "effector" proteins. Taken together, the various combinations of known polysaccharides and proteins provide ample scope for fine-tuning infection-thread development to the large array of plant hosts.
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