**2.1. Specificity of legume-rhizobial symbiosis**

shape, which can be round, pear-shaped, Y- or X-like, depending on specific symbiotic system. After the aforesaid differentiation, the synthesis of nitrogenase (the enzyme catalyzing

*Sym37* – stages on which symbiosis is blocked in case of corresponding pea mutants/genotypes.

**Figure 1.** General scheme of RN symbiosis formation and functioning in pea. From left to right: three major stages of symbiosis, namely preinfection, root colonization/nodule morphogenesis, and nitrogen fixation. F – flavonoids excret‐ ed by the plant, NF – Nod-factors excreted by nodule bacteria, IT – infection thread, B – bacteroids. *Sym10* and *Sym2*,

Bacteroids are embedded into a membrane structure named symbiosome, which are derived from membrane vesicle originating from ID. They are organelle-like units of plant cell responsible for nitrogen fixation. Symbiosome formation as well as bacteroid differentiation is induced by plant. Peri-bacteroid membrane (PBM) that surrounds bacteroids is an active interface of RN symbiosis where exchange of metabolites between symbionts occurs [19, 20]. Plant cells containing symbiosomes also undergo the deep differentiation, increasing the amount of their membrane structures (endoplasmic reticulum and the Golgi complex), which participate in the development of PBM and biosynthetic processes. Many proteins associated

The developmental program described above is typical only for evolutionary advanced legumes belonging to the inverted repeat–lacking clade (IRLC) of Papilionoideae, such as *Medicago*, *Pisum*, or *Trifolium* (clover). They form so-called "indeterminate" nodules which are characterized by stable apical meristem and division into histological zones with constantly renewed N2-fixing zone. Rhizobia in these nodules undergo terminal bacteroid differentiation and cannot revert to free-living form [21, 22]. Other legumes such as *Lotus* or *Phaseolus* (bean),

) and other proteins involved in nitrogen fixation is activated in

reduction of N2 into NH4

bacterial cells [19].

138 Plants for the Future

+

with nitrogen fixation appear in these cells *de novo*.

Root-nodule symbiosis is well known as highly specific plant-microbe interaction. According to the early surveys of symbiotic specificity [25], legumes were suggested to comprise a range of taxonomically restricted cross-inoculation groups (CIG) within which the free crossinoculation occurs, while the species from different groups do not cross-inoculate.

The best studied examples of this classification are represented by four CIG: "*Trifolium* – *Rhizobium leguminosarum* bv. *trifolii*," "*Pisum*, *Vicia*, *Lathyrus*, *Lens* – *R. leguminosarum* bv. *viciae*," "*Galega* – *R. galegae*," "*Medicago*, *Melilotus*, *Trigonella* – *Sinorhizobium meliloti*, *S. medicae*." However, it was demonstrated later that such strictly defined specificity is limited to the herbage papilionoid legumes growing in temperate zones and representing the so-called Galegoid complex [26, 27]. Other legumes, including the majority of tropical species, tend to broad their symbiotic specificity, where cross-inoculation is possible between tribes, subfami‐ lies, and even with non-legume plant *Parasponia* [28].

The analysis of CIG structure for both strictly and broadly specific legumes has shown that plant specificity towards rhizobia has good correlation with plant taxonomy on the genus or tribe level. It was also revealed that specificity of nodule formation does not correlate with symbiotic efficiency, i.e., efficiency of nitrogen fixation: several bacterial strains form normal nodules with one plant species, and are inactive (not able to fix nitrogen, Fix- ) with another [26]. This could be due to the fact that nodulation is an early stage of symbiosis similar (and supposedly related) to pathogenic interaction, and is based on strict cross-activation of plant and bacterial genes ("gene-for-gene" interaction), while nitrogen fixation occurs on the later stages for which "gene-for-gene" interaction is not typical.

Moreover, it is specificity that makes possible the natural selection of effective **symbiotic pairs**, but not the single "symbiotically effective" plant or single "symbiotically effective" microorganism. On the other side, specificity of legume-rhizobial symbiosis should be somewhat associated with nitrogen-fixing intensity, upon which is based the ecological efficiency of cooperation; otherwise it would not be an evolutionary advantage. The majority of "Galegoid complex" members have both narrow specificity and effective nitrogen fixation, suggesting that these two features are connected, though specificity of recognition is obviously not the only condition required for effective symbiosis.

It is also important to note that the range of potential symbiotic partners can vary for both bacteria and plants. Symbiotic pair *Trifolium*–*Rhizobium leguminosarum* bv. *trifolii* represents one side of this continuum, as they are the only possible partners for each other. On the opposite side are *Phaseolus vulgaris* and *Vigna unguiculata*, which are able to exchange their symbionts with many unrelated legume species [25]. In bacteria, the *Sinorhizobium fredii* strain NGR234 was shown to interact with more than 120 plant species from all three Fabaceae subfamilies, as well as with *Parasponia*, thus being the most "unscrupulous" strain known so far [29]. The most striking feature of this strain is that its genome, although not particularly large (6.9 Mbp), encodes more different secretion systems than any other known rhizobia and probably most known bacteria [30]. These, among others, include type III and type IV secretion systems which allow bacteria to direct effector proteins or DNA into the cytoplasm of their eukaryotic hosts. There seems to be a correlation between the host range of rhizobia and the number of speci‐ alized protein secretion systems they have, as "classic" narrow-host-range rhizobia such as *S. meliloti* and *R. leguminosarum* carry neither type III nor type IV secretion systems. Furthermore, NGR234 is shown to secrete a large family of NFs that are variously 3-O, 4-O, or 6-O carba‐ moylated, which are N-methylated, and which carry a 2-O-methyl-fucose residue that may be either 3-O sulfated or 4-O acetylated (see below) [29]. Since no other rhizobia synthesize such a large family of NFs, it should be proposed as one of the main aspects contributing to the broad host range of NGR234 [17, 31]. Another possible aspect is that NGR234 not only treats the legume root to a large palette of NFs, but that their concentration is much higher than in even very closely related rhizobia [32].

#### **2.2. Initial steps of rhizobium-legume symbiosis**

The specificity of legume-rhizobia interactions is expressed mostly during the preinfection stage when rhizobia recognize the roots of appropriate host plants and colonize their surfaces. When the root-excreted signals (in particular, flavonoids) are perceived by bacteria, they activate the bacterial nodulation genes (*nod*/*nol*/*noe*) [13]. These genes control the synthesis of lipo-chito-oligosaccharidic (LCO) nodulation factors (Nod-factors) which induce the early stages of RN symbiosis development. NFs represent the unique group of bacterial signal molecules not known outside legume-rhizobia symbiosis. They are among the most potent developmental regulators: their effect is expressed at concentrations merely of 10–8 – 10–12 M. The core structure of these molecules, common for all rhizobia species, consists of 3-6 residues of N-acetylglucosamine and of a fatty acid (acyl) chain (Figure 2). The type of symbiotic specificity is dependent mainly on the chemical modifications in NF structures [14-16]. However, a sufficient impact to the host specificity of RN symbiosis can also be made by the interactions between bacterial surface molecules (some polysaccharides and proteins) [33, 34] and the lectins located on the root hair surfaces, as well as by means of NFs secretion [35].

n =2 or 3; regarding Afghan peas, see below (3.2).

somewhat associated with nitrogen-fixing intensity, upon which is based the ecological efficiency of cooperation; otherwise it would not be an evolutionary advantage. The majority of "Galegoid complex" members have both narrow specificity and effective nitrogen fixation, suggesting that these two features are connected, though specificity of recognition is obviously

It is also important to note that the range of potential symbiotic partners can vary for both bacteria and plants. Symbiotic pair *Trifolium*–*Rhizobium leguminosarum* bv. *trifolii* represents one side of this continuum, as they are the only possible partners for each other. On the opposite side are *Phaseolus vulgaris* and *Vigna unguiculata*, which are able to exchange their symbionts with many unrelated legume species [25]. In bacteria, the *Sinorhizobium fredii* strain NGR234 was shown to interact with more than 120 plant species from all three Fabaceae subfamilies, as well as with *Parasponia*, thus being the most "unscrupulous" strain known so far [29]. The most striking feature of this strain is that its genome, although not particularly large (6.9 Mbp), encodes more different secretion systems than any other known rhizobia and probably most known bacteria [30]. These, among others, include type III and type IV secretion systems which allow bacteria to direct effector proteins or DNA into the cytoplasm of their eukaryotic hosts. There seems to be a correlation between the host range of rhizobia and the number of speci‐ alized protein secretion systems they have, as "classic" narrow-host-range rhizobia such as *S. meliloti* and *R. leguminosarum* carry neither type III nor type IV secretion systems. Furthermore, NGR234 is shown to secrete a large family of NFs that are variously 3-O, 4-O, or 6-O carba‐ moylated, which are N-methylated, and which carry a 2-O-methyl-fucose residue that may be either 3-O sulfated or 4-O acetylated (see below) [29]. Since no other rhizobia synthesize such a large family of NFs, it should be proposed as one of the main aspects contributing to the broad host range of NGR234 [17, 31]. Another possible aspect is that NGR234 not only treats the legume root to a large palette of NFs, but that their concentration is much higher than in

The specificity of legume-rhizobia interactions is expressed mostly during the preinfection stage when rhizobia recognize the roots of appropriate host plants and colonize their surfaces. When the root-excreted signals (in particular, flavonoids) are perceived by bacteria, they activate the bacterial nodulation genes (*nod*/*nol*/*noe*) [13]. These genes control the synthesis of lipo-chito-oligosaccharidic (LCO) nodulation factors (Nod-factors) which induce the early stages of RN symbiosis development. NFs represent the unique group of bacterial signal molecules not known outside legume-rhizobia symbiosis. They are among the most potent developmental regulators: their effect is expressed at concentrations merely of 10–8 – 10–12 M. The core structure of these molecules, common for all rhizobia species, consists of 3-6 residues of N-acetylglucosamine and of a fatty acid (acyl) chain (Figure 2). The type of symbiotic specificity is dependent mainly on the chemical modifications in NF structures [14-16]. However, a sufficient impact to the host specificity of RN symbiosis can also be made by the interactions between bacterial surface molecules (some polysaccharides and proteins) [33, 34] and the lectins located on the root hair surfaces, as well as by means of NFs secretion [35].

not the only condition required for effective symbiosis.

140 Plants for the Future

even very closely related rhizobia [32].

**2.2. Initial steps of rhizobium-legume symbiosis**

**Figure 2.** Example of Nod-factor excreted by *Rhizobium leguminosarum* bv. *viciae* strain TOM nodulating Afghan peas.

Rhizobia possess a wide range of genes involved in the early stages of nodulation, i.e., the NFs production [36]. Genes which are common to all rhizobia – *nodA*, *nodB*, *nodC*, and their regulator *nodD* – are responsible for NF core structure synthesis [37]. The other genes specific for particular species or strains control various modifications of signaling molecule. The difference in the spectrum of hosts possible for microsymbiont to interact with is based on the variety of combinations of different *nod*-genes. For example, presence of gene *nodE*, which encodes protein similar to fatty acid synthase in several genera of rhizobia, provides modifi‐ cation of fatty acid moiety on the nonreducing end of NF molecule, thereby affecting the ability of bacteria to nodulate certain plant species [38, 39]. Genes *nodH, nodP*, and *nodQ* found in *Sinorhizobium meliloti* control the specific NF modification – the O-sulfation of reducing end – which makes it recognizable for *Medicago* receptors [40]. Overall, each strain of rhizobia is characterized by specific set of *nod*-genes, which together form the "molecular key" suitable for plant receptor. It is significant to note that most rhizobia secrete an assortment of NFs varying in their structure instead of just one particular kind [41, 42]. Thereby, the symbiotic success of bacteria could be directly connected with diversity of NFs they are able to produce, and "molecular key" rather becomes the "set of lock picks," with secretion systems and surface molecules being additional tools in it (see above).

By perceiving the NF, plant starts various processes in root tissues. In particular, signaling molecule is required for the activation of plant genes in the epidermis cells and pericycle, as well as for mitotic reactivation of cortical cells and the formation of IT. Genes responsible for proper NF reception were first discovered in mutants of *Lotus japonicus* lacking any response to NFs [43, 44]. These genes were named *NFR1* and *NFR5*, for Nod-Factor Receptor. Cloning of these genes revealed that they encode receptor-like kinases comprising LysM domains (LysM-RLK). LysM domains occur in a variety of proteins in bacteria and eukaryotes and have been shown to bind glycan-containing ligands (such as chitin) [45]. They consist of a repetition of a small motif typically containing from 44 to 65 amino acid residues – the LysM sequence, or LysM module [46, 47]. One LysM sequence has a βααβ secondary structure with the two helices packing onto the same side of an antiparallel β sheet. Multiple LysM modules in a protein are often separated by small Ser-, Thr-, and Asn-rich intervening sequences [48].

Only in plants are LysM domains associated with a kinase-like domain [49] forming two main LysM-RLK gene families: the LYK family and the LYR family. All the LysM-RLKs are predicted to contain three LysM modules, although these modules exhibit a high degree of divergence, both within a protein and between proteins. It is considered that the initial function of LysM-RLKs has been recognition of chitin-based signal molecules produced by hostile microbes (termed as MAMPs ("microbe-associated molecular patterns") or PAMPs ("pathogenassociated molecular patterns")), similar to the function of CERK1 receptor-like kinase from *Arabidopsis thaliana* [2]. Based on microsyntenies between genomic regions around LysM-RLK genes in legumes and non-legumes (*A.thaliana*, rice) plants, it has been speculated that these genes are the descendants of a common ancestor [50]. Zhang et al. (2007) [51] proposed that in Leguminosae LysM-RLKs have undergone further duplication and diversification, with some LysM-RLKs acquiring the ability to perceive bacterial NFs, leading to mutually beneficial endosymbiosis with rhizobia. One aspect of this diversification is the adaptation of extracel‐ lular LysM domains to recognize specific structures of NFs, while another being evolution of the intracellular kinase domains to switch the signals from cascades inducing defense re‐ sponses to symbiotic gene cascades. Recently, the function of NFRs as NF receptors was confirmed by demonstration of their ability to directly bind NF molecule *in vitro* [52].

In *Medicago* and pea, which belong to IRLC (see above), NF perception seems to be more complicated than in *Lotus*. Genes orthologous to *NFR1* and *NFR5* were identified in *Medicago truncatula* (*LYK3* and *NFP*) and in *Pisum sativum* (*Sym37* and *Sym10*), with careful description of corresponding mutant phenotypes [44, 53-55]. While phenotype of *nfp* and *sym10* mutants (in *Medicago* and pea, respectively) coincided with that of *nfr5* mutants in *Lotus*, mutations in genes *lyk3* and *sym37* (orthologs of *NFR1*) led to significantly different phenotype – successful penetration of bacteria into root hair with subsequent block of IT progress, instead of complete absence of responses to rhizobia [55, 56]. These data support the "two-receptor" model of Nodfactor perception proposed more than 20 years ago [40]. According to this model, which was developed on the base of the infection phenotype of several *S. meliloti nod* mutants, there are two different types of NF receptors – the "recognition" (or "signaling") receptor inducing early responses with high affinity for Nod-factor and low requirements toward its structure, and the "entry" receptor that controls penetration of bacteria into plant cell and has more stringent demands [40].

It is significant to note that NFR5 (and its homologs, NFP in *Medicago* and Sym10 in *Pisum*) lacks the independent kinase activity and thus can function properly only in complex with active kinase (which is suggested to be NFR1) [52]. It can be assumed, based on the above, that in general the "recognition" receptor (NFR5, NFP or Sym10) perceives NF and afterwards forms complex with another receptor possessing kinase activity (NFR1, LYK3 or Sym37, respectively), thus constituting the "entry" receptor. Still, results of genome and transcriptome sequencing in *Lotus*, *Medicago* and pea show that legumes possess more than 10 genes of receptor kinases similar by structure to the aforementioned ones. So, the system of NF receptors could be actually much more complicated, suggesting that the overall mechanism of NF perception is probably even more intricate than was thought before.
