**2. Legume-rhizobial symbiosis: An example of highly integrated plantmicrobe system**

Nitrogen is an essential component of all living systems, since it is part of the most important biological molecules – DNA and proteins. Molecular nitrogen (N2) in the atmosphere, despite being abundant, is extremely chemically inert and thus cannot be used by the majority of organisms, causing them to compete for more accessible nitrogen sources. Leguminous plants are able to grow in the soil/substrate without any combined nitrogen due to the fixation of atmospheric nitrogen by their symbiotic nodule bacteria (collectively called rhizobia) [4]. Nitrogen fixation occurs within special plant organs – root nodules (or, in some associations, also stem nodules). Development of these organs represents a well-organized process based on the tightly coordinated expression of specialized symbiotic plant and bacterial genes. The legume nodules provide an ecological niche for bacteria, as well as structure for metabolic/ signal exchange between the partners and for the control of symbionts by the hosts [5].

impacts from different sources, so they developed complex signaling pathways to effectively detect these impacts and adequately respond to them [1]. Various microorganisms, which are constantly present in the environment, form one of the major factors affecting the life cycle of plants [2, 3]. Although many plant-associated microbes are pathogens that impair plant growth and reproduction, there are also a lot of beneficial (mutualistic) microorganisms able to provide plants with nutrition and additional defense mechanisms. Cooperation with such microor‐ ganisms constitutes the universal and highly effective strategy of plants' ecological adaptation, so they tend to form long-lasting associations, which sometimes grow into highly integrated symbioses where one or both partners can develop novel features useful for their survival. Establishing of such symbiotic relationships involves the complicated developmental pro‐ grams implemented under the joint control by plant and microbial partners and based on the

Leguminous plants (or Legumes, family Fabaceae) are known to form symbioses with extremely broad range of beneficial soil microorganisms (BSM), representing examples of almost all plant-microbe mutualistic systems. One of the most ecologically important and wellstudied legume-beneficial symbioses is root nodule (RN) symbiosis (symbiotic association with nitrogen-fixing bacteria). Compared with other interactions of legumes with BSM, RN symbioses demonstrate high level of genetic and metabolic integrity, which implies, *inter alia*, highly specific mutual recognition of partners. As legume plant plays a central role in establishing of RN symbiosis, performing functions of initiation, coordination, and regulation of all developmental processes, it possesses complex receptor system capable of accurate identification of microsymbiotic partner. In this chapter, we describe the mechanisms of plantmicrobe recognition during initial steps of RN symbiosis using the interaction of model legumes – pea (*Pisum sativum* L.), barrel medic (*Medicago truncatula* Gaertn.), and *Lotus japonicus* (Regel.) K. Larsen – with rhizobia as an example. We pay particular attention to symbiotic system of *P. sativum* since pea, besides its importance as a model object of genetics, is also a valuable crop plant. Hence, in conclusion, we discuss the potential to use obtained knowledge for optimizing the broad spectrum of plant adaptive functions and to improve the

**2. Legume-rhizobial symbiosis: An example of highly integrated plant-**

Nitrogen is an essential component of all living systems, since it is part of the most important biological molecules – DNA and proteins. Molecular nitrogen (N2) in the atmosphere, despite being abundant, is extremely chemically inert and thus cannot be used by the majority of organisms, causing them to compete for more accessible nitrogen sources. Leguminous plants are able to grow in the soil/substrate without any combined nitrogen due to the fixation of atmospheric nitrogen by their symbiotic nodule bacteria (collectively called rhizobia) [4]. Nitrogen fixation occurs within special plant organs – root nodules (or, in some associations, also stem nodules). Development of these organs represents a well-organized process based on the tightly coordinated expression of specialized symbiotic plant and bacterial genes. The

cross-regulation of their genes.

136 Plants for the Future

sustainability of legume crop production.

**microbe system**

Family Fabaceae contains about 19,000 species divided between three subfamilies (Caesalpi‐ nioideae, Mimosoideae, and Papilionoideae), with more than 700 genera of worldwide distribution [6]. With a single exception (*Parasponia*, family Ulmaceae), the ability for symbio‐ ses with rhizobia is restricted to Fabaceae, although in eight related dicotyledonous families (Rosid I clade) an ability to form nodules with the nitrogen-fixing actinomycete *Frankia* is known [7].

By contrast to legumes, their nitrogen-fixing microsymbionts do not constitute a taxonomically coherent group of organisms. The majority of rhizobia belong to the α-proteobacteria previ‐ ously assigned to the *Rhizobiaceae* family solely on the basis of their ability to nodulate the legumes (e.g., *Azorhizobium*, *Bradyrhizobium*, *Mesorhizobium*, *Rhizobium*, *Sinorhizobium*). In the last years, several non-rhizobial symbionts capable of forming nodules and fixing nitrogen in legume roots have been documented. According to modern conception, bacteria that can form RN symbiotic associations (about 44 species of 12 genera) are not clustered in any common lineage, instead being distributed in the classes α-proteobacteria and β-proteobacteria (close to *Burkholderia*, *Cupriavidus* and *Ralstonia*) and dispersed over nine monophyletic groups along with taxa that do not contain legume symbionts [8]. Recently, some γ-proteobacteria (belong‐ ing to *Escherichia*, *Enterobacter*, and *Pseudomonas* genera) have been discovered that can also form nitrogen-fixing nodules with the legumes [9]. All these bacteria (collectively still referred to as rhizobia) vary significantly in their overall genome structure, location of "symbiotic" (*sym*) genes, their molecular organization and regulation [10, 11]. However, a particular legume plant can find the appropriate rhizobial partner (species, or even strain) due to the fine-tuned mechanism of molecular interaction.

The development of nitrogen-fixing nodule is complex process that is traditionally divided into three major stages: preinfection, root colonization/nodule morphogenesis, and nitrogen fixation. On the first stage, the mutual recognition of partners occurs. The interaction between micro- and macrosymbiont begins with the activation of bacterial *nod*-genes under the influence of flavonoid molecules secreted by the plant root [12, 13]. *nod*-genes provide the synthesis of the main bacterial signaling molecule called Nod-factor (NF), which is crucial for identification of microsymbiont [14-16]. After the proper reception of Nod-factor, plant activates two parallel processes: bacterial penetration into root hair cells via so-called infection thread (IT), and differentiation of nodule from the root cortex. IT is a special structure generated by invagination of plant cell membrane, covered with plant-derived cell wall and filled with matrix produced by both plant and bacteria. It grows into root hair cell and then to the cortex where nodule tissues are formed (Figure 1) [17].

The key stage of nodule development is conversion of bacteria into the form of intracellular symbionts through endocytosis-like process. Herein, the distal area of IT transforms into structure called infection droplet (ID), which releases membrane vesicles containing bacteria into plant cytoplasm. After leaving IT, rhizobia for some time retain their size and shape, subsequently differentiating into a specific form called bacteroids [18]. Compared to free-living bacteria, bacteroids have significantly (about 3-7 times) increased size and more complex

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

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 reduction of N2 into NH4 + ) and other proteins involved in nitrogen fixation is activated in bacterial cells [19].

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

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), however, form morphologically more simple "determinate" nodules, where apical meristem exists only for several days, nitrogen-fixing zone is not strongly expressed, and infected (N2 fixing) cells intermingle with noninfected ones [21]. Bacteroids in determinate nodules show no sign of terminal differentiation as they usually maintain their normal bacterial size, genome content, and reproductive capacity lacking from those in indeterminate nodules [22].

Several Papilionoideae members, such as *Arachis* and *Stylosanthes*, demonstrate the reductive scheme of nodule development: rhizobia invade roots through the cracks of epidermis, and instead of IT they are brought into cytoplasm by the direct endocytosis from intercellular space [6, 23]. Even more primitive morphology of symbiosis is typical for members of Caesalpinioi‐ deae subfamily, as they lack endocytosis step, and nitrogen fixation occurs within modified persistent ITs called "fixation threads" [24]. This is also relevant for evolutionary primitive woody plants from Papilionoideae: *Andira* and *Hymenolobium*, and for *Parasponia*.

Such a complicated system of biological nitrogen fixation will work properly only when suitable partners meet each other in soil. This rendezvous becomes possible owing to reciprocal molecular signal exchange, which is not exhaustively studied to date.
