**4. Legume nodules: The perfect environment for rhizobia nitrogen fixation**

Nodulation is a complex process orchestrated by a multitude of bacteria and plant signals (reviewed in Ferguson and Mathesius 2003; Ferguson et al., 2010). The process is initiated by plant roots secreting flavonoid molecules into the soil. This attracts compatible rhizobia and concomitantly stimulates them to synthesize a highly specific signal molecule called Nod factor. The rhizobia strain that is compatible with soybean is *Bradyrhizobium japonicum*. The plant perceives Nod factor via LysM receptors on the root. In soybean, these receptors are called GmNFR1 and GmNFR5 (Indrasumunar et al., 2010, 2011). Nod factor perception trig‐ gers a subsequent signaling cascade that is required for proper nodule establishment. The known legume and rhizobia genes/signals that are involved in this signaling cascade have recently been thoroughly reviewed in Ferguson (2012).

The presence of the rhizobia together with their Nod factor signal molecule initiates the nod‐ ulation infection process. Root hair penetration is the most common form of rhizobia inva‐ sion. The bacteria attach to emerging root hairs, which begin to deform and eventually encapsulate some of the bacteria, which are continuously dividing (Callaham and Torrey 1981; Turgeon and Bauer 1985). This process happens in as little as 6 – 8 h post-inoculation (Yao and Vincent 1969; Bhuvaneswari et al., 1981; Bhuvaneswari and Solheim 1985; Turgeon and Bauer 1982, 1985). Specialized structures, called infection threads, begin to form and provide a passage way for the bacteria to enter the root (reviewed by Gage 2004). These in‐ fection threads are predominately comprised of plant cell wall components and they permit the bacteria to continue proliferating within the host plant.

As the process of rhizobia infection occurs, cortical cells in the root begin to divide and eventually give rise to the nodule primordium (Calvert et al., 1984; Mathews et al., 1989). The position of the nodule primordium is typically adjacent to the radial cells of the xylem, and away from the phloem. This positioning is thought to be largely dependent on plant hormone levels, namely gradients of the gaseous hormone, ethylene (Heidstra et al., 1997; Gresshoff et al., 2009; Lohar *et al.,* 2009). Additional tissues, including vascular tissues and central nodule tissues that are composed of both invaded and non-invaded cells, also devel‐ op to form the nodule structure (Newcomb et al., 1979; Calvert et al., 1984; Ferguson and Reid 2005).

Infection threads initiating in the root hair eventually grow and extend towards the dividing nodule primordium located in the root cortex. Once there, rhizobia located at the tip of the infection threads are released into an infection droplet that separates and is released into the cytoplasm of the host cell. Within the cytoplasm, the rhizobia are encapsulated by a special‐ ized plant-derived membrane, known as the peribacteroid membrane, making what is com‐ monly referred to as the symbiosome (Udvardi and Day 1997).

Ultimately, the dividing bacteria differentiate into what are known as bacteroids, which are highly specialized and whose main purpose is to fix atmospheric di-nitrogen gas. Inside the mature nodule, the bacteroids use a nitrogenase enzyme complex to fix the di-nitrogen into forms of nitrogen that the plant can use, such as ammonia. The ammonia, which is toxic to the plant, is then quickly converted into compounds such as glutamate or ureides that are non-toxic and are safely transported throughout the plant. Legume nodules provide the ide‐ al setting for this process as they establish a peripheral oxygen barrier, via physical and met‐ abolic barriers, to create a low-oxygen environment that is essential for nitrogenase activity to occur.

The nodules formed on the roots of soybean plants are referred to as 'determinate' nodules. They are spherical and lack a persistent meristem, unlike indeterminate nodule structures that can form on other legume species, particularly those from temperate growing regions (Ferguson et al., 2010). The life-span of a soybean nodule is typically a few weeks, after which they senesce and are replaced by new nodule structures developing on the growing root system. Following nodule senescence, the bacteroids can re-differentiate and become new inoculum for the soil (Gresshoff and Rolfe 1978).
