Role Of Flotillins And Flavonoids

For a successful establishment of compatible rhizobial-legume symbioses, plant roots should support bacterial infection via facilitation of progression of infection threads (ITs). Formation of IT further leads to formation of functional N2-fixing nodules, as a result of a series of molecular and physio-chemical events (Freiberg et al., 1997). Haney and Long (2010) have reported requirement of plant flotillin-like genes (FLOTs) expressed during S. meliloti infection by its host legume M. truncatula. Earlier, flotillins have been reported in other organisms playing roles in viral pathogenesis, endocytosis and membrane shaping. Haney and Long (2010) identified seven FLOT genes in the M. truncatula genome and showed that two, FLOT2 and

Days post inoculation (dpi)

Fig. 1. Demonstration of expression of FLOTs which is up-regulated during nodulation, and this regulation depends on the Nod factor. Expression of individual FLOT genes was measured by quantitative RT-PCR. Each FLOT expression level was normalised to an internal actin control. Study was done in wild-type S. meliloti Rm1021.Reproduced from Haney and Long, 2010 with permission.

Days post inoculation (dpi)

Fig. 1. Demonstration of expression of FLOTs which is up-regulated during nodulation, and this regulation depends on the Nod factor. Expression of individual FLOT genes was measured by quantitative RT-PCR. Each FLOT expression level was normalised to an internal actin control. Study was done in wild-type S. meliloti Rm1021.Reproduced from Haney and Long, 2010 with permission.

FLOT4, are strongly up-regulated during early symbiotic events (Fig. 1). The rate of up-regulation depends on bacterial Nod factor and the plant's ability to perceive the Nod factor. Data from microscopy suggest that M. truncatula FLOT2 and FLOT4 localise to membrane microdomains. Upon rhizobial inoculation, FLOT4 uniquely becomes localised to the tips of elongating root hairs. Silencing FLOT2 and FLOT4 gene expression reveals a non-redundant requirement for both genes in IT initiation and nodule formation. FLOT4 is uniquely required for IT elongation, and FLOT4 localises to IT membranes. Thus, the work of Haney and Long (2010) reveals a critical role of plant flotillins in symbiotic bacterial infection.

A complex signal exchange between macrosymbiont and microsymbiont initiates the nodulation process (Den Herder et al., 2007; Jones et al., 2007): upon perception of flavonoids exuded by host roots, rhizobia switch on their nodulation genes (Cooper, 2007), thus forming lipochitooligosaccharide molecules, designated as nodulation factors (NFs; Deakin and Broughton, 2009; D'Haeze and Holesters, 2002). NFs are essential for bacterial invasion and induction of cortical cell division to form nodule organs (Geurts and Bisseling, 2002). In fact, root infection by rhizobia is a multi-step process. It is initiated by certain pre-infection events occurring in the rhizosphere and progresses further under control of several factors and genes (Fig. 2). Plant root secretes exudates to which rhizobia respond by positive chemotaxis and move towards localised sites on the legume roots (Barbour et al., 1991;

Flotillins

Fig. 2. Schematic representation of the interaction between Rhizobium species and legume roots. Plant secretes flavonoids which induces nod genes in Rhizobium. Nod factors induce root-hair activation, cortical cell division, facilitation of infection process, increased flavonoid production and flotillins.

Fig. 2. Schematic representation of the interaction between Rhizobium species and legume roots. Plant secretes flavonoids which induces nod genes in Rhizobium. Nod factors induce root-hair activation, cortical cell division, facilitation of infection process, increased flavonoid production and flotillins.

Caetano-Annol├ęs and Gresshoff, 1991). Both Bradyrhizobium and Sinorhi-zobium spp. are attracted by amino acids, dicarboxylic acids and very low concentrations of excreted components such as flavonoids present in the exudates (Peters and Verma, 1990). Physiological conditions have been found to influence the attachment capacity of R. leguminosarum bv. viciae to pea root hairs (Smit et al., 1989). However, lectins are also involved in the rhizobial attachment (Kijne et al., 1988; Kijne, 1992). For R. leguminosarum bv. viciae, a Ca2+-dependent adhesin, called rhicadhesin, mediates the initial direct attachment to pea root-hair surfaces (Vesper et al., 1987). For cap formation, the firm attachment step, fibrillous appendages of (brady)rhizo-bia appear to be involved. These appendages can be cellulose fibrils (R. leguminosarum) or proteinous fimbriae (Bradyrhizobium japonicum) (Ho et al., 1990). Other non-protein bacterial macromolecules might also be involved. In all Rhizobium-plant interactions studied so far, the active substances have been identified as lipooligosaccharides, also called Nod factors. These Nod factors are synthesised by some of the nodulation genes (Spaink et al., 1991; Truchet et al., 1991). It has been shown that Medicago GRAStype protein-nodulation signalling pathway 1 (NSP1; Smit et al., 2005) and 2 (NSP2; Kalo et al., 2005), which are essential for all known Nod factor-induced changes in gene expression, are involved. NSP1 is constitutively expressed, and so it acts as a primary transcriptional regulator mediating all known Nod factor-induced transcriptional responses; it is therefore named as Nod factor response (Smit et al., 2005). NSP2 encodes a GRAS protein essential for Nod factor signalling. NSP2 functions downstream of Nod factor-induced calcium spiking and a calcium/calmodulin-dependent protein kinase.

The host plant reacts by depositing new cell wall material around the lesion made by Rhizobium infection in the form of an inwardly growing tube. The tube is filled with proliferating bacteria surrounded by a matrix and becomes an IT. The IT grows towards the inner tangential wall of the root-hair cell tip by a process of tip growth. Concomitant with formation of the IT, particular cortical cells divide to form a nodule primordium and the IT grows towards these primordia (Wood and Newcomb, 1989). While the meristem is active, rhizobia are released from the ITs into the plant-cell cytoplasm (Brewin, 1991; Hirsch, 1992); in tropical legumes (e.g. soybean), a nodule meristem is induced in the root outer cortex, and the bacteria are released into actively dividing meristematic cells, with each daughter cell receiving rhizobia (Newcomb, 1981). Den Herder et al. (2007) have reviewed other mechanisms of infection. A protein, remorin, has been shown to interact with symbiotic receptors and regulate bacterial infections (Lefebvre et al., 2010). Intercellular bacterial microcolonies or infection pockets (IPs) are created in the outer cortex, from where ITs guide the bacteria towards the nodule primordium (Den Herder et al., 2006).

Oxidative burst-like phenomena have been observed as a primary response in the interaction of S. meliloti with alfalfa (M. sativa) where superoxide and H2O2 are produced (Santos et al., 2001). In alfalfa roots, recognition of compatible NFs rapidly stimulates localised production of superoxide. This response is absent in the non-nodulating plant mutant and does not make infections1-1 (dmi1-1), which is impaired in the NF signal-transduction pathway (Ramu et al., 2002). Symbiotic bacteria overcome the plant's defence by activating antioxidant enzymes (Jamet et al., 2003; Santos et al.,

1999, 2000). Also, several plant genes related to protection against oxidative stress are differentially expressed during nodulation.

After successful establishment of bacteroids in the nodules, symbiotic nitrogen fixation (SNF) takes place in specialised bacterial cells with the help of bacterial enzyme nitrogenase which catalyses the following reaction:

N2 + 8H+ + 8e- + 16Mg - ATP = 2NH3 + H2 + 16Mg - ADP + 16Pi

Nitrogenase consists of two components, the homodimeric Fe protein, encoded by nifH, and the tetrameric molybdenum-iron (Mo-Fe) protein, encoded by nifD and nifK, which contains the Mo-Fe cofactor. In symbiosis, ammonium is exported to and assimilated in the plant, which in turn supplies the bacteria with carbon sources to provide energy for the nitrogenase reaction. The structure of a mature nodule develops to meet the requirements set by this nutrient exchange between both the symbiotic partners.

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