Nodulation of Mimosa spp. by the β-Proteobacterium Ralstonia taiwanensis

Abstract

Several beta-proteobacteria have been isolated from legume root nodules and some of these are thought to be capable of nodulating and fixing N2. However, in no case has there been detailed studies confirming that they are the active symbionts. Here, Ralstonia taiwanensis LMG19424, which was originally isolated from Mimosa pudica nodules, was transformed to carry the green fluorescent protein (gfp) reporter gene before being used to inoculate axenically-grown seedlings of M. pudica and M. diplotricha. Plants were harvested at various intervals for 56 days after inoculation, then examined for evidence of infection and nodule formation. Nodulation of both Mimosa spp. was abundant, and acetylene reduction assays confirmed that nodules had nitrogenase activity. Confocal laser scanning microscopy (CLSM) showed that fresh M. pudica nodules with nitrogenase activity had infected cells containing bacteroids expressing gfp. In parallel, fixed and embedded nodules from both Mimosa spp. were sectioned for light and electron microscopy, followed by immunogold labeling with antibodies raised against gfp and nitrogenase Fe (nifH) protein. Significant immunolabeling with these antibodies confirmed that R. taiwanensis LMG19424 is an effective N2-fixing symbiont of Mimosa spp. Both species were infected via root hairs and, in all respects, the nodule ontogeny and development was similar to that described for other mimosoid legumes. The nodules were indeterminate with a persistent meristem, an invasion zone containing host cells being invaded via prominent infection threads, and an N2-fixing zone with infected cells containing membrane-bound symbiosomes.

Experimental evolution of nodule intracellular infection in legume symbionts

“…Nodule cell infection capacity was however lower than that of the reference M. pudica symbiont, C. taiwanensis (Chen et al, 2003). Indeed, less bacteria were routinely isolated per nodule (Figure 2), there is a larger variance in number of infected nodule cells ( Figure 1b) and of recovered bacteria ( Figure 2) and the nodules did not fix nitrogen.…”

“…C. taiwanensis is a nitrogen fixing symbiont of Mimosa pudica. As most rhizobia, it invades host roots by means of root hair infection threads (ITs), which are initiated from micro-colonies entrapped within curled root hairs (Chen et al, 2003). Extending and ramifying ITs bring bacteria to the root cortex where nodules are simultaneously developing (Gage, 2004;Oldroyd et al, 2011).…”

Biosynthesis of branched-chain amino acids is essential for effective symbioses between betarhizobia and Mimosa pudica

“…Although it was generally accepted that legumes were nodulated exclusively by relatives of Rhizobium in the class Alphaproteobacteria, the so-called 'alpharhizobia', over the last 10 years there have been an increasing number of reports of legumes being nodulated by members of the Betaproteobacteria (the so-called 'betarhizobia') (Gyaneshwar et al, 2011). Cupriavidus taiwanensis LMG19424 and Burkholderia phymatum STM815 were among the first identified nodulating betaproteobacterial strains (Gyaneshwar et al, 2011) and both are highly effective nitrogen-fixing symbionts of Mimosa species (Chen et al, 2003(Chen et al, , 2005Elliott et al, 2007;dos Reis et al, 2010).…”

“…Nodulation is a multistep process (van Rhijn & Vanderleyden, 1995;Chen et al, 2003;Gibson et al, 2008;Oldroyd & Downie, 2008), in which rhizobia (alpha or beta) in the rhizosphere infect legume roots via root hairs and are ultimately released into cells of the developing nodule primordium via endocytosis. Inside a nodule cell, the bacteria are enclosed in symbiosomes, in which they differentiate into bacteroids (Chen et al, 2003).…”

“…Inside a nodule cell, the bacteria are enclosed in symbiosomes, in which they differentiate into bacteroids (Chen et al, 2003). The C4-dicarboxylate intermediates of the tricarboxylic acid cycle, succinate, fumarate and malate, have generally been accepted as the principal carbon and energy sources supplied by the plant to bacteroids (Stowers, 1985).…”