Identifying abnormalities in symbiotic development between Trifolium spp. and Rhizobium leguminosarum bv. trifolii leading to sub-optimal and ineffective nodule phenotypes.

BACKGROUND AND AIMS: Legumes overcome nitrogen limitations by entering into a mutualistic symbiosis with N(2)-fixing bacteria (rhizobia). Fully compatible associations (effective) between Trifolium spp. and Rhizobium leguminosarum bv. trifolii result from successful recognition of symbiotic partners in the rhizosphere, root hair infection and the formation of nodules where N(2)-fixing bacteroids reside. Poorly compatible associations can result in root nodule formation with minimal (sub-optimal) or no (ineffective) N(2)-fixation. Despite the abundance and persistence of strains in agricultural soils which are poorly compatible with the commercially grown clover species, little is known of how and why they fail symbiotically. The aims of this research were to determine the morphological aberrations occurring in sub-optimal and ineffective clover nodules and to determine whether reduced bacteroid numbers or reduced N(2)-fixing activity is the main cause for the Sub-optimal phenotype. METHODS: Symbiotic effectiveness of four Trifolium hosts with each of four R. leguminosarum bv. trifolii strains was assessed by analysis of plant yields and nitrogen content; nodule yields, abundance, morphology and internal structure; and bacteroid cytology, quantity and activity. KEY RESULTS: Effective nodules (Nodule Function 83-100 %) contained four developmental zones and N(2)-fixing bacteroids. In contrast, Sub-optimal nodules of the same age (Nodule Function 24-57 %) carried prematurely senescing bacteroids and a small bacteroid pool resulting in reduced shoot N. Ineffective-differentiated nodules carried bacteroids aborted at stage 2 or 3 in differentiation. In contrast, bacteroids were not observed in Ineffective-vegetative nodules despite the presence of bacteria within infection threads. CONCLUSIONS: Three major responses to N(2)-fixation incompatibility between Trifolium spp. and R. l. trifolii strains were found: failed bacterial endocytosis from infection threads into plant cortical cells, bacteroid differentiation aborted prematurely, and a reduced pool of functional bacteroids which underwent premature senescence. We discuss possible underlying genetic causes of these developmental abnormalities and consider impacts on N(2)-fixation of clovers.

Visualization of symbiotic tissue in intact root nodules of Vicia tetrasperma using GFP-marked Rhizobium leguminosarum bv. viciae.

In rhizobial symbiosis with legume plant hosts, the symbiotic tissue in the root nodules of indeterminate type is localized to the basal part of the nodule where the symbiotic zones contain infected cells (IC) interspersed with uninfected cells (UC) that are devoid of rhizobia. Although IC are easily distinguished in nodule sections using standard histochemical techniques, their observation in intact nodules is hampered by nodule tissue characteristics. Tagging of Rhizobium leguminosarum bv. viciae strain 128C30 with a constitutively expressed gene for green fluorescent protein (nonshifted mutant form cycle3) in combination with the advantages of the tiny nodules formed by Vicia tetrasperma (L.) SCHREB . allowed for vital observation of symbiotic tissue using fluorescence microscopy. Separation of a red-shifted background channel and digital image stacking along z-axis enabled us to construct a nodule image in a classical fluorescence microscopy of nodules exceeding 1 mm in diameter. In parallel, visualization of nodule bacteria inside the symbiotic tissue by confocal microscopy at the excitation wavelength 488 nm clearly distinguished IC/UC pattern in the nodule virtual sections and revealed red-shifted fluorescence of nonrhizobial origin. This signal was located on the periphery of IC and increased with their degradation, thus suggesting accumulation of secondary metabolites, presumably flavonoids. The simultaneous detection of bacteria and secondary metabolites can be used for monitoring changes to intact nodule physiology in the model legumes. The advantage of V. tetrasperma as a suggested laboratory model for pea cross-inoculation group has been demonstrated.

Rhizobium cellulase CelC2 is essential for primary symbiotic infection of legume host roots.

The rhizobia-legume, root-nodule symbiosis provides the most efficient source of biologically fixed ammonia fertilizer for agricultural crops. Its development involves pathways of specificity, infectivity, and effectivity resulting from expressed traits of the bacterium and host plant. A key event of the infection process required for development of this root-nodule symbiosis is a highly localized, complete erosion of the plant cell wall through which the bacterial symbiont penetrates to establish a nitrogen-fixing, intracellular endosymbiotic state within the host. This process of wall degradation must be delicately balanced to avoid lysis and destruction of the host cell. Here, we describe the purification, biochemical characterization, molecular genetic analysis, biological activity, and symbiotic function of a cell-bound bacterial cellulase (CelC2) enzyme from Rhizobium leguminosarum bv. trifolii, the clover-nodulating endosymbiont. The purified enzyme can erode the noncrystalline tip of the white clover host root hair wall, making a localized hole of sufficient size to allow wild-type microsymbiont penetration. This CelC2 enzyme is not active on root hairs of the nonhost legume alfalfa. Microscopy analysis of the symbiotic phenotypes of the ANU843 wild type and CelC2 knockout mutant derivative revealed that this enzyme fulfils an essential role in the primary infection process required for development of the canonical nitrogen-fixing R. leguminosarum bv. trifolii-white clover symbiosis.

Mutagenesis of the carboxy terminal protease CtpA decreases desiccation tolerance in Rhizobium leguminosarum.

To better understand the role of proteases in Rhizobium leguminosarum biovar viciae, a gene with homology to the carboxy-terminal protease (CtpA), which belongs to a novel group of serine proteases, was studied. The ctpA gene was cloned and mutated using allelic exchange and a gusA reporter gene was used to study ctpA expression. Mutational analysis shows that ctpA is critical for the viability of R. leguminosarum when cells are grown on complex semi-solid media but is dispensable when cells are grown in complex liquid media and that this is likely due to an increase in susceptibility to desiccation on semi-solid media. The ctpA mutant also displayed an increased sensitivity to detergents, indicating an alteration in the permeability of the cell envelope. This is the first characterization of a ctpA gene within the Rhizobiaceae and the first report of a ctpA mutant that exhibits an increased sensitivity to desiccation.

Investigations of Rhizobium biofilm formation.

The development of nitrogen-fixing nodules of the Rhizobium-legume symbiosis, especially the early stages of root hair deformation and curling, infection thread formation, and nodule initiation, has been well studied from a genetic standpoint. In contrast, the factors important for the colonization of surfaces by rhizobia, including roots-an important prerequisite for nodule formation-have not been as thoroughly investigated. We developed conditions for analyzing the ability of two fast-growing rhizobia, Sinorhizobium meliloti and Rhizobium leguminosarum bv. viciae, to produce biofilms on abiotic surfaces such as glass, plastic microtiter plates, sand and soil as a prelude to characterizing the genes important for aggregation and attachment. Factors involved in adherence to abiotic surfaces are likely to be used in rhizobial attachment to legume root cells. In this report, we show that S. meliloti exopolysaccharide-deficient mutants as well as exopolysaccharide overproducers exhibit reduced biofilm phenotypes that show parallels with their nodulation abilities. We also investigated two flagella-less S. meliloti mutants and found them to have reduced biofilming capabilities. To investigate whether there was a symbiotic phenotype, we tested one of the Fla- mutants on two different S. meliloti hosts, alfalfa and white sweetclover, and found that nodule formation was significantly delayed on the latter.

Use of differential fluorescence induction and optical trapping to isolate environmentally induced genes.

The techniques of differential fluorescence induction (DFI) and optical trapping (OT) have been combined to allow the identification of environmentally induced genes in single bacterial cells. Designated DFI-OT, this technique allows the in situ isolation of genes driving the expression of green fluorescent protein (Gfp) using temporal and spatial criteria. A series of plasmid-based promoter probe vectors (pOT) was developed for the construction of random genomic libraries that are linked to gfpUV or egfp. Bacteria that do not express Gfp on laboratory medium (i.e. non-fluorescent) were inoculated into the environment, and induced genes were detected with a combined fluorescence/optical trapping microscope. Using this selection strategy, rhizosphere-induced genes with homology to thiamine pyrophosphorylase (thiE) and cyclic glucan synthase (ndvB) were isolated. Other genes were expressed late in the stationary phase or as a consequence of surface-dependent growth, including fixND and metX, and a putative ABC transporter of putrescine. This strategy provides a unique ability to combine spatial, temporal and physical information to identify environmental regulation of bacterial gene expression.

[Study on intergenera fusion of protoplasts from Rhizobium leguminosorum and Sinorhizobium xinjiangnesis].

Penicillin and chloromycetin were regarded as the sign of resistance to antibodies of R. leguminosorum USDA2370 and S. xinjiangnesis CCBAU110 respectively. Using the protoplast fusion technique, USDA2370 and CCBAU110 were successfully fused. Fusion hybrid can inoculate in the leguminous of parental strains respectively. There were apparent differences between parents and fusion hybrid in cell morphology, colony and pattern of whole-cell protein. The values of DNA homology between fusion hybrid and USDA2370 and CCBAU110 were 56.6% and 10.2% respectively.

Adaptation to nutrient starvation in Rhizobium leguminosarum bv. phaseoli: analysis of survival, stress resistance, and changes in macromolecular synthesis during entry to and exit from stationary phase.

The nitrogen-fixing bacterium Rhizobium leguminosarum bv. phaseoli often has to survive long periods of starvation in the soil, when not in a useful symbiotic relationship with leguminous plants. We report that it can survive carbon, nitrogen, and phosphorus starvation for at least 2 months with little loss of viability. Upon carbon starvation, R. leguminosarum cells were found to undergo reductive cell division. During this period, they acquired the potential for long-term starvation-survival, levels of protein, DNA, and RNA synthesis were decreased to base levels, and pool mRNA was stabilized. The starved cells are ready to rapidly restart growth when nutrients become available. Upon addition of fresh nutrients, there is an immediate increase in the levels of macromolecular synthesis, pool mRNA destabilizes, and the cultures enter exponential growth within 5 to 8 h. The starved cells were cross-protected against pH, heat, osmotic, and oxidative shock. These results provide evidence for a general starvation response in R. leguminosarum similar to that previously found in other bacteria such as Escherichia coli and Vibrio sp.