Early recognition in the Rhizobium meliloti-alfalfa symbiosis: root exudate factor stimulates root adsorption of homologous rhizobia

Abstract

Adsorption of Rhizobium meliloti to alfalfa roots before their infection and nodule formation shows the specificity of the symbiotic association (G. Caetano-Anolles and G. Favelukes, Appl. Environ. Microbiol. 52:377-382, 1986). The time course of specific adsorption of R. meliloti (103 to 104 cells per ml) to roots shows an initial lag period of 3 h, suggesting that either or both symbionts must become conditioned for the adsorption process. Preincubation of R. meliloti L5-30 for 3 h with dialyzed alfalfa root exudate (RE) markedly increased early adsorption of rhizobia to alfalfa roots. The activity in RE was linked to a nondialyzable, thermolabile, trypsin-sensitive factor(s), very different from the root-exuded flavonoid compounds also involved in early Rhizobium-legume interactions. The lack of activity in the RE from plants grown in 5 mM N03 suggested its negative regulation by the nitrogen nutritional status of the plant. Preincubation of R. meliloti with heterologous clover RE did not stimulate adsorption of rhizobial cells to roots. A short pretreatment of RE with homologous (but not heterologous) strains eliminated the stimulatory activity from solution. The stimulation of adsorption of R. meliloti to alfalfa roots was strongly dependent on the growth phase of the rhizobia, being greater at the late exponential stage. Nevertheless, the capacity of R. meliloti L5-30 to eliminate from solution the stimulatory activity in RE appeared to be constitutive in the rhizobia. The low concentration of rhizobial cells used in these experiments was critical to detect the stimulation of adsorption. The early interaction of spontaneously released alfalfa root macromolecular factor(s) and free-living R. meliloti, which shows the specificity and regulatory properties characteristic of infection and nodulation, would be an initial recognition event in the rhizosphere which triggers the process of symbiotic association.Soil bacteria of the genera Rhizobium and Bradyrhizobium associate symbiotically with roots of leguminous plants to fix nitrogen. In the process of the symbiotic association, bacteria infect the roots through their root hairs and participate in the joint formation of a specialized root structure, the nodule, within which the rhizobia, differentiated into bacteroids, utilize plant-provided carbon compounds and reduce atmospheric nitrogen for its assimilation by the plant (2, 29).A remarkable feature of these associations is their high degree of selectivity, whereby only certain pairs of rhizobial strains and legume species are able to form effective nodules. As an example, Rhizobium meliloti infects the roots of alfalfa and R. trifolii infects the roots of white clover, but crosswise pairings are not compatible.Starting with the young growing root as it penetrates the soil populated by free-living rhizobia, the early events of the symbiotic process-collectively designated as preinfection-take place with the bacterial cells still outside the root tissues: they comprise rhizobial chemotactic approach to the ...

Stimulation of adhesiveness, infectivity, and competitiveness for nodulation of Bradyrhizobium japonicum by its pretreatment with soybean seed lectin

“…SBL is released into the soybean root exudate [14,15], as AAG [13] and PHA (A. Lodeiro, 1994, Ph.D. Thesis, UNLP). However, a role for this lectin in establishing a bridge between the plant and bacterial surfaces [30] seems unlikely, since: (1) stimulation occurred with a very small amount of SBL molecules per bacterium (see Fig.…”

“…YMB-grown rhizobia were pretreated for 72 h (unless otherwise indicated) in N-free Fa ®hraeus solution at 28³C with no shaking at a density of ca. 10 7 CFU ml 31^a ccording to the protocol of Halverson and Stacey [5,14]^in the presence of either di¡erent lectins or samples from the SBL puri¢cation procedure; control rhizobia were always pretreated in the same conditions, without protein. After this pretreatment period, rhizobia were diluted about 1U10 3 into fresh Fa ®hraeus solution to study their adhesiveness, infectivity, or competitiveness.…”

“…However, a root-exuded protein that stimulates S. meliloti adhesiveness was described [12] and a preliminary indication suggested that this might be the alfalfa agglutinin (AAG) [13]. Both the alfalfa root-exuded protein, and SBL are present at very low concentration in the respective root exudates [12^15] and thus a dilute rhizobial population as well as a prolonged preincubation time before exposure of rhizobia to plants are required in order to observe a signi¢cant stimulatory e¡ect on adhesiveness [12] or infectivity [5,14]. Moreover, stimulation of infectivity by SBL required de novo RNA and protein synthesis in the rhizobia during the preincubation time [5].…”

Improved Soybean Root Association of N-Starved Bradyrhizobium japonicum

“…Total counts of visible microcolonies on all primary roots, expressed as the percentage of the total number of CFU present during the incubation, represent the adsorption index (%A). Stimulation of adsorption is the difference in %A between protein-treated and control rhizobia as a percentage of the %A of control rhizobia (30,55).…”

“…In addition, some plant lectins, such as soybean lectin (SBL), are released into the rhizosphere (52,55) and specifically stimulate rhizobial adsorption and infection (30,55). Although the mechanism of plant lectin action remains obscure, its role in restricting rhizobium-host specificity range was demonstrated in studies with transgenic plants (15,52).…”