Alfalfa root nodule invasion efficiency is dependent on Sinorhizobium meliloti polysaccharides

Abstract

The soil bacterium Sinorhizobium meliloti is capable of entering into a nitrogen-fixing symbiosis with Medicago sativa (alfalfa). Particular low-molecular-weight forms of certain polysaccharides produced by S. meliloti are crucial for establishing this symbiosis. Alfalfa nodule invasion by S. meliloti can be mediated by any one of three symbiotically important polysaccharides: succinoglycan, EPS II, or K antigen (also referred to as KPS). Using green fluorescent protein-labeled S. meliloti cells, we have shown that there are significant differences in the details and efficiencies of nodule invasion mediated by these polysaccharides. Succinoglycan is highly efficient in mediating both infection thread initiation and extension. However, EPS II is significantly less efficient than succinoglycan at mediating both invasion steps, and K antigen is significantly less efficient than succinoglycan at mediating infection thread extension. In the case of EPS II-mediated symbioses, the reduction in invasion efficiency results in stunted host plant growth relative to plants inoculated with succinoglycan or K-antigen-producing strains. Additionally, EPS II- and K-antigen-mediated infection threads are 8 to 10 times more likely to have aberrant morphologies than those mediated by succinoglycan. These data have important implications for understanding how S. meliloti polysaccharides are functioning in the plant-bacterium interaction, and models are discussed.

FIG. 1

(A) Structure of the succinoglycan repeating unit. The asterisk indicates the position of a second succinyl modification (45) in some repeating units. (B) The structure of the EPS II repeating unit. (C) The structure of the K-antigen repeating unit. Pse, pseudaminic acid. (D) Schematic diagram of a curled root hair (CRH) and the three indices scored in GFP-based plant assays: a colonized curled root hair (CCRH), a colonized curled root hair with initiated infection thread (IT), and a colonized curled root hair with extended infection thread (adapted from reference 8).

FIG. 2

Kinetics of colonized curled root hair (CRH) formation, infection thread (IT) initiation, and infection thread extension on alfalfa plants inoculated with various S. meliloti strains. Sets of at least 36 plants were inoculated with Rm1021 (succinoglycan [SG]-producing strain) or Rm9000 (EPS II-producing strain) (A) or with Rm41 (succinoglycan- and K-antigen [KPS]-producing strain) or AK631 (K-antigen-producing strain) (B). The numbers of colonized curled root hairs, initiated infection threads, and extended infection threads per plant were recorded for 12 days. The colonized curled root hair count includes those with initiated and extended infection threads, and the initiated infection thread count includes extended infection threads. Standard error of the mean calculations were performed with the mean daily values from at least 3 groups of 12 plants. The standard errors for the time points were nonoverlapping.

FIG. 3

Efficiencies of nodule invasion by various S. meliloti strains. Sets of at least 36 plants were inoculated with different strains and scored on day 12 for numbers of colonized curled root hairs (CCRHs), numbers of initiated infection threads (ITs), and numbers of extended infection threads. (A) The relative efficiencies of colonized curled root hair formation were computed by normalizing the numbers of colonized curled root hairs to that of Rm1021 (for Rm7210, Rm9000, and Rm9011) or Rm41 (for AK631 and PP674). (B) The efficiency of infection thread initiation is the percentage of curled root hairs colonized by a particular strain that initiate an infection thread. (C) The efficiency of infection thread extension is the percentage of curled root hairs colonized by a particular strain that have infection threads that are extended (reach the base of the root hair cell). The error bars represent the standard errors of the means computed using the mean values from at least three groups of 12 plants.

FIG. 4

Fluorescence microscopy analyses of infection thread formation mediated by various S. meliloti polysaccharides. All images are composite images of GFP-expressing S. meliloti cells (green) and root hair cells (red). (A) Typical succinoglycan-mediated extended infection thread formed by Rm1021. The infection thread extends from the colonized, curled root hair to the base of the root hair cell. (B) Aborted succinoglycan-mediated infection thread with a densely packed pocket of bacteria near the terminus. (C) Colonized, curled root hair formed by Rm7210, an exoY210::Tn5 mutant of Rm1021 that fails to produce a symbiotically active polysaccharide. (D and E) Aborted, aberrant EPS II-mediated infection threads present on plants inoculated with Rm9000. (F and G) Extended, aberrant, K-antigen-mediated infection threads on plants inoculated with AK631.

FIG. 5

Models for the perception of succinoglycan (SG), EPS II, and K antigen (KPS) by a host plant and subsequent signal transduction resulting in nodule invasion by S. meliloti. (A) A three-polysaccharide, one-receptor model. (B) A three-polysaccharide, three-receptor model in which the three signal transduction pathways feed into a common signal transduction pathway. (C) A three-polysaccharide, three-receptor model in which the three signal transduction pathways are independent of one another.