Succinoglycan is required for initiation and elongation of infection threads during nodulation of alfalfa by Rhizobium meliloti

Abstract

Rhizobium meliloti Rm1021 must be able to synthesize succinoglycan in order to invade successfully the nodules which it elicits on alfalfa and to establish an effective nitrogen-fixing symbiosis. Using R. meliloti cells that express green fluorescent protein (GFP), we have examined the nature of the symbiotic deficiency of exo mutants that are defective or altered in succinoglycan production. Our observations indicate that an exoY mutant, which does not produce succinoglycan, is symbiotically defective because it cannot initiate the formation of infection threads. An exoZ mutant, which produces succinoglycan without the acetyl modification, forms nitrogen-fixing nodules on plants, but it exhibits a reduced efficiency in the initiation and elongation of infection threads. An exoH mutant, which produces symbiotically nonfunctional high-molecular-weight succinoglycan that lacks the succinyl modification, cannot form extended infection threads. Infection threads initiate at a reduced rate and then abort before they reach the base of the root hairs. Overproduction of succinoglycan by the exoS96::Tn5 mutant does not reduce the efficiency of infection thread initiation and elongation, but it does significantly reduce the ability of this mutant to colonize the curled root hairs, which is the first step of the invasion process. The exoR95::Tn5 mutant, which overproduces succinoglycan to an even greater extent than the exoS96::Tn5 mutant, has completely lost its ability to colonize the curled root hairs. These new observations lead us to propose that succinoglycan is required for both the initiation and elongation of infection threads during nodule invasion and that excess production of succinoglycan interferes with the ability of the rhizobia to colonize curled root hairs.

FIG. 1

Fluorescence microscopy analyses of nodule invasion by GFP-expressing cells of the wild-type R. meliloti strain Rm1021 and its succinoglycan (exo) mutants. (A) Composite image of a colonized curled root hair (red) with an extended infection thread filled with the wild-type bacterial cells (green). The infection thread started from the Shepherd’s crook of the curled root hair and has reached the base of the root hair. (B, C, and F) Images of a curled root hair that has been colonized by the exoY210 mutant showing a composite image of a curled root hair with a fluorescent bacterial colony (green) in the Shepherd’s crook (B), the Shepherd’s crook of the curled root hair (C), and the composite phase-contrast and fluorescence image, which demonstrated that there is no infection thread inside the root hair (F). (D) Composite image of a root hair colonized by the exoZ341 mutant, which had developed an aborted infection thread. The infection thread started from a very small colony inside the curled root hair and aborted in the middle of the root hair with a swollen end. (E) Composite image of a curled root hair colonized by the exoZ341 mutant showing a large fluorescent bacterial colony in the middle of a curled root hair. No infection thread can be seen. (G) Fluorescence image of a nodule from a plant inoculated with GFP-expressing wild-type strain Rm1021 showing the outline of the nodule and individual nodule epidermal cells. (H) Fluorescence image of a nodule from a plant inoculated with GFP-expressing cells of the exoY mutant showing patches of bacterial cells on the surface of the nodule and bright bacterial colonies within the nodule epidermis. Images of root hairs (A to E) were photographed with a magnification of ×400. Images of root nodules (G and H) were photographed with a magnification of ×100.

FIG. 2

Schematic representation of invading R. meliloti bacterial cells that were blocked at different stages of infection thread formation, showing a curled root hair (Shepherd’s crook), a colonized curled root hair, a colonized curled root hair with an initiated infection thread, and a colonized curled root hair with an infection thread extending to the base of the root hair.

FIG. 3

Kinetics of infection thread (IT) formation on alfalfa plants inoculated with the wild type or the exoY mutant. Sets of 24 plants were inoculated with the wild-type strain or the exoY210 mutant. The average number of colonized curled root hairs (CRH) (A), initiated infection threads (B), and extended infection threads (C) were recorded daily for 12 days. The colonized CRH includes those with initiated and extended infection threads and initiated infection threads include extended infection threads.

FIG. 4

Efficiency of nodule invasion by the wild-type strain, Rm1021, and exo mutants. Sets of 24 plants were each inoculated with different strains and scored on day 10 for the number of colonized curled root hairs (CRH) (A), initiation of infection thread (IT) formation (B), and elongation of infection threads (C). (A) The relative efficiency of colonization of the curled root hairs was calculated by comparing the number of curled root hairs colonized by a mutant per plant to that colonized by the wild-type strain. The wild-type strain is considered 100% efficient in colonizing curled root hairs. (B) The efficiency of initiation of infection thread formation is the percentage of curled root hairs colonized by a given strain that initiate infection thread formation. (C) The efficiency of infection thread elongation is the percentage of curled root hairs colonized by a given strain that develop extended infection threads.