Infection and invasion of roots by symbiotic, nitrogen-fixing rhizobia during nodulation of temperate legumes

Abstract

Bacteria belonging to the genera Rhizobium, Mesorhizobium, Sinorhizobium, Bradyrhizobium, and Azorhizobium (collectively referred to as rhizobia) grow in the soil as free-living organisms but can also live as nitrogen-fixing symbionts inside root nodule cells of legume plants. The interactions between several rhizobial species and their host plants have become models for this type of nitrogen-fixing symbiosis. Temperate legumes such as alfalfa, pea, and vetch form indeterminate nodules that arise from root inner and middle cortical cells and grow out from the root via a persistent meristem. During the formation of functional indeterminate nodules, symbiotic bacteria must gain access to the interior of the host root. To get from the outside to the inside, rhizobia grow and divide in tubules called infection threads, which are composite structures derived from the two symbiotic partners. This review focuses on symbiotic infection and invasion during the formation of indeterminate nodules. It summarizes root hair growth, how root hair growth is influenced by rhizobial signaling molecules, infection of root hairs, infection thread extension down root hairs, infection thread growth into root tissue, and the plant and bacterial contributions necessary for infection thread formation and growth. The review also summarizes recent advances concerning the growth dynamics of rhizobial populations in infection threads.

FIG. 1.

Overview of the nodulation process in plants that form indeterminate nodules. (A) One form of Nod factor synthesized by S. meliloti. The upper arrow indicates the acetyl group added by NodL, and the lower arrow indicates the lipid moiety, the length and degree of saturation of which is modified by NodF and NodE. (B) Diagrammatic cross section of a root, showing gradients of an activating factor at protoxylem poles (blue) and an inhibitor at protophloem poles (red). Together such gradients may determine which root cells can become activated in response to infecting rhizobia. Nodules are typically formed next to the protoxylem poles, which are at the ends of the Y-shaped structure depicted in the center the diagram, rather than above the protophloem poles, which are depicted as ovals. (C) An epidermal cell and two underlying outer cortical cells. The epidermal cell has a nucleus positioned across from the place where a new root hair will form. (D) Root hair initiation in the epidermal cell. (E) Binding of a rhizobial cell to a type I root hair and activation of the underlying cortical cells in response to Nod factor. (F) Continued growth of the root hair, shown as stage II. (G) Curling of the stage II root hair under the influence of Nod factor and growth of a rhizobial microcolony in the curl. The underlying cortical cells have become polarized, and cytoplasmic bridges (PITs) have formed and are shown in grey. (H) Infection thread initiation. (I) Growth of the infection thread down the root hair. The nucleus moves down the root hair in front of the thread. (J) Fusion of the infection thread with the epidermal cell wall and growth of rhizobia into the intracellular space between the epidermal cell and the underlying cortical cell. (K) Growth of the infection thread through PITs in the outer cortical cells. (L) Enlarged view of the root hair shown in panel I. The curl has been unrolled to show that topologically, the bacteria in the infection thread are still outside the root hair. The plant cell wall and plant cell membrane are shown as black and dashed lines, respectively. Microtubules are located between the nucleus and the infection thread tip (blue). Actin cables are depicted as orange strands. These are likely to be found where indicated in the diagram because cytoplasmic streaming is seen in these areas during the progression of infection threads down root hairs. Bacteria are topologically outside the root until they later bud from the tip of the thread and enter nodule cells as membrane-enclosed bacteria. (M) Diagram showing root tissues and a young nodule not yet emerged from the root. The derivation of nodule tissues from root tissues is indicated. (N) A longitudinal section of 10-day-old alfalfa nodule. The nodule was infected with GFP-expressing S. meliloti, and the infection thread network can be seen behind the meristem region of the nodule. The initial infection site that gave rise to the bacteria in the nodule can be seen on the nodule periphery at the left. Propidium iodide (red) was used to counterstain the plant tissue. The root from which the nodule emerged is seen in cross-section at the right.

FIG. 2.

Root hair morphology on uninoculated and inoculated roots. (A to C) Typical root hairs from zones I, II, and III, respectively, of an uninoculated alfalfa plant. (D) Diagram of an alfalfa seedling, showing the locations of root hair zones I, II, and III. (E to G) Photographs showing how root hair responses to S. meliloti can vary along the length of a single root. The three images were taken from a single inoculated seedling at the locations indicated on the central diagram.

FIG. 3.

Infection threads in alfalfa root hairs. (A) A growing infection thread, with an active column of cytoplasm between the nucleus (arrow) and the tip region of the thread (arrowhead). The actual tip of the thread cannot be discerned because it is obscured by the cytoplasmic column or because it has not yet been enclosed with thick, mature cell wall. The inset shows the nucleus and its associated column of active cytoplasm. (B) An infection thread that has terminated growth. Note that the tip is easily discerned and the tip region has only very thin cytoplasmic streams connected to it (inset).

FIG. 4.

Infection threads induced by an exoF mutant of S. meliloti. (A) Two swollen and misshapen infection threads induced by a GFP-expressing exoF mutant of S. meliloti. Arrows point to sections of the threads that are particularly swollen. (B) Another example of an infection thread induced by the exoF mutant. For infection threads induced by wild-type S. meliloti on alfalfa, see Fig. 5.

FIG. 5.

Examples of mixed infection threads following coinoculation of alfalfa with red- and green-fluorescent bacteria. (A) Infection threads containing only gfp-expressing or DsRed-expressing S. meliloti. (B) A sectored infection thread in which the mixed population gave rise to a series of sectors which increase in length along the thread. The tipmost sector advanced into the epidermal cell body, branched (top arrow), and penetrated the underlying cell (bottom arrow), leaving the distal sectors behind. (C) A root hair infected with dual infection threads. Each thread contains sectors of gfp-expressing and DsRed-expressing bacteria. (D) A jumbled type of mixed infection thread. gfp-expressing and DsRed-expressing cells appear randomly mixed throughout the infection thread. (E) Dual infection threads inside a root hair. The threads began as jumbled-type threads but later gave rise to green sectors at their tips (arrows show the sectors in each thread). (Reprinted from reference .)

FIG. 6.

Model of infection thread growth. (A). A computer model was used to simulate sectored infection thread growth. For these cases, it was assumed that the thread started with six alternating bright and dark sectors of equal size. The doubling time for the growing bacteria (those 60 μm or less from the growing tip of the thread) was 4 h. Each bar is a representation of the length of the thread and its six sectors at each time point. Growth of the thread is exponential until it reaches 60 μm in length (dashed line) and becomes linear thereafter. Once the tipmost sector is more than 60 μm in length, it will be the only sector which continues to increase in size (see the last bar as an example). (B) Same as panel A, except the size of the growth zone is 20 μm. The thread grows at the same rate as for panel A while the thread is less than 20 μm long. After that, only cells in the tipmost 20 μm contribute to subsequent growth, and the overall rate of thread extension is lower than in the case where the growth zone is 60 μm long. (C). Same as panel A, except bacteria entering the thread are not assumed to have strictly alternated in terms of fluorescence. The entry pattern shown was dark-light-dark-dark-dark-light. This gives a total of four sectors, with the large dark sector in the middle of the thread having arisen from the loading of three dark cells in a row.

FIG. 7.

Architecture of the infection thread network inside a 10-day-old alfalfa nodule. An alfalfa nodule, induced by wild-type S. meliloti strain Rm1021, was fixed, embedded, and sliced into 1-μm-thick sections. Fifteen sections were dyed to reveal the bacteria and plant cell structures, photographed, and reassembled into a three-dimensional volume. (A) Projection of all 15 sections onto a single plane. The nuclei in each section are outlined in red, and the infection threads are filled in with green. The infection thread network appears to be polarized and growing toward the meristem, which is in the left side of the image. (B) Three-dimensional reconstruction of the nuclei and infection threads, showing a volume from the central part of the data set shown in panel A.