339Development 125, 339-349 (1998)Printed in Great Britain © The Company of Biologists Limited 1998DEV0146

Nod factor internalization and microtubular cytoskeleton changes occur

concomitantly during nodule differentiation in alfalfa

Antonius C. J. Timmers, Marie-Christine Auriac, Françoise de Billy and Georges Truchet*

Laboratoire de Biologie Moléculaire des Relations Plantes-Microorganismes, CNRS-INRA, BP 27, 31326 Castanet-TolosanCédex, France*Author for correspondence (e-mail: truchet@toulouse.inra.fr)

Accepted 20 November 1997: published on WWW 13 January 1998

Reorganization of the plant cytoskeleton is thought to playan important role during nodule ontogeny. In situimmunolocalisation of tubulin reveals that importantcytoskeletal changes, implying a transient disorganizationfollowed by a newly patterned reorganization, occur inindeterminate and determinate nodules. In alfalfa nodules,cytoskeletal changes closely parallel the symbioticdifferentiation features related to cell infection, bacterialrelease, endopolyploidization, cell enlargement, cell spatial

organization and organelle ultrastructure and positioning.Moreover, the fact that microtubule disorganization can becorrelated with Nod factor internalization in centralinfected cells suggests that Nod factors are possiblyinvolved in the control of cytoskeletal changes which directthe differentiation of bacteria-containing cells.

Key words: Microtubular cytoskeleton, Nod factors, Noduledifferentiation, Rhizobium meliloti, Alfalfa

SUMMARY

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INTRODUCTION

The symbiotic interactions between soil bacteria of the genRhizobium, Azorhizobium or Bradyrhizobium (here referreto as rhizobia) and plants of the Leguminosae family resultthe formation of nodules, new organs in which the bactereduce nitrogen into ammonia which can subsequently utilized by the plant.

Nodule organogenesis starts with a molecular dialogbetween symbionts and takes place through a seriesdevelopmental stages. Rhizobia produce Nod factors (NFs),synthesis of which is under the control of nodulation (nod)genes which are transcribed in the presence of plant flavonoChemical studies have shown that NFs from all rhizobspecies are lipochitooligosaccharides consisting of a backbof N-acetylglucosamine residues which is decorated on its tterminal residues (reviewed by Dénarié et al., 1996; Lon1996; Schultze and Kondorosi, 1996; Spaink, 1996). Earhizobial species possesses a characteristic set of nod genesthat specifies the length of the backbone and the nature ofdecorations at both ends of the molecule, thus making the Nspecific for a given plant host (Roche et al., 1991). NFs signal molecules involved in most of the early developmenresponses which are elicited by the corresponding bacteEarly rhizobia-dependent responses lead to various celluevents such as root hair induction and deformations, plinfection by means of tubular structures called infectiothreads and the formation of a nodule meristem whose actiensures nodule growth (reviewed by Newcomb, 1981; Brew1991; Roth and Stacey, 1991; Hirsch, 1992; Kijne, 1992).

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In legumes such as alfalfa and vetch, the nodule meristremains active for several weeks, thus leading to the formatof elongated indeterminate nodules comprising central aperipheral tissues. Histologically, central tissues are organizedinto five well-defined zones: the apical meristematic zone I, tprefixing (infection) zone II, the starch-rich interzone II-III, thenitrogen-fixing zone III and the proximal senescent zone IThese central zones are surrounded by a parenchyma, vascbundles, an endodermis and a cortex (Vasse et al., 1990). differentiation of the central cells depends on whether the hcells are invaded or remain bacteria-free. Fully differentiateinvaded cells are dramatically enlarged in size, highpolyploid and filled with nitrogen-fixing bacteroids (Truchet1978; Vasse et al., 1990). In contrast, non-invaded cells aresmall size and their DNA content remains monosomat(Truchet, 1978). Different developmental features characterdeterminate nodules formed, for example, on the roots siratro or soybean. In such nodules, meristematic activity transient and the central cells differentiate almosimultaneously (Newcomb, 1981). As a result, round-shapmature determinate nodules increase in size by cenlargement and all central cells are more or less at simstages of differentiation.

The plant cytoskeleton mediates several functions in livincells (reviewed by Seagull, 1989; Cyr and Palevitz, 1995). Tcytoskeleton is dynamic in the sense that it continualrearranges by virtue of its two major componentsmicrofilaments and microtubules which assemble andisassemble in response to extracellular and intracellustimuli. This property can be correlated with fundament

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cellular traits that the cytoskeleton is thought to regulate, sas, for example, cell division, cell shape and polarity, ctrafficking and the spatial organization of the cytoplas(reviewed by Seagull, 1989; Goddard et al., 1994). Ainvolvement of the plant cytoskeleton during early stagesnodulation has been suggested by studies showing cytoskeletal reorganizations occur at the tip of root hairs treawith Nod factors (Allen et al., 1994) or in the root cortex Vicia hirsuta either infected by its specific symbion(Bakhuizen, 1988) or treated with Nod factors (Van Spronset al., 1995). Moreover, if one considers the general rolesthe cytoskeleton in the context of symbiotic responses sucroot hair induction and deformation (Ardourel et al., 1994activation of cortical cells (Van Brussel et al., 1992; Ardouret al., 1994) which enter the cell cycle but arrest in G2 (Yanget al., 1994), oriented growth of an infection network in platissues (Bakhuizen, 1988; Ridge, 1992; Van Brussel et 1992), formation of nodulation-related division cente(Truchet et al., 1991) and the cell enlargement and DNendopolyploidization typical of invaded central cells (Truche1978), then it is reasonable to anticipate that variations in cytoskeletal structure are likely to be involved in many of tsymbiosis-related steps directing nodule development.

Very little is known about the early molecular mechanismthrough which the host plant responds to rhizobial infectionNF treatment. The current hypothetical model proposes tNFs bind to plasmalemma-located receptors (Bono et al., 19Niebel et al., 1997), followed by subsequent signtransduction. Data indicate that Rhizobium melilotiNFs inducea depolarization of the plasma membrane potential (Ehrhaet al., 1992; Felle et al., 1995), cytoskeletal changes (Allenal., 1994) and calcium spiking (Ehrhardt et al., 1996) in alfaroot hairs. In contrast, it has been shown that Rhizobiumleguminosarumbv. trifolii NFs are internalized specifically intoclover root hairs (Philip-Hollingsworth et al., 1997). In thistudy, we show that the microtubular cytosketon (MCdramatically reorganizes in differentiating infected cells both indeterminate and determinate nodules. In alfalfa noduMC changes initiate in the nodule zone where rhizobial Nfactors are internalized in infected cells and strongly correlwith symbiosis-specific cell differentiation traits. Thus, Ninternalization, MC changes and cell differentiation are tighcoupled in the course of nodule development.

MATERIALS AND METHODS

Bacterial strains and plant assaysThe list of bacterial strains and plant species used in this study is gin Table 1. Respective bacterial and plant growth conditions weredescribed by the references listed in Table 1. Spontaneous andinduced nodulation in alfalfa were achieved as described by Trucet al. (1989) and Truchet et al. (1991), respectively.

Microscopic methodsNodules harvested at different developmental stages depending onodulation type were processed for histological or ultrastructuobservations as described by Vasse et al. (1990).

Immunolocalization of microtubulesIn situ visualization of the microtubular cytoskeleton was as followNodules were fixed in 4% formaldehyde (prepared fro

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paraformaldehyde) in microtubule stabilizing buffer (MSB) consistinof 60 mM Pipes, 10 mM EGTA, 1 mM MgCl2, 0.1% Triton X-100 and10% DMSO (pH 6.9, 30 minutes at room temperature) followed bysubsequent 30 minute fixation in 4% formaldehyde in phosphabuffered saline (PBS) pH 7.4. After rinsing with PBS, the samples winfiltrated with sucrose up to 1 M in PBS, and cut into 8 and 50 µmthick sections at −20°C, using a MICROM HM500 M cryostat.Sections were deposited on poly-L-lysine-coated slides or in smcontainers, and tubulin immunolocalized by subsequent incubatwith monoclonal anti-α-tubulin (Sigma T-5168) and an anti-mouseIgG-FITC antibody (Boehringer Mannheim, 821462). After staining Evans blue (0.1% in PBS), to quench autofluorescence, the labsections were mounted in glycerol containing 1% 1,4-diazabicyc(2.2.2) octane as an anti-fading agent and 4,6-diamidinophenylindole as a nuclear stain and viewed by laser confomicroscopy (Zeiss, LSM 410 Invert). Images were recorded eithfrom a single focal plane with an average thickness of 0.5 µm or as anextended focus in which several confocal planes were superimpoThe gain and offset were chosen in such a way that all the 255 gvalues were used resulting in 0 for the background (i.e. the slwithout section) and 255 for the maximum fluorescence in tspecimen. As a result of the chosen gain and offset, the scaling ofpseudo-color look up table is comparable for all images. Images wdisplayed, after background subtraction, as false colour imagindicating increasing fluorescence intensity on a colour scale rangfrom blue to red.

Immunolocalization of Nod factorsRabbit anti-NF polyclonal antibody was prepared by Biocyte(Marseille, France) using R. melilotipurified NodRm-IV(S,C16:2) asimmunogen. Immunserum was tested by ELISA according to standprocedures (Engvall and Perlmann, 1971). For immunocytochemisseveral protocols were tested. Optimal results regarding labeefficiency and preservation of cellular structure were obtained by fixithe specimen for 1 hour in 2% formaldehyde (prepared froparaformaldehyde) and 0.5% glutaraldehyde in 0.12 M cacodylate 7.2, or 2.5% glutaraldehyde in 0.2 M cacodylate pH 7.2 respectivfor light and electron microscopy. After subsequent dehydration wethanol and infiltration with LR white resin, polymerization waperformed at low temperature under UV irradiation. For labeling, 1 µmsemithin sections stuck onto poly-L-lysine-coated slides or ultrathsections picked up on nickel grids covered with formvar were fiincubated overnight at 4°C in 10% goat serum, 0.5% Triton X-100.5% Tween 20 in Tris-buffered saline (TBS) to prevent non-specbinding of antibodies. For immunolocalization at the histological levesections were incubated either overnight at 4°C or for 2 hours at 3with anti-NF immunserum diluted 1:50 in TBS. After rinsing in TBSincubation was continued with anti-rabbit IgG antibody:1 nm go(BioCell) for 1 hour at room temperature. The signal was amplified using the BioCell silver enhancing kit. Sections were then stainbriefly in 0.02% toluidine blue, mounted in DPX (BDH LaboratorieSupplies) and observed by bright field or dark field microscopy with Olympus Vanox light microscope. For immunolabeling at thultrastructural level, sections were incubated as described above the immunserum diluted 1:10 and an anti-rabbit IgG:20 nm gold wused as secondary antibody. Ultrathin sections were stained with uracetate and lead citrate and observed with a Hitachi H600 transmiselectron microscope.

Quantification of anti-Nod factor labeling intensityThe intensity of NF immunolabeling in different nodule zones wquantified by silver grain counting on labeled 1 µm thick semithinsections. The region of interest was identified under bright field optat 100× magnification and outlined on the screen. The quantity of ligreflected by each area was then measured under fluorescent illumination using a computer-based image analysis system accordto the procedure described by Blanchard et al. (1993) and conve

341NF internalization and cytoskeletal changes in nodules

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roid positioning in alfalfa nodules. Bright-field microscopy.of a 3 week-old wild-type nodule showing the meristematic zone I (smallall star), the interzone II-III (large asterisk) and the nitogen-fixing zone

nges in morphology of the invaded cells in zone II. Cells are small anderisk) and enlarge to become round-shaped in the proximal region (star). dots) at the cell periphery, and vacuolar parceling are seen. Bar, 25 µm.toplasm of invaded cells. Bacteroids are randomly orientated in proximalne II-III (large asterisk) and zone III (star), the outermost bacteroids arell wall, while those in a central location remain randomly distributed. Noteon at the interface zone II-interzone II-III. Bar, 100 µm.

Table 1. Plants and bacterial strains used in this studyPlants Bacteria Relevant characteristics* References

Medicago sativa Rhizobium melilotiRCR 2011 WT, IN Ardourel et al. 1994M. sativa R. melilotiGMI6390, Nod factor overproducing strain WT, IN Roche et al. 1991M. sativa R. melilotiGMI6371, Nod factor non producing strain Nod−, nodA− Roche et al. 1991M. sativa R. meliloti fixK− GMI942 Fix−, IN Foussard et al. 1997M. sativa R. meliloti exoA− Fix−, IN Leigh et al. 1985Trifolium repens R. leguminosarumbv trifolii ANU843 WT, IN Djordjevic et al. 1985Vicia sativa R. leguminosarumbv viciae248 WT, IN Van Brussel et al. 1986Macroptilium atropurpureum Rhizobium sp. NGR234 WT, DN Lewin et al. 1990

*DN, determinate nodule; Fix−, non-fixing nodule; IN, indeterminate nodule; WT, wild type.

into the number of grains per square µm. Background grain densitymeasured on the resin but out of the section was finally subtrafrom the grain density of each region to obtain the density per zdue to NF immunolabeling.

RESULTS

Variations in shape, cytoplasmic organization andultrastructural differentiation in invaded cells ofalfalfa nodulesSpeculating that cellular features occurring during nodugrowth might reflect cytoskeletal changes (see Introductiowe first studied, in detail, the histological and ultrastructudifferentiation and the cytoplasmic organization of central cein alfalfa nodules. The terminology used in this study describe the histological organization of alfalfa nodules and ultrastructural stages inbacteroid differentiation isas described by Vasse et al.(1990).

Invaded cells enlargeisodiametrically in the distalpart of zone II and becomeround shaped in proximalzone II, interzone II-III andzone III (Fig. 1A-C).Simultaneously, theclustering of cell organelles(plastids and mitochondria)and bacteroids at the cellperiphery, shows that amodification in spatialorganization takes place inzone II, particularly in theproximal part of this zone(Fig. 1B). Interestingly, ascells mature, the spatialpositioning of bacteroidschanges from a randomorientation in the cells of theprefixing zone II (Fig. 1B,C),to a precise positioningwhich depends on bacteroidlocation in the interzone II-IIIand zone III. In the two latterzones, the bacteroids locatedat the cell periphery are

Fig. 1.Cell morphology and bacte(A) Longitudinal semithin section asterisk), the prefixing zone II (smIII (large star). Bar, 50 µm. (B) Chaisodiametric in the distal part (astThe clustering of bacteroids (blue(C) Bacteroid positioning in the cyzone II (small asterisk). In interzoorientated perpendicular to the cethe increase in bacteroid elongati

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orientated perpendicular to the cell wall, whilst those in the cenremain randomly distributed within the cytoplasm (Fig. 1C).

The positioning and the ultrastructural differentiation of ceorganelles and of bacteroids were studied in more detail electron microscopy. Both plastids (as undifferentiateproplastids) and rod-shaped mitochondria were found to randomly distributed within the plant cytoplasm of bacteria-frecells observed either in the meristematic zone I or in the modistal border of zone II where the infection network develop(Fig. 2A). Progressive changes in ultrastructure and positioniof organelles occur after the release of bacteria along tprefixing zone II (Fig. 2B). First proplastids, and thenmitochondria increase in size (Fig. 2B) and in length (Fig. 2C,Dand move to the periphery of the cytoplasm where they orientparallel to, and in close contact with, the plasmalemmparticularly at intercellular spaces (Fig. 2E). Very shormicrotubules are often seen in the limited cytoplasmic spa

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between the organelles and the plasmalemma (Fig. 2F). similar way, actively dividing bacteroids (type I bacteroidVasse et al., 1990) and bacteroids which have ceased to dand have started to differentiate (type II bacteroids; Vasse e1990), also migrate to the cell periphery where they were srandomly orientated in the cytoplasm (Fig. 2C,E).

Fig. 2. Ultrastructure andpositioning of cell organellesand bacteroids in invaded cellsof zone II. Transmissionelectron microscopy. (A) Cellsat the distal border of zone IIin which cell organelles arerandomly distributed. Thearrowhead points to aninfection thread. Bar, 5 µm.(B) Bacterial release ofrhizobia at the unwalledextremity of an infectionthread. Remnants of the threadcell wall (arrows), releasedbacteroids (arrowheads) andenlarging plastids (white star)are seen. Bar, 2.5 µm.(C) Distal zone II. Progressiveclustering of plastids (largearrows), mitochondria (smallarrows) and type 2 bacteroids(lower cell) at the cellperiphery. Note the randompositioning of type 1bacteroids in the upper cell.Bar, 5 µm. (D) Detail of a cellin distal zone II, showing cellorganelle and bacteroidclustering at the periphery ofthe cell and abundantendoplasmic reticulum profiles(arrowheads) in the cytoplasm.Bar, 1 µm. (E) Cell organellesin proximal zone II. Note theelongation of plastids (arrows)parallel to and in close contactwith the cell wall andmitochondria (arrowheads)applied against plastids. Bar, 1µm. (F) Microtubules(arrowheads) at the interfacebetween a plastid (whiteasterik) and the cell wall (star).Bar, 1 µm.

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Changes continued in interzone II-III. Here, we noted thexceptional elongation of both mitochondria and plastids, whicaccumulate very large starch granules in their stroma (Fig. 3see Vasse et al., 1990) and we confirmed our observation, mby light microscopy, that the spatial positioning of type III andtype IV bacteroids (in interzone II-III and zone III, respectively

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Fig. 3. Ultrastructure and positioning of cell organelles andbacteroids in invaded cells of zone III. Transmission electronmicroscopy. (A) Part of a nitrogen-fixing cell in zone III. Positioninof type IV bacteroids perpendicular to the cell wall. Bar, 5 µm.(B) Microtubules (arrowheads) in contact with a type IV bacteroidsectioned tangentially at one pole. Bar, 1 µm. (C) Proximalinefficient zone III. Small-sized plastids (arrows) and mitochondria(arrowheads) at the cell periphery. The star indicates the section oinfection thread. Bar, 1 µm.

Vasse et al., 1990), indeed depends on their location (Fig. compare with Fig. 1C). Interestingly, the positioning of thoutermost bacteroids often correlated with the presencemicrotubules orientated parallel to, and often in close contwith, symbiosomes containing nitrogen-fixing type IVbacteroids (Fig. 3B). Such a distribution is progressively lostproximal zone III (not shown), a nodule region where boplastids and mitochondria gradually lose their elongated foand return to being rod-shaped (Fig. 3C).

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Major microtubular cytoskeleton changes areobserved during cell differentiation in alfalfanodules Immunolocalization of MC in alfalfa enabled thedifferentiation features described above to be correlated wivariations in cytoskeletal structure. As expected, meristemacells fluoresced strongly, while a loss in staining due to thprogressive disorganization of both endoplasmic and corticmicrotubules was observed in cell layers of the prefixing zonII (Fig. 4A-C). Such a MC disorganization continuesthroughout the entire zone II to the point where only very lowlevel staining can be detected at the periphery of the moproximal cells of this zone (Fig. 4D).

A signal is suddenly recovered at the interzone II-III, a zonwhich differentiates from cell to cell in indeterminate nodule(Vasse et al, 1990). In this zone, a fluorescent band underlinithe cell periphery without interruption (Fig. 4D), indicates thaa cortical cytoskeleton has reformed. Moreover, short radimicrotubular appendages are seen, which then develcentripetally from the cell wall into the cytoplasm of eachinvaded cell. As cells mature, the number and the length these appendages increase significantly. In the cells of zone radial appendages are orientated in the cell cytoplasm like tspokes of a bicycle wheel (Fig. 4E), i.e. parallel to thindividual outermost bacteroids (compare Figs 1C, 3A and 4EThese appendages which most likely correspond to thoobserved in close contact with symbiosomes (see Fig. 3B), not extend to the central region of the invaded cells whebacteroids are randomly oriented in the cytoplasm (compaFigs 1C and 4E). Such an organization of the endoplasmic Mis progressively lost in the most proximal part of the nodulewhere a new decrease in fluorescence is observed (Fig. 4Fis worth mentioning that (i) MC changes described above anot related to the nitrogen-fixing capacity of nodules sincsimilar variations were observed in equivalent zones of Fix−

alfalfa nodules elicited by a R. meliloti fixK mutant (Vasse etal., 1990; not shown), and (ii) no labeling was seen in controwhere the primary antibody was omitted, thus confirming thspecificity of the labeling (not shown).

Microtubular cytoskeleton changes are commontraits of nodule differentiation and are rhizobia-relatedTo examine whether MC changes are general traits of nodudifferentiation, we studied MC in various indeterminate andeterminate nodules. Changes identical to those describabove in alfalfa, i.e. transient microtubule depolymerizatiofollowed by a newly patterned reorganization, were observein clover and vetch indeterminate nodules (not shown). progressive MC disorganization was also seen in developideterminate siratro nodules (6-10 days after inoculation) frothe outer meristematic cells, which fluoresced strongly, to thcells of the central region where labeling could hardly bdetected (Fig. 4G). In this legume, MC reformed in thnitrogen-fixing cells of fully differentiated nodules. Howeverthe signal was restricted to the cell periphery (Fig. 4Hindicating that the cortical cytoskeleton, but not theendoplasmic cytoskeleton, reorganizes in determinanodules.

To determine whether cytoskeletal changes are rhizobirelated, we studied the cytoskeleton in bacteria-free nodul

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Fig. 4. Microtubular cytoskeletal rearrangements in nodules. Laser confocal microscopy. Fig. 4E, F and G are extended focus compositions of10 sections each separated by 1 µm. The others are single focal plane images. (A-F) Three-week old alfalfa nodules. (A) Longitudinal sectionshowing a significant decrease in signal in zone II (asterisk). Bar, 25 µm. (B) Decrease in labeling in the distal part of a nodule, frommeristematic zone I (star) to the proximal part of zone II (asterisk). Arrows indicate the middle region of zone II. Bar, 50 µm. (C) Faint stainingat the periphery of the cells in proximal zone II. Bar, 25 µm. (D) Signal at the transition between proximal zone II (asterisk) and interzone II-III(star). Bar, 25 µm. (E) Newly formed microtubules which radiate from the cell cortex to the cytoplasm in nitrogen-fixing cells of zone III. Bar,20 µm. (F) Loss in fluorescence in the proximal cells of zone III. Bar, 25 µm. (G) and (H) Siratro nodules. (G) Eight-day old nodule. Decreasein fluorescence with a centripetal gradient. Bar, 100 µm. (H) Three-week old nodule. Labeling restricted to the cell periphery. Bar, 25 µm.(I) Two-week old alfalfa nodule elicited by a R. meliloti exoA mutant. A fluorescent signal is observed both in the meristematic region (star) andin the central uniform tissue (asterisk). Bar, 50 µm.

induced either by a R. meliloti exoAmutant (Leigh et al., 1985)or by NFs purified from R. meliloti (Truchet et al., 1991) orfollowing spontaneous development (NAR nodules; Truche

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Fig. 5. Nod factor immunolocalization in alfalfa nodules. (A,E) Dark-field microscopy; (B-D,F) bright-field microscopy. (A,B) Samelongitudinal section of a 2-week-old alfalfa nodule. NF internalization (bright area in A; dark area in B) in zone II. Note the decrease in signalfrom prefixing zone II (asterisks) to nitrogen-fixing zone III (stars). The arrows point to infection threads. Aspecific labeling is seen on thenodule endodermis (large arrow) and amyloplasts (arrowheads). Bars, 10 µm. (C) Labeling in the invaded cells of zone II. The arrows point toinfection threads. Bar, 25 µm. (D) NF immunolocalization on bacteroids (arrowheads) and in the vacuole (star) of a cell in proximal zone III.Bar, 100 µm. (E) Serial section to A and B. Control assay using preimmune serum. Aspecific labeling on nodule endodermis (arrow) andamyloplasts (arrowheads). Bar, 10 µm. (F) Serial section to (A and B). Control assay using NF-adsorbed immunserum. Note the absence oflabeling on infection threads (arrows) and in zone II (asterisk). Bar, 10 µm.

throughout the differentiation of these three nodule typeindicating that MC changes require the presence of bacterianodules (Fig. 4I).

Nod factors are immunolocalized in alfalfa nodulesOur observation that MC changes are rhizobia-dependent (above) together with results showing that nod genes are

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transcribed in rhizobia that are still enclosed in infectiothreads (Schlaman et al., 1991), prompted us to examwhether MC changes could be correlated with the presenceNFs in central nodule cells. Therefore, rabbit polyclonaantibodies directed against purified NFs of R. meliloti wereprepared and their reactivity verified by ELISA. Briefly, wefound that coated NFs reacted with antiserum in a time- a

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concentration-dependent manner (data not shown). Out of sera tested, the one which gave the highest positive signal a concentration of NFs as low as 10−9 M, was used for theexperiments. We then controlled the specificity of the immuserum by inoculating axenic plants with R. meliloti strainsknown either to overproduce NFs (strain GMI6390, Rocheal., 1991), or to be unable to synthesize NFs (strain GMI63Roche et al., 1991). Immunostaining of plants 2 days poinoculation showed that the NF-producing strain fluorescstrongly while no signal was detected on alfalfa inoculatwith rhizobia which do not synthesize NFs (not shown). Thresult indicated clearly that the antibodies were specific NFs and did not immunoreact with plant and bacterial surfacomponents.

NFs were immunolocalized in wild-type alfalfa noduleelicited by the wild type R. melilotiRCR 2011 strain. By lightmicroscopy, the strongest signal was observed in prefixing zII, particularly associated with the infection threads, whilelower, but still significant labeling, was detected in thcytoplasm of the invaded cells (Fig. 5A-C). In contrast, lolevels of staining were found in interzone II-III and zone I(Fig. 5A and 5B). This variation was confirmed by quantifyinthe density of gold particles per surface unit (µm2), inconditions where the highly reactive infection threads and laplastids were excluded from the scanned regions. We founprefixing zone II, a higher density than in meristematic zonand distal nitrogen-fixing zone III, respectively (Fig. 6Detailed observations showed that NFs are internalized incytoplasm of the infected cells in zone II (Fig. 5C). Therealso a constant, albeit discrete, level of immunolabeliassociated with bacteroids at all stages of their differentiat(Fig. 5D). The labeling of bacteroids might account for tslight, but significant (t=2.8, P<0.025), increase in gold particledensity detected in the cells of zone III as compared to meristematic cells (Fig. 6). Moreover, a signal was aobserved in the large vacuoles of the most proximal cells

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zone III (Fig. 5D). This could be due to an accumulation in thvacuole of NFs released from bacteroids which undergodegeneration process in the vacuole of infected cells in tregion of the nodule (Truchet and Coulomb, 1973). Finally, aunexpectedly, labeling was seen on starch granuaccumulating in amyloplasts and on the cell wall of endodermcells (Fig. 5A and 5B). This latter labeling, which variedstrongly between assays, could be due either to the recogniby the immune serum of epitopes localised at these sites anto non-specific labeling. Comparative studies between assand controls omitting the primary antibodies (not shown), usithe preimmune serum (Fig. 5E, compare with 5A), or wheimmune serum was exhaustively adsorbed with purified Nprior to the staining procedure (Fig. 5F, compare with 5B) leopen the two possibilites. However, in contrast to the labeliin infection threads and cell cytoplasm in zone II, it seems veunlikely that labeling on the endodermis accounts for thpresence of NFs, since this tissue is totally bacteria-free (Vaet al., 1990). NF immunolocalization was confirmed at thultrastructural level (not shown).

DISCUSSION

In this paper, we provide experimental evidence of Ninternalization and MC architectural rearrangements durinodule differentiation. That similar changes are observed different nodule types and depend on bacterial release indicthat cytoskeleton changes are symbiosis-specific and rhizobdependent. Our results lead to several conclusions ahypotheses concerning the general role that the cytoskeleplays in mediating plant cell differentiation and suggest thNF internalization and MC changes are correlated durinodule maturation.

The cytoskeleton in relation to nodule infection andbacterial release in host cellsIn indeterminate nodules, MC changes occur in nodule cenzones in which at least six major cellular changes are obsermicroscopically. The first cellular event which determines thindividualization of the prefixing zone II is the penetration omeristem-derived cells by the growing part of the infectionetwork. A number of studies either provide direct evidence imply that the cytoskeleton is involved in infection threagrowth: (i) microtubules are present at the original site penetration (Ridge and Rolfe, 1985); (ii) microtubuleinterconnect the host cell nucleus to the growing infectiothread tip (Bakhuizen, 1988); (iii) cytoskeleton-determinecytoplasmic bridges guide the growth of infection threads otheir way down to the nodule primordium (Van Brussel et a1992; Bakhuizen, 1988); (iv) the cytoskeleton plays a majrole in cell wall formation (reviewed by Seagull, 1989; Cyr1994), and infection threads are cell wall limited structure(Callaham and Torrey, 1981) and (v) an interesting model hbeen proposed in which Rhizobium infection through rohairs may result from the mobilization of normal root hair tigrowth machinery, including the cytoskeleton, at the infectiosite (Ridge, 1992). Taken together, these and our data indicthat cytoskeletal integrity and functions are sufficientlpreserved to sustain infection thread growth at the meristeprefixing zone II interface of developing nodules.

The second cellular event is the release of bacteria in the

n

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cytoplasm which is restricted to the distal part of prefixing zoII (Vasse et al., 1990). The signals that trigger bacterial releare not known. However, the fact that release occurs exclusiat unwalled parts of infection threads (Roth and Stacey, 19has suggested the possibility of an enzymatic degradation othread cell wall either by enclosed rhizobia (Truchet aCoulomb, 1973) or by a mechanism which would involve tmobilization of the endoplasmic reticulum (Roth and Stac1991) which is particularly abundant in the region of bacterrelease (Truchet and Coulomb, 1973; this study). Our resopen up another possibility. We hypothesize that the relemight also be a consequence of discrete and local variationthe endoplasmic cytoskeleton in the direct vicinity of thgrowing tip of infection threads, i.e. at sites where the developcell wall is not yet completely organized. As a consequensome of the cytoskeleton-controlled functions, such as cell wbuilding (for example), could be locally modified, or slackeneto an extent and for a period of time sufficient to allow bacterrelease through an opened door at infection extremities.

The cytoskeleton in relation to cell morphology andendopolyploidyThe third cellular event observed in prefixing zone II is thonce invaded, host cells enlarge isodiametrically in distal zoII, leading to a round-shaped morphology in the proximal ceof this zone. The cytoskeleton plays an active role in celongation and cell shape control by directing the ordesynthesis of cellulose microfibrils via a microtubule/plasmmembrane/cell wall functional continuum (reviewed bSeagull, 1989; Giddings and Staehelin, 1991; Williamso1991; Cyr, 1994). Thus the isodiametric enlargement of invaded cells in the distal part of zone II, should indicate thin this region, the cortical array maintains its function in cwall building necessary for cell enlargement. In contrast, changes in both cell shape and morphology in zone II andarrest in cell enlargement occurring in the central nodtissues (this study) might result from the progressive lostcytoskeletal integrity observed in the infected nodule cellsmight seem contradictory to hypothesize that in distal cellszone II the cytoskeleton still functions efficiently in plant cewall organization whilst infection thread cell wall formation impaired. However, as stated above, we propose that lochanges in the cytoskeleleton centered around the extremian infection branch and which are not discernable microscopy, locally modify cytoskeleton functioning.

The fourth cellular event is that simultaneously to cenlargement, endopolyploidy takes place gradually aexclusively in the invaded cells of prefixing zone II iindeterminate pea and alfalfa nodules (Truchet, 1978). Tcytoskeleton is subjected to a series of specific rearrangemthat are essential for karyokinesis and cytokinesis to procsuccessively (reviewed by Seagull, 1989; Goddard et 1994). The fact that, in nodules, the impairment of these tcellular events occurs simultaneously to cytoskeledisorganization raises the question of the mechanisms tharesponsible for DNA amplification. Particularly relevant to ostudy are the results showing that drug-induced microtubdepolymerization is sufficient to initiate both DNA synthesand the entry of human and animal fibroblast-like cells into proliferative cycle (Crossin and Carney, 1981; Bershadskyal., 1996) and that microtubule depolymerization is direc

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responsible for DNA synthesis (Crossin and Carney, 1981)would be interesting to determine if a similar mechanism involved in the establishment of the polyploid gradient in thprefixing zone II of indeterminate nodules.

The cytoskeleton in relation to cytoplasmic spatialorganization and ultrastructural differentiationThe fifth cellular event concerns the major variations in thspatial distribution and the positioning of both organelles abacteroids in the infected cells. The capacity of the cytoskeleto mediate the movement and distribution of organelles in a cis supported by several correlative studies (reviewed by Cole Lippincott-Schwartz, 1995). For example, the distribution oorganelles frequently parallel that of the cytoskeleton and the of drugs which depolymerise microtubules or microfilamenresults in modified spatial distribution of organelles (Yaffe et a1996). Our data are in agreement with these studies: the randistribution of cell organelles and bacteroids in the most discells of zone II is associated with an organized cytoskeleton; progressive migration of organelles and bacteroids to tperiphery of the cells in proximal zone II occurs simultaneousto endoplasmic MC disorganization. Moreover, our studsuggests that the positioning of both cell organelles abacteroids is microtubule-mediated. Thus, in zone III, thpeculiar positioning of the outermost bacteroids, perpendicuto the cell wall, parallels that of radial microtubules originatinfrom the cortical cytoskeleton and radiating centripetally to innregions of the cytoplasm. In plants, a radial cytoskeleton patthas been described in meristematic and elongating c(Bakhuizen et al., 1985; Baluska et al., 1992), and in tsyncytial stage during endosperm development (Brown et 1994). Generally, radial patterns are thought to originate frothe nuclear periphery (Lambert, 1993) and to provide an ancfor the nucleus at its appropriate position during cedifferentiation. Similar functions for the radial cytoskeleton initrogen-fixing cells are unlikely. In nodules, radial arrayobviously originate in the cell cortex, then elongate into the ccytoplasm as cells mature and are not connected to otstructures in the cytoplasm. Thus, we provide further evidenfor the presence of microtubule organizing centers (MTOCs)the cortex of plant cells (Liu et al., 1994; Panteris et al., 199The situation is different in mature determinate nodules in whithe random distribution of large symbiosomes containing mabacteroids in the cell cytoplasm (Newcomb, 1981) is agreement with our data showing that a cytoplasmcytoskeleton does not reform in the invaded cells of nitrogefixing siratro nodules.

The sixth cellular event concerns the ultrastructural changwhich characterize cell organelles and bacteroids in invaded ceBy correlating the present data with our previous results bacteroid differentiation in alfalfa nodules (Vasse et al., 1990)clearly appears that bacteroids (i) divide exclusively in the ceof distal zone II, where the MC is microscopically observed, (stop dividing and elongate moderately in the second half of proximal zone II where the MC disorganizes and (iii) increadramatically in size in a cell to cell fashion (Vasse et al., 199correlating with the differentiation of the interzone II-III wherecortical MC reforms. Interestingly, we found that cell organelleundergo similar variations in the same regions of the nodubeing small-sized in distal zone II, increasing moderately in sin proximal zone II and reaching their maximal size in interzon

348

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II-III, before returning to a smaller size in zone III. Thmechanisms which control the division of cell organelles apoorly understood, although it is generally admitted that thereno link between the mechanism that divides the mother cell the dividing organelle (reviewed by Warren and Wickner, 199Despite the difficulty in deducing a dynamic relationship simpfrom morphological features, our results strongly suggest thatMC plays a role in bacteroid and cell organelle division aelongation. This hypothesis implies that bacteroids are perceas endogenous organelles. Microtubules mediate the distribuof cell organelles via membrane-binding microtubule-basmotor proteins such as kinesin and cytoplasmic dynein (Yaffeal., 1996). As bacteroids are enclosed by a plasmalemma-derperibacteroid membrane (Roth and Stacey, 1989), we anticipthat the host cell senses intracellular bacteroids as normal organelles via the connection of cytoskeleton microtubuelements and associated proteins with the peribactermembrane.

Nod factor internalization and cytoskeletal changesIn this paper, we show that NFs are immunolocalized throughthe central tissues of alfalfa nodules from the prefixing zoneto the proximal part of zone III. The fact that the strongest sigis observed in infection threads is in agreement with previostudies indicating that nod genes are expressed in bacterenclosed in infection threads (Schlaman et al., 1991; F. MailG. Truchet and J. Dénarié, unpublished data). Moreover, in zII, a specific signal is also detected in the cytoplasm of infected plant cells indicating that NFs are internalized. Finaa weak but reproducible labeling is associated with bacteroindependent of their differentiation stage and histologiclocation. This result is in contradiction with previous daindicating that in vetch and alfalfa nodules, nod genes are notexpressed in intracellular bacteroids (Schlaman et al., 1991Maillet, G. Truchet and J. Dénarié, unpublished data) asuggests that NFs are synthesized at a low level by bactero

Our data and the recent results showing that R.leguminosarum bv. trifolii NFs are internalized in clover roothairs (Philip-Hollingsworth et al., 1997) indicate that Ninternalization can take place in different tissues amenableinfection. At the moment, we do not know how NFinternalization occurs. It might involve a simple diffusioprocess, or be receptor-mediated. Putative high-affinity Nreceptors could be located in the plasma membrane (Bono e1995; Niebel et al., 1997), a membrane which also surrouinfection threads and bacteroids (Hirsch, 1992; Kijne, 1992; Fig. 2B). The possible coupling of NF reception aninternalization is also supported by the recent report showthat purified R. leguminosarumbv. trifolii NFs are internalizedinto the root hairs of clover, the homologous host, but not alfalfa, an heterologous host for this species (PhiliHollingsworth et al., 1997). Finally, our observation that NFs aonly internalized in the distal region of alfalfa nodules whilthey are synthesized by bacteroids throughout their endifferentiation process, suggests that the mechanism(s) direcNF internalization change(s) during nodule maturation.

Identifying the nature of the causal mechanism(s) resultin cytoskeletal disorganization is a major challenge raisedthis study. Our data suggest that NF internalization and Mdisorganization are coupled events. Firstly, NFs aimmunolocalized in the cytoplasm of invaded cells of th

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prefixing zone II, where the MC disorganizes, but not in thcytoplasm of cells in nitrogen-fixing zone III, where thecytoskeleton reforms. This result highlights the spatiotemporcorrelation between the two processes. Secondly, the MC is disorganized in bacteria-free alfalfa nodules indicating that Mdisorganization requires the presence of bacteria. Thirdidentical MC disorganization occurs in nodules of differentypes induced by rhizobial strains which are taxonomicaldistant (Martinez-Romero and Caballero-Mellado, 1996), bshare the capacity to produce NFs belonging to the sachemical family and displaying similar biological activities(reviewed by Dénarié et al., 1996; Long, 1996; Schultze aKondorosi, 1996; Spaink, 1996). From these considerationscan reasonably be hypothesized that the causal mechanismresponsible for cytoskeletal disorganization in nodules common to all rhizobia and that NFs, or a structural componeof NFs, are (is) this common link.

In summary, this paper reports several correlations betweNF internalization, MC changes and the particuladifferentiation of the invaded central cells in alfalfa nodulesWe propose that, once internalized, rhizobial NFs are involvin cytoskeletal changes accompanying cell differentiation. validation of this model requires a detailed study of the direeffects of purified NFs on cytoskeletal architecture.

We are very grateful to Fabienne Maillet and Jean Dénarié fproviding purified R. melilotiNod factors, Philippe Cochard for advicein using laser confocal microscopy and Michel Petitprez for help in golabel quantification. We thank very much D. Barker, P. Boistard andDénarié for their constructive comments regarding the manuscript aC. Gough and D. Barker for English reviewing. A. C. J. T. was supportby BIOTECH and TMR postdoctoral fellowships and this work wafunded by grants from the European Community BIOTECH Programm(PTP CT93-0400) and TMR Programme (FMRX-CT96-0039).

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