These two authors contributed equally to this work
An unusual infection mechanism and nodule morphogenesis in white lupin (Lupinus albus)
Article first published online: 7 JUN 2004
Volume 163, Issue 2, pages 371–380, August 2004
How to Cite
González-Sama, A., Lucas, M. M., De Felipe, M. R. and Pueyo, J. J. (2004), An unusual infection mechanism and nodule morphogenesis in white lupin (Lupinus albus). New Phytologist, 163: 371–380. doi: 10.1111/j.1469-8137.2004.01121.x
- Issue published online: 7 JUN 2004
- Article first published online: 7 JUN 2004
- Received: 17 February 2004 Accepted: 15 April 2004; doi: 10.1111/j.1469-8137.2004.01121.x
- Lupinus albus;
- white lupin;
- Mesorhizobium loti;
- nitrogen-fixing symbiosis
- • The infection of white lupin (Lupinus albus) roots and the early stages in organogenesis of the lupinoid nodule are characterized in detail in this work.
- • Immunolabelling of Bradyrhizobium sp. (Lupinus) ISLU16 and green fluorescent protein labelling of Mesorhizobium loti NZP2037, two strains that induce nodulation in L. albus, allowed us to monitor the infection and morphogenesis process. Light and transmission electron microscopy, low-temperature scanning electron microscopy, fluorescence and confocal microscopy were employed.
- • Rhizobia penetrated the root intercellularly at the junction between the root hair base and an adjacent epidermal cell. Bacteria invaded the subepidermal cortical cell immediately beneath the root hair through structurally altered cell wall regions. The newly infected cell divided repeatedly to form the central infected zone of the young nodule. Bacteria seemed to be equally distributed between the daughter cells.
- • A new mode of direct epidermal infection and an unusual morphogenesis for indeterminate nodules lead to the formation of the lupinoid nodule with unique characteristics.
Soil bacteria of the genera Rhizobium, Sinorhizobium, Bradyrhizobium, Mesorhizobium, Azorhizobium and Allorhizobium (Wang & Martínez-Romero, 2000), collectivelly referred to as rhizobia, establish a symbiotic association with leguminous plants that results in the development of nitrogen-fixing root nodules. This plant–microbe interaction is a complex process that starts with the exchange of specific recognition signals between both symbionts (Hirsch, 1992). Legume roots exude flavonoid compounds that induce the synthesis and the secretion of rhizobial lipo-chito-oligosaccharides, the so-called Nod factors (Long, 1989; Dénariéet al., 1996). These molecules act as mitogens stimulating the cell division in the root cortex that leads to the formation of a nodule primordium and the progressive differentiation of specialized cells and tissues. Concomitantly, the bacteria enter the root cortex to infect nodule primordium cells, after which rhizobia differentiate to their nitrogen-fixing form, the bacteroid (Brewin, 1991).
Rhizobia have been reported to infect legume roots by three different mechanisms involving both intracellular and intercellular routes (Sprent, 1989). The best-studied mode of infection takes place through the root hair and involves the formation of an intracellular tube-like structure bounded by a plant membrane, the infection thread. Infection threads contain the proliferating bacteria embedded in an infection matrix and grow inwardly until reaching the nodule primordium where the bacteria are released (VandenBosch et al., 1989; Brewin, 1991; Rae et al., 1992; van Spronsen et al., 1994). Cell invasion occurs by a process resembling endocytosis from unwalled infection droplets that arise from the tip of the infection threads. Within the host cell, bacteria are enclosed by a plant-derived membrane, the peribacteroid membrane (Roth & Stacey, 1989; Kijne, 1992).
Rhizobia can also invade the root cortex through an intercellular route via natural wounds caused by the splitting of the epidermis and the emergence of young lateral roots or adventitious roots (Hadri et al., 1998). This alternative mode of infection, known as crack entry, has been described in (sub)tropical legumes, such as Arachis hypogaea (Chandler, 1978), Stylosanthes (Chandler et al., 1982), and Aeschynomene (Alazard & Duhoux, 1990), where no infection threads are observed and the intercellular rhizobia invade the cortical cells through structurally altered cell walls. In Sesbania rostrata (Dreyfus & Dommergues, 1981; Tsien et al., 1983; Ndoye et al., 1994) and Neptunia (Subba-Rao et al., 1995), the infection leads to the formation of intercellular infection pockets, which give rise to intracellular infection threads.
A third mode of infection has been described in the tree legume Mimosa scabrella (Sprent, 1989). Rhizobia penetrate directly between undamaged epidermal cells by dissolution of the middle lamella of the radial cell walls and invade the host cells through infection thread-like structures (de Faria et al., 1988). Unlike M. scabrella, M. pudica and M. diplotricha have a root hair infection process (Chen et al., 2003).
Two main types of legume root nodules have been described according to the persistence of meristematic activity and the cell layers of root cortex involved in the initial rhizobia-induced cell divisions (Brewin, 1991; Hadri et al., 1998). Temperate legumes (Pisum, Medicago, Trifolium or Vicia) usually develop cylindrical-shaped indeterminate nodules, which are initiated in the inner root cortex and maintain a persistent apical meristem from which all nodule tissues originate. Consecutive developmental zones can be observed in the infected central tissue of a mature nodule, corresponding to a gradient of symbiotic differentiation from the apical meristem to the basal tissues attached to the root (Vasse et al., 1990; Timmers et al., 2000). Determinate nodules are characteristic of (sub)tropical legumes such as Phaseolus, Glycine, Lotus or Vigna. These nodules originate from cell division in the outer cortex (the hypodermis) and do not have a persistent meristem. The meristematic activity ceases at an early stage of development and the subsequent growth is caused by cell expansion, resulting in spherical-shaped nodules (Tatéet al., 1994; Patriarca et al., 1996).
Lupin, a temperate legume and the major grain legume crop in Australia, is mainly used in the production of feed. Lupin seeds are rich in proteins, lipids and alkaloids and have specific uses in the pharmaceutical industry. Lupinus albus, the white lupin, establishes an effective symbiosis with the slow-growing rhizobium, Bradyrhizobium sp. (Lupinus). Lupin nodules are recognized as a unique subtype of indeterminate nodules, the so-called lupinoid nodules, characterized by the broadening of the connection to the root as the nodule grows, causing both connection and nodule to surround the root (Bergersen, 1982; Corby et al., 1983). Moreover, other particular characteristics distinguish them from all other indeterminate nodules: their initiation takes place in the outer cortex, the infected central tissue does not show the typical zonation and does not have uninfected interstitial cells, and the basal-laterally located meristematic region contains bacteroids (Golinowski et al., 1987).
Although infection thread-like structures have been occasionally found in white lupin nodules (James et al., 1997), these rarely observed structures do not explain the initial process of root infection. The aim of this work was to characterize the early events of nodulation in Lupinus albus in order to elucidate features of infection, cell invasion and early nodule organogenesis that lead to the formation of a unique type of root nodule.
Materials and Methods
Bacterial strains, growth and transformation
Bradyrhizobium sp. (Lupinus) strain ISLU16 was grown at 28°C with gentle shaking in a yeast extract-mannitol (YEM) liquid medium (Vincent, 1970). Mesorhizobium loti strain NZP2037 was kindly supplied by Dr HP Spaink. Competent M. loti cells were transformed by electroporation with plasmid pHC60 (Cheng & Walker, 1998), using a Bio-Rad E. coli Pulser Electroporator (Hercules, CA, USA) according to standard protocols (Sambrook et al., 1989). Mesorhizobium loti NZP2037 harbouring plasmid pHC60, thus expressing the green fluorescent protein (GFP), was grown in YEM medium supplemented with tetracycline (12 µg ml−1).
Plant material and growth conditions
White lupin (Lupinus albus L. cv. Multolupa) seeds were surface sterilized in 1% HgCl2 with gentle shaking for 5 min, rinsed several times in distilled water and allowed to imbibe for 2 h. Seeds were sowed in pots containing sterilized vermiculite. Three days after sowing, seedlings were inoculated with suspensions of either Bradyrhizobium sp. (Lupinus) ISLU16 or M. loti NZP2037 (pHC60). Plants were grown in a growth chamber under controlled conditions at 60% relative humidity, 25°C/15°C (day/night) temperature and 16-h light photoperiod. Plants were watered with distilled water for 1 wk and thereafter with nitrogen-free nutrient solution (Hoagland & Arnon, 1938) at one fourth and one half strength during the second and the third week, respectively.
Light and transmission electron microscopy and immunolabelling
The infection process was monitored daily (1–14 d after inoculation, dai) using light microscopy (LM) and transmission electron microscopy (TEM). Root, primordia and nodule samples were collected and immediately fixed by vacuum infiltration for 2 h at room temperature with 2.5% glutaraldehyde in 0.05 m Na cacodylate buffer, pH 7.4. After fixing and rinsing with the same buffer, tissues were dehydrated in an ethanol series and progressively embedded in LR White resin (London Resin Co., Reading, UK). Resin was polymerised at 60°C for 24 h.
Serial semithin (1 µm) and ultrathin (70 nm) sections were cut with a Reichert Ultracut S ultramicrotome (Vienna, Austria) fitted with a diamond knife. Semithin sections for LM were stained with 0.1% (w/v) basic fuchsine in 5% (v/v) ethanol or with 1% (w/v) toluidine blue in aqueous 1% sodium borate for direct observation with a Zeiss Axiophot photomicroscope (Oberkochen, Germany). Ultrathin sections for TEM were contrasted with 2% aqueous uranyl acetate and lead citrate (Reynolds, 1963). TEM observations were performed with a STEM LEO 910 electron microscope (Oberkochen, Germany) at accelerating voltage of 80 kV, equipped with a Gatan Bioscan 792 digital camera (Pleasanton, CA, USA).
Immunogold labelling reactions were performed on semithin sections for LM and on ultrathin sections for TEM according to the procedure of Lucas et al. (1992) and Vivo et al. (1989), respectively. A rabbit polyclonal antibody raised against whole Bradyrhizobium sp. (Lupinus) ISLU16 cells (Lucas, 1992) was used as primary antibody (1 : 30 000 dilution). Goat antirabbit IgG coupled to colloidal gold (5 nm for LM, 15 nm for TEM, British Biocell International, Cardiff, UK) was used as secondary antibody. Controls were performed in the absence of primary antibody. Silver enhancement reactions were carried out for LM (Silver Enhancing Kit, British Biocell International, Cardiff, UK) and tissue was stained with 0.1% (w/v) basic fuchsine in 5% (v/v) ethanol.
Low-Temperature Scanning Electron Microscopy (LTSEM)
Small portions of inoculated roots were attached to a sampler for LTSEM with a special adhesive (Gurr®, OCT, BDH, Poole, UK) and immediately cryofixed by immersion in slush nitrogen (−196°C), vacuum-cryo-transferred to a −90°C chamber and gold coated. The samples were then transferred to the observation chamber of a Zeiss Digital Scanning Microscope DSM960 (Oberkochen, Germany) where they were observed at low temperature with secondary and back-scattered electrons.
Fluorescence and confocal microscopy
Small portions of roots inoculated with GFP-labelled M. loti were collected (1–14 dai) and embedded in 6% agarose in PBS buffer, pH 7,4 (Sambrook et al., 1989). Sections were cut at 50 µm with a vibratome and observed under blue light with a Zeiss Axiophot fluorescence photomicroscope (Oberkochen, Germany). The filter set for fluorescence microscopy consisted of a 450–490 nm band-pass excitation filter and a barrier filter with 590-nm long pass cutoff. Propidium iodide-stained sections were observed and photographed with a Leitz DM IRB inverted epifluorescence microscope connected to a Leica TCS 4D confocal system (Bannockburn, IL, USA). GFP was excited with the 488-nm laser line, and the barrier filter had a 525-nm cutoff. Propidium iodide-stained tissue was excited with the 568-nm laser line, and the barrier filter had a 590-nm long pass cutoff. Serial optical sections for both excitation wavelengths were performed and confocal images were reconstructed and combined with the confocal system software.
GFP labelling of rhizobia
Attempts to transform the typical lupin symbiont Bradyrhizobium sp. (Lupinus), in order to obtain GFP-labelled bacteria, were unsuccessful. Transformation procedures such as conjugal mating, heat shock, electroporation and freeze-thaw method (Hofgen & Willmitzer, 1988) were performed with an array of GFP plasmids. Different bacterial strains and experimental conditions were employed.
Conversely, a Mesorhizobium loti strain (NZP2037) that nodulates lupin roots was readily transformed by electroporation with plasmid pHC60 (Cheng & Walker, 1998). This plasmid constitutively expresses a variant of the green fluorescent protein (GFP-S65T) and contains a 0.8-kb fragment of the stabilization region from the broad-host-range plasmid RK2 (Weinstein et al., 1992), that permits its maintenance in rhizobial cells in planta with no antibiotic selection. Mesorhizobium loti NZP2037 cells harbouring plasmid pHC60 strongly expressed GFP-S65T during nodulation, thus allowing visualization of the bacteria on the root surface and within the root cortex. We used both GFP-labelled M. loti, an incompatible strain leading to nonfunctional nodules, and Bradyrhizobium sp. (Lupinus) to monitor infection and early nodule morphogenesis.
Colonization of Lupinus albus roots
Rhizobial colonization of the root surface was already evident 4 d after inoculation (dai). Rhizobia accumulated preferentially in the root hair portion of the main root, c. 10–15 mm from the root apex. Rhizobia were attracted to specific areas of the root where they actively proliferated, probably in response to the secretion of mucilaginous exudates by the plant. Bacteria were often found embedded in this mucilaginous material (Fig. 1a), which was shown to be polysaccharide by basic fuchsine staining (Fig. 1b,c). Rhizobia had to penetrate the mucigel layer in order to gain access to the root cells. Once on the root surface, rhizobia accumulated mainly in the junctions of root hair cells and adjacent epidermal cells (Fig. 1c,d). To verify unequivocally the presence of Bradyrhizobium sp. (Lupinus) ISLU16, specific immunolocalization reactions were performed. Fig. 1(b,c) show the peripheral immunolabelling, as visualized by the silver enhancement technique under light microscopy. Neither infection threads nor curled root hairs were observed, thus suggesting an alternative mode of infection. It was possible, however, to observe some noncurled root hairs being colonized by rhizobia.
GFP-labelled Mesorhizobium loti colonized the root surface in the same manner and laser confocal microscopy revealed that colonization was superficial and bacteria did not penetrate the root hair cell (Fig. 1d).
Infection process and nodule initiation
A cortical cell directly beneath a root hair was the first cell to be infected in over 50 infection events analysed. Bacteria entered the root between the cell walls of the root hair base and an adjacent epidermal cell, and accumulated between the cell walls on the surface of an outer cortical cell (Fig. 2a). Occasionally, some rhizobia could be found in the broad intercellular spaces between epidermal and outer cortex cells (Fig. 2b). Rhizobial invasion was concomitant with the induction of cell divisions in the outer cortex of the root, immediately below the root hair. The first cell divisions could be detected 5–6 d after inoculation with either Bradyrhizobium sp. (Lupinus) or Mesorhizobium loti (Fig. 2c,d). At this time, bacterial accumulations could be observed inside cell wall swellings in the cortical cell underneath the root hair. Rhizobia were released from such structures into the cytoplasm of the cortical cell through structurally altered cell walls (Fig. 3). The initially infected cell divided rapidly and repeatedly. Up to 6–8 freshly divided cells could be observed within the primitive cell wall, each containing a small number of bacteria. The adjoining noninfected cortical cells divided too (Fig. 3a). Transmission electron microscopy ultrastructural observations revealed that the altered cell wall structures were composed of a fibrilar, electron-dense matrix, in which bacteria were embedded (Fig. 3b,c). Release of rhizobia from this matrix into the cytosol of the cortical cell surrounded by a membrane could be observed (Fig. 3c). Additionally, a small number of bacteria could be found between cell walls in a less dense matrix (Fig. 3d). Fig. 3(e,f) shows an altered cell wall region containing immunolabelled bacteria in the proximity of the plant cell nucleus.
Approximately 8–9 dai, the cell divisions in the outer cortex were accompanied by cell divisions in deeper layers of the root cortex, immediately beneath the infection focus (Fig. 4a). The structure of the nodule primordium 11–12 dai resembled that of a mature nodule; it consisted of a peripheral tissue, the nodule cortex, surrounding the central infected zone (Fig. 4b). The development of primordia into nodules resulted from the repeated division of both infected (Fig. 4c,d) and noninfected cells (Fig. 4e). At this developmental stage an intense division of the infected cells was observed. Some cells in the most external layers of the infected zone and some bordering noninfected cells in the nodule cortex continued to divide, thus allowing continuous growth of the nodule. Infected cells contained a cytoplasm rich in small vacuoles and an enlarged nucleus with a prominent nucleolus. Intracellular rhizobia actively divided inside their host cells. Within the cells, bacteria were usually localized near the cell walls. However, at the onset of cytokinesis they were found near the nucleus (Fig. 4c,d). During the division process, rhizobia appeared to be equally distributed between the daughter cells. During the early stages of nodule morphogenesis, M. loti-induced nodules were indistinguishable from those induced by Bradyrhizobium sp. (Lupinus) (Fig. 4f,g).
Differential immunolabelling of Bradyrhizobium sp. (Lupinus) strain ISLU16 and GFP labelling of Mesorhizobium loti strain NZP2037 allowed us to follow and describe in detail the initial steps of root infection and nodule morphogenesis in Lupinus albus. The two bacterial strains showed no differences in infection mechanism or in the organogenesis process during the early stages of nodule development. However, M. loti NZP2037 is an incompatible strain and the subsequent development of M. loti-induced nodule primordia was aborted with no functional nodules being formed.
Following inoculation with Bradyrhizobium, it was possible to see some slightly curled root hairs in L. albus roots, but the typical curled shepherd's crooks could not be observed and no intracellular infection threads were found at any stage of the infection process. Short infection threads similar to those found in Glycine (James et al., 1991) and Phaseolus (Rae et al., 1992), containing little or no infection matrix, have been described in the central infected zone of young nodules of L. angustifolius (Robertson et al., 1978; Tang et al., 1992; Tang et al., 1993) and L. albus (James et al., 1997). However, those infection structures were too scarce and sporadic to explain the early steps of the infection process. Additionally, L. albus nodules developed along the entire length of the root and were not associated with the bases of lateral roots, indicating that bacteria did not enter via crack infection.
Rhizobia colonized the root surface and accumulated in certain areas of the root where an active excretion of mucilaginous material was present. Some root hairs were surface-colonized by the bacteria, perhaps causing the weakening of the cell wall structure and the separation of the neighbouring epidermal cells in these sites, thus easing bacterial entry. Rhizobia penetrated intercellularly at the junction between the root hair base and the adjoining epidermal and cortical cells. This penetration resembled the infection mechanism described in the roots of the tree legume Mimosa scabrella, which does not develop root hairs and proceeds through direct epidermal entry between any two contiguous cells (de Faria et al., 1988). The infection process has been studied in other Lupinus spp. In L. luteus, a root hair infection process has been described in which bacteria enter a curled root hair, but no infection thread structures are observed (Lotocka et al., 2000). In that study, bacteria penetrated the root hair, and subsequently other cortical cells, through structurally altered cell walls. By contrast with L. luteus, no infected root hairs were observed in L. albus. In L. angustifolius, nodule primordia have been observed adjacent to enlarged root hairs, and although some rarely observed, short structures resembling infection threads have been described (Tang et al., 1993), the infection process seems to be similar to that reported here for L. albus.
Bacteria invaded the outer cortical cell located below the root hair through altered regions of the cell wall. The rhizobia were immersed in an infection matrix. The infection structures described here are similar to those formed during intracellular penetration by crack infection in the tropical legumes Arachis (Chandler, 1978), Stylosanthes (Chandler et al., 1982) and Aeschynomene (Alazard & Duhoux, 1990), all of which produce nodules of the aeschynomenoid type, always associated with lateral roots. In those legumes, cell invasion involves changes in the structure of the cell wall. The penetration sites in L. albus roots were found sometimes close to the nucleus of the infected cell, in the same manner that has been reported for the infection of Arachis roots (Chandler, 1978).
The infection of lupin roots and the development of the lupin nodule display some features that are distinctive of indeterminate nodules, while others are characteristic of the determinate nodule type. A peculiar feature of the lupin indeterminate nodule is that the infected zone originates from the division of a single infected outer cortical cell. This process is common to all other Lupinus spp. studied (Tang et al., 1992, 1993; Lotocka et al., 2000). A small number of bacteria gain access to the cell and multiply within the cytosol. During host cell division, rhizobia appear to be equally distributed between the daughter cells, giving rise to the central infected zone of nodule characterized by the absence of uninfected cells as described in aeschynomenoid nodules. It is likely that the infected cell regulates the maximum number and the intracellular distribution of bacterial cells during cytokinesis, such that rhizobia do not interfere with the plant cell division process. The small symbiosome size, containing a single rod-shaped bacteroid, may also facilitate the process. Various cell organelles are equally segregated between the daughter cells during plant cell division; their movement is directed by the cytoskeleton (Warren & Wickner, 1996). In fact, the peribacteroid membrane of symbiosomes displays some tonoplast membrane features (Roth & Stacey, 1989) and it can be hypothesised that the plant cell senses young symbisomes as cell organelles. Although some information regarding the cytoskeleton during the early stages of nodulation is available (Whitehead et al., 1998; Timmers et al., 1999; Davison & Newcomb, 2001a,b), the role of the cytoskeleton in the movement and segregation of symbiosomes remains to be investigated. The lupinoid nodule constitutes a unique model for study of the division process in cells containing prokaryotic organisms.
The authors thank Drs G. Walker and H.-P. Cheng for kindly providing plasmid pHC60, Dr H.P. Spaink for kindly providing Mesorhizobium loti strain NZP2037 and Dr F. Temprano for his information on lupin nodulation by M. loti. We also thank F. Pinto, S. Lapole, C. de Mesa and C. Morcillo for technical assistance. We thank Dr E. Fedorova and Dr W. Sanders for their suggestions and critical reading of the manuscript. This work was supported by grant BIO2001-2355 from MCYT to MRF and grant AGL2001-2093 from MCYT to JJP. AG-S was recipient of a MCYT predoctoral fellowship.
- 1990. Development of stem nodules in a tropical forage legume, Aeschynomene afraspera. Journal of Experimental Botany 0: 1199–1206. , .
- 1982. Root nodules of legumes: structure and functions. Chichester, UK: Research Studies Press. .
- 1991. Development of the legume root nodule. Annual Review of Cell Biology 7: 191–226. .
- 1978. Some observations on infection of Arachis hypogaea L. by Rhizobium. Journal of Experimental Botany 29: 749–755. .
- 1982. Infection and root-nodule development in Stylosanthes species by Rhizobium. Journal of Experimental Botany 33: 47–57. , , .
- 2003. Nodulation of Mimosa spp. by the beta-proteobacterium Ralstonia taiwanensis. Molecular Plant–Microbe Interactions 16: 1051–1061. , , , , .
- 1998. Succinoglycan is required for initiation and elongation of infection threads during nodulation of alfalfa by Rhizobium meliloti. Journal of Bacteriology 180: 5183–5191. , .
- 1983. Taxonomy. In: BroughtonWJ, ed. Nitrogen fixation , vol. 3 : legumes. Oxford, UK: Oxford University Press, 1–35. , , .
- 2001a. Changes in actin microfilament arrays in developing pea root nodule cells. Canadian Journal of Botany 79: 767–776. , .
- 2001b. Organization of microtubules in developing pea root nodule cells. Canadian Journal of Botany 79: 777–786. , .
- 1996. Rhizobium lipochitooligosaccharides nodulation factors: signalling molecules mediating recognition and morphogenesis. Annual Review of Biochemistry 65: 503–535. , , .
- 1981. Nitrogen-fixing nodules induced by Rhizobium on the stem of the tropical legume Sesbania rostrata. FEMS Microbiology Letters 10: 313–317. , .
- 1988. Entry of rhizobia into roots of Mimosa scabrella Bentham occurs between epidermal cells. Journal of General Microbiology 134: 2291–2296. , , .
- 1987. The morphogenesis of lupine root nodules during infection by Rhizobium lupini. Acta Societatia Botanicarum Poloniae 56: 687–703. , , .
- 1998. Diversity of root nodulation and rhizobial infection processes. In: SpainkHP, KondorosiA, HooykaasPJJ, eds. The rhizobiaceae, molecular biology of model plant-associated bacteria. Dordrecht, The Netherlands: Kluwer Academic Publishers, 347–360. , , , .
- 1992. Developmental biology of legume nodulation. New Phytologist 122: 211–237. .
- 1938. The water culture method for growing plants without soil. California Agricultural Experimental Station Circle, no. 347. , .
- 1988. Storage of competent cells for Agrobacterium tumefaciens transformation. Nucleic Acid Research 16: 9877–9877. , .
- 1997. Temporal relationships between nitrogenase and intercellular glycoprotein in developing white lupin nodules. Annals of Botany 69: 493–503. , , , .
- 1991. Intercellular location of glycoprotein in soybean nodules: effects of altered rhizosphere oxygen concentration. Plant, Cell & Environment 14: 467–476. , , , .
- 1992. The Rhizobium infection process. In: StaceyG, BurrisRH, EvansHJ, eds. Biological nitrogen fixation. New York, USA: Chapman and Hall, 349–398. .
- 1989. Rhizobium-legume nodulation: life together in the underground. Cell 56: 203–214. .
- 2000. Formation and abortion of root nodule primordia in Lupinus luteus L. Acta Biologica Cracoviensia 42: 87–102. , , , .
- 1992. Influencia de la fertilización fosfatada sobre la simbiosis Lupinus albus L.-Bradyrhizobium sp. (Lupinus). Producción y competitividad. Madrid, Spain: Editorial Universidad Complutense de Madrid. .
- 1992. Application of immunolabelling techniques to Bradyrhizobium dual occupation in Lupinus nodules. Journal of Plant Physiology 140: 84–91. , , .
- 1994. Root nodulation of Sesbania rostrata. Journal of Bacteriology 176: 1060–1068. , , , , .
- 1996. Down-regulation of the Rhizobium ntr system in the determinate nodule of Phaseolus vulgaris indentifies a specific developmental zone. Molecular Plant–Microbe Interactions 9: 243–251. , , , , , .
- 1992. Structure and growth of infection threads in the legume simbiosis with Rhizobium leguminosarum. Plant Journal 2: 385–395. , , .
- 1963. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. Journal of Cell Biology 17: 208–213. .
- 1978. Membranes in lupin root nodules. I. The role of Golgi bodies in the biogenesis of infection threads and peribacteroid membranes. Journal of Cell Science 30: 129–149. , , , .
- 1989. Bacterium release into host cells of nitrogen-fixing soybean nodules: the symbiosome membrane comes from three sources. European Journal of Cell Biology 49: 13–23. , .
- 1989. Molecular cloning: a laboratory manual, 2nd edn. New York, USA: Cold Springer Harbor Laboratory Press. , , .
- 1989. Which steps are essential for the formation of functional legumes nodules. New Phytologist 111: 129–153. .
- 1994. Cell wall degradation during infection thread formation by the root nodule bacterium Rhizobium leguminosarum is a two step process. European Journal of Cell Biology 64: 88–94. , , , .
- 1995. The unique root-nodule symbiosis between Rhizobium and the aquatic legume Neptunia natans (L.f.) Druce. Planta 196: 311–320. , , , , , , .
- 1993. Anatomical and ultrastructural observations on infection of Lupinus angustifolius L. by Bradyrhizobium sp. Journal of Computer-Assisted Microscopy 5: 47–51. , , .
- 1992. Microscopic evidence on how iron deficiency limits nodule initiation in Lupinus angustifolius L. New Phytologist 121: 457–467. , , , .
- 1994. Development of Phaseolus vulgaris root nodules. Molecular Plant–Microbe Interactions 7: 582–589. , , , , .
- 1999. Refined analysis of early symbiotic steps of the Rhizobium–Medicago interaction in relationship with microtubular cytoskeleton rearrangements. Development 126: 3617–3628. , , .
- 2000. Saprophytic intracellular rhizobia in alfalfa nodules. Molecular Plant–Microbe Interactions 13: 1204–1213. , , , , , , .
- 1983. Initial stages in the morphogenesis of nitrogen-fixing stem nodules of Sesbania rostrata. Journal of Bacteriology 156: 888–897. , , .
- 1989. Common components of the infection thread matrix and the intercellular space identified by immunocytochemical analysis of pea nodules and uninfected roots. EMBO Journal 8: 967–978. , , , , , .
- 1990. Correlation between ultrastructural differentiation of bacteroids and nitrogen fixation in alfalfa nodules. Journal of Bacteriology 172: 4295–4306. , , , .
- 1970. A manual for the practical study of root-nodule bacteria. Oxford, UK: Blackwell Scientific Publications, IBP Handbook no. 15. .
- 1989. Leghemoglobin in lupin plants (Lupinus albus L. cv. Multolupa). Plant Physiology 90: 452–457. , , , .
- 2000. Phylogeny of root and stem-nodule bacteria associated with legumes. In: TriplettEW, ed. Prokaryotic nitrogen fixation: a model system for analysis of a biology process. Wymondham, UK: Horizon Scientific Press, 177–186. , .
- 1996. Organelle inheritance. Cell 84: 395–400. , .
- 1992. A region of the broad-host-range plasmid RK2 causes stable in planta inheritance of plasmids in Rhizobium meliloti cells isolated from alfalfa root nodules. Journal of Bacteriology 174: 7486–7489. , , .
- 1998. Cytoskeleton arrays in the cells of soybean root nodules: the role of actin microfilaments in the organisation of symbiosomes. Protoplasma 203: 1194–1205. , , .