红豆草根瘤侵入线的超微结构研究

用透射电镜对红豆草根瘤侵入线的超微结构进行了观察研究.结果表明,(1)红豆草根瘤侵入线由胞间隙和胞间层细胞壁内陷形成,它们的体积较小,多为管状,基质丰富,含菌很少,常有分叉和1个以上的基质区,而且不同基质区的电子密度、细菌数量和侵入线壁厚度都不相同.(2)红豆草根瘤的侵入线十分丰富,它们不仅大量存在于根瘤分生细胞和幼龄侵染细胞中,也经常出现在发育成熟的侵染细胞内.(3)红豆草根瘤中有一种近似圆形的特殊结构,表面由一层膜包围,其内电子密度较低且无固定结构,且只位于侵染细胞的细胞质中,常在侵入线附近,从不出现在侵染细胞的液泡内和非侵染细胞里面.     

Localization of acid phosphatase activity in the apoplast of pea (Pisum sativum L.) root nodules grown under phosphorus deficiency

ACPase activity was localized in the apoplast of pea root nodules under phosphorus deficiency. Pea plants (Pisum sativum L. cv. Sze ciotygodniowy) where inoculated with Rhizobium leguminosarum bv. viciae 248 and were cultured on nitrogen-free medium with phosphate (−N/+P) or phosphate-deficient (−N/−P) one. In comparison with control nodules, P-deficient nodules showed the increase of ACPase activity in plant cell walls and the infection threads. The increase in bacterial ACPase activity under P-deficiency may reflect higher demand for inorganic phosphorus that is necessary for bacteria multiplication within the infection threads. The increase of ACPase activity in nodule apoplast under P stress may enlarge the availability of phosphate for plant and bacteria.     

Plant defence and delayed infection of alfalfa pseudonodules induced by an exopolysaccharide (EPS I)-deficient Rhizobium meliloti mutant

Mutants of the symbiotic soil bacterium Rhizobium meliloti that fail to synthesize the acidic exopolysaccharide EPS I were unable to induce infected root nodules on Medicago sativa L. (alfalfa). These strains, however, elicited pseudonodules that contained no infection threads or bacteroids. The cortical cell walls of the pseudonodules were abnormally thick and incrusted with an autofluorescent material. Parts of these cell walls and wall appositions contained callose. Biochemical analysis of nodules induced by the EPS I-deficient R. meliloti mutant revealed an increase of phenolic compounds bound to the nodule cell walls when compared with the wild-type strain. These microscopic and biochemical data indicated that a general plant defence response against the EPS I-deficient mutant of R. meliloti was induced in alfalfa pseudonodules. Following prolonged incubation with the EPS I-deficient R. meliloti mutant, the defence system of the alfalfa plant could be overcome by the rhizobium mutant. In the case of the delayed infections, the mutants colonized lobes of the pseudonodules, but the infection threads in these nodules had an abnormal morphology. They were greatly enlarged and did not contain the typical gum-like matrix inside. The bacteria were tightly packed. Based on the mechanism of phytopathogenic interactions, we propose that EPS I or a related compound may act as a suppressor of the alfalfa plant defence system, enabling R. meliloti to infect the plant.     

Infection and Nodule Development in Aotus ericoides (Vent.) G. Don, A Woody Native Australian Legume

Infection and nodule development were studied by light and electronmicroscopy in Aotus ericoides, a woody native Australian legume,inoculated with a slow-growing field isolate of Rhizobium. Rhizobiabound to straight, but not deformed, root hairs, as detectedby immunofluorescence. Neither markedly curled root hairs norroot hairs with infection threads were seen. Nodules were indeterminate(astragaloid), with a peripheral meristematic layer, few vasculartraces and both infected and uninfected cells in the centralinfected zone. Infection threads containing contorted bacteriawere present throughout the nodule. Swollen, rod-shaped bacteriain infected cells were in groups in vesicles bounded by plasmalemma-derivedperibacteroid membranes. Senescence in infected cells was associatedwith accumulation of a fibrillar matrix inside peribacteroidmembranes, distortion of bacteria and destruction of most cytoplasmiccontents of the bacteria and host cells; however, most bacterialand plant membranes and plant cell walls remained intact. Ineffectivenesswas associated with relatively little, short-lived infectedtissue. Events in infection and nodule development were similarto those in most herbaceous legumes but showed characters ofboth determinate and indeterminate nodules. Key words: Bacteroids, Legume, Nitrogen-fixing, Nodule, Rhizobium     

Effects of Boron on Rhizobium-Legume Cell-Surface Interactions and Nodule Development

Boron (B) is an essential micronutrient for the development of nitrogen-fixing root nodules in pea (Pisum sativum). By using monoclonal antibodies that recognize specific glycoconjugate components implicated in legume root-nodule development, we investigated the effects of low B on the formation of infection threads and the colonization of pea nodules by Rhizobium leguminosarum bv viciae. In B-deficient nodules the proportion of infected host cells was much lower than in nodules from plants supplied with normal quantities of B. Moreover, the host cells often developed enlarged and abnormally shaped infection threads that frequently burst, releasing bacteria into damaged host cells. There was also an over-production of plant matrix material in which the rhizobial cells were embedded during their progression through the infection thread. Furthermore, in a series of in vitro binding studies, we demonstrated that the presence of B can change the affinity with which the bacterial cell surface interacts with the peribacteroid membrane glycocalyx relative to its interaction with intercellular plant matrix glycoprotein. From these observations we suggest that B plays an important role in mediating cell-surface interactions that lead to endocytosis of rhizobia by host cells and hence to the correct establishment of the symbiosis between pea and Rhizobium.     

A block in the endocytosis of Rhizobium allows cellular differentiation in nodules but affects the expression of some peribacteroid membrane nodulins

A transposon-induced mutant (T8-1) of Bradyrhizobium japonicum (61A76) was unable to develop into the nitrogen-fixing endosymbiotic form, the bacteroid. Comparison between this mutant and T5-95, an ineffective (non-nitrogen fixing, Fix^-) mutant, confirmed that the process of bacteroid development is a distinct phase of differentiation of the endosymbiont and is independent of nitrogen fixation activity. The T8-1 mutant was able to induce normal-size nodules which differentiated two plant cell types and contained numerous infection threads. However, the infected cells were devoid of bacteroids. Electron microscopy revealed that the ends of the infection threads were broken down in a normal manner once the thread had penetrated the cells, but the mutant was not internalized by endocytosis. The lack of peribacteroid membrane (PBM) in nodules induced by this mutant was correlated with a reduced level of expression of plant genes coding for PBM nodulins. These genes were expressed in the T5-95 mutant, showing that the low expression in T8-1 was not due to the lack of nitrogen fixation. One of the PBM nodulins, nodulin-26, was found at normal levels in the nodules which lack PBM, suggesting that there are at least two developmental stages in PBM biosynthesis. These data suggest that a coordination of plant and Rhizobium gene expression is required for the release and internalization of bacteria into the PBM compartments of infected cells of nodules.author for correspondence     

Rhizobium meliloti nodulation genes allow Agrobacterium tumefaciens and Escherichia coli to form pseudonodules on alfalfa.

Regions of the Rhizobium meliloti symbiotic plasmid (20 to 40 kilobase pairs long) containing nodulation (nod) genes were transferred to Agrobacterium tumefaciens or Escherichia coli by conjugation. The A. tumefaciens and E. coli transconjugants elicited root hair curling and the formation of ineffective pseudonodules on inoculated alfalfa plants. A tumefaciens elicited pseudonodules formed at a variable frequency, ranging from 15 to 45%, irrespective of the presence of the Ti plasmid. These pseudonodules developed characteristic nodule meristems, and in some nodules, infection threads were found within the interior of nodules. Infrequently, infection threads penetrated deformed root hairs, but these threads were found only in a minority of nodules. There was no evidence of bacterial release from the infection threads. In addition to being found within threads, agrobacteria were also found in intercellular spaces and within nodule cells that had senesced . In the latter case, the bacteria appeared to invade the nodule cells independently of infection threads and degenerated at the same time as the senescing host cells. No peribacteroid membranes enclosed any agrobacteria , and no bacteroid differentiation was observed. In contrast to the A. tumefaciens-induced pseudonodules , the E. coli-induced pseudonodules were completely devoid of bacteria; infection threads were not found to penetrate root hairs or within nodules. Our results suggest that relatively few Rhizobium genes are involved in the earliest stages of nodulation, and that curling of root hairs and penetration of bacteria via root hair infection threads are not prerequisites for nodule meristem formation in alfalfa.     

Development of N2-fixing nodules on the wetland legume Lotus uliginosus exposed to conditions of flooding

Seeds of the wetland legume, Lotus uliginosus , were germinated and grown in vermiculite which was either continuously flooded or well-drained. Plants from both treatments were infected by Mesorhizobium loti strain DUS341 via a 'classical' root hair pathway, although some flooded plants appeared to be infected via enlarged epidermal cells. Subsequent to infection by M. loti , nodule meristems, which had developed within the root outer cortex, were penetrated by infection threads that released bacteria into the meristematic cells. The infection threads and infection droplets were immunogold labelled with monoclonal antibodies (MAC265 and MAC236) that recognize epitopes (at approx. 155/170 and 170/210 kDa, respectively) on a glycoprotein component of the matrix that surrounded the bacteria within the threads or droplets. Although labelling of infection threads or infection droplets with MAC236 was stronger than that with MAC265, both antibodies strongly labelled material occluding intercellular spaces in the cortices of developing nodules that had not yet expressed nitrogenase (as determined by a lack of signal after immunogold labelling with an antibody raised against nitrogenase component II). After 60 d, nitrogenase activity, shoot and root dry weights, and nodule fresh weight per plant did not differ between the treatments. After a further 30 d submergence, the flooded stems developed extensive aerenchyma and there was profuse development of (nodulated) adventitious roots. Nodules also formed at the junction of adventitious roots and the subtending stem and these were connected vascularly to a small stalk of tissue which gave rise to both a nodule and an adventitious root. The flooded nodules had prominent lenticels, and possible air pathways from the atmosphere to the nitrogen-fixing bacteroids are discussed.     

The pea (Pisum sativum L.) genes sym33 and sym40 control infection thread formation and root nodule function

Two novel non-allelic mutants that were unable to fix nitrogen (Fix^?) were obtained after EMS (ethyl methyl sulfonate) mutagenesis of pea (Pisum sativum L.). Both mutants, SGEFix^?–1 and SGEFix^?–2, form two types of nodules: SGEFix^?–1 forms numerous white and some pink nodules, while mutant SGEFix^?–2 forms white nodules with a dark pit at the distal end and also some pinkish nodules. Both mutations are monogenic and recessive. In both lines the manifestation of the mutant phenotype is associated with the root genotype. White nodules of SGEFix^?–1 are characterised by hypertrophied infection threads and infection droplets, mass endocytosis of bacteria, abnormal morphological differentiation of bacteroids, and premature degradation of nodule symbiotic structures. The structure of the pink nodules of SGEFix^?–1 does not differ from that of the parental line, SGE. White nodules of SGEFix^?–2 are characterised by “locked” infection threads surrounded with abnormally thick plant cell walls. In these nodules there is no endocytosis of bacteria into host-cell cytoplasm. The pinkish nodules of SGEFix^?–2 are characterised by virtually undifferentiated bacteroids and premature degradation of nodule tissues. Thus, the novel pea symbiotic genes, sym40 and sym33, identified after complementation analysis in SGEFix^?–1 and SGEFix^?–2 lines, respectively, control early nodule developmental stages connected with infection thread formation and function.     

Structure and growth of infection threads in the legume symbiosis with Rhizobium leguminosarum

Specific antibodies and enzyme–gold probes were used to study the structure and development of infection threads in nodules induced by Rhizobium leguminosarum on the roots of Vicia, Pisum and Phaseolus. In Pisum nodules, the tubular infection thread wall contains polysaccharides antigenically similar to those of the cell wall, including cellulose, xyloglucan, methyl-esterified pectin and non-esterified pectin, but none of these wall components is present around the infection droplet structures from which bacteria are internalized by plant plasma membrane. As reported previously for pea nodules, the luminal matrix of infection threads and infection droplets contains a plant glycoprotein; this glycoprotein is also secreted by infected and uninfected cortical cells of a Vicia root at the earliest stages of nodule initiation. Synthesis of a transcellular infection thread apparently involves reorganized deposition of components normally targeted to the cell wall, and infection thread growth is orientated anticlinally through the outer cortex in the same plane observed for the deposition of new cell walls following mitosis. Both the development of infection threads in the outer cortex and the initiation of cell division in the inner cortex are preceded by a similar process of cell reactivation involving centralization of nuclei and the development of anticlinal transvacuolar strands. It is therefore suggested that the two Rhizobium-induced processes of infection thread growth and cortical cell division may both be consequences of a similar plant cell response in the inner and outer root cortex, respectively. Phaseolus nodules contained only short intracellular infection structures which terminated within individual cells and contained no luminal matrix material. The differences in infection thread structure between Pisum and Phaseolus nodules may reflect differences in ontogeny between “indeterminate” and “determinate” nodule meristems.     

The development and structure of root nodules on bambara groundnut [Voandzeia (Vigna)subterranea]

The infection of Vigna subterranea (formerly Voandzeia subterranea) by Bradyrhizobium strain MAO 113 (isolated from V. subterranea) was examined by light and transmission electron microscopy. Bacteria accumulated on the epidermis close to root hairs, and subsequently entered the latter via infection threads. Most of the steps involved in nodule formation were generally characteristic of determinate nodules, such as those which form on the closely related V. radiata. For example, nodule meristems were induced beneath the root epidermis adjacent to infected root hairs, but prior to infection of the meristem by rhizobia. Moreover, after the infection of some of the meristematic cells by the infection threads, and the release of the rhizobia into membrane-bound vesicles, the infection process ceased and dissemination of the rhizobia was by division of already-infected host cells. However, there were some aspects of this process in V. subterranea which have been more commonly described in indeterminate nodules. These include long infection threads entering a number of cells within the meristems simultaneously and a matrix within infection threads which was strongly labelled with immunogold monoclonal antibodies, MAC236 and MAC265, which recognize epitopes on an intercellular glycoprotein. The MAC236 and MAC265 antibodies also recognized material in the unwalled infection droplets surrounding bacteria which were newly-released from the infection threads. The amount of labelling shown was more characteristic of the long infection threads seen in indeterminate nodules such as pea (Pisum sativum) and Neptunia plena. The structure of mature V. subterranea nodules was similar to that described for other determinate nodules such as Glycine max, Vigna unguiculata and V.radiata, i.e. they were spherical and the infected zone consisted of both infected and uninfected cells. Surrounding the infected tissue was an inner cortex of uninfected cell layers containing the putative components of an oxygen diffusion barrier (including glycoprotein-occluded intercellular spaces), and an outer cortex with cells containing calcium oxalate crystals.     

The development and structure of root nodules on bambara groundnut [Voandzeia (Vigna)subterranea]

The infection of Vigna subterranea (formerly Voandzeia subterranea) by Bradyrhizobium strain MAO 113 (isolated from V. subterranea) was examined by light and transmission electron microscopy. Bacteria accumulated on the epidermis close to root hairs, and subsequently entered the latter via infection threads. Most of the steps involved in nodule formation were generally characteristic of determinate nodules, such as those which form on the closely related V. radiata. For example, nodule meristems were induced beneath the root epidermis adjacent to infected root hairs, but prior to infection of the meristem by rhizobia. Moreover, after the infection of some of the meristematic cells by the infection threads, and the release of the rhizobia into membrane-bound vesicles, the infection process ceased and dissemination of the rhizobia was by division of already-infected host cells. However, there were some aspects of this process in V. subterranea which have been more commonly described in indeterminate nodules. These include long infection threads entering a number of cells within the meristems simultaneously and a matrix within infection threads which was strongly labelled with immunogold monoclonal antibodies, MAC236 and MAC265, which recognize epitopes on an intercellular glycoprotein. The MAC236 and MAC265 antibodies also recognized material in the unwalled infection droplets surrounding bacteria which were newly-released from the infection threads. The amount of labelling shown was more characteristic of the long infection threads seen in indeterminate nodules such as pea (Pisum sativum) and Neptunia plena. The structure of mature V. subterranea nodules was similar to that described for other determinate nodules such as Glycine max, Vigna unguiculata and V.radiata, i.e. they were spherical and the infected zone consisted of both infected and uninfected cells. Surrounding the infected tissue was an inner cortex of uninfected cell layers containing the putative components of an oxygen diffusion barrier (including glycoprotein-occluded intercellular spaces), and an outer cortex with cells containing calcium oxalate crystals.     

Use of green fluorescent protein to visualize the early events of symbiosis between Rhizobium meliloti and alfalfa (Medicago sativa).

A gene encoding a variant of green fluorescent protein (GFP) of Aequorea victoria was put under the control of a promoter which is constitutive in Rhizobium meliloti. The heterologous GFP gene was expressed at high levels during all stages of symbiosis, allowing R. meliloti cells to be visualized as they grew in the rhizosphere, on the root surface, and inside infection threads. In addition, nodules that were infected with bacteria which were synthesizing GFP fluoresced when illuminated with blue light. GFP-tagged bacteria could be seen inside infection threads, providing the opportunity to measure the growth rate and determine the patterns of growth of R. meliloti residing inside its host plant.     

nip, a symbiotic Medicago truncatula mutant that forms root nodules with aberrant infection threads and plant defense-like response

To investigate the legume-Rhizobium symbiosis, we isolated and studied a novel symbiotic mutant of the model legume Medicago truncatula, designated nip (numerous infections and polyphenolics). When grown on nitrogen-free media in the presence of the compatible bacterium Sinorhizobium meliloti, the nip mutant showed nitrogen deficiency symptoms. The mutant failed to form pink nitrogen-fixing nodules that occur in the wild-type symbiosis, but instead developed small bump-like nodules on its roots that were blocked at an early stage of development. Examination of the nip nodules by light microscopy after staining with X-Gal for S. meliloti expressing a constitutive GUS gene, by confocal microscopy following staining with SYTO-13, and by electron microscopy revealed that nip initiated symbiotic interactions and formed nodule primordia and infection threads. The infection threads in nip proliferated abnormally and very rarely deposited rhizobia into plant host cells; rhizobia failed to differentiate further in these cases. nip nodules contained autofluorescent cells and accumulated a brown pigment. Histochemical staining of nip nodules revealed this pigment to be polyphenolic accumulation. RNA blot analyses demonstrated that nip nodules expressed only a subset of genes associated with nodule organogenesis, as well as elevated expression of a host defense-associated phenylalanine ammonia lyase gene. nip plants were observed to have abnormal lateral roots. nip plant root growth and nodulation responded normally to ethylene inhibitors and precursors. Allelism tests showed that nip complements 14 other M. truncatula nodulation mutants but not latd, a mutant with a more severe nodulation phenotype as well as primary and lateral root defects. Thus, the nip mutant defines a new locus, NIP, required for appropriate infection thread development during invasion of the nascent nodule by rhizobia, normal lateral root elongation, and normal regulation of host defense-like responses during symbiotic interactions.     

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

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.     

Infection and Invasion of Roots by Symbiotic, Nitrogen-Fixing Rhizobia during Nodulation of Temperate Legumes

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.     

CERBERUS, a novel U-box protein containing WD-40 repeats, is required for formation of the infection thread and nodule development in the legume–Rhizobium symbiosis

Endosymbiotic infection of legume plants by Rhizobium bacteria is initiated through infection threads (ITs) which are initiated within and penetrate from root hairs and deliver the endosymbionts into nodule cells. Despite recent progress in understanding the mutual recognition and early symbiotic signaling cascades in host legumes, the molecular mechanisms underlying bacterial infection processes and successive nodule organogenesis are still poorly understood. We isolated a novel symbiotic mutant of Lotus japonicus , cerberus , which shows defects in IT formation and nodule organogenesis. Map-based cloning of the causal gene allowed us to identify the CERBERUS gene, which encodes a novel protein containing a U-box domain and WD-40 repeats. CERBERUS expression was detected in the roots and nodules, and was enhanced after inoculation of Mesorhizobium loti . Strong expression was detected in developing nodule primordia and the infected zone of mature nodules. In cerberus mutants, Rhizobium colonized curled root hair tips, but hardly penetrated into root hair cells. The occasional ITs that were formed inside the root hair cells were mostly arrested within the epidermal cell layer. Nodule organogenesis was aborted prematurely, resulting in the formation of a large number of small bumps which contained no endosymbiotic bacteria. These phenotypic and genetic analyses, together with comparisons with other legume mutants with defects in IT formation, indicate that CERBERUS plays a critical role in the very early steps of IT formation as well as in growth and differentiation of nodules.     

Molecular genetics of Rhizobium Meliloti symbiotic nitrogen fixation

The application of recombinant DNA techniques to the study of symbiotic nitrogen fixation has yielded a growing list of Rhizobium meliloti genes involved in the processes of nodulation, infection thread formation and nitrogenase activity in nodules on the roots of the host plant, Medicago sativa (alfalfa). Interaction with the plant is initiated by genes encoding sensing and motility systems by which the bacteria recognizes and approaches the root. Signal molecules, such as flavonoids, mediate a complex interplay of bacterial and plant nodulation genes leading to entry of the bacteria through a root hair. As the nodule develops, the bacteria proceed inward towards the cortex within infection threads, the formation of which depends on bacterial genes involved in polysaccharide synthesis. Within the cortex, the bacteria enter host cells and differentiate into forms known as bacteroids. Genes which encode and regulate nitrogenase enzyme are expressed in the mature nodule, together with other genes required for import and metabolism of carbon and energy sources offered by the plant.