RESUMEN
In indeterminate alfalfa nodules, the establishment of the senescent zone IV, in which both symbionts undergo simultaneous degeneration, has been considered, until now, as the end point of the symbiotic interaction. However, we now describe an additional zone, zone V, proximal to the senescent zone IV and present in alfalfa nodules more than 6 weeks old. In zone V, a new round of bacterial release occurs from remaining infection threads, leading to the reinvasion of plant cells that have completely senesced. These intracellular rhizobia are rod shaped and do not display the ultrastructural differentiation features of bacteroids observed in the more distal zones of the nodule. Interestingly, we have found that oxygen is available in zone V at a concentration compatible with both bacterial development and nitrogen fixation gene expression in newly released rhizobia. However, this expression is not correlated with acetylene reduction. Moreover, the pattern of nifH expression in this zone, as well as new data relating to expression in zone II, strongly suggest that nifH transcription in the nodule is under the control of a negative regulator in addition to oxygen. Our results support the conclusion that zone V is an ecological niche where intracellular rhizobia take advantage of the interaction for their exclusive benefit and live as parallel saprophytic partners. The demonstration of such an advantage for rhizobia in nodules was the missing evidence that Rhizobium-legume interactions are indeed symbiotic and, in particular, suggests that benefits to the two partners are associated with different developmental stages within the nodule.
Asunto(s)
Medicago sativa/microbiología , Raíces de Plantas/microbiología , Rhizobiaceae/aislamiento & purificación , Acetileno/metabolismo , Proteínas Bacterianas/biosíntesis , Ecosistema , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Fijación del Nitrógeno/genética , SimbiosisRESUMEN
In situ immunolocalization of tubulin revealed that important rearrangements occur during all the early symbiotic steps in the Medicago/R. meliloti symbiotic interaction. Microtubular cytoskeleton (MtC) reorganizations were observed in inner tissues, first in the pericycle and then in the inner cortex where the nodule primordium forms. Subsequently, major MtC changes occurred in outer tissues, associated with root hair activation and curling, the formation of preinfection threads (PITs) and the initiation and the growth of an infection network. From the observed sequence of MtC changes, we propose a model which aims to better define, at the histological level, the timing of the early symbiotic stages. This model suggests the existence of two opposite gradients of cell differentiation controlling respectively the formation of division centers in the inner cortex and plant preparation for infection. It implies that (i) MtC rearrangements occur in pericycle and inner cortex earlier than in the root hair, (ii) the infection process proceeds prior to the formation of the nodule meristem, (iii) the initial primordium prefigures the future zone II of the mature nodule and (iv) the nodule meristem derives from the nodule primordium. Finally, our data also strongly suggest that in alfalfa PIT differentiation, a stage essential for successful infection, requires complementary signaling additional to Nod factors.
Asunto(s)
Citoesqueleto/fisiología , Medicago sativa/fisiología , Microtúbulos/fisiología , Rhizobium/fisiología , Simbiosis/fisiología , Diferenciación Celular , Citoesqueleto/ultraestructura , Medicago sativa/citología , Medicago sativa/microbiología , Meristema , Microtúbulos/ultraestructura , Raíces de PlantasRESUMEN
Sinorhizobium meliloti nodulation factors (NFs) elicit a number of symbiotic responses in alfalfa (Medicago sativa) roots. Using a semiquantitative nodulation assay, we have shown that chemically synthesized NFs trigger nodule formation in the same range of concentrations (down to 10(-10) M) as natural NFs. The absence of O-sulfate or O-acetate substitutions resulted in a decrease in morphogenic activity of more than 100-fold and approximately 10-fold, respectively. To address the question of the influence of the structure of the N-acyl chain, we synthesized a series of sulfated tetrameric lipo-chitooligosaccharides (LCOs) having fatty acids of different lengths and with unsaturations either conjugated to the carbonyl group (2E) or located in the middle of the chain (9Z). A nonacylated, sulfated chitin tetramer was unable to elicit nodule formation. Acylation with short (C8) chains rendered the LCO active at 10(-7) M. The optimal chain length was C16, with the C16-LCO being more than 10-fold more active than the C12- and C18-LCOs. Unsaturations were important, and the diunsaturated 2E,9Z LCO was more active than the monounsaturated LCOs. We discuss different hypotheses for the role of the acyl chain in NF perception.
Asunto(s)
Lipopolisacáridos/química , Lipopolisacáridos/farmacología , Medicago sativa/efectos de los fármacos , Rhizobiaceae/química , Secuencia de Carbohidratos , Lipopolisacáridos/síntesis química , Medicago sativa/crecimiento & desarrollo , Medicago sativa/microbiología , Datos de Secuencia Molecular , Relación Estructura-Actividad , SimbiosisRESUMEN
Two Medicago truncatula nodulin genes putatively encoding proteins structurally related to two classes of proteins commonly associated with plant defense reactions have been characterized. MtN1 is homologous to two small, cysteine-rich, pathogen-inducible proteins from pea (pI39 and pI230), whereas MtN13 is closely related to the PR10 family of pathogenesis-related proteins. We show that neither MtN1 nor MtN13 is induced in leaves in response to pathogenic bacteria, and that both are exclusively expressed during nodulation. In situ hybridization experiments as well as Northern (RNA) studies of interactions between M. truncatula and either wild-type Rhizobium meliloti or mutants deficient in infection establish that MtN1 is associated with the infection process, while MtN13 represents the first specific marker described for the nodule outer cortex. Possible roles for MtN1 and MtN13 are discussed. We also present the identification of another member of the PR10 family, designated as MtPR10-1, whose regulation is strikingly different from that observed for MtN13, being constitutively expressed in roots and pathogen-inducible in leaves.
Asunto(s)
Genes de Plantas , Medicago sativa/genética , Proteínas de la Membrana , Proteínas de Plantas/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN Complementario/genética , ADN de Plantas/genética , Regulación de la Expresión Génica de las Plantas , Hibridación in Situ , Medicago sativa/microbiología , Datos de Secuencia Molecular , Pseudomonas/patogenicidad , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Planta/genética , ARN de Planta/metabolismo , Homología de Secuencia de Aminoácido , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/fisiología , Simbiosis/genética , Xanthomonas/patogenicidadRESUMEN
Reorganization of the plant cytoskeleton is thought to play an important role during nodule ontogeny. In situ immunolocalisation of tubulin reveals that important cytoskeletal changes, implying a transient disorganization followed by a newly patterned reorganization, occur in indeterminate and determinate nodules. In alfalfa nodules, cytoskeletal changes closely parallel the symbiotic differentiation features related to cell infection, bacterial release, endopolyploidization, cell enlargement, cell spatial organization and organelle ultrastructure and positioning. Moreover, the fact that microtubule disorganization can be correlated with Nod factor internalization in central infected cells suggests that Nod factors are possibly involved in the control of cytoskeletal changes which direct the differentiation of bacteria-containing cells.
Asunto(s)
Medicago sativa/citología , Proteínas de Microtúbulos/análisis , Microtúbulos/ultraestructura , Raíces de Plantas/citología , Diferenciación Celular , Citoplasma/química , Orgánulos/ultraestructura , Raíces de Plantas/química , Raíces de Plantas/microbiología , Raíces de Plantas/ultraestructura , RhizobiumRESUMEN
Symbiotic nitrogen fixation involves the development of specialized organs called nodules within which plant photosynthates are exchanged for combined nitrogen of bacterial origin. To determine the importance of bacterial nitrogen metabolism in symbiosis, we have characterized a key regulator of this metabolism in Rhizobium meliloti, the uridylylatable P(II) protein encoded by glnB. We have constructed both a glnB null mutant and a point mutant making nonuridylylatable P(II). In free-living conditions, P(II) is required for expression of the ntrC-dependent gene glnII and for adenylylation of glutamine synthetase I. P(II) is also required for efficient infection of alfalfa but not for expression of nitrogenase. However alfalfa plants inoculated with either glnB mutant are nitrogen-starved in the absence of added combined nitrogen. We hypothesize that P(II) controls expression or activity of a bacteroid ammonium transporter required for a functional nitrogen-fixing symbiosis. Therefore, the P(II) protein affects both Rhizobium nitrogen metabolism and alfalfa nodule development.
Asunto(s)
Proteínas Bacterianas/metabolismo , Medicago sativa/crecimiento & desarrollo , Medicago sativa/microbiología , Fijación del Nitrógeno , Sinorhizobium meliloti/metabolismo , Proteínas Bacterianas/genética , Cartilla de ADN/genética , Genes Bacterianos , Medicago sativa/metabolismo , Microscopía Electrónica , Modelos Biológicos , Mutación , Nitrógeno/metabolismo , Nitrogenasa/metabolismo , Proteínas PII Reguladoras del Nitrógeno , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/ultraestructura , SimbiosisRESUMEN
Symbiotic bacteria of the genus Rhizobium synthesize lipo-chitooligosaccharides, called Nod factors (NFs), which act as morphogenic signal molecules on legume hosts. The common nodABC genes, present in all Rhizobium species, are required for the synthesis of the core structure of NFs. NodC is an N-acetylglucosaminyltransferase, and NodB is a chitooligosaccharide deacetylase; NodA is involved in N-acylation of the aminosugar backbone. Specific nod genes are involved in diverse NF substitutions that confer plant specificity. We transferred to R. tropici, a broad host-range tropical symbiont, the ability to nodulate alfalfa, by introducing nod genes of R. meliloti. In addition to the specific nodL and nodFE genes, the common nodABC genes of R. meliloti were required for infection and nodulation of alfalfa. Purified NFs of the R. tropici hybrid strain, which contained chitin tetramers and were partly N-acylated with unsaturated C16 fatty acids, were able to elicit nodule formation on alfalfa. Inactivation of the R. meliloti nodABC genes suppressed the ability of the NFs to nodulate alfalfa. Studies of NFs from nodA, nodB, nodC, and nodI mutants indicate that (i) NodA of R. meliloti, in contrast to NodA of R. tropici, is able to transfer unsaturated C16 fatty acids onto the chitin backbone and (ii) NodC of R. meliloti specifies the synthesis of chitin tetramers. These results show that allelic variation of the common nodABC genes is a genetic mechanism that plays an important role in signaling variation and in the control of host range.
Asunto(s)
Aciltransferasas/genética , Amidohidrolasas/genética , Proteínas Bacterianas/genética , N-Acetilglucosaminiltransferasas/genética , Sinorhizobium meliloti/genética , Acilación , Aciltransferasas/biosíntesis , Aciltransferasas/química , Amidohidrolasas/biosíntesis , Amidohidrolasas/química , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Secuencia de Carbohidratos , Genotipo , Medicago sativa/microbiología , Datos de Secuencia Molecular , N-Acetilglucosaminiltransferasas/biosíntesis , N-Acetilglucosaminiltransferasas/química , Plásmidos , Reacción en Cadena de la Polimerasa , Sinorhizobium meliloti/fisiología , Supresión Genética , SimbiosisRESUMEN
Six nod box regulatory sequences are present in the Rhizobium meliloti genome. We have analysed the DNA region located downstream of nod box n6, and identified three open reading frames, designated nolQa, nolQb and nolS. LacZ fusions in these ORFs are not induced by classical nod gene inducers, which indicates that their expression either is not under the control of the nod box, or involves another regulatory mechanism acting in conjunction with the NodD/nod box regulatory circuit. Mutations in this n6 locus result in a delay in nodule formation on a particular host, Medicago lupulina. As this region is not strictly conserved among different R. meliloti strains, nolQa, nolQb and nolS may constitute auxiliary nodulation genes, for which the selection pressure is limited to particular host plants.
Asunto(s)
Genes Bacterianos , Sinorhizobium meliloti/genética , Regulación Bacteriana de la Expresión Génica , Medicago sativa/genética , Datos de Secuencia MolecularRESUMEN
In Rhizobium meliloti, the genes required for nodulation of legume hosts are under the control of DNA regulatory sequences called nod boxes. In this paper, we have characterized three host-specific nodulation genes, which form a flavonoid-inducible operon down-stream of the nod box n5. The first gene of this operon is identical to the nodL gene identified by Baev and Kondorosi (1992) in R. meliloti strain AK631. The product of the second gene, NoeA, presents some homology with a methyltransferase. nodL mutants synthesize Nod factors lacking the O-acetate substituent. In contrast, in strains carrying a mutation in either noeA or noeB, no modification in Nod-factor structure or production could be detected. On particular hosts, such as Medicago littoralis, mutants of the n5 operon showed a very weak nodule-forming ability, associated with a drastic decrease in the number of infection threads, while nodulation of Medicago truncatula or Melilotus alba was not affected. Thus, nodL noeA and noeB are host-specific nodulation genes. By using a gain-of-function approach, we showed that the presence of nodL, and hence of O-acetylated Nod factors, is a major prerequisite for confering the ability to nodulate alfalfa upon the heterologous bacterium Rhizobium tropici.
Asunto(s)
Genes Bacterianos/genética , Medicago sativa/microbiología , Operón , Sinorhizobium meliloti/genética , Acetilación , Acetiltransferasas/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Carbohidratos , Mapeo Cromosómico , Lipopolisacáridos/biosíntesis , Lipopolisacáridos/química , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Raíces de Plantas/microbiología , Secuencias Reguladoras de Ácidos Nucleicos , Regulón , Análisis de Secuencia de ADN , Especificidad de la Especie , SimbiosisRESUMEN
The symbiotic pattern of expression of Rhizobium meliloti N2-fixation genes is tightly coupled with the histological organization of the alfalfa root nodule and thus is under developmental control. N2-fixation gene expression is induced very sharply at a particular zone of the nodule called interzone II-III that precedes the zone where N2 fixation takes place. We show here that this coupling can be disrupted, hereby resulting in ectopic expression of N2-fixation genes in the prefixing zone II of the nodule. Uncoupling was obtained either by using a R. meliloti strain in which a mutation rendered N2-fixation gene expression constitutive with respect to oxygen in free-living bacterial cultures or by placing nodules induced by a wild-type R. meliloti strain in a microoxic environment. These results implicate oxygen as a key determinant of the symbiotic pattern of N2-fixation gene expression.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Fijación del Nitrógeno/genética , Sinorhizobium meliloti/fisiología , Aerobiosis , Electrofisiología , Medicago sativa/microbiología , Microelectrodos , Oxígeno/metabolismo , Raíces de Plantas , Mutación Puntual , Mapeo Restrictivo , Sinorhizobium meliloti/genéticaRESUMEN
The nucleotide sequence of the Rhizobium meliloti (Rm) fliP gene was determined. Rm strains carrying insertions within this gene were non-motile, lacked flagella and formed normal N2-fixing root nodules on alfalfa. The FliP protein showed similarity to several bacterial gene products involved in pathogenicity in both plant and animal pathogens. It is likely that all of these proteins share a common functional role in the secretion of specific proteins from bacterial cells.
Asunto(s)
Proteínas Bacterianas/genética , Genes Bacterianos/genética , Sinorhizobium meliloti/genética , Secuencia de Aminoácidos , Bacterias/genética , Bacterias/patogenicidad , Medicago sativa/microbiología , Datos de Secuencia Molecular , Mutagénesis Insercional , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Simbiosis , Virulencia/genéticaRESUMEN
Rhizobium meliloti produces lipochitooligosaccharide nodulation NodRm factors that are required for nodulation of legume hosts. NodRm factors are O-acetylated and N-acylated by specific C16-unsaturated fatty acids. nodL mutants produce non-O-acetylated factors, and nodFE mutants produce factors with modified acyl substituents. Both mutants exhibited a significantly reduced capacity to elicit infection thread (IT) formation in alfalfa. However, once initiated, ITs developed and allowed the formation of nitrogen-fixing nodules. In contrast, double nodF/nodL mutants were unable to penetrate into legume hosts and to form ITs. Nevertheless, these mutants induced widespread cell wall tip growth in trichoblasts and other epidermal cells and were also able to elicit cortical cell activation at a distance. NodRm factor structural requirements are thus clearly more stringent for bacterial entry than for the elicitation of developmental plant responses.
Asunto(s)
Lipopolisacáridos/metabolismo , Medicago sativa/microbiología , Oligosacáridos/metabolismo , Raíces de Plantas/microbiología , Sinorhizobium meliloti/fisiología , Simbiosis/fisiología , Bioensayo , Secuencia de Carbohidratos , Comunicación Celular/fisiología , Genes Bacterianos , Lipopolisacáridos/química , Espectrometría de Masas , Modelos Biológicos , Datos de Secuencia Molecular , Mutación , Oligosacáridos/química , Oligosacáridos/genética , Raíces de Plantas/citología , Receptores de Superficie Celular , Sinorhizobium meliloti/genética , Especificidad de la Especie , Simbiosis/genéticaRESUMEN
Extracellular lipo-oligosaccharides of Rhizobium, known as Nod factors, play a key role in the molecular signal exchange which leads to the specific nitrogen-fixing symbiotic association between the soil microbe and its host legume. The biological activity of Nod factors and their perception by the host plant during the earliest stages of the Rhizobium/legume interaction have been studied using transgenic alfalfa carrying a fusion between the promoter of the early nodulin gene MtENOD12 and the beta-glucuronidase (GUS) reporter gene. Histochemical staining has shown that GUS accumulates specifically in the differentiating root epidermis, prior to and during root hair emergence, within 2-3 h following the addition of purified Rhizobium meliloti Nod factors. This precocious transcriptional activation of the MtENOD12 gene, reminiscent of that observed after inoculation with intact Rhizobium, implies that the Nod factor signal can be perceived at a developmental stage preceding root hair formation. GUS activity can be detected following treatment with a wide range of R. meliloti Nod factor concentrations down to 10(-13) M, and furthermore, this rapid response to the bacterial elicitor appears to be non-systemic. Significantly, MtENOD12-GUS expression is not observed after inoculation with a R. meliloti nodH mutant which synthesizes exclusively non-sulphated Nod factors. Indeed purified Nod factors which lack the sulphate substituent are approximately 1000-fold less active than their sulphated counterparts. Thus, the triggering of ENOD12 transcription in the alfalfa root epidermis is a rapid molecular response which is subject to the same host-specificity determinant (Nod factor sulphation) that governs the interaction between alfalfa and its bacterial symbiont.
Asunto(s)
Lipopolisacáridos/farmacología , Medicago sativa/genética , Proteínas de la Membrana , Proteínas de Plantas/genética , Sinorhizobium meliloti/metabolismo , Secuencia de Carbohidratos , Expresión Génica/efectos de los fármacos , Genes de Plantas , Genes Reporteros , Glucuronidasa/genética , Lipopolisacáridos/química , Medicago sativa/microbiología , Datos de Secuencia Molecular , Estructura Molecular , Plantas Modificadas Genéticamente , Simbiosis , Transcripción Genética/efectos de los fármacosRESUMEN
The tropical legume Sesbania rostrata can be nodulated by Azorhizobium caulinodans on both its stem and its root system. Here we investigate in detail the process of root nodulation and show that nodules develop exclusively at the base of secondary roots. Intercellular infection leads to the formation of infection pockets, which then give rise to infection threads. Concomitantly with infection, cortical cells of the secondary roots dedifferentiate, forming a meristem which has an "open-basket" configuration and which surrounds the initial infection site. Bacteria are released from the tips of infection threads into plant cells via "infection droplets," each containing several bacteria. Initially, nodule differentiation is comparable to that of indeterminate nodules, with the youngest meristematic cells being located at the periphery and the nitrogen-fixing cells being located at the nodule center. Because of the peculiar form of the meristem, Sesbania root nodules develop uniformly around a central axis. Nitrogen fixation is detected as early as 3 days following inoculation, while the nodule meristem is still active. Two weeks after inoculation, meristematic activity ceases, and nodules then show the typical histology of determinate nodules. Thus, root nodule organogenesis in S. rostrata appears to be intermediate between indeterminate and determinate types.
Asunto(s)
Fabaceae/microbiología , Plantas Medicinales , Rhizobiaceae/patogenicidad , Diferenciación Celular , Fabaceae/anatomía & histología , Fabaceae/ultraestructura , Morfogénesis , Especificidad de Órganos , Rhizobiaceae/ultraestructura , Factores de Tiempo , Clima TropicalAsunto(s)
Fabaceae/embriología , Lipopolisacáridos/metabolismo , Plantas Medicinales , Rhizobium/metabolismo , Transducción de Señal , Simbiosis , Secuencia de Carbohidratos , Fabaceae/genética , Fabaceae/metabolismo , Fabaceae/microbiología , Lipopolisacáridos/biosíntesis , Datos de Secuencia Molecular , Fijación del NitrógenoRESUMEN
To study the molecular responses of the host legume during early stages of the symbiotic interaction with Rhizobium, we have cloned and characterized the infection-related early nodulin gene MtENOD12 from Medicago truncatula. In situ hybridization experiments have shown that, within the indeterminate Medicago nodule, transcription of the MtENOD12 gene begins in cell layers of meristematic origin that lie ahead of the infection zone, suggesting that these cells are undergoing preparation for bacterial infection. Histochemical analysis of transgenic alfalfa plants that express an MtENOD12 promoter-beta-glucuronidase gene fusion has confirmed this result and further revealed that MtENOD12 gene transcription occurs as early as 3 to 6 hr following inoculation with R. meliloti in a zone of differentiating root epidermal cells which lies close to the growing root tip. It is likely that this transient, nodulation (nod) gene-dependent activation of the ENOD12 gene also corresponds to the preparation of the plant for bacterial infection. We anticipate that this extremely precocious response to Rhizobium will provide a valuable molecular marker for studying early signal exchange between the two symbiotic organisms.
Asunto(s)
Genes de Plantas , Medicago sativa/genética , Proteínas de la Membrana , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/genética , Sinorhizobium meliloti/genética , Secuencia de Aminoácidos , Secuencia de Bases , Diferenciación Celular/genética , Clonación Molecular , Regulación de la Expresión Génica , Glucuronidasa , Medicago sativa/crecimiento & desarrollo , Datos de Secuencia Molecular , Fijación del Nitrógeno/genética , Proteínas de Plantas/aislamiento & purificación , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Regiones Promotoras Genéticas , Homología de Secuencia de Ácido Nucleico , Sinorhizobium meliloti/fisiología , Simbiosis , Transcripción GenéticaRESUMEN
The symbiosis between Rhizobium and legumes is highly specific. For example, R. meliloti elicits the formation of root nodules on alfalfa and not on vetch. We recently reported that R. meliloti nodulation (nod) genes determine the production of acylated and sulfated glucosamine oligosaccharide signals. We now show that the biochemical function of the major host-range genes, nodH and nodPQ, is to specify the 6-O-sulfation of the reducing terminal glucosamine. Purified Nod factors (sulfated or not) from nodH+ or nodH- strains exhibited the same plant specificity in a variety of bioassays (root hair deformations, nodulation, changes in root morphology) as the bacterial cells from which they were purified. These results provide strong evidence that the molecular mechanism by which the nodH and nodPQ genes mediate host specificity is by determining the sulfation of the extracellular Nod signals.
Asunto(s)
Lipopolisacáridos/metabolismo , Rhizobium/genética , Sulfotransferasas/metabolismo , Secuencia de Aminoácidos , Clonación Molecular , Genes Bacterianos , Medicago sativa/microbiología , Datos de Secuencia Molecular , Mapeo Restrictivo , Alineación de Secuencia , Sulfatos , Sulfotransferasas/genética , SimbiosisRESUMEN
The interaction between Rhizobium lipopolysaccharide (LPS) and white clover roots was examined. The Limulus lysate assay indicated that Rhizobium leguminosarum bv. trifolii (hereafter called R. trifolii) released LPS into the external root environment of slide cultures. Immunofluorescence and immunoelectron microscopy showed that purified LPS from R. trifolii 0403 bound rapidly to root hair tips and infiltrated across the root hair wall. Infection thread formation in root hairs was promoted by preinoculation treatment of roots with R. trifolii LPS at a low dose (up to 5 micrograms per plant) but inhibited at a higher dose. This biological activity of LPS was restricted to the region of the root present at the time of exposure to LPS, higher with LPS from cells in the early stationary phase than in the mid-exponential phase, incubation time dependent, incapable of reversing inhibition of infection by NO3- or NH4+, and conserved among serologically distinct LPSs from several wild-type R. trifolii strains (0403, 2S-2, and ANU843). In contrast, infections were not increased by preinoculation treatment of roots with LPSs from R. leguminosarum bv. viciae strain 300, R. meliloti 102F28, or members of the family Enterobacteriaceae. Most infection threads developed successfully in root hairs pretreated with R. trifolii LPS, whereas many infections aborted near their origins and accumulated brown deposits if pretreated with LPS from R. meliloti 102F28. LPS from R. leguminosarum 300 also caused most infection threads to abort. Other specific responses of root hairs to infection-stimulating LPS from R. trifolii included acceleration of cytoplasmic streaming and production of novel proteins. Combined gas chromatography-mass spectroscopy and proton nuclear magnetic resonance analyses indicated that biologically active LPS from R. trifolii 0403 in the early stationary phase had less fucose but more 2-O-methylfucose, quinovosamine, 3,6-dideoxy-3-(methylamino)galactose, and noncarbohydrate substituents (O-methyl, N-methyl, and acetyl groups) on glycosyl components than did inactive LPS in the mid-exponential phase. We conclude that LPS-root hair interactions trigger metabolic events that have a significant impact on successful development of infection threads in this Rhizobium-legume symbiosis.
Asunto(s)
Fabaceae/microbiología , Lipopolisacáridos/metabolismo , Plantas Medicinales , Rhizobium/metabolismo , Simbiosis , Western Blotting , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Fabaceae/metabolismo , Fabaceae/ultraestructura , Técnica del Anticuerpo Fluorescente , Microscopía ElectrónicaRESUMEN
Rhizobium meliloti trc genes controlling the catabolism of trigonelline, a plant secondary metabolite often abundant in legumes, are closely linked to nif-nod genes on the symbiotic megaplasmid pSym [Boivin, C., Malpica, C., Rosenberg, C., Denarie, J., Goldman, A., Fleury, V., Maille, M., Message, B., and Tepfer, D. (1989). In Molecular Signals in the Microbe-Plant Symbiotic and Pathogenic Systems. (Berlin: Springer-Verlag), pp. 401-407]. To investigate the role of trigonelline catabolism in the Rhizobium-legume interaction, we studied the regulation of trc gene expression in free-living and in endosymbiotic bacteria using Escherichia coli lacZ as a reporter gene. Experiments performed with free-living bacteria indicated that trc genes were organized in at least four transcription units and that the substrate trigonelline was a specific inducer for three of them. Noninducing trigonelline-related compounds such as betaines appeared to antagonize the inducing effect of trigonelline. None of the general or symbiotic regulatory genes ntrA, dctB/D, or nodD seemed to be involved in trigonelline catabolism. trc fusions exhibiting a low basal and a high induced [beta]-galactosidase activity when present on pSym were used to monitor trc gene expression in alfalfa tissue under symbiotic conditions. Results showed that trc genes are induced during all the symbiotic steps, i.e., in the rhizosphere, infection threads, and bacteroids of alfalfa, suggesting that trigonelline is a nutrient source throughout the Rhizobium-legume association.