Nodule Infection Research Articles

Nodulation of Aeschynomene afraspera and A. indica by Photosynthetic Bradyrhizobium Sp. Strain ORS285: The Nod-Dependent Versus the Nod-Independent Symbiotic Interaction

Here, we present a comparative analysis of the nodulation processes of Aeschynomene afraspera and A. indica that differ in their requirement for Nod factors (NF) to initiate symbiosis with photosynthetic bradyrhizobia. The infection process and nodule organogenesis was examined using the green fluorescent protein-labeled Bradyrhizobium sp. strain ORS285 able to nodulate both species. In A. indica, when the NF-independent strategy is used, bacteria penetrated the root intercellularly between axillary root hairs and invaded the subepidermal cortical cells by invagination of the host cell wall. Whereas the first infected cortical cells collapsed, the infected ones immediately beneath kept their integrity and divided repeatedly to form the nodule. In A. afraspera, when the NF-dependent strategy is used, bacteria entered the plant through epidermal fissures generated by the emergence of lateral roots and spread deeper intercellularly in the root cortex, infecting some cortical cells during their progression. Whereas the infected cells of the lower cortical layers divided rapidly to form the nodule, the infected cells of the upper layers gave rise to an outgrowth in which the bacteria remained enclosed in large tubular structures. Together, two distinct modes of infection and nodule organogenesis coexist in Aeschynomene legumes, each displaying original features.

Composite Actinorhizal Plants with Transgenic Roots for the Study of Symbiotic Associations with Frankia

More than 200 species of dicotyledonous plants belonging to eight different families and 24 genera can establish actinorhizal symbiosis with the nitrogen-fixing soil actinomycete Frankia. Compared to the symbiotic interaction between legumes and rhizobia, little is known about the molecular basis of the infection process and nodule formation in actinorhizal plants. Here, we review a gene transfer system based on Agrobacterium rhizogenes that opens the possibility to rapidly analyze the function of candidate symbiotic genes. The transformation protocol generates “composite plants” that consist of a nontransgenic aerial part with transformed hairy roots. Composite plants have already been obtained in three different species of actinorhizal plants, including the tropical tree species Casuarina glauca, the Patagonian shrub Discaria trinervis, and the nonwoody plant Datisca glomerata. The potential of this technique to advancing our understanding of the molecular mechanisms underlying infection by Frankia is demonstrated by functional analyses of symbiotic genes.

Early signaling in actinorhizal symbioses.

Nitrogen-fixing root nodulation, confined to four plant orders, encompasses more than 14,000 Leguminosae species, and approximately 200 actinorhizal species forming symbioses with rhizobia (Rhizobium, Bradyrhizobium, etc) and Frankia bacterial species, respectively. While several genetic components of the host-symbiont interaction have been identified in legumes, little is known about the genetic bases of actinorhizal symbiosis. However, we recently demonstrated the existence of common symbiotic signaling pathways in actinorhizals and legumes. Moreover, important data on the identification of flavonoids as plant signaling compounds and the role for auxins during Frankia infection process and nodule organogenesis have been acquired. All together these results lead us to propose a unified model for symbiotic exchange and genetic control of actinorhizal symbiosis.

Exopolysaccharides produced by the symbiotic nitrogen-fixing bacteria of leguminosae

The process of biological nitrogen fixation (BNF), performed by symbiotic nitrogen fixing bacteria with legume species, commonly known as α and β rhizobia, provides high sustainability for the ecosystems. Its management as a biotechnology is well succeeded for improving crop yields. A remarkable example of this success is the inoculation of Brazilian soybeans with Bradyrhizobium strains. Rhizobia produce a wide diversity of chemical structures of exopolysaccharides (EPS). Although the role of EPS is relatively well studied in the process of BNF, their economic and environmental potential is not yet explored. These EPS are mostly species-specific heteropolysaccharides, which can vary according to the composition of sugars, their linkages in a single subunit, the repeating unit size and the degree of polymerization. Studies have showed that the EPS produced by rhizobia play an important role in the invasion process, infection threads formation, bacteroid and nodule development and plant defense response. These EPS also confer protection to these bacteria when exposed to environmental stresses. In general, strains of rhizobia that produce greater amounts of EPS are more tolerant to adverse conditions when compared with strains that produce less. Moreover, it is known that the EPS produced by microorganisms are widely used in various industrial activities. These compounds, also called biopolymers, provide a valid alternative for the commonly used in food industry through the development of products with identical properties or with better rheological characteristics, which can be used for new applications. The microbial EPS are also able to increase the adhesion of soil particles favoring the mechanical stability of aggregates, increasing levels of water retention and air flows in this environment. Due to the importance of EPS, in this review we discuss the role of these compounds in the process of BNF, in the adaptation of rhizobia to environmental stresses and in the process of soil aggregation. The possible applications of these biopolymers in industry are also discussed.

Enzymatic Activity of the Soybean Ecto-Apyrase GS52 Is Essential for Stimulation of Nodulation

Nitrogen is an essential nutrient for plant growth. In the Rhizobium-legume symbiosis, root nodules are the sites of bacterial nitrogen fixation, in which atmospheric nitrogen is converted into a form that plants can utilize. While recent studies suggested an important role for the soybean (Glycine max) ecto-apyrase GS52 in rhizobial root hair infection and root nodule formation, precisely how this protein impacts the nodulation process remains undetermined. In this study, the biochemical characteristics of the GS52 enzyme were investigated. Computer modeling of the GS52 apyrase structure identified key amino acid residues important for catalytic activity, which were subsequently mutagenized. Although the GS52 enzyme exhibited broad substrate specificity, its activity on pyrimidine nucleotides and diphosphate nucleotides was significantly higher than on ATP. This result was corroborated by structural modeling of GS52, which predicted a low specificity for the adenine base within the substrate-binding pocket of the enzyme. The wild-type enzyme and its inactive mutant forms were expressed in soybean roots in order to evaluate the importance of GS52 enzymatic activity for nodulation. The results indicated a clear correlation between GS52 enzymatic activity and nodule number. Altogether, our study indicates that the catalytic activity of the GS52 apyrase, likely acting on extracellular nucleotides, is critical for rhizobial infection and nodulation.

Análise das trocas de implantes mamários nos últimos cinco anos na Clínica Ivo Pitanguy

Background: The breast augmentation surgery was the most performed in Brazil in 2008. With the evolution of the technique and the quality of the implants, complications have diminished, leading to a more secure procedure. Objective: To assess the profile of patients submitted to implant exchange in the last five years at Ivo Pitanguy Clinic, analyzing the causes that led to implant removal. Methods: We have analyzed all patients submitted to exchange, removal or replacement of mammary implants in the last five years, at a total of 59 patients, 68 procedures and 129 breasts operated. Results: The time interval from the inclusion to the removal of the implants ranged from one month to 34 years; 44 patients have first operated at our service and 24 at other services. Forty three implants were, before the exchange, made of polyurethane, 13 texturized and six smooth. The major cause of the exchange was esthetic, with 50 cases, followed by capsular contracture, 13 cases and five cases of infection, rupture or breast nodule. Conclusion: The major reason of implants exchange was esthetic, differing from the literature. The capsular contracture and infection rates, at this study, were comparable to several others.

A dominant function of CCaMK in intracellular accommodation of bacterial and fungal endosymbionts.

In legumes, Ca2+/calmodulin-dependent protein kinase (CCaMK) is a component of the common symbiosis genes that are required for both root nodule (RN) and arbuscular mycorrhiza (AM) symbioses and is thought to be a decoder of Ca2+ spiking, one of the earliest cellular responses to microbial signals. A gain-of-function mutation of CCaMK has been shown to induce spontaneous nodulation without rhizobia, but the significance of CCaMK activation in bacterial and/or fungal infection processes is not fully understood. Here we show that a gain-of-function CCaMKT265D suppresses loss-of-function mutations of common symbiosis genes required for the generation of Ca2+ spiking, not only for nodule organogenesis but also for successful infection of rhizobia and AM fungi, demonstrating that the common symbiosis genes upstream of Ca2+ spiking are required solely to activate CCaMK. In RN symbiosis, however, CCaMKT265D induced nodule organogenesis, but not rhizobial infection, on Nod factor receptor (NFRs) mutants. We propose a model of symbiotic signaling in host legume plants, in which CCaMK plays a key role in the coordinated induction of infection thread formation and nodule organogenesis.

Reduced nodulation in alfalfa induced by arsenic correlates with altered expression of early nodulins

Arsenic (As) reduces legume nodulation by affecting the first stages of the symbiotic interaction, which causes a 90% decrease in rhizobial infections. In this paper, we examine molecular mechanisms underlying this toxic effect, using the model system Medicago sativa–Sinorhizobium. In the presence and absence of As, the expression patterns of seven nodulin genes, markers for the different events leading to nodule formation, were analyzed by RT-PCR and by real-time RT-PCR. A significant decrease was observed, especially from days 1–5 after the inoculation, in the expression of four early nodulins: the genes coding the Nod factor receptor ( nork), the transcription factor NIN and the markers for infection progression ( N6) and nodule organogenesis ( Enod2). On the contrary, the expression of markers for primordium initiation ( Enod40) and differentiation ( ccs52) was not significantly altered. Finally, the expression of a marker for nitrogen fixation ( Legbrc) was also reduced, probably due to the reduction in nodule number induced by As. These results suggest that As affects the expression of nodulation genes that have been associated with processes that take place in the epidermis and the outer cortical cells, and that the expression of genes associated with events that take place in the inner cortical cells is less affected. This is the first report showing changes in the expression of nodulin genes induced by the presence of any toxic metal(loid).

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.

Competitiveness of a native Rhizobium leguminosarum biovar trifolii strain for nodule occupancy is manifested during infection

The stages in the nodulation process that determined the competitiveness of R. leguminosarum bv. trifolii (Rlt) strain 20–15, which proved to be highly competitive for nodulation in Iceland fields tests over several years, is analysed. White clover (Trifolium repens L.) roots were inoculated with inoculum mixtures containing three strains (Rlt 20-15, Rlt 8-9 and Rlt 32-28) in different proportions and cell densities. Competitiveness in root colonization, formation of infection threads and nodule development was assessed for Rlt 20-15 and its weakest competitor, Rlt 32-28. ERIC-polymerase chain reaction (PCR) DNA fingerprinting was used to identify inoculated strains recovered from root surfaces and individual nodules. GFP or DsRed tagged strains were used to determine identity in root hairs and nodules. Both strains colonized the root equally at all inoculum ratios tested. But, Rlt 20-15 initiated significantly more infection threads and formed more nodules than Rlt 32-28. These results show that Rlt 20-15 expresses its nodulation competitiveness during infection, either at infection thread initiation or during successive growth in the infection threads. The data presented support earlier observations that this strain competed well in the field in spite of its inferior ability to survive in the soil.

Simultaneous Erythema Nodosum and Erythema Multiforme After Local Lidocaine Injection

To report a case of simultaneous erythema nodosum and erythema multiforme after local lidocaine injection. A 33-year-old female experienced coexisting erythema nodosum and erythema multiforme after lidocaine spray was used for upper gastrointestinal endoscopy. The reaction was exacerbated after localized injection of 2% lidocaine for a skin biopsy. An objective causality assessment revealed that an adverse drug reaction was highly probable. Lidocaine is the most frequently used local anesthetic agent. The coexistence of erythema nodosum and erythema multiforme has been reported in lepromatous leprosy, Yersinia enterocolítica and Yersinia pseudotuberculosis gastrointestinal infection (both with diarrhea), histoplasmosis, hepatitis C, and milkeŕs nodule, but not with lidocaine. If clinicians observe similar lesions after lidocaine exposure, they should suspect an adverse drug reaction and exercise caution in the administration of amide anesthetics to patients with a documented history of adverse reaction to lidocaine.

Cloning and identification ofopa22, a new gene involved in nodule formation byMesorhizobium huakuii

Using Tn5-sacB insertion mutagenesis, 3000 mutants were obtained from Mesorhizobium huakuii 7653R. Eight nodulation-defective mutants were screened by plant nodulation experiments. The DNA sequences of the contiguous region from the Tn5 insertion site were determined by thermal asymmetric interlaced PCR. A new gene was cloned and designated opa22, as judged from its structural and functional homology. Sequence analysis indicated that opa22 was composed of 774 nucleotides and encoded a protein of 257 amino acids. RPS-BLAST analysis of the Opa22 protein showed a sequence similarity (88.9%) to the opacity protein and related to surface antigens of the bacterial outer membrane, which can mediate various pathogen-host cell interactions and promote invasion. Our results from root hair curling experiments suggested that expression of the opa22 gene might occur at the stage of infection thread formation and nodule development. The complement stain HK24 was able to restore the nodule forming ability of the mutant.

Visualization of nodulation gene activity on the early stages of Rhizobium leguminosarum bv. viciae symbiosis.

A technique was optimized for the in situ detection of nodulation (nod) gene activity in Rhizobium leguminosarum bv. viciae symbiosis with compatible plant hosts Vicia tetrasperma (L.) SCHREB. and Pisum sativum L. The transcription of nodABC-lacZ fusion was visualized as beta-galactosidase (beta-Gal) activity after reaction with the chromogenic substrate X-Gal and subsequent light microscopy, while the background of the indigenous beta-Gal activity of rhizobia and the host plant was eliminated by glutaraldehyde treatment. V. tetrasperma was suggested as a suitable model plant for pea cross-inoculation group due to its advantages over the common model of V. hirsuta (L.) S.F. GRAY: compactness of the plant, extremely small seeds, fast development and stable nodulation under laboratory conditions. In the roots of both plants, a certain extent of nod gene activity was detectable in all rhizobia colonizing the rhizoplane. In pea 1 d after inoculation (d.a.i.), the maximum was localized in the region of emerging root hairs (RH) later (3 and 6 d.a.i.) shifting upwards from the root tip. Nodulation genes sustained full expression even in the infection threads inside the RH and the root cortex, independently of their association with nodule primordia. Comparison of two pea symbiotic mutant lines, Risnod25 and Risnod27, with the wild type did not reveal any differences in the RH formation, RH curling response and rhizoplane colonization. Both mutants appeared to be blocked at the infection thread initiation stage and in nodule initiation, consistent with the phenotype caused by other mutant alleles in the pea sym8 locus. Judging from the nod gene expression level and pattern in the rhizoplane, flavonoid response upon inoculation is preserved in both pea mutants, being independent of infection thread and nodule initiation.

Lipopolysaccharides as a communication signal for progression of legume endosymbiosis

Establishment of a successful symbiosis between rhizobia and legumes results from an elaborate molecular dialogue between both partners. Bacterial nodulation (Nod) factors are indispensable for initiating plant responses, whereas bacterial surface polysaccharides are important for infection progression and nodule development. The mutant ORS571-oac2 of Azorhizobium caulinodans, affected in its surface polysaccharides, provokes a defective interaction with its host Sesbania rostrata. ORS571-oac2 induced structures with retarded development and continued generation of infection centers and organ primordia, leading to multilobed ineffective nodules. Bacterial development throughout the interaction occurred without major defects. A functional bidirectional complementation was obtained upon coinfection of ORS571-oac2 and a Nod factor-deficient mutant, indicating that the Fix- phenotype of ORS571-oac2-induced nodules resulted from the absence of a positive signal from ORS571-oac2. Indeed, the Fix- phenotype could be complemented by coinoculation of ORS571-oac2 with lipopolysaccharides (LPSs) purified from A. caulinodans. Our data show that Nod factors and LPSs are consecutive signals in symbiosis. Nod factors act first to trigger the onset of the nodulation and invasion program; LPSs inform the plant to proceed with the symbiotic interaction and to develop a functional fixation zone.

Microscopical studies of Arachis pintoi root nodule with special reference to bacteroids and oleosomes at different stages of plant growth and nodule development

Nitrogen fixation in legume root nodules are believed to be supported by the supply of carbon compounds derived from host cells. The presence of storage organelles such as oleosomes in the infected cells may have an advantage as a supplementary source of carbon and energy as has been suggested for peanut ( Arachis hypogaea) root nodules. The present investigation explores the development of symbiosis in a wild diploid species of Arachis in relation to the bacteroids and oleosomes. Microscopical observations at different developmental stages of nodule and host plant have revealed that oleosomes were present only during early stages of infection process and nodule development before the establishment of symbiosis, and during senescence. Oleosomes were absent in the infected cells of effective nodules of A. pintoi whereas similar cells in the nodules of other wild diploid species of Arachis ( A. batizocoi, A. duranensis) and tetraploid A. hypogaea show presence of oleosomes. Another interesting feature in this species was the reversion of spherical bacteroids into rod-forms within the confines of the senescent nodule tissue.

Transgenic Lotus japonicus with an ethylene receptor gene Cm-ERS1/H70A enhances formation of infection threads and nodule primordia.

Ethylene inhibits the establishment of symbiosis between rhizobia and legumes. To examine how and when endogenous ethylene inhibits rhizobial infection and nodulation, we produced transgenic Lotus japonicus carrying the mutated melon ethylene receptor gene Cm-ERS1/H70A that confers ethylene insensitivity and fixes the transgene in the T(3) generation. The resultant transgenic plants showed reduced ethylene sensitivity because of 1-aminocyclopropane-1-carboxylate resistance and increased flowering duration, probably due to a dominant negative mechanism. When inoculated with Mesorhizobium loti, transgenic plants showed markedly higher numbers of infection threads and nodule primordia on their roots than did either wild-type or azygous plants during the early stage of cultivation period as well as during later stages, when the number of mature nodules had reached a steady state. In addition, transcripts of NIN, a gene governing infection thread formation, increased in the inoculated transgenic plants as compared with the wild-type plants. The infection responses of transgenic plants were similar to those of wild-type plants treated with ethylene inhibitors. These results imply that the endogenous ethylene in L. japonicus roots inhibits rhizobial infection at the primary nodulation, probably via NIN gene, and suggest that ethylene perception assists negative feedback regulation of secondary nodule initiation.

Four developmental stages identified by genetic dissection of pea ( Pisum sativum L.) root nodule morphogenesis

Root nodule structural organisation of nine pea Fix − mutants representing seven symbiotic loci has been studied. This set of mutants has revealed lesions at four developmental stages in the pea– Rhizobium symbiosis. (i) Mutant RisFixA is affected in infection thread differentiation in nodule tissue, infection droplet formation, bacteroid differentiation and nodule persistence. (ii) Allelic mutants RisFixL and RisFixO ( sym32) are blocked in bacteroid differentiation. (iii) Mutants RisFixM ( sym26), RisFixN, RisFixQ ( sym27), RisFixT ( sym26) show premature degradation of symbiotic structures. (iv) Mutant RisFixV shows similar defects in nodule persistence but, in addition, this mutant is characterised by an abnormal and severe increase in the thickness of infection thread walls during the process of infection thread ‘maturation’ and senescence. Combining our new data with previously published studies of pea mutant phenotypes has allowed us to create an integrated scheme for the sequential functioning of the late pea symbiotic genes identified and characterised to date.

Nitrate- and Inoculation-enhanced Ethylene Biosynthesis in Soybean Roots as a Possible Mediator of Nodulation Control

Root ethylene biosynthesis has been studied in soybean ( Glycine max [L.] Merr.) cv. Bragg plants and its supernodulating (nts382 and nts1007) and non-nodulating (nod49 and nod139) mutants. Regardless of NO 3 treatment, inoculation with Bradyrhizobium japonicum significantly increased root ethylene evolution rate, reaching a plateau between 24 and 48 h after inoculation, with the rates being significantly higher in 8 mmol L −1 fed roots (high) than in those given 1 mmol L -1 (low) during the time of experiment. This Inoculation Stimulated Ethylene Release (ISER) response appears to be related to the infection process and nodule development, as treatment with Ag + (an inhibitor of ethylene action) at the moment of inoculation markedly increased nodule number of Bragg plants under both high and low NO 3 concentrations. Compared with the parental Bragg, the near-isogenic nodulating mutants used in this study showed normal ethylene biosynthesis ability (ethylene evolution and ACC oxidase activity), although significant quantitative differences were detected among them. Whether these differences are causally related to the nodulation phenotype is not known. Our previous observations on the involvement of endogenous ethylene in the control of nodule number in alfalfa are therefore also applicable to soybean, a determinate nodule type legume. The results further suggest that effects other than an alteration of ethylene biosynthesis might have also been caused by the nts mutations.

Stem Nodulation in Legumes: Diversity, Mechanisms, and Unusual Characteristics

Rhizobia can establish a nitrogen-fixing symbiosis with plants of the Leguminosae family. They elicit on their host plant the formation of new organs, called nodules, which develop on the roots. A few aquatic legumes, however, can form nodules on their stem at dormant root primordia. The stem-nodulating legumes described so far are all members of the genera Aeschynomene, Sesbania, Neptunia, and Discolobium. Their rhizobial symbionts belong to four genera already described: Rhizobium, Bradyrhizobium, Sinorhizobium, and Azorhizobium. This review summarizes our current knowledge on most aspects of stem nodulation in legumes, the infection process and nodule development, the characterization and unusual features of the associated bacteria, and the molecular genetics of nodulation. Potential use as green manure in lowland rice of these stem-nodulating legumes, giving them agronomical importance, is also discussed.

Genistein accumulation in soybean (Glycine max[L.] Merr.) root systems under suboptimal root zone temperatures

Genistein, as a plant-to-bacteria signal, plays an important role in the establishment of the soybean {Glycine max [L.] Merr.)-Bradyrhizobium japonicum nitrogen-fixing symbiosis. It is essential to the development of effective root nodules and responsible for inducing the nod genes of B. japonicum. Because suboptimal root zone temperature (RZT) delays infection and early nodule development, and decreases plant nodule number, and genistein addition overcomes some of this, it is reasonable to hypothesize that suboptimal RZT disrupts the inter-organismal signal exchange by inhibiting genistein synthesis. Four experiments were conducted to test these hypotheses. The results of these studies indicated that: (1) when soybean plants were germinated and maintained at RZTs ranging from 13 to 17CC, root genistein concentration and content per plant were lower than those of plants with roots maintained at RZTs above 17C; (2) when plants were germinated at an optimal RZT (25 °C) then transferred to RZTs below 17°C, and acclimated for a few days, root genistein concentration and content per plant were higher than those of plants with roots maintained either at optimal RZT, or transferred to RZT above 17 °C, although by the end of the experiment, the genistein concentration of root systems at below 17°C RZT appeared to be declining to values below those of plants with above 17 °C RZT; (3) the root genistein concentration increased before the onset of nitrogen fixation and decreased thereafter; and (4) part of the effect of RZTs on genistein content per plant root system was from reductions in genistein concentration at lower RZTs, and part was due to decreased plant root growth.