Grafting between model legumes demonstrates roles for roots and shoots in determining nodule type and host/rhizobia specificity

Previous grafting experiments have demonstrated that legume shoots play a critical role in symbiotic development of nitrogen-fixing root nodules by regulating nodule number. Here, reciprocal grafting experiments between the model legumes Lotus japonicus and Medicago truncatula were carried out to investigate the role of the shoot in the host-specificity of legume-rhizobia symbiosis and nodule type. Lotus japonicus is nodulated by Mesorhizobium loti and makes determinate nodules, whereas M. truncatula is nodulated by Sinorhizobium meliloti and makes indeterminate nodules. When inoculated with M. loti, L. japonicus roots grafted on M. truncatula shoots produced determinate nodules identical in appearance to those produced on L. japonicus self-grafted roots. Moreover, the hypernodulation phenotype of L. japonicus har1-1 roots grafted on wild-type M. truncatula shoots was restored to wild type when nodulated with M. loti. Thus, L. japonicus shoots appeared to be interchangeable with M. truncatula shoots in the L. japonicus root/M. loti symbiosis. However, M. truncatula roots grafted on L. japonicus shoots failed to induce nodules after inoculation with S. meliloti or a mixture of S. meliloti and M. loti. Instead, only early responses to S. meliloti such as root hair tip swelling and deformation, plus induction of the early nodulation reporter gene MtENOD11:GUS were observed. The results indicate that the L. japonicus shoot does not support normal symbiosis between the M. truncatula root and its microsymbiont S. meliloti, suggesting that an unidentified shoot-derived factor may be required for symbiotic progression in indeterminate nodules.     

Gibberellin controls the nodulation signaling pathway in Lotus japonicus

Root nodule formation is regulated by several plant hormones, but the details of the regulation of the nodulation signaling pathway are largely unknown. In this study, the role of gibberellin (GA) in the control of root nodule symbiosis was investigated at the physiological and genetic levels in Lotus japonicus . Exogenous application of biologically active GA, GA₃, inhibited the formation of infection threads and nodules, which was counteracted by the application of a biosynthesis inhibitor of GA, Uniconazole P. Nod factor-induced root hair deformation was severely blocked in the presence of GA, which was phenocopied by nsp2 mutants. The number of spontaneous nodules triggered by the gain-of-function mutation of calcium/calmodulin-dependent kinase (CCaMK) or the lotus histidine kinase 1 (LHK1) was decreased upon the addition of GA; moreover, the overexpression of the gain-of-function mutation of L. japonicus , SLEEPY1, a positive regulator of GA signaling, resulted in a reduced nodule number, without other aspects of root development being affected. These results indicate that higher GA signaling levels specifically inhibit the nodulation signaling pathway. Nod factor-dependent induction of NSP2 and NIN was inhibited by exogenous GA. Furthermore, the cytokinin-dependent induction of NIN was suppressed by GA. From these results, we conclude that GA inhibits the nodulation signaling pathway downstream of cytokinin, possibly at NSP2, which is required for Nod factor-dependent NIN expression. These results clarify the roles of GA in the nodulation signaling pathway, and in relation to the cytokinin signaling pathway for nodulation in L. japonicus .     

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.     

Regulation of nodulation in Alnus incana-Frankia symbiosis

We have studied regulation of nodulation in Alnus incana (L.) Moench using double inoculations in plastic pouches and a slide technique to observe root hair deformation. Initially, the distribution of nodules between main and lateral roots appeared quite constant, independent of the concentration of inoculum (1 to 250 μg of crushed nodules plant⁻¹). Susceptibility to infection after the second inoculation was restricted to lateral roots after the initial infections developed. When pre-existing nodules were excised before the second inoculation, subsequent nodules appeared to arise where infections had arrested at stages earlier than actual nodule emergence. We observed that root hairs formed postinoculation were very crowded and short with a pronounced deformation. No nodules were found later on this region of the root, suggesting a loss of susceptibility in this region. Split-root experiments with delays between inoculation of the first and second side of the root system showed irreversible, systemic inhibition of nodulation on the second side starting between 3 and 6 days after the inoculation of the first side. Only when compatible, infective strains were used in the first inoculation, was nodule formation inhibited after the second inoculation. We conclude that autoregulation of nodulation operates in Alnus incana and on a time scale similar to what is found in some legumes.     

Abscisic acid inhibition of root nodule initiation in Pisum sativum

Summary The effect of exogenous abscisic acid (ABA) on root nodule formation in Pisum sativum cv. Alaska was examined. ABA supplied to the roots at 1.9×10^-6M reduced the number of nodules/plant 61% without affecting root or shoot growth. The first noticeable inhibition of nodulation occurred at 3.8×10^-7M ABA. ABA at a concentration of 1.9×10^-6M inhibited neither root hair formation nor infection of root hairs by Rhizobium leguminosarum. Similar numbers of infection threads penetrated the cortex in both control and treated plants. ABA concentrations of 3.8×10^-6M had no effect on the doubling time or maximum density of R. leguminosarum in pure cultures. Normal nodule formation involves a polyploid cortical proliferation. This response to rhizobial infection can be imitated by culturing 1-mm pea-root segments on a medium containing 4.7×10^-6M kinetin. Under these conditions a highly significant reduction in the number of polyploid mitoses after 72 h is produced by 3.8×10^-8M ABA. A maximum inhibition of 68% was found with 3.8×10^-6M ABA. A similar range of ABA concentrations also inhibited the cytokinin-induced cell division in soybean callus. It is concluded that ABA reduces the number of root nodules/plant by inhibiting the cortical cell divisions required for root nodule formation.     

Rhizobium infection and nodule development in soybean are affected by exposure of the cotyledons to light

The initiation of Rhizobium infections and the development of nodules on the primary root of soybean Glycine max L. Merr cv Williams seedlings are strongly affected by exposure of the cotyledons/hypocotyls to light. Seedlings in plastic growth pouches were inoculated with R. japonicum in dim light and the position of the root tip of each seedling was marked on the face of the pouch. The pouches were covered and kept in the dark for various times before exposing the upper portions of the plants (cotyledons and hypocotyls) to light. Maximum nodulation occurred if the plants were kept in the dark until 1 day after inoculation. The exposure of plants to light 2 days before inoculation reduced the number of nodules by 50% while the number of nodules was reduced by 70% if the plants were kept in the dark until 7 days after inoculation. Anatomical studies revealed that exposure to light prior to inoculation reduced both the number of infection centers with visible infection threads and the number of infections which developed nodule meristems. Plants kept in the dark for 7 days after inoculation formed a normal number of infection threads above the root tip mark, but very few of these infections developed a nodule meristem. It appears that light stimulates soybean to produce substances which can both inhibit the formation of infection threads and enhance the development of nodules from established infection threads. The effects of light on nodulation appear to be expressed independently of the Rhizobium-induced suppression of nodule formation in younger regions of the root.     

豇豆根瘤菌NGR234和紫云英根瘤菌109感染紫云英的比较研究

利用光学和电子显微镜对紫云英根瘤菌菌株109和广宿主的快生型根瘤菌菌株NGR234感染温带型豆科植物紫云英进行了研究,结果表明根瘤菌感染紫云英是通过在根毛中形成侵染线的途径。电子显微镜研究揭示了固氮根瘤中细胞内侵染线的存在。接种二天后,首先可观察到根毛的卷曲或分枝。接种四至五天后,在每株植物卷曲的根毛中可看到侵染线。接种八至十天后的植株出现肉眼可见的根瘤。菌株NGR234能够在紫云英上诱导根毛的卷曲,侵染线和根瘤的形成,但所形成的根瘤却未能固氮,根瘤中无明显的类菌体区,但有少数包有细菌的侵染线。NGR234抗抗菌素的衍生菌均未能使紫云英结瘤。将NGR234的共生质粒转移至三叶草、苜蓿、豌豆、快生型大豆根瘤菌和农杆菌,亦未能使这些细菌获得紫云英上结瘤的能力。     

Nodule formation is stimulated by the ethylene inhibitor aminoethoxyvinylglycine

Previous researchers found that formation and function of nitrogen-fixing nodules on legume roots were severely inhibited by addition of exogenous ethylene. Nodule formation by Rhizobium meliloti on Medicago sativa was stimulated twofold when the ethylene biosynthesis inhibitor aminoethoxyvinylglycine (AVG) was added with the inoculum. Stimulation of nodule formation by AVG showed a similar concentration dependence as inhibition of ethylene biosynthesis, suggesting that the primary action of AVG is the inhibition of ethylene biosynthesis. When AVG was added 2 to 3 days after inoculation, the number of nodules formed was still increased. On a per plant basis, however, the average nitrogen fixation was unchanged by AVG treatment and was independent of nodule number.     

Nodulation of Glycine max by Six Bradyrhizobium japonicum Strains with Different Competitive Abilities

The root nodule locations of six Bradyrhizobium japonicum strains were examined to determine if there were any differences which might explain their varying competitiveness for nodule occupancy on Glycine max. When five strains were added to soybeans in plastic growth pouches in equal proportions with a reference strain (U.S. Department of Agriculture, strain 110), North Carolina strain 1028 and strain 110 were the most competitive for nodule occupancy, followed by U.S. Department of Agriculture strains 122, 76, and 31 and Brazil strain 587. Among all strains, nodule double occupancy was 17% at a high inoculum level (10⁷ CFU pouch⁻¹) and 2% at a low inoculum level (10⁴ CFU pouch⁻¹). The less competitive strains increased their nodule representation by an increase in the doubly occupied nodules at the high inoculum level. Among all strains, the number of taproot and lateral root nodules was inversely related at both the high and low inoculum levels (r = −0.62 and −0.69, respectively; P = 0.0001). This inverse relationship appeared to be a result of the plant host control of bacterial infection. Among each of the six strains, greater than 95% of the taproot nodules formed at the high inoculum density were located on 25% of the taproot length, the nodules centering on the position of the root tip at the time of inoculation. No differences among the six strains were observed in nodule initiation rates as measured by taproot nodule position. Taproot nodules were formed in the symbiosis before lateral root nodules. One of the poorly competitive strains (strain 76) occupied three times as many taproot nodules as lateral root nodules when competing with strain 110 (nodules were harvested from 4-week-old plants). Among these six wild-type strains of B. japonicum, competitive ability evidently is not related to nodule initiation rates.     

Effect of time of inoculation on in vitro ectomycorrhizal colonization and nodule initiation in Acacia holosericea seedlings

The complex interactions that occur in systems with more than one type of symbiosis were studied using one isolate of Bradyrhizobium sp. and the ectomycorrhizal fungus Pisolithus tinctorius (Pers.) Coker and Couch inoculated on to the roots of Acacia holosericea A. Cunn. ex G. Don in vitro. After a single inoculation with Bradyrhizobium sp., bacteria typically entered the roots by forming infection threads in the root hair cells via the curling point of the root hair and/ or after intercellular penetration. Sheath formation and intercellular penetration were observed on Acacia roots after a single inoculation with Pisolithus tinctorius but no radial elongation of epidermal cells. Simultaneous inoculation with both microorganisms resulted in nodules and ectomycorrhiza on the root system, occasionally on the same lateral root. On lateral roots bearing nodules and ectomycorrhiza, the nodulation site was characterized by the presence of a nodule meristem and the absence of an infection thread; sheath formation and Hartig net development occurred regularly in the region of the roots adjacent to nodules. Prior inoculation with Bradyrhizobium sp. did not inhibit ectomycorrhizal colonization in root segments adjacent to nodules in which nodule meristems and infection threads were clearly present. Conversely, in ectomycorrhizae inoculated by bacteria, the nodule meristem and the infection thread were typically absent. These results show that simultaneous inoculation with both microorganisms inhibits infection thread development, thus conferring an advantage on fungal hyphae in the competition for infection sites. This suggests that fungal hyphae can modify directly and/or indirectly the recognition factors leading to nodule meristem initiation and infection thread development.     

Genetic suppressors of the Lotus japonicus har1-1 hypernodulation phenotype

Lotus japonicus har1 mutants respond to inoculation with Mesorhizobium loti by forming an excessive number of nodules due to genetic lesions in the HAR1 autoregulatory receptor kinase gene. In order to expand the repertoire of mutants available for the genetic dissection of the root nodule symbiosis (RNS), a screen for suppressors of the L. japonicus har1-1 hypernodulation phenotype was performed. Of 150,000 M2 plants analyzed, 61 stable L. japonicus double-mutant lines were isolated. In the context of the har1-1 mutation, 26 mutant lines were unable to form RNS, whereas the remaining 35 mutant lines carried more subtle symbiotic phenotypes, either forming white ineffective nodules or showing reduced nodulation capacity. When challenged with Glomus intraradices, 18 of the 61 suppressor lines were unable to establish a symbiosis with this arbuscular mycorrhiza fungus. Using a combined approach of genetic mapping, targeting induced local lesions in genomics, and sequencing, all non-nodulating mutant lines were characterized and shown to represent new alleles of at least nine independent symbiotic loci. The class of mutants with reduced nodulation capacity was of particular interest because some of them may specify novel plant functions that regulate nodule development in L. japonicus. To facilitate mapping of the latter class of mutants, an introgression line, in which the har1-1 allele was introduced into a polymorphic background of L. japonicus ecotype MG20, was constructed.     

Nodulation inhibition by Rhizobium leguminosarum multicopy nodABC genes and analysis of early stages of plant infection.

During analysis of early events in the infection and nodulation of Vicia hirsuta roots inoculated with normal and mutant strains of Rhizobium leguminosarum and strains containing cloned nodulation (nod) genes, a number of novel observations were made. (i) Alternating zones of curled and straight root hairs were seen on roots of V. hirsuta inoculated with the wild-type strain of R. leguminosarum. This phasing of root hair curling was not seen if plants were grown under continuous light or continuous dark conditions. (ii) Reduced nodulation and delayed nodule initiation was observed with a strain carrying a Tn5 mutation in the nodE gene. In addition the phased root hair curling was absent, and root hair curling was observed along the length of the root. (iii) The nodABC genes cloned on a multicopy plasmid in a wild-type strain inhibited nodulation but induced a continuous root hair curling response. Those few nodules that eventually formed were found to contain bacteria which had lost the plasmid carrying the nodABC genes. (iv) With a strain of Rhizobium cured of its indigenous symbiotic plasmid, but containing the cloned nodABCDEF genes, continuous root hair curling on V. hirsuta was observed. However, no infection threads were observed, and surprisingly, it did appear that initial stages of nodule development occurred. Observations of thin sections of these early developing nodules indicated that early nodule meristematic divisions may have occurred but that no bacteria were found within the nodules and no infection threads were observed either within the nodule bumps or within any of the root hairs. It was concluded that for normal infections to occur, precise regulation of the nod genes is required and that overexpression of the root hair curling genes inhibits the normal infection process.     

Early Events in the Infection of Soybean (Glycine max L. Merr) by Rhizobium japonicum: I. LOCALIZATION OF INFECTIBLE ROOT CELLS

The infectible cells of soybean roots appear to be located at any given time just above the zone of root elongation and just below the position of the smallest emergent root hairs. The location of infectible cells on the primary root at the time of inoculation was inferred from the position of subsequent nodule development, correcting for displacement of epidermal cells due to root elongation. Marks were made on the seedling growth pouches at the time of inoculation to indicate the position of the root tip and the zones of root hair development. Virtually all of the seedlings developed nodules on the primary root above the marks made at the root tips at the time of inoculation. None of the plants formed nodules on the root where mature root hairs were present at the time of inoculation. These results and profiles of nodulation frequency indicate that the location of infectible cells is developmentally restricted. When inoculations were delayed for intervals of 1 to 4 hours after marking the positions of the root tips, progressively fewer nodules were formed above the root tip marks, and the uppermost of these nodules were formed at progressively shorter distances above the marks. These results indicate that the infectibility of given host cells is a transient property that appears and then is lost within a few hours. The results also indicate that host responses leading to infection and nodulation are triggered or initiated in less than 2 hours after inoculation. The extent of nodulation above the root tip mark increased in proportion to the logarithm of the number of bacteria in the inoculum.     

New nodulation mutants responsible for infection thread development in Lotus japonicus

Legume plants develop specialized root organs, the nodules, through a symbiotic interaction with rhizobia. The developmental process of nodulation is triggered by the bacterial microsymbiont but regulated systemically by the host legume plants. Using ethylmethane sulfonate mutagenesis as a tool to identify plant genes involved in symbiotic nodule development, we have isolated and analyzed five nodulation mutants, Ljsym74-3, Ljsym79-2, Ljsym79-3, Ljsym80, and Ljsym82, from the model legume Lotus japonicus. These mutants are defective in developing functional nodules and exhibit nitrogen starvation symptoms after inoculation with Mesorhizobium loti. Detailed observation revealed that infection thread development was aborted in these mutants and the nodules formed were devoid of infected cells. Mapping and complementation tests showed that Ljsym74-3, and Ljsym79-2 and Ljsym79-3, were allelic with reported mutants of L. japonicus, alb1 and crinkle, respectively. The Ljsym82 mutant is unique among the mutants because the infection thread was aborted early in its development. Ljsym74-3 and Ljsym80 were characterized as mutants with thick infection threads in short root hairs. Map-based cloning and molecular characterization of these genes will help us understand the genetic mechanism of infection thread development in L. japonicus.     

Spontaneous nodules induce feedback suppression of nodulation in alfalfa

A small subpopulation of alfalfa (Medicago saliva L.) plants grown without fixed nitrogen can develop root nodules in the absence of Rhizobium. Cytological studies showed that these nodules were organized structures with no inter- or intracellular bacteria but with the histological characteristics of a normal indeterminate nodule. Few if any viable bacteria were recovered from the nodules after surface sterilization, and when the nodular content was used to inoculate alfalfa roots no nodulation was observed. These spontaneous nodules were formed mainly on the primary roots in the region susceptible to Rhizobium infection between 4 and 6 d after seed imbibition. Spontaneous nodules appeared as early as 10 d after germination and emerged at a rate comparable to normal nodules. The formation of spontaneous nodules on the primary root suppressed nodulation in lateral roots after inoculation with R. meliloti RCR2011. Excision of spontaneous nodules at inoculation eliminated the suppressive response. Our results indicate that the presence of Rhizobium is not required for nodule organogenesis and the elicitation of feedback regulation of nodule formation in alfalfa.Abbreviation RT root tip This work was supported by an endowment to the Racheff Chair of Excellence of the University of Tennessee, and the Soybean Promotion Board, Haskinsville, Tenn., USA. We are indebted to Noel Gerahty for performing the acetylene-reduction assays, and Dr. E.T. Graham for allowing the use of microscope facilities.