Endomycorrhizal and rhizobial symbiosis: How much do they share?

Abstract

Legume plants enter two important endosymbioses – with soil fungi, forming phosphorus acquiring arbuscular mycorrhiza (AM), and with nitrogen-fixing bacteria, leading to the formation of nitrogen-fixing root nodules. Both symbioses have been studied extensively because these symbioses have great potential for agricultural applications. Although 80% of all living land plants form AM, the nitrogen-fixing root nodule symbiosis with rhizobia is almost exclusively restricted to legumes. Despite varying degree of differences in the morphological responses induced by both endosymbionts in the host plants, significant similarities in the development of both fungal and bacterial symbioses have been reported. The signal perception and signal transduction cascades that initiate nodulation and mycorrhization in legumes partially overlap. Legume genes have been identified that are required for the establishment of both AM and root nodule symbiosis and are referred to as the common SYM genes. Genetic dissection of the common SYM signal transduction pathway required for bacterial and fungal root endosymbiosis has not only unraveled the players involved but also provided a first glimpse at conservation and specialization of signaling cascades essential for nodulation and mycorrhiza development. Based on the observation of common signaling cascades, it is tempting to speculate that the root nodule symbiosis, where fossil records date back to the late Cretaceaous, adopted and subsequently modified more ancient signal transduction pathways leading to AM formation, having already been in place 400 million years ago. This review discusses the common aspects of recognition of mycorrhizal fungi and Rhizobium by the host, and further signal transduction that leads to an effective symbiosis.     

Soil Bacterial Diversity Responses to Root Colonization by an Ectomycorrhizal Fungus are not Root-Growth-Dependent

The hypothesis tested in this present study was that the ectomycorrhizosphere effect on the bacterial community was not root-growth-dependent. The impacts of ectomycorrhizal infection (Pisolithus albus COI007) and a chemical fertilization to reproduce the fungal effect on root growth were examined on (1) the structure of bacterial community and (2) fluorescent pseudomonad and actinomycete populations in the mycorrhizosphere of Acacia auriculiformis using both culture-independent and culture-dependent methods. A. auriculiformis plants were grown in disinfested soil in pots with or without addition of the ectomycorrhizal fungus or N/P/K fertilization (to reproduce the fungal effect on root growth) for 4 months and then transferred to 20-L pots filled with nondisinfested sandy soil. The fungal and fertilizer applications significantly improved the plant growth after 4-month culture in the disinfested soil. In the nondisinfested cultural substrate, these positive effects on plant growth were maintained. The total soil microbiota was significantly different within the treatments as revealed from DNA analysis [denaturing gradient gel electrophoresis (DGGE)]. The structure of fluorescent pseudomonad populations was also affected by fungal and fertilizer applications. In contrast, no qualitative effect was observed for the actinomycete communities within each treatment, but fungal inoculation significantly decreased the number of actinomycetes compared to the fertilizer application treatment. These results show that the mycorrhizosphere effect is not root-growth-dependent but is mainly due to the presence of the ectomycorrhizal fungus and more particularly to the extramatrical mycelium.     

Investigation of Four Classes of Non-nodulating White Sweetclover (Melilotus alba annua Desr.) Mutants and Their Responses to Arbuscular-Mycorrhizal Fungi

The nitrogen-fixing symbiosis between Rhizobiaceae and legumes is one of the best-studied interactions established between prokaryotes and eukaryotes. The plant develops root nodules in which the bacteria are housed, and atmospheric nitrogen is fixed into ammonia by the rhizobia and made available to the plant in exchange for carbon compounds. It has been hypothesized that this symbiosis evolved from the more ancient arbuscular mycorrhizal (AM) symbiosis, in which the fungus associates with roots and aids the plant in the absorption of mineral nutrients, particularly phosphate. Support comes from several fronts: 1) legume mutants where Nod(-) and Myc(-) co-segregate, and 2) the fact that various early nodulin (ENOD) genes are expressed in legume AM. Both strongly argue for the idea that the signal transduction pathways between the two symbioses are conserved. We have analyzed the responses of four classes of non-nodulating Melilotus alba (white sweetclover) mutants to Glomus intraradices (the mycorrhizal symbiont) to investigate how Nod(-) mutations affect the establishment of this symbiosis. We also re-examined the root hair responses of the non-nodulating mutants to Sinorhizobium meliloti (the nitrogen-fixing symbiont). Of the four classes, several sweetclover sym mutants are both Nod(-) and Myc(-). In an attempt to decipher the relationship between nodulation and mycorrhiza formation, we also performed co-inoculation experiments with mutant rhizobia and Glomus intraradices on Medicago sativa, a close relative of M. alba. Even though sulfated Nod factor was supplied by some of the bacterial mutants, the fungus did not complement symbiotically defective rhizobia for nodulation.     

Competition between rhizobia under different environmental conditions affects the nodulation of a legume

Mutualistic symbiosis and nitrogen fixation of legume rhizobia play a key role in ecological environments. Although many different rhizobial species can form nodules with a specific legume, there is often a dominant microsymbiont, which has the highest nodule occupancy rates, and they are often known as the “most favorable rhizobia”. Shifts in the most favorable rhizobia for a legume in different geographical regions or soil types are not well understood. Therefore, in order to explore the shift model, an experiment was designed using successive inoculations of rhizobia on one legume. The plants were grown in either sterile vermiculite or a sandy soil. Results showed that, depending on the environment, a legume could select its preferential rhizobial partner in order to establish symbiosis. For perennial legumes, nodulation is a continuous and sequential process. In this study, when the most favorable rhizobial strain was available to infect the plant first, it was dominant in the nodules, regardless of the existence of other rhizobial strains in the rhizosphere. Other rhizobial strains had an opportunity to establish symbiosis with the plant when the most favorable rhizobial strain was not present in the rhizosphere. Nodule occupancy rates of the most favorable rhizobial strain depended on the competitiveness of other rhizobial strains in the rhizosphere and the environmental adaptability of the favorable rhizobial strain (in this case, to mild vermiculite or hostile sandy soil). To produce high nodulation and efficient nitrogen fixation, the most favorable rhizobial strain should be selected and inoculated into the rhizosphere of legume plants under optimum environmental conditions.     

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 CCaMK^T265D 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, CCaMK^T265D 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.     

豆科植物共生结瘤的分子基础和调控研究进展

豆科植物与根瘤菌共生互作的结果导致了一个新的植物器官――根瘤的形成, 根瘤菌生活在根瘤中, 它们具有将氮气转化为能被植物同化的氨的能力。该文阐述了根瘤的形成过程和类型, 并主要以模式豆科植物蒺藜苜蓿(Medicago truncatula)和日本百脉根(Lotus japonicus)为例, 对近年来共生结瘤过程中宿主植物对根瘤菌结瘤因子的识别和信号传递、侵入线形成和固氮的分子基础, 以及宿主植物对根瘤形成的自主调控机制、环境中氮素营养对结瘤的影响研究进行了综述, 指出当前豆科植物与根瘤菌共生互作研究存在的问题, 并对今后的研究方向作了分析与展望。     

Suppression of arbuscular mycorrhizal colonization and nodulation in split-root systems of alfalfa after pre-inoculation and treatment with Nod factors

Roots of legumes establish symbiosis with arbuscular mycorrhizal fungi (AMF) and nodule-inducing rhizobia. The existing nodules systemically suppress subsequent nodule formation in other parts of the root, a phenomenon termed autoregulation. Similarly, mycorrhizal roots reduce further AMF colonization on other parts of the root system. In this work, split- root systems of alfalfa (Medicago sativa) were used to study the autoregulation of symbiosis with Sinorhizobium meliloti and the mycorrhizal fungus Glomus mosseae. It is shown that nodulation systemically influences AMF root colonization and vice versa. Nodules on one half of the split-root system suppressed subsequent AMF colonization on the other half. Conversely, root systems pre-colonized on one side by AMF exhibited reduced nodule formation on the other side. An inhibition effect was also observed with Nod factors (lipo-chito-oligosaccharides). NodSm-IV(C16:2, S) purified from S. meliloti systemically suppressed both nodule formation and AMF colonization. The application of Nod factors, however, did not influence the allocation of (14)C within the split-root system, excluding competition for carbohydrates as the regulatory mechanism. These results indicate a systemic regulatory mechanism in the rhizobial and the arbuscular mycorrhizal association, which is similar in both symbioses.     

Effects of Co-Inoculation with Arbuscular Mycorrhizal Fungi and Rhizobia on Fungal Occupancy in Chickpea Root and Nodule Determined by Real-Time PCR

Legume roots in nature are usually colonized with rhizobia and different arbuscular mycorrhizal fungi (AMF) species. Light microscopy that visualizes the presence of AMF in roots is not able to differentiate the ratio of each AMF species in the root and nodule tissues in mixed fungal inoculation. The purpose of this study was to characterize the dominant species of mycorrhiza in roots and nodules of plants co-inoculated with mycorrhizal fungi and rhizobial strains. Glomus intraradices (GI), Glomus mosseae (GM), their mix (GI + GM), and six Mesorhizobium ciceri strains were used to inoculate chickpea. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to assess occupancy of these fungal species in roots and nodules. Results showed that GI molecular ratio and relative density were higher than GM in both roots and nodules. These differences in molecular ratio and density between GI and GM in nodules were three folds higher than roots. The results suggested that M. ciceri strains have different effects on nodulation and mycorrhizal colonization pattern. Plants with bacterial S3 and S1 strains produced the highest root nodulation and higher fungal density in both the roots and nodules.     

A novel nuclear protein interacts with the symbiotic DMI3 calcium- and calmodulin-dependent protein kinase of Medicago truncatula

Many higher plants establish symbiotic relationships with arbuscular mycorrhizal (AM) fungi that improve their ability to acquire nutrients from the soil. In addition to establishing AM symbiosis, legumes also enter into a nitrogen-fixing symbiosis with bacteria known as rhizobia that results in the formation of root nodules. Several genes involved in the perception and transduction of bacterial symbiotic signals named "Nod factors" have been cloned recently in model legumes through forward genetic approaches. Among them, DMI3 (Doesn't Make Infections 3) is a calcium- and calmodulin-dependent kinase required for the establishment of both nodulation and AM symbiosis. We have identified, by a yeast two-hybrid system, a novel protein interacting with DMI3 named IPD3 (Interacting Protein of DMI3). IPD3 is predicted to interact with DMI3 through a C-terminal coiled-coil domain. Chimeric IPD3::GFP is localized to the nucleus of transformed Medicago truncatula root cells, in which split yellow fluorescent protein assays suggest that IPD3 and DMI3 physically interact in Nicotiana benthamiana. Like DMI3, IPD3 is extremely well conserved among the angiosperms and is absent from Arabidopsis. Despite this high level of conservation, none of the homologous proteins have a demonstrated biological or biochemical function. This work provides the first evidence of the involvement of IPD3 in a nuclear interaction with DMI3.     

Nod factor-independent ‘crack-entry’ symbiosis in dalbergoid legume Arachis hypogaea

Dalbergoids are typified by crack-entry symbiosis which is evidenced to be Nod Factor (NF)-independent in several Aeschynomene legumes. Natural symbionts of the dalbergoid legume Arachis hypogaea are always NF-producing, prompting us to check whether symbiosis in this legume could also be NF-independent. For this, we followed the symbiosis with two NF-containing bradyrhizobial strains – SEMIA6144, a natural symbiont of Arachis and ORS285, a versatile nodulator of Aeschynomene legumes, along with their corresponding nodulation (nod) mutants. Additionally, we investigated NF-deficient bradyrhizobia like BTAi1, a natural symbiont of Aeschynomene indica and the WBOS strains that were natural endophytes of Oryza sativa, collected from an Arachis-Oryza intercropped field. While SEMIA6144ΔnodC was non-nodulating, both ORS285 and ORS285ΔnodB could induce functional nodulation, although with lower efficiency than SEMIA6144. On the other hand, all the NF-deficient strains – BTAi1, WBOS2 and WBOS4 showed comparable nodulation with ORS285 indicating Arachis to harbour an NF-independent mechanism of symbiosis. Intriguingly, symbiosis in Arachis, irrespective of whether it was NF-dependent or independent, was always associated with the curling or branching of the rosette root hairs at the lateral root bases. Thus, despite being predominantly described as an NF-dependent legume, Arachis does retain a vestigial, less-efficient form of NF-independent symbiosis.     

Agrobacterium strains isolated from root nodules of common bean specifically reduce nodulation by Rhizobium gallicum

In a previous work, we showed that non-nodulating agrobacteria strains were able to colonize root nodules of common bean. Both rhizobia and agrobacteria co-existed in the infected nodules. No impact on symbiosis was found in laboratory conditions when using sterile gravel as a support for growth. In this study, soil samples originating from different geographic and agronomic regions in Tunisia were inoculated with a mixture of agrobacteria strains isolated previously from root nodules of common bean. A significant effect on nodulation and vegetal growth of common bean was observed. Characterization of nodulating rhizobia and comparison with non-inoculated controls showed a biased genetic structure. It seemed that Rhizobium gallicum was highly inhibited, whereas nodulation by Sinorhizobium medicae was favored. Co-inoculation of non-sterile soils with R. gallicum and agrobacteria confirmed these findings. In vitro antibiosis assays indicated that agrobacteria exercised a significant antagonism against R. gallicum.     

Rhizobial factors required for stem nodule maturation and maintenance in Sesbania rostrata-Azorhizobium caulinodans ORS571 symbiosis

The molecular and physiological mechanisms behind the maturation and maintenance of N(2)-fixing nodules during development of symbiosis between rhizobia and legumes still remain unclear, although the early events of symbiosis are relatively well understood. Azorhizobium caulinodans ORS571 is a microsymbiont of the tropical legume Sesbania rostrata, forming N(2)-fixing nodules not only on the roots but also on the stems. In this study, 10,080 transposon-inserted mutants of A. caulinodans ORS571 were individually inoculated onto the stems of S. rostrata, and those mutants that induced ineffective stem nodules, as displayed by halted development at various stages, were selected. From repeated observations on stem nodulation, 108 Tn5 mutants were selected and categorized into seven nodulation types based on size and N(2) fixation activity. Tn5 insertions of some mutants were found in the well-known nodulation, nitrogen fixation, and symbiosis-related genes, such as nod, nif, and fix, respectively, lipopolysaccharide synthesis-related genes, C(4) metabolism-related genes, and so on. However, other genes have not been reported to have roles in legume-rhizobium symbiosis. The list of newly identified symbiosis-related genes will present clues to aid in understanding the maturation and maintenance mechanisms of nodules.     

Manipulation of Ethylene Synthesis in Roots Through Bacterial ACC Deaminase for Improving Nodulation in Legumes

Symbiotic association between rhizobia and legumes results in the development of unique structures on roots, called nodules. Nodulation is a very complex process involving a variety of genes that control NOD factors (bacterial signaling molecules), which are essential for the establishment, maintenance and regulation of this process and development of root nodules. Ethylene is an established potent plant hormone that is also known for its negative role in nodulation. Ethylene is produced endogenously in all plant tissues, particularly in response to both biotic and abiotic stresses. Exogenous application of ethylene and ethylene-releasing compounds are known to inhibit the formation and functioning of nodules. While inhibitors of ethylene synthesis or its physiological action enhance nodulation in legumes, some rhizobial strains also nodulate the host plant intensively, most likely by lowering endogenous ethylene levels in roots through their 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity. Co-inoculation with ACC deaminase containing plant growth promoting rhizobacteria plus rhizobia has been shown to further promote nodulation compared to rhizobia alone. Transgenic rhizobia or legume plants with expression of bacterial ACC deaminase could be another viable option to alleviate the negative effects of ethylene on nodulation. Several studies have well documented the role of ethylene and bacterial ACC deaminase in development of nodules on legume roots and will be the primary focus of this critical review.     

Dynamic of the genetic structure of bacterial and fungal communities at different developmental stages of Medicago truncatula Gaertn. cv. Jemalong line J5

The genetic structure of bacterial and fungal communities was characterized in the rhizosphere of Medicago truncatula Gaertn. cv. Jemalong line J5 at five developmental stages (three vegetative and two reproductive stages), and in three compartments (bulk soil, rhizosphere soil and root tissues). The genetic structure of microbial communities was determined by cultivation-independent methods using directly extracted DNA that was characterized by automated ribosomal intergenic spacer analysis (ARISA). Principal component analyses (PCA) indicate that, for all developmental stages, the genetic structure of microbial communities differed significantly by compartment, with a major shift in the community in root tissues corresponding to the most intimate compartment with the plant. Differences were also recorded during plant development, the most significant being observed during the transition between vegetative and reproductive stages. Throughout this period, plants were shown to establish the highest level of symbiotic association (mycorrhization, nodulation) with arbuscular mycorrhizal fungi and Rhizobia. During the reproductive stages, the dynamics of the genetic structure differed between bacterial and fungal communities. At the last reproductive stage, the genetic structure of bacterial communities became close to that recorded during the first vegetative stages, suggesting a resilience phenomenon, whereas the genetic structure of fungal communities remained different from the vegetative stages and also from the early reproductive stages, suggesting a persistence of the rhizosphere effect.     

Nod factor perception during infection thread growth fine-tunes nodulation

Bacterial nodulation factors (NFs) are essential signaling molecules for the initiation of a nitrogen-fixing symbiosis in legumes. NFs are perceived by the plant and trigger both local and distant responses, such as curling of root hairs and cortical cell divisions. In addition to their requirement at the start, NFs are produced by bacteria that reside within infection threads. To analyze the role of NFs at later infection stages, several phases of nodulation were studied by detailed light and electron microscopy after coinoculation of adventitious root primordia of Sesbania rostrata with a mixture of Azorhizobium caulinodans mutants ORS571-V44 and ORS571-X15. These mutants are deficient in NF production or surface polysaccharide synthesis, respectively, but they can complement each other, resulting in functional nodules occupied by ORS571-V44. The lack of NFs within the infection threads was confirmed by the absence of expression of an early NF-induced marker, leghemoglobin 6 of S. rostrata. NF production within the infection threads is shown to be necessary for proper infection thread growth and for synchronization of nodule formation with bacterial invasion. However, local production of NFs by bacteria that are taken up by the plant cells at the stage of bacteroid formation is not required for correct symbiosome development.     

Fine mapping of the Rj4 locus,a gene controlling nodulation specificity in soybean

Leguminous plants have the ability to make their own nitrogen fertilizer by forming a root nodule symbiosis with nitrogen-fixing soil bacteria, collectively called rhizobia. This biological process plays a critical role in sustainable agriculture because it reduces the need for external nitrogen input. One remarkable property of legume–rhizobial symbiosis is its high level of specificity, which occurs at both inter- and intra-species levels and takes place at multiple phases of the interaction, ranging from initial bacterial infection and nodulation to late nodule development associated with nitrogen fixation. Knowledge of the molecular mechanisms controlling symbiotic specificity will facilitate the development of new crop varieties with improved agronomic potential for nitrogen-fixing symbiosis. In this report, we describe fine mapping of the Rj4 locus, a gene controlling nodulation specificity in soybean (Glycine max). The Rj4 allele prevents the host plant from nodulation with many strains of Bradyrhizobium elkanii, which are frequently present in soils of the southeastern USA. Since B. elkanii strains are poor symbiotic partners of soybean, cultivars containing an Rj4 allele are considered favorable. We have delimited the Rj4 locus within a 57-kb genomic region on soybean chromosome 1. The data reported here will facilitate positional cloning of the Rj4 gene and the development of genetic markers for marker-assisted selection in soybean.     

Enhanced phytoremediation of soils contaminated with PAHs by arbuscular mycorrhiza and rhizobium

Greenhouse experiment was conducted to evaluate the potential effectiveness of a legume (Sesbania cannabina), arbuscular mycorrhizal fungi (AMF) (Glomus mosseae), and rhizobia (Ensifer sp.) symbiosis for remediation of Polycyclic aromatic hydrocarbons (PAHs) in spiked soil. AMF and rhizobia had a beneficial impact on each other in the triple symbiosis. AMF and/or rhizobia significantly increased plant biomass and PAHs accumulation in plants. The highest PAHs dissipation was observed in plant + AMF + rhizobia treated soil, in which >97 and 85–87% of phenanthrene and pyrene, respectively, had been degraded, whereas 81–85 and 72–75% had been degraded in plant-treated soil. During the experiment, a relatively large amount of water-soluble phenolic compounds was detected in soils of AMF and/or rhizobia treatment. It matches well with the high microbial activity and soil enzymes activity. These results suggest that the mutual interactions in the triple symbiosis enhanced PAHs degradation via stimulating both microbial development and soil enzyme activity. The mutual interactions between rhizobia and AMF help to improve phytoremediation efficiency of PAHs by S. cannabina.     

Plant phenology and genetic variability in root and nodule development strongly influence genetic structuring of Rhizobium leguminosarum biovar viciae populations nodulating pea

The symbiotic relationships between legumes and their nitrogen (N(2))-fixing bacterial partners (rhizobia) vary in effectiveness to promote plant growth according to both bacterial and legume genotype. To assess the selective effect of host plant on its microsymbionts, the influence of the pea (Pisum sativum) genotype on the relative nodulation success of Rhizobium leguminosarum biovar viciae (Rlv) genotypes from the soil populations during plant development has been investigated. Five pea lines were chosen for their genetic variability in root and nodule development. Genetic structure and diversity of Rlv populations sampled from nodules were estimated by molecular typing with a marker of the genomic background (rDNA intergenic spacer) and a nodulation gene marker (nodD region). Differences were found among Rlv populations related to pea genetic background but also to modification of plant development caused by single gene mutation. The growth stage of the host plant also influenced structuring of populations. A particular nodulation genotype formed the majority of nodules during the reproductive stage. Overall, modification in root and nodule development appears to strongly influence the capacity of particular rhizobial genotypes to form nodules.     

氮肥和水分条件对长喙田菁生长、结瘤和固氮的影响

 盆栽试验研究了长喙田菁(Sesbania rostrata) 茎瘤固氮根瘤菌（Azorhizobium caulinodans）共生体系在不同水分和无机氮肥条件下的生长、结瘤和氮积累。水分处理为: 不浸水、浅浸水(土壤水分为田间持水量)和深浸水, 氮肥处理分别为每千克土施加无机氮肥10 mg、20 mg和40 mg。结果表明,水分条件对共生体系有较大影响, 浸水环境虽然抑制了根瘤的形成, 却促进了茎瘤的结瘤作用和长喙田菁根系的生长, 田间持水量状态下共生体系能获得最大的生物量和氮累积量; 无机氮肥对共生体系的影响受水分供应的影响较大,实验氮肥施用浓度范围内,长喙田菁的结瘤作用对复合态氮无负敏感反应,明显有别于一般根瘤体系的复合态氮反应。     

Epidermal and cortical roles of NFP and DMI3 in coordinating early steps of nodulation in Medicago truncatula

Legumes have evolved the capacity to form a root nodule symbiosis with soil bacteria called rhizobia. The establishment of this symbiosis involves specific developmental events occurring both in the root epidermis (notably bacterial entry) and at a distance in the underlying root cortical cells (notably cell divisions leading to nodule organogenesis). The processes of bacterial entry and nodule organogenesis are tightly linked and both depend on rhizobial production of lipo-chitooligosaccharide molecules called Nod factors. However, how these events are coordinated remains poorly understood. Here, we have addressed the roles of two key symbiotic genes of Medicago truncatula, the lysin motif (LysM) domain-receptor like kinase gene NFP and the calcium- and calmodulin-dependent protein kinase gene DMI3, in the control of both nodule organogenesis and bacterial entry. By complementing mutant plants with corresponding genes expressed either in the epidermis or in the cortex, we have shown that epidermal DMI3, but not NFP, is sufficient for infection thread formation in root hairs. Epidermal NFP is sufficient to induce cortical cell divisions leading to nodule primordia formation, whereas DMI3 is required in both cell layers for these processes. Our results therefore suggest that a signal, produced in the epidermis under the control of NFP and DMI3, is responsible for activating DMI3 in the cortex to trigger nodule organogenesis. We integrate these data to propose a new model for epidermal/cortical crosstalk during early steps of nodulation.