Partner choice in Medicago Truncatula–Sinorhizobium symbiosis

In nitrogen-fixing symbiosis, plant sanctions against ineffective bacteria have been demonstrated in previous studies performed on soybean and yellow bush lupin, both developing determinate nodules with Bradyrhizobium sp. strains. In this study, we focused on the widely studied symbiotic association Medicago truncatula–Sinorhizobium meliloti, which forms indeterminate nodules. Using two strains isolated from the same soil and displaying different nitrogen fixation phenotypes on the same fixed plant line, we analysed the existence of both partner choice and plant sanctions by performing split-root experiments. By measuring different parameters such as the nodule number, the nodule biomass per nodule and the number of viable rhizobia per nodule, we showed that M. truncatula is able to select rhizobia based on recognition signals, both before and after the nitrogen fixation step. However, no sanction mechanism, described as a decrease in rhizobia fitness inside the nodules, was detected. Consequently, even if partner choice seems to be widespread among legumes, sanction of non-effective rhizobia might not be universal.     

The involvement of Medicago truncatula non-specific lipid transfer protein N5 in the control of rhizobial infection

Cysteine-rich proteins seem to play important regulatory roles in Medicago truncatula/Sinorhizobium meliloti symbiosis. In particular, a large family of nodule-specific cysteine-rich (NCR) peptides is crucial for the differentiation of nitrogen-fixing bacteroids. The Medicago truncatula N5 protein (MtN5) is currently the only reported non-specific lipid transfer protein necessary for successful rhizobial symbiosis; in addition, MtN5 shares several characteristics with NCR peptides: a small size, a conserved cysteine-rich motif, an N-terminal signal peptide for secretion and antimicrobial activity. Unlike NCR peptides, MtN5 expression is not restricted to the root nodules and is induced during the early phases of symbiosis in root hairs and nodule primordia. Recently, MtN5 was determined to be involved in the regulation of root tissue invasion; while, it was dispensable for nodule primordia formation. Here, we discuss the hypothesis that MtN5 participates in linking the progression of bacterial invasion with restricting the competence of root hairs for infection.     

Detection of S-Nitrosothiol and Nitrosylated Proteins in Arachis hypogaea Functional Nodule: Response of the Nitrogen Fixing Symbiont

To detect the presence of NO, ROS and RNS in nodules of crack entry legumes, we used Arachis hypogaea functional nodule. The response of two cognate partner rhizobia was compared towards NO and GSNO using S. meliloti and Bradyrhizobium sp NC921001. ROS, NO, nitrosothiol and bacteroids were detected by fluorescence microscopy. Redox enzymes and thiol pools were detected biochemically. Nitrosothiols were found to be present but ROS and NO were absent in A. hypogaea nodule. A number of S-nitrosylated proteins were also detected. The total thiol pool and most of the redox enzymes were low in nodule cytosolic extract but these were found to be high in the partner microorganisms indicating partner rhizobia could protect the nodule environment against the nitrosothiols. Both S. meliloti and Bradyrhizobium sp NC921001 were found to contain GSNO reductase. Interestingly, there was a marked difference in growth pattern between S. meliloti and Bradyrhizobium sp in presence of sodium nitroprusside (SNP) and S-nitrosoglutathione (GSNO). Bradyrhizobium sp was found to be much more tolerant to NO donor compounds than the S. meliloti. In contrast, S. meliloti showed resistance to GSNO but was sensitive to SNP. Together our data indicate that nodule environment of crack entry legumes is different than the nodules of infection mode entry in terms of NO, ROS and RNS. Based on our biochemical characterization, we propose that exchange of redox molecules and reactive chemical species is possible between the bacteroid and nodule compartment.     

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

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

Single-plant,Sterile Microcosms for Nodulation and Growth of the Legume Plant Medicago truncatula with the Rhizobial Symbiont Sinorhizobium meliloti

Rhizobial bacteria form symbiotic, nitrogen-fixing nodules on the roots of compatible host legume plants. One of the most well-developed model systems for studying these interactions is the plant Medicago truncatula cv. Jemalong A17 and the rhizobial bacterium Sinorhizobium meliloti 1021. Repeated imaging of plant roots and scoring of symbiotic phenotypes requires methods that are non-destructive to either plants or bacteria. The symbiotic phenotypes of some plant and bacterial mutants become apparent after relatively short periods of growth, and do not require long-term observation of the host/symbiont interaction. However, subtle differences in symbiotic efficiency and nodule senescence phenotypes that are not apparent in the early stages of the nodulation process require relatively long growth periods before they can be scored. Several methods have been developed for long-term growth and observation of this host/symbiont pair. However, many of these methods require repeated watering, which increases the possibility of contamination by other microbes. Other methods require a relatively large space for growth of large numbers of plants. The method described here, symbiotic growth of M. truncatula/S. meliloti in sterile, single-plant microcosms, has several advantages. Plants in these microcosms have sufficient moisture and nutrients to ensure that watering is not required for up to 9 weeks, preventing cross-contamination during watering. This allows phenotypes to be quantified that might be missed in short-term growth systems, such as subtle delays in nodule development and early nodule senescence. Also, the roots and nodules in the microcosm are easily viewed through the plate lid, so up-rooting of the plants for observation is not required.     

Nodule morphology,symbiotic specificity and association with unusual rhizobia are distinguishing features of the genus Listia within the southern African crotalarioid clade Lotononis s.l.

Background and Aims

The legume clade Lotononis sensu lato (s.l.; tribe Crotalarieae) comprises three genera: Listia, Leobordea and Lotononis sensu stricto (s.s.). Listia species are symbiotically specific and form lupinoid nodules with rhizobial species of Methylobacterium and Microvirga. This work investigated whether these symbiotic traits were confined to Listia by determining the ability of rhizobial strains isolated from species of Lotononis s.l. to nodulate Listia, Leobordea and Lotononis s.s. hosts and by examining the morphology and structure of the resulting nodules.

Methods

Rhizobia were characterized by sequencing their 16S rRNA and nodA genes. Nodulation and N₂ fixation on eight taxonomically diverse Lotononis s.l. species were determined in glasshouse trials. Nodules of all hosts, and the process of infection and nodule initiation in Listia angolensis and Listia bainesii, were examined by light microscopy.

Key Results

Rhizobia associated with Lotononis s.l. were phylogenetically diverse. Leobordea and Lotononis s.s. isolates were most closely related to Bradyrhizobium spp., Ensifer meliloti, Mesorhizobium tianshanense and Methylobacterium nodulans. Listia angolensis formed effective nodules only with species of Microvirga. Listia bainesii nodulated only with pigmented Methylobacterium. Five lineages of nodA were found. Listia angolensis and L. bainesii formed lupinoid nodules, whereas nodules of Leobordea and Lotononis s.s. species were indeterminate. All effective nodules contained uniformly infected central tissue. Listia angolensis and L. bainesii nodule initials occurred on the border of the hypocotyl and along the tap root, and nodule primordia developed in the outer cortical layer. Neither root hair curling nor infection threads were seen.

Conclusions

Two specificity groups occur within Lotononis s.l.: Listia species are symbiotically specific, while species of Leobordea and Lotononis s.s. are generally promiscuous and interact with rhizobia of diverse chromosomal and symbiotic lineages. The seasonally waterlogged habitat of Listia species may favour the development of symbiotic specificity.     

Determination of symbiotic nodule occupancy in the model Vicia tetrasperma using a fluorescence scanner

Background

Fluorescent tagging of nodule bacteria forming symbioses with legume host plants represents a tool for vital tracking of bacteria inside the symbiotic root nodules and monitoring changes in gene activity. The constitutive expression of heterologous fluorescent proteins, such as green fluorescent protein (GFP), also allows screening for nodule occupancy by a particular strain. Imaging of the fluorescence signal on a macro-scale is associated with technical problems due to the robustness of nodule tissues and a high level of autofluorescence.

Scope

These limitations can be reduced by the use of a model species with a fine root system, such as Vicia tetrasperma. Further increases in the sensitivity and specificity of the detection and in image resolution can be attained by the use of a fluorescence scanner. Compared with the standard CCD-type cameras, the availability of a laser source of a specified excitation wavelength decreases non-specific autofluorescence while the photomultiplier tubes in emission detection significantly increase sensitivity. The large scanning area combined with a high resolution allow us to visualize individual nodules during the scan of whole root systems. Using a fluorescence scanner with excitation wavelength of 488 nm, a band-pass specific emission channel of 532 nm and a long-pass background channel of 555 nm, it was possible to distinguish nodules occupied by a rhizobial strain marked with one copy of cycle3 GFP from nodules colonized by the wild-type strain.

Conclusions

The main limitation of the current plant model and GFP with the wild-type emission peak at 409 nm is a sharp increase in root autofluorescence below 550 nm. The selectivity of the technique can be enhanced by the use of red-shifted fluorophores and the contrasting labelling of the variants, provided that the excitation (482 nm) and emission (737 nm) maxima corresponding to root chlorophyll are respected.     

Morphogenesis of lucerne root nodules incited by Rhizobium meliloti in the presence of combined nitrogen

Combined light and transmission electron microscopy were used to examine the effect of nitrate on the development of root nodules in lucerne (alfalfa, Medicago sativa L.) following induction by the nitrogen-fixing symbiont, Rhizobium meliloti. The timing of NO ₃ ^- addition was varied in order to study its effect on all of the recognized morphogenetic steps of nodule formation. Roots of plants inoculated in the presence of 18 mM NO ₃ ^- had straight root hairs which were devoid of adherent rhizobia and infection threads, and developed no nodules. However, nodules were formed on roots if 18 mM NO ₃ ^- was added 5 d after inoculation. At this time, the initiation of nodule primordia had already commenced in the root cortex. The histology and ultrastructure of young nodules which had developed for 5 d in the absence of NO ₃ ^- and another 5 d in the presence of 18 mM NO ₃ ^- resembled nodules developing under N-free conditions, except that in the infection threads within the infection zone of the nodule 1) some bacteria tended to loose their normal shape and gain more electron density, indicating premature degradation, and 2) the matrix of the infection threads was abnormally enlarged. In the presence of high NO ₃ ^- levels in the medium, lysis and degeneration of the bacteria released from the infection threads were observed in the infection and bacteroid zones of developing nodules, indicative of premature senescence. On the other hand, the nodule meristems continued to proliferate even after 12 d of exposure of 18 mM NO ₃ ^- . This was the only morphogenetic step of root nodulation which was insensitive to levels of combined nitrogen that completely prevented infection if present at the time of inoculation. These data indicate that all of the recognized steps of root nodule morphogenesis in which the bacteria play a key role are sensitive to the inhibitory effect of combined nitrogen.     

Enhanced nodule initiation on alfalfa by wild-typeRhizobium meliloti co-inoculated withnod gene mutants and other bacteria

Nodule formation on alfalfa (Medicago sativa L.) roots was determined at different inoculum dosages for wild-typeRhizobium meliloti strain RCR2011 and for various mutant derivatives with altered nodulation behavior. The number of nodules formed on the whole length of the primary roots was essentially constant regardless of initial inoculum dosage or subsequent bacterial multiplication, indicative of homeostatic regulation of total nodule number. In contrast, the number of nodules formed in just the initially susceptible region of these roots was sigmoidally dependent on the number of wild-type bacteria added, increasing rapidly at dosages above 5·10³ bacteria/plant. This behavior indicates the possible existence of a threshold barrier to nodule initiation in the host which the bacteria must overcome. When low dosages of the parent (10³ cells/plant) were co-inoculated with 10⁶ cells/plant of mutants lacking functionalnodA, nodC, nodE, nodF ornodH genes, nodule initiation was increased 10- to 30-fold. Analysis of nodule occupancy indicated that these mutants were able to help the parent (wild-type) strain initiate nodules without themselves occupying the nodules. Co-inoculation withR. trifolii orAgrobacterium tumefaciens cured of its Ti plasmid also markedly stimulated nodule initiation by theR. meliloti parent strain. Introduction of a segment of the symbiotic megaplasmid fromR. meliloti intoA. tumefaciens abolished this stimulation.Bradyrhizobium japonicum and a chromosomal Tn5 nod^- mutant ofR. meliloti did not significantly stimulate nodule initiation when co-inoculated with wild-typeR. meliloti. These results indicate that certainnod gene mutants and members of theRhizobiaceae may produce extracellular signals that supplement the ability of wild-typeR. meliloti cells to induce crucial responses in the host.Abbreviations EH emergent root hairs - kb kilobase - RDU relative distance unit - RT root tip This is journal article No. 188-87 of the Ohio Agricultural Research and Development Center     

Endophytic occupation of peanut root nodules by opportunistic Gammaproteobacteria

Several bacterial isolates were recovered from surface-sterilized root nodules of Arachis hypogaea L. (peanut) plants growing in soils from Córdoba, Argentina. The 16S rDNA sequences of seven fast-growing strains were obtained and the phylogenetic analysis showed that these isolates belonged to the Phylum Proteobacteria, Class Gammaproteobacteria, and included Pseudomonas spp., Enterobacter spp., and Klebsiella spp. After storage, these strains became unable to induce nodule formation in Arachis hypogaea L. plants, but they enhanced plant yield. When the isolates were co-inoculated with an infective Bradyrhizobium strain, they were even found colonizing pre-formed nodules. Analysis of symbiotic genes showed that the nifH gene was only detected for the Klebsiella-like isolates and the nodC gene could not be amplified by PCR or be detected by Southern blotting in any of the isolates. The results obtained support the idea that these isolates are opportunistic bacteria able to colonize nodules induced by rhizobia.     

Symbiotic diversity of Ensifer meliloti strains recovered from various legume species in Tunisia

Ensifer meliloti (formerly Sinorhizobium meliloti) was first considered as a specific microsymbiont of Medicago, Melilotus and Trigonella. However, strains of E. meliloti were recovered from root nodules of various legume species and their symbiotic status still remains unclear. Here, we further investigate the specificity of these strains. A collection of 47 E. meliloti strains isolated in Tunisia from root nodules of Medicago truncatula, Medicago sativa, Medicago ciliaris, Medicago laciniata, Medicago marina, Medicago scutellata, Phaseolus vulgaris, Cicer arietinum, Argyrolobium uniflorum, Lotus creticus, Lotus roudairei, Ononis natrix, Retama raetam, Genista saharae, Acacia tortilis, Hedysarum carnosum and Hippocrepis bicontorta were examined by REP-PCR fingerprinting, PCR-RFLPs of the 16S-23S rDNA IGS, the nifH gene and nifD-K intergenic spacer, and sequencing of 16S rRNA and nodA genes. Their nodulation range was also assessed by cross-inoculation experiments. No clear correlation was found between chromosomal backgrounds and host plants of origin. The nodulation polyvalence of the species E. meliloti was associated with a high symbiotic heterogeneity. On the basis of PCR-RFLP data from the nifH gene and nifD-K intergenic spacer, E. meliloti strains isolated from non-Medicago legumes harboured distinct genes and possessed wider host ranges. Some strains did not nodulate Medicago species. On the basis of nodA phylogeny, the majority of the Tunisian strains, including strains from Medicago, harboured distinct nodA alleles more related to those found in E. medicae than those found in E. meliloti. However, more work is still needed to characterize this group further. The diversity observed among M. laciniata isolates, which was supported by nodA phylogeny, nifH typing and the efficiency profile on M. ciliaris, indicated that what was thought to be bv. medicaginis is certainly heterogeneous.     

Identifying abnormalities in symbiotic development between Trifolium spp. and Rhizobium leguminosarum bv. trifolii leading to sub-optimal and ineffective nodule phenotypes

Background and Aims

Legumes overcome nitrogen limitations by entering into a mutualistic symbiosis with N₂-fixing bacteria (rhizobia). Fully compatible associations (effective) between Trifolium spp. and Rhizobium leguminosarum bv. trifolii result from successful recognition of symbiotic partners in the rhizosphere, root hair infection and the formation of nodules where N₂-fixing bacteroids reside. Poorly compatible associations can result in root nodule formation with minimal (sub-optimal) or no (ineffective) N₂-fixation. Despite the abundance and persistence of strains in agricultural soils which are poorly compatible with the commercially grown clover species, little is known of how and why they fail symbiotically. The aims of this research were to determine the morphological aberrations occurring in sub-optimal and ineffective clover nodules and to determine whether reduced bacteroid numbers or reduced N₂-fixing activity is the main cause for the Sub-optimal phenotype.

Methods

Symbiotic effectiveness of four Trifolium hosts with each of four R. leguminosarum bv. trifolii strains was assessed by analysis of plant yields and nitrogen content; nodule yields, abundance, morphology and internal structure; and bacteroid cytology, quantity and activity.

Key Results

Effective nodules (Nodule Function 83–100 %) contained four developmental zones and N₂-fixing bacteroids. In contrast, Sub-optimal nodules of the same age (Nodule Function 24–57 %) carried prematurely senescing bacteroids and a small bacteroid pool resulting in reduced shoot N. Ineffective-differentiated nodules carried bacteroids aborted at stage 2 or 3 in differentiation. In contrast, bacteroids were not observed in Ineffective-vegetative nodules despite the presence of bacteria within infection threads.

Conclusions

Three major responses to N₂-fixation incompatibility between Trifolium spp. and R. l. trifolii strains were found: failed bacterial endocytosis from infection threads into plant cortical cells, bacteroid differentiation aborted prematurely, and a reduced pool of functional bacteroids which underwent premature senescence. We discuss possible underlying genetic causes of these developmental abnormalities and consider impacts on N₂-fixation of clovers.     

Influence of Nematodes and Light Sources on Growth and Nodulation of Soybean

The influence of nematodes on nodulation of soybean varied according to their modes of parasitism. In the greenhouse, nodule formation was stimulated by the endoparasites, Meloidogyne hapla and Pratylenchus penetrans, but was inhibited slightly by the ectoparasite, Belonolaimus longicaudatus. In an experiment under controlled conditions in a phytotron, Heterodera glycines severely inhibited nodule formation, whereas plants inoculated with B. longicaudatus and P. penetrans had more nodules per g root than nematode-free plants. Nitrogen-fixing capacity, however, was inhibited by all three nematode species. Different light sources used in the phytotron experiment also influenced growth and nodulation of soybean. A fluorescent plus incandescent light regime resulted in plants with the greatest shoot weight, pod number, and nodules per g root. Plants grown under Lucalox lamps had excessive stem elongation.     

Characterization of a copper-resistant symbiotic bacterium isolated from Medicago lupulina growing in mine tailings

A root nodule bacterium, Sinorhizobium meliloti CCNWSX0020, resistant to 1.4 mM Cu²⁺ was isolated from Medicago lupulina growing in mine tailings. In medium supplied with copper, this bacterium showed cell deformation and aggregation due to precipitation of copper on the cell surface. Genes similar to the copper-resistant genes, pcoR and pcoA from Escherichia coli, were amplified by PCR from a 1.4-Mb megaplasmid. Inoculation with S. meliloti CCNWSX0020 increased the biomass of M. lupulina grown in medium added 0 and 100 mg Cu²⁺ kg⁻¹ by 45.8% and 78.2%, respectively, and increased the copper concentration inside the plant tissues grown in medium supplied with 100 μM Cu²⁺ by 39.3%, demonstrating that it is a prospective symbiotic system for bioremediation purposes.     

Influence of Location, Host Cultivar, and Inoculation on the Composition of Naturalized Populations of Rhizobium meliloti in Medicago sativa Nodules

A phage typing system was used to evaluate the composition of indigenous populations of Rhizobium meliloti inhabiting nodules of Medicago sativa cultivars grown with and without inoculation at two field sites during 1983 and 1984. Soil at both locations contained established populations of R. meliloti at planting. Analysis of 1,920 nodule isolates revealed 55 unique phage types of indigenous R. meliloti at one site and 65 indigenous types at the other location. The distributions of phage types differed markedly between locations. At one site, the nodule population was dominated by two phage types; seven others occurred consistently but at lower frequency, and the remainder were encountered infrequently. No indigenous types predominated at the other location, although nine occurred more frequently than the remaining types. Indigenous R. meliloti predominated in nodules from inoculated plots at both sites, with inoculant recovery varying between 10 and 38% in each of two years. The frequency of occurrence of particular phage types at one location was significantly influenced by both M. sativa cultivar and inoculation. At this location, the interaction of cultivar and inoculation on the incidence of phage types suggests that the presence of an inoculant strain differentially affected nodule occupancy of M. sativa cultivars by members of the indigenous R. meliloti population. At both sites, the frequency of specific phage types differed between years. The data emphasize the importance of understanding the ecology and characteristics of indigenous Rhizobium populations as a prerequisite for elucidating problems of inoculant establishment and persistence in competitive situations.     

Nodules Initiated by Rhizobium meliloti Exopolysaccharide Mutants Lack a Discrete, Persistent Nodule Meristem

Infection of alfalfa with Rhizobium meliloti exo mutants deficient in exopolysaccharide results in abnormal root nodules that are devoid of bacteria and fail to fix nitrogen. Here we report further characterization of these abnormal nodules. Tightly curled root hairs or shepherd's crooks were found after inoculation with Rm 1021-derived exo mutants, but curling was delayed compared with wild-type Rm 1021. Infection threads were initiated in curled root hairs by mutants as well as by wild-type R. meliloti, but the exo mutant-induced threads aborted within the peripheral cells of the developing nodule. Also, nodules elicited by Rm 1021-derived exo mutants were more likely to develop on secondary roots than on the primary root. In contrast with wild-type R. meliloti-induced nodules, the exo mutant-induced nodules lacked a well defined apical meristem, presumably due to the abortion of the infection threads. The relationship of these findings to the physiology of nodule development is discussed.     

Histopathology of Antarctic krill, Euphausia superba, bearing black spots

Small black spots have been noticed on the cephalothorax of Antarctic krill, Euphausia superba, since January, 2001. To study the nature of the black spots, the krill were sampled in the winter of 2003, 2006, and 2007 in the South Georgia region, the Antarctic Ocean. Histological observations revealed that the black spots were melanized nodules that were composed of hemocytes surrounding either bacteria or amorphous material. In the 2007 samples, 42% of the krill had melanized nodules. Most of the nodules had an opening on the body surface of the krill. A single melanized nodule often contained more than one type of morphologically distinct bacterial cell. Three bacteria were isolated from these black spots, and classified into either Psychrobacter or Pseudoalteromonas based on the sequences of 16S rRNA genes. More than three bacterial species or strains were also confirmed by in situ hybridization for 16S rRNA. The melanized nodules were almost always accompanied by a mass of atypical, large heteromorphic cells, which were not observed in apparently healthy krill. Unidentified parasites were observed in some of the krill that had melanized nodules. These parasites were directly surrounded by the large heteromorphic cells. Histological observations suggested that these heteromorphic cells were attacking the parasites. These results suggest the possibility that the krill had been initially affected by parasite infections, and the parasitized spots were secondary infected by environmental bacteria after the parasites had escaped from the host body.     

Spatiotemporal cytokinin response imaging and ISOPENTENYLTRANSFERASE 3 function in Medicago nodule development

Most legumes can establish a symbiotic association with soil rhizobia that trigger the development of root nodules. These nodules host the rhizobia and allow them to fix nitrogen efficiently. The perception of bacterial lipo-chitooligosaccharides (LCOs) in the epidermis initiates a signaling cascade that allows rhizobial intracellular infection in the root and de-differentiation and activation of cell division that gives rise to the nodule. Thus, nodule organogenesis and rhizobial infection need to be coupled in space and time for successful nodulation. The plant hormone cytokinin (CK) contributes to the coordination of this process, acting as an essential positive regulator of nodule organogenesis. However, the temporal regulation of tissue-specific CK signaling and biosynthesis in response to LCOs or Sinorhizobium meliloti inoculation in Medicago truncatula remains poorly understood. In this study, using a fluorescence-based CK sensor (pTCSn::nls:tGFP), we performed a high-resolution tissue-specific temporal characterization of the sequential activation of CK response during root infection and nodule development in M. truncatula after inoculation with S. meliloti. Loss-of-function mutants of the CK-biosynthetic gene ISOPENTENYLTRANSFERASE 3 (IPT3) showed impairment of nodulation, suggesting that IPT3 is required for nodule development in M. truncatula. Simultaneous live imaging of pIPT3::nls:tdTOMATO and the CK sensor showed that IPT3 induction in the pericycle at the base of nodule primordium contributes to CK biosynthesis, which in turn promotes expression of positive regulators of nodule organogenesis in M. truncatula.

Precise spatial and temporal characterization of cytokinin (CK) responses reveals the function of the CK biosynthesis gene ISOPENTENYLTRANSFERASE 3 during nodule development in Medicago truncatula.