Symbiotic Plant-microbe Interactions Research Articles

'Friend versus foe'-does autophagy help regulate symbiotic plant-microbe interactions and can it be manipulated to improve legume cultivation?

Autophagy is a genetically regulated, eukaryotic catabolic pathway that responds to internal and external cellular signals. In plants, it plays crucial roles in development, and responses to abiotic and biotic stresses. Due to its role in limiting the hypersensitive response, research on the molecular mechanisms of autophagic signalling pathways in plant-microbe interactions has primarily focused on plant-pathogen responses. Although there is substantially less information on the role of autophagy signalling in symbiotic plant-microbe interactions, there is accumulating evidence that it is also a key regulator of mutualistic plant-microbe interactions. Here, we review recent progress on the roles of autophagy in symbiotic plant interactions and discuss potential future research directions. Once understood, the central role that autophagy plays within pathogenic and symbiotic plant-microbe interactions has significant potential application for crop improvement. Manipulating autophagy in legume crops could help support crop growth with reduced levels of fertiliser application while maintaining yields with increased protein content in the harvest.

Realizing the Full Potential of Advanced Microscopy Approaches for Interrogating Plant-Microbe Interactions

Microscopy has served as a fundamental tool for insight and discovery in plant-microbe interactions for centuries. From classical light and electron microscopy to corresponding specialized methods for sample preparation and cellular contrasting agents, these approaches have become routine components in the toolkit of plant and microbiology scientists alike to visualize, probe and understand the nature of host-microbe relationships. Over the last three decades, three-dimensional perspectives led by the development of electron tomography, and especially, confocal techniques continue to provide remarkable clarity and spatial detail of tissue and cellular phenomena. Confocal and electron microscopy provide novel revelations that are now commonplace in medium and large institutions. However, many other cutting-edge technologies and sample preparation workflows are relatively unexploited yet offer tremendous potential for unprecedented advancement in our understanding of the inner workings of pathogenic, beneficial, and symbiotic plant-microbe interactions. Here, we highlight key applications, benefits, and challenges of contemporary advanced imaging platforms for plant-microbe systems with special emphasis on several recently developed approaches, such as light-sheet, single molecule, super-resolution, and adaptive optics microscopy, as well as ambient and cryo-volume electron microscopy, X-ray microscopy, and cryo-electron tomography. Furthermore, the potential for complementary sample preparation methodologies, such as optical clearing, expansion microscopy, and multiplex imaging, will be reviewed. Our ultimate goal is to stimulate awareness of these powerful cutting-edge technologies and facilitate their appropriate application and adoption to solve important and unresolved biological questions in the field. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

A rulebook for peptide control of legume–microbe endosymbioses

Plants engage in mutually beneficial relationships with microbes, such as arbuscular mycorrhizal fungi or nitrogen-fixing rhizobia, for optimized nutrient acquisition. In return, the microbial symbionts receive photosynthetic carbon from the plant. Both symbioses are regulated by the plant nutrient status, indicating the existence of signaling pathways that allow the host to fine-tune its interactions with the beneficial microbes depending on its nutrient requirements. Peptide hormones coordinate a plethora of developmental and physiological processes and, recently, various peptide families have gained special attention as systemic and local regulators of plant-microbe interactions and nutrient homeostasis. In this review, we identify five 'rules' or guiding principles that govern peptide function during symbiotic plant-microbe interactions, and highlight possible points of integration with nutrient acquisition pathways.

Native arbuscular mycorrhizal fungi improve growth, biomass yield, and phosphorus nutrition of sorghum in saline and sodic soils of the semi–arid region

Exploiting symbiotic plant-microbe interactions with arbuscular mycorrhizal fungi (AMF) adapted to hostile soil can be a promising approach for enhancing crop productivity and tolerance to salinity and sodicity-related stresses in salt-affected agroecosystems. This study was conducted to characterize the native mycorrhizal ecotype and its responsiveness to sorghum in saline and sodic soils under controlled conditions. The AMF spore density in sodic soil under the rice-wheat cropping system was greater than in the sorghum-based systems. The spore density was greater under sodic compared to saline soils. The sequence of the amplified fungal ribosomal DNA of the 18 S region of the isolated culture from the rice-wheat system under sodic soil conditions showed the Funneliformis mosseae and Funneliformis geosporum as the dominant AMF species. The colonization and arbuscular abundance of Funneliformis sps. inoculated sorghum was greater for sodic and normal soil, respectively. Plant height and fresh and dry biomass of AMF inoculated plants were greater in normal soil followed by sodic and saline soils. Sodic soil showed a greater increase in root-to-shoot ratio compared to saline soils. The P content, P uptake, and K + / Na + were greater in AMF inoculated soils. The increase in Olsen’s-P in AMF inoculated soils was in the order of normal > sodic > saline soils. Sodic soils showed a maximum 15–35 fold increase in the EE-GRSP and DE GRSP because of AMF inoculation ( P ≤ 0.05 ). Dehydrogenase and alkaline phosphatase enzymes were greater in AMF inoculated ( P < 0.05 ) . The soil electrical conductivity , glomalin, and arbuscular abundance alone explained about 76 % variability in the plant response to AMF inoculation in these soils. This study concludes that the use of native AMF with the important cropping system can be an agronomically sound option to cope with abiotic stress in salt-affected soils. • AMF spore density greater in sodic soils and rice-wheat cropping system. • AMF identified as Funneliformis mosseae and F. geosporum. • Inoculation increased growth and biomass yield. • AMF inoculation increased P content, P uptake, and K+ / Na+ ratio. • 15–35 fold increase in glomalin content in inoculated soils.

Cyclophilins and Their Functions in Abiotic Stress and Plant-Microbe Interactions.

Plants have developed a variety of mechanisms and regulatory pathways to change their gene expression profiles in response to abiotic stress conditions and plant–microbe interactions. The plant–microbe interaction can be pathogenic or beneficial. Stress conditions, both abiotic and pathogenic, negatively affect the growth, development, yield and quality of plants, which is very important for crops. In contrast, the plant–microbe interaction could be growth-promoting. One of the proteins involved in plant response to stress conditions and plant–microbe interactions is cyclophilin. Cyclophilins (CyPs), together with FK506-binding proteins (FKBPs) and parvulins, belong to a big family of proteins with peptidyl-prolyl cis-trans isomerase activity (Enzyme Commission (EC) number 5.2.1.8). Genes coding for proteins with the CyP domain are widely expressed in all organisms examined, including bacteria, fungi, animals, and plants. Their different forms can be found in the cytoplasm, endoplasmic reticulum, nucleus, chloroplast, mitochondrion and in the phloem space. They are involved in numerous processes, such as protein folding, cellular signaling, mRNA processing, protein degradation and apoptosis. In the past few years, many new functions, and molecular mechanisms for cyclophilins have been discovered. In this review, we aim to summarize recent advances in cyclophilin research to improve our understanding of their biological functions in plant defense and symbiotic plant–microbe interactions.

Detection of Diverse N-Acyl Homoserine Lactone Signalling Molecules Among Bacteria Associated with Rice Rhizosphere.

Bacterial communities communicate, regulate and coordinate their cooperative activities and physiological process by releasing, sensing and responding to small diffusible signal molecules such as acyl homoserine lactones (AHLs), auto-inducing oligo-peptides (AIPs) and autoinducer-2, a process referred to as Quorum sensing (QS). The QS mediated communication in rhizosphere associated bacterial communities significantly influence traits governing plant-microbe interactions. This study aimed to identify AHL-mediated QS signals in bacterial communities associated with rice rhizosphere using two AHL biosensors reporter strains Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NTL4 (pZLR4). Approximately 375 bacterial isolates isolated from rice rhizosphere and screened using both the biosensors, detected 49 (13%) AHLpositive isolates. The BOX-Polymerase Chain reaction (BOX-PCR) fingerprinting profiles of the 49 AHL positive isolates represented 11 distinct cluster groups. Subsequent 16S rRNA gene sequence analysis identified 11 different species affiliated to two different phyla; predominantly γ-proteobacteria, representing 5 genera and 1 genus in α-proteobacteria. Thin-layer chromatography (TLC) analysis detected diverse AHL profiles among the 11 AHL positive isolates with both substituted and unsubstituted acyl side chains of C4, C6 and C8 carbon. Further, AHL production in Acinetobacter lactucae, Aeromonas popoffii, Serratia oryzae, and Rhizobium wuzhouense is being reported for the first time. Detection of diverse AHLs from different groups of rhizobacteria associated with rice indicates that these signalling molecules may be involved in the regulation of rhizobacterial behaviour and symbiotic plant-microbe interactions. Future research on the role of AHLs in trans-kingdom communication particularly plant-microbe interaction using synthetic microbial community will enable in evaluating and developing potential plant specific bioproducts.

Bioactivity-guided isolation of antimicrobial metabolite from Xylaria sp.

Symbiotic plant-microbe metabolic interactions not only have beneficial effects on plants but also contribute to rich, unmatched and complex chemical biodiversity with biological potential. Systematic delineated bioprospecting of fungal diversity associated with Ficus pumila Linn (Moraceae) for antimicrobial metabolite revealed Xylaria sp. FPL-25(M). The present study describes bioactivity guided fractionation prioritized for antimicrobial potential. Thus, chemical investigation of culture broth of Xylaria sp. FPL-25(M) by bioactivity guided fractionation with spectroscopic techniques revealed bioactive metabolite xylobovide-9-methyl ester. The xylobovide-9-methyl ester exhibited broad-spectrum antimicrobial activity. However, Gram-positive bacteria and fungi were more susceptible than Gram-negative bacteria. The present study results represent bioassay-based screening strategy which facilitates rapid, efficient and reliable approach for endophytic strain prioritization for novel bioactive molecules.

Plant-Microbe Symbiosis: What Has Proteomics Taught Us?

Beneficial microbes have a positive impact on the productivity and fitness of the host plant. A better understanding of the biological impacts and underlying mechanisms by which the host derives these benefits will help to address concerns around global food production and security. The recent development of omics-based technologies has broadened our understanding of the molecular aspects of beneficial plant-microbe symbiosis. Specifically, proteomics has led to the identification and characterization of several novel symbiosis-specific and symbiosis-related proteins and post-translational modifications that play a critical role in mediating symbiotic plant-microbe interactions and have helped assess the underlying molecular aspects of the symbiotic relationship. Integration of proteomic data with other "omics" data can provide valuable information to assess hypotheses regarding the underlying mechanism of symbiosis and help define the factors affecting the outcome of symbiosis. Herein, an update is provided on the current and potential applications of symbiosis-based "omic" approaches to dissect different aspects of symbiotic plant interactions. The application of proteomics, metaproteomics, and secretomics as enabling approaches for the functional analysis of plant-associated microbial communities is also discussed.

Papers of note in Science 356 (6340)

This week’s articles highlight new research that identifies a microRNA decay pathway that promotes cell cycle progression; an interactive atlas of the subcellular localization of thousands of human proteins, which may help refine signaling networks; and a review of symbiotic plant-microbe interactions.

Role of Plant Genotype and Soil Conditions in Symbiotic Plant-Microbe Interactions for Adaptation of Plants to Cadmium-Polluted Soils

We highlighted some of the key problems associated with the use of beneficial microorganisms for improving adaptation of plants to soils, polluted with heavy metals (HMs), especially Cd. Inoculation of pea line SGE and its Cd-tolerant mutant SGECdt with nodule bacteria Rhizobium leguminosarum bv. viciae demonstrated that nodulation process may be disturbed at Cd concentrations below threshold toxicity levels for each partner and the plant genotype plays a major role in nodulation under Cd stress. A comparative mathematical analysis of available information about Cd tolerance, accumulation of HMs (Cd, Cr, Cu, Ni, Pb, Sr and Zn), response to mycorrhizal fungus Glomus sp. and 15 phenotypic traits of 99 pea varieties revealed that (1) the Cd-sensitive varieties were more efficient in exploring the protective potential of symbiosis to compensate their deficit in Cd tolerance and (2) correlations between the studied traits exist and can be helpful for selection of plant-microbe systems adapted to polluted soils. In pot experiment with 11 varieties of Indian mustard, the plant growth-promoting effect of rhizobacterium Variovorax paradoxus 5C-2 negatively correlated with Cd tolerance and shoot Cd concentration of the plants grown in Cd-supplemented soil. In an outdoor pot experiment, inoculation of willow with the ectomycorrhizal fungus Pisolithus tinctorius and a cocktail of rhizobacteria stimulated root exudation, decreased soil pH and increased Cd mobilization in soil and Cd uptake by plants, but decreased plant growth at a moderate contamination level (25 mg Cd kg−1). Opposite effects were observed in highly contaminated soil (77 mg Cd kg−1). We propose a preliminary systematic framework of interactions between these factors that determine the success of microbial inoculation aimed at improving crop performance on HM-polluted soils or enhancing phytoremediation.

A fluorometric assay for trehalose in the picomole range.

BackgroundTrehalose is a non-reducing disaccharide that is used as an osmolyte, transport sugar, carbon reserve and stress protectant in a wide range of organisms. In plants, trehalose 6-phosphate (Tre6P), the intermediate of trehalose biosynthesis, is thought to be a signal of sucrose status. Trehalose itself may play a role in pathogenic and symbiotic plant-microbe interactions, in responses to abiotic stress and in developmental signalling, but its precise functions are unknown. A major obstacle to investigating its function is the technical difficulty of measuring the very low levels of trehalose usually found in plant tissues, as most of the established trehalose assays lack sufficient specificity and/or sensitivity.ResultsA kinetic assay for trehalose was established using recombinant Escherichia coli cytoplasmic trehalase (treF), which was shown to be highly specific for trehalose. Hydrolysis of trehalose to glucose is monitored fluorometrically and the trehalose content of the tissue extract is determined from an internal calibration curve. The assay is linear for 0.2-40 pmol trehalose, and recoveries of trehalose were ≥88%. A. thaliana Col-0 rosettes contain about 20–30 nmol g-1FW of trehalose, increasing to about 50–60 nmol g-1FW in plants grown at 8°C. Trehalose is not correlated with sucrose content, whereas a strong correlation between Tre6P and sucrose was confirmed. The trehalose contents of ear inflorescence primordia from the maize ramosa3 mutant and wild type plants were 6.6±2.6 nmol g-1FW and 19.0±12.7 nmol g-1FW, respectively. The trehalose:Tre6P ratios in the ramosa3 and wild-type primordia were 2.43±0.85 and 6.16±3.45, respectively.ConclusionThe fluorometric assay is highly specific for trehalose and sensitive enough to measure the trehalose content of very small amounts of plant tissue. Chilling induced a 2-fold accumulation of trehalose in A. thaliana rosettes, but the levels were too low to make a substantial quantitative contribution to osmoregulation. Trehalose is unlikely to function as a signal of sucrose status. The abnormal inflorescence branching phenotype of the maize ramosa3 mutant might be linked to a decrease in trehalose levels in the inflorescence primordia or a downward shift in the trehalose:Tre6P ratio.

Nitric oxide in legume–rhizobium symbiosis

Nitric oxide (NO) is a gaseous signaling molecule with a broad spectrum of regulatory functions in plant growth and development. NO has been found to be involved in various pathogenic or symbiotic plant–microbe interactions. During the last decade, increasing evidence of the occurrence of NO during legume–rhizobium symbioses has been reported, from early steps of plant–bacteria interaction, to the nitrogen-fixing step in mature nodules. This review focuses on recent advances on NO production and function in nitrogen-fixing symbiosis. First, the potential plant and bacterial sources of NO, including NO synthase-like, nitrate reductase or electron transfer chains of both partners, are presented. Then responses of plant and bacterial cells to the presence of NO are presented in the context of the N 2-fixing symbiosis. Finally, the roles of NO as either a regulatory signal of development, or a toxic compound with inhibitory effects on nitrogen fixation, or an intermediate involved in energy metabolism, during symbiosis establishment and nodule functioning are discussed.

Identification of legume RopGEF gene families and characterization of a Medicago truncatula RopGEF mediating polar growth of root hairs

Root hairs play important roles in the interaction of plants with their environment. Root hairs anchor the plant in the soil, facilitate nutrient uptake from the rhizosphere, and participate in symbiotic plant-microbe interactions. These specialized cells grow in a polar fashion which gives rise to their elongated shape, a process mediated in part by a family of small GTPases known as Rops. RopGEFs (GEF, guanine nucleotide exchange factor) activate Rops to effect tip growth in Arabidopsis pollen and root hairs, but the genes mediating tip growth in legumes have not yet been characterized. In this report we describe the Rop and RopGEF gene families from the model legume Medicago truncatula and from the crop legume soybean. We find that one member of the M. truncatula gene family, MtRopGEF2, is required for root hair development because silencing this gene by RNA interference affects the cytosolic Ca2+ gradient and subcellular structure of root hairs, and reduces root hair growth. Consistent with its role in polar growth, we find that a GFP::MtRopGEF2 fusion protein localizes in the apex of emerging and actively growing root hairs. The amino terminus of MtRopGEF2 regulates its ability to interact with MtRops in yeast, and regulates its biological activity in vivo.

The B-3 Ethylene Response Factor MtERF1-1 Mediates Resistance to a Subset of Root Pathogens inMedicago truncatulawithout Adversely Affecting Symbiosis with Rhizobia

The fungal necrotrophic pathogen Rhizoctonia solani is a significant constraint to a range of crops as diverse as cereals, canola, and legumes. Despite wide-ranging germplasm screens in many of these crops, no strong genetic resistance has been identified, suggesting that alternative strategies to improve resistance are required. In this study, we characterize moderate resistance to R. solani anastomosis group 8 identified in Medicago truncatula. The activity of the ethylene- and jasmonate-responsive GCC box promoter element was associated with moderate resistance, as was the induction of the B-3 subgroup of ethylene response transcription factors (ERFs). Genes of the B-1 subgroup showed no significant response to R. solani infection. Overexpression of a B-3 ERF, MtERF1-1, in Medicago roots increased resistance to R. solani as well as an oomycete root pathogen, Phytophthora medicaginis, but not root knot nematode. These results indicate that targeting specific regulators of ethylene defense may enhance resistance to an important subset of root pathogens. We also demonstrate that overexpression of MtERF1-1 enhances disease resistance without apparent impact on nodulation in the A17 background, while overexpression in sickle reduced the hypernodulation phenotype. This suggests that under normal regulation of nodulation, enhanced resistance to root diseases can be uncoupled from symbiotic plant-microbe interactions in the same tissue and that ethylene/ERF regulation of nodule number is distinct from the defenses regulated by B-3 ERFs. Furthermore, unlike the stunted phenotype previously described for Arabidopsis (Arabidopsis thaliana) ubiquitously overexpressing B-3 ERFs, overexpression of MtERF1-1 in M. truncatula roots did not show adverse effects on plant development.

Characterisation of microbial communities colonising the hyphal surfaces of arbuscular mycorrhizal fungi

Arbuscular mycorrhizal fungi (AMF) are symbiotic soil fungi that are intimately associated with the roots of the majority of land plants. They colonise the interior of the roots and the hyphae extend into the soil. It is well known that bacterial colonisation of the rhizosphere can be crucial for many pathogenic as well as symbiotic plant-microbe interactions. However, although bacteria colonising the extraradical AMF hyphae (the hyphosphere) might be equally important for AMF symbiosis, little is known regarding which bacterial species would colonise AMF hyphae. In this study, we investigated which bacterial communities might be associated with AMF hyphae. As bacterial-hyphal attachment is extremely difficult to study in situ, we designed a system to grow AMF hyphae of Glomus intraradices and Glomus proliferum and studied which bacteria separated from an agricultural soil specifically attach to the hyphae. Characterisation of attached and non-attached bacterial communities was performed using terminal restriction fragment length polymorphism and clone library sequencing of 16S ribosomal RNA (rRNA) gene fragments. For all experiments, the composition of hyphal attached bacterial communities was different from the non-attached communities, and was also different from bacterial communities that had attached to glass wool (a non-living substratum). Analysis of amplified 16S rRNA genes indicated that in particular bacteria from the family of Oxalobacteraceae were highly abundant on AMF hyphae, suggesting that they may have developed specific interactions with the fungi.

Global consequences of phosphatidylcholine reduction in Bradyrhizobium japonicum

Phosphatidylcholine (PC) is the major phospholipid in eukaryotic membranes. In contrast, it is found in only a limited number of bacteria including members of the Rhizobiales. Here, PC is required for pathogenic and symbiotic plant-microbe interactions, as shown for Agrobacterium tumefaciens and Bradyrhizobium japonicum, respectively. Two different phospholipid N-methyltransferases, PmtA and PmtX1, convert phosphatidylethanolamine (PE) to PC by three consecutive methylation reactions in B. japonicum. PmtA mainly catalyzes the first methylation reaction converting PE to monomethyl PE, which then serves as substrate for PmtX1 performing the last two methylation reactions. Disruption of the pmtA gene results in a significantly reduced PC content causing a defect in symbiosis with the soybean host. A genome-wide survey for differentially expressed genes in the pmtA mutant with a custom-made Affymetrix gene chip revealed that PC reduction affects transcription of a strictly confined set of genes. Among the 11 up regulated genes were pmtX3 and pmtX4, which code for isoenzymes of PmtA. The expression of two typical two-component systems, a MarR-like regulator and two proteins of a RND-type (resistance nodulation cell division) efflux system were differentially expressed in the pmtA mutant. Our data suggests that a decrease in the PC content of B. japonicum membranes induces a rather specific transcriptional response involving three different transcriptional regulators all involved in the regulatory fine-tuning of a RND-type transport system.

Multiple Phospholipid N -Methyltransferases with Distinct Substrate Specificities Are Encoded in Bradyrhizobium japonicum

Phosphatidylcholine (PC) is the major phospholipid in eukaryotic membranes. In contrast, it is found in only a few prokaryotes including members of the family Rhizobiaceae. In these bacteria, PC is required for pathogenic and symbiotic plant-microbe interactions, as shown for Agrobacterium tumefaciens and Bradyrhizobium japonicum. At least two different phospholipid N-methyltransferases (PmtA and PmtX) have been postulated to convert phosphatidylethanolamine (PE) to PC in B. japonicum by three consecutive methylation reactions. However, apart from the known PmtA enzyme, we identified and characterized three additional pmt genes (pmtX1, pmtX3, and pmtX4), which can be functionally expressed in Escherichia coli, showing different substrate specificities. B. japonicum expressed only two of these pmt genes (pmtA and pmtX1) under all conditions tested. PmtA predominantly converts PE to monomethyl PE, whereas PmtX1 carries out both subsequent methylation steps. B. japonicum is the first bacterium known to use two functionally different Pmts. It also expresses a PC synthase, which produces PC via condensation of CDP-diacylglycerol and choline. Our study shows that PC biosynthesis in bacteria can be much more complex than previously anticipated.

The application of proteomics to plant biology: a review

The term proteomics, although still less than a decade old, is becoming commonplace in the vocabulary of biologists. Advances made in yeast and humans have been remarkable, sustained by equally remarkable progress in mass spectrometry, bioinformatics, and separation techniques. Progress in plants has been more recent, much of it in the model organisms Arabidopsis thaliana (L.) Heynh. and rice ( Oryza sativa L.), reflecting the tremendous advantage of a complete genomic sequence for proteomics endeavours. Other plants have also been the subject of investigation and this review deals with recent progress in proteomics under three main subheadings: total proteome studies, stress and post-translational modifications, and symbiotic plant–microbe interactions. Examples from the current literature are used to illustrate how proteomics can be used by itself or as part of a larger strategy to gain insight into the functioning of plants at the molecular level.

Azide resistant mutants of Acetobacter diazotrophicus and Azospirillum brasilense increase yield and nitrogen content of cotton

Evolution of symbiotic plant-microbe interactions has provided mankind a powerful and environment-friendly means to increase yield of agricultural crops. Here, we report that some azide resistant mutants of two microbial strains can significantly enhance the productivity of cotton varieties, as an attractive and cheap biological substitute of chemical fertilizers, for improved yield of an important cash crop, without any untoward impacts. Sodium azide resistant mutants were isolated from each strain of Azospirillum brasilense and Acetobacter diazotrophicus on different concentrations of sodium azide ranging from 5–60µg/ml. These azide resistant mutants were assessed for their performance on cotton (varieties H-117, HD-123) for various parameters. Inoculation of cottonseeds with mutants obtained better results than inoculation with their respective parental strains. Azide resistant mutants, when used as biofertilizers, showed increased plant height, early flowering, more yield, and high biomass and total nitrogen content. They also increased, in cotton genotypes, the indole acetic acid production and ammonia excretion due to high nitrogenase activity.

Responses of a model legume Lotus japonicus to lipochitin oligosaccharide nodulation factors purified from Mesorhizobium loti JRL501.

Lotus japonicus has been proposed as a model legume for molecular genetic studies of symbiotic plant-microbe interactions leading to the fixation of atmospheric nitrogen. Lipochitin oligosaccharides (LCOs), or Nod factors, were isolated from the culture of Mesorhizobium loti strain JRL501 (MAFF303099), an efficient microsymbiont of L. japonicus B-129 cv. Gifu. High-performance liquid chromatography and mass spectrometric analyses allowed us to identify at least five different structures of LCOs that were produced by JRL501. The major component was NodMl-V(C18:1, Me, Cb, AcFuc), an N-acetyl-glucosamine pentamer in which the nonreducing residue is N-acylated with a C18:1 acyl moiety, N-methylated, and carries a carbamoyl group and the reducing N-acetylglucosamine residue is substituted with 4-O-acetyl-fucose. Additional novel LCO structures bearing fucose instead of acetyl-fucose at the reducing end were identified. Mixtures of these LCOs could elicit abundant root hair deformation on L. japonicus roots at a concentration of 10(-7) to 10(-9) M. Spot inoculation of a few nanograms of LCOs on L. japonicus roots induced the formation of nodule primordia in which the early nodulin genes, ENOD40 and ENOD2, were expressed in a tissue-specific manner. We also observed the formation of a cytoplasmic bridge (preinfection thread) in the swollen outermost cortical cells. This is the first description of cytoplasmic bridge formation by purified LCOs alone in a legume-forming determinate nodules.