Faculty & Staff

Start and Promotion Dates

  • Assistant Professor: 2004
  • Associate Professor: 2010
  • Full Professor: 2014


University of Lyon (France) B.S. Molecular and Cellular Biology 1995
University of Toulouse (France) M.S. Plant Cellular and Molecular Biology 1997
University of Toulouse (France) Ph.D. Plant Cellular and Molecular Biology 2002

Areas of Study

Plant-Microbe Symbiotic Interactions

Research Overview

Understanding how symbiotic (beneficial) associations between plants and microbes develop is an important biological question that is particularly relevant in modern agriculture and economy.

Our laboratory seeks to understand and manipulate the molecular mechanism controlling symbiotic associations between plants and microbes.

We transfer information gained from model plants such as Medicago truncatula to crops such as soybean, rice and corn in order to take full advantage of the fantastic opportunities offered by these beneficial associations to our agriculture.

Our goal is to use microbes better to maintain the sustainability of our agriculture by protecting the environment over the long term and reducing costs for food, feed and biofuel production.


Microbiology 335: The Microbiome of Plants, Animals, and Humans 
Agronomy 339: Plant Biotechnology Principles and Techniques


Microbiology Doctoral Training Program
Plant Breeding and Plant Genetics
Cellular and Molecular Biology
Biotechnology Training Program
Genetics Training Program
Plant Pathology Graduate Program
Agronomy Graduate Program
Agroecology Graduate Program


  • 2015, Vilas Faculty Mid-Career Investigator Award 
  • 2014, Rothermel Bascom Professorship of Agronomy 
  • 2009, Honored Instructors Award, University Housing, University of Wisconsin-Madison 
  • 1998, « Agrégation » in Biochemistry and Biotechnology, French National Education, Competitive entry in the French National Education System for permanent teaching positions
  • 1993, ENS-Lyon Fellow, French National Education, Ecole Normale Supérieure de Lyon (ENS-Lyon)

Lab Personnel

Picture of Alptekin
Burcu Alptekin
Picture of Carrera
Cristobal Carrera
Grad Student
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Rubens Diogo
Picture of Dohr
Shannon Dohr
Research Intern
Picture of Espejel Venado
Rafael Espejel Venado
Picture of Infante
Valentina Infante
Grad Student
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Junko Maeda
Research Specialist
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Bren Myers
Research Specialist
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Biswajit Samal
Picture of Singh
TJ Singh
Honorary Associate
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Manish Tiwari

Research Papers

  • Kates HR, O'Meara BC, LaFrance R, Stull GW, James EK, Liu SY, Tian Q, Yi TS, Conde D, Kirst M, Ané JM, Soltis DE, Guralnick RP, Soltis PS, Folk RA (2024) Shifts in evolutionary lability underlie independent gains and losses of root-nodule symbiosis in a single clade of plants. Nature communications 15((1)):4262 PMC7268803 · Pubmed · DOI

    Root nodule symbiosis (RNS) is a complex trait that enables plants to access atmospheric nitrogen converted into usable forms through a mutualistic relationship with soil bacteria. Pinpointing the evolutionary origins of RNS is critical for understanding its genetic basis, but building this evolutionary context is complicated by data limitations and the intermittent presence of RNS in a single clade of ca. 30,000 species of flowering plants, i.e., the nitrogen-fixing clade (NFC). We developed the most extensive de novo phylogeny for the NFC and an RNS trait database to reconstruct the evolution of RNS. Our analysis identifies evolutionary rate heterogeneity associated with a two-step process: An ancestral precursor state transitioned to a more labile state from which RNS was rapidly gained at multiple points in the NFC. We illustrate how a two-step process could explain multiple independent gains and losses of RNS, contrary to recent hypotheses suggesting one gain and numerous losses, and suggest a broader phylogenetic and genetic scope may be required for genome-phenome mapping.

  • Pereira WJ, Boyd J, Conde D, Triozzi PM, Balmant KM, Dervinis C, Schmidt HW, Boaventura-Novaes C, Chakraborty S, Knaack SA, Gao Y, Feltus FA, Roy S, Ané JM, Frugoli J, Kirst M (2024) The single-cell transcriptome program of nodule development cellular lineages in Medicago truncatula. Cell reports 43((2)):113747 · Pubmed · DOI

    Legumes establish a symbiotic relationship with nitrogen-fixing rhizobia by developing nodules. Nodules are modified lateral roots that undergo changes in their cellular development in response to bacteria, but the transcriptional reprogramming that occurs in these root cells remains largely uncharacterized. Here, we describe the cell-type-specific transcriptome response of Medicago truncatula roots to rhizobia during early nodule development in the wild-type genotype Jemalong A17, complemented with a hypernodulating mutant (sunn-4) to expand the cell population responding to infection and subsequent biological inferences. The analysis identifies epidermal root hair and stele sub-cell types associated with a symbiotic response to infection and regulation of nodule proliferation. Trajectory inference shows cortex-derived cell lineages differentiating to form the nodule primordia and, posteriorly, its meristem, while modulating the regulation of phytohormone-related genes. Gene regulatory analysis of the cell transcriptomes identifies new regulators of nodulation, including STYLISH 4, for which the function is validated.

  • Wolf ESA, Vela S, Wilker J, Davis A, Robert M, Infante V, Venado RE, Voiniciuc C, Ané JM, Vermerris W (2023) Identification of genetic and environmental factors influencing aerial root traits that support biological nitrogen fixation in sorghum. G3 (Bethesda, Md.) 14((3)): PMC2931336 · Pubmed · DOI

    Plant breeding and genetics play a major role in the adaptation of plants to meet human needs. The current requirement to make agriculture more sustainable can be partly met by a greater reliance on biological nitrogen fixation by symbiotic diazotrophic microorganisms that provide crop plants with ammonium. Select accessions of the cereal crop sorghum (Sorghum bicolor (L.) Moench) form mucilage-producing aerial roots that harbor nitrogen-fixing bacteria. Breeding programs aimed at developing sorghum varieties that support diazotrophs will benefit from a detailed understanding of the genetic and environmental factors contributing to aerial root formation. A genome-wide association study of the sorghum minicore, a collection of 242 landraces, and 30 accessions from the sorghum association panel was conducted in Florida and Wisconsin and under 2 fertilizer treatments to identify loci associated with the number of nodes with aerial roots and aerial root diameter. Sequence variation in genes encoding transcription factors that control phytohormone signaling and root system architecture showed significant associations with these traits. In addition, the location had a significant effect on the phenotypes. Concurrently, we developed F2 populations from crosses between bioenergy sorghums and a landrace that produced extensive aerial roots to evaluate the mode of inheritance of the loci identified by the genome-wide association study. Furthermore, the mucilage collected from aerial roots contained polysaccharides rich in galactose, arabinose, and fucose, whose composition displayed minimal variation among 10 genotypes and 2 fertilizer treatments. These combined results support the development of sorghums with the ability to acquire nitrogen via biological nitrogen fixation.

  • Ferrer-Orgaz S, Tiwari M, Isidra-Arellano MC, Pozas-Rodriguez EA, Vernié T, Rich MK, Mbengue M, Formey D, Delaux PM, Ané JM, Valdés-López O (2023) Early Phosphorylated Protein 1 is required to activate the early rhizobial infection program. The New phytologist 241((3)):962-968 · Pubmed · DOI

    No abstract available.

  • Venkataraman M, Yñigez-Gutierrez A, Infante V, MacIntyre A, Fernandes-Júnior PI, Ané JM, Pfleger B (2023) Synthetic Biology Toolbox for Nitrogen-Fixing Soil Microbes. ACS synthetic biology 12((12)):3623-3634 PMC2861460 · Pubmed · DOI

    The soil environment adjacent to plant roots, termed the rhizosphere, is home to a wide variety of microorganisms that can significantly affect the physiology of nearby plants. Microbes in the rhizosphere can provide nutrients, secrete signaling compounds, and inhibit pathogens. These processes could be manipulated with synthetic biology to enhance the agricultural performance of crops grown for food, energy, or environmental remediation, if methods can be implemented in these nonmodel microbes. A common first step for domesticating nonmodel organisms is the development of a set of genetic engineering tools, termed a synthetic biology toolbox. A toolbox comprises transformation protocols, replicating vectors, genome engineering (e.g., CRISPR/Cas9), constitutive and inducible promoter systems, and other gene expression control elements. This work validated synthetic biology toolboxes in three nitrogen-fixing soil bacteria: Azotobacter vinelandii , Stutzerimonas stutzeri ( Pseudomonas stutzeri ), and a new isolate of Klebsiella variicola . All three organisms were amenable to transformation and reporter protein expression, with several functional inducible systems available for each organism. S. stutzeri and K. variicola showed more reliable plasmid-based expression, resulting in successful Cas9 recombineering to create scarless deletions and insertions. Using these tools, we generated mutants with inducible nitrogenase activity and introduced heterologous genes to produce resorcinol products with relevant biological activity in the rhizosphere.

  • Gopalakrishnan Meena M, Lane MJ, Tannous J, Carrell AA, Abraham PE, Giannone RJ, Ané JM, Keller NP, Labbé JL, Geiger AG, Kainer D, Jacobson DA, Rush TA (2023) A glimpse into the fungal metabolomic abyss: Novel network analysis reveals relationships between exogenous compounds and their outputs. PNAS nexus 2((10)):pgad322 PMC374315 · Pubmed · DOI

    Fungal specialized metabolites are a major source of beneficial compounds that are routinely isolated, characterized, and manufactured as pharmaceuticals, agrochemical agents, and industrial chemicals. The production of these metabolites is encoded by biosynthetic gene clusters that are often silent under standard growth conditions. There are limited resources for characterizing the direct link between abiotic stimuli and metabolite production. Herein, we introduce a network analysis-based, data-driven algorithm comprising two routes to characterize the production of specialized fungal metabolites triggered by different exogenous compounds: the direct route and the auxiliary route. Both routes elucidate the influence of treatments on the production of specialized metabolites from experimental data. The direct route determines known and putative metabolites induced by treatments and provides additional insight over traditional comparison methods. The auxiliary route is specific for discovering unknown analytes, and further identification can be curated through online bioinformatic resources. We validated our algorithm by applying chitooligosaccharides and lipids at two different temperatures to the fungal pathogen Aspergillus fumigatus . After liquid chromatography-mass spectrometry quantification of significantly produced analytes, we used network centrality measures to rank the treatments' ability to elucidate these analytes and confirmed their identity through fragmentation patterns or in silico spiking with commercially available standards. Later, we examined the transcriptional regulation of these metabolites through real-time quantitative polymerase chain reaction. Our data-driven techniques can complement existing metabolomic network analysis by providing an approach to track the influence of any exogenous stimuli on metabolite production. Our experimental-based algorithm can overcome the bottlenecks in elucidating novel fungal compounds used in drug discovery.

  • Chakraborty S, Venkataraman M, Infante V, Pfleger BF, Ané JM (2023) Scripting a new dialogue between diazotrophs and crops. Trends in microbiology 32((6)):577-589 PMC7733420 · Pubmed · DOI

    Diazotrophs are bacteria and archaea that can reduce atmospheric dinitrogen (N) into ammonium. Plant-diazotroph interactions have been explored for over a century as a nitrogen (N) source for crops to improve agricultural productivity and sustainability. This scientific quest has generated much information about the molecular mechanisms underlying the function, assembly, and regulation of nitrogenase, ammonium assimilation, and plant-diazotroph interactions. This review presents various approaches to manipulating N fixation activity, ammonium release by diazotrophs, and plant-diazotroph interactions. We discuss the research avenues explored in this area, propose potential future routes, emphasizing engineering at the metabolic level via biorthogonal signaling, and conclude by highlighting the importance of biocontrol measures and public acceptance.

  • Keller-Pearson M, Bortolazzo A, Willems L, Smith B, Peterson A, Ané JM, Silva EM (2023) A Dual Transcriptomic Approach Reveals Contrasting Patterns of Differential Gene Expression During Drought in Arbuscular Mycorrhizal Fungus and Carrot. Molecular plant-microbe interactions : MPMI 36((12)):821-832 · Pubmed · DOI

    While arbuscular mycorrhizal (AM) fungi are known for providing host plants with improved drought tolerance, we know very little about the fungal response to drought in the context of the fungal-plant relationship. In this study, we evaluated the drought responses of the host and symbiont, using the fungus Rhizophagus irregularis with carrot ( Daucus carota ) as a plant model. Carrots inoculated with spores of R. irregularis DAOM 197198 were grown in a greenhouse. During taproot development, carrots were exposed to a 10-day water restriction. Compared with well-watered conditions, drought caused diminished photosynthetic activity and reduced plant growth in carrot with and without AM fungi. Droughted carrots had lower root colonization. For R. irregularis , 93% of 826 differentially expressed genes (DEGs) were upregulated during drought, including phosphate transporters, several predicted transport proteins of potassium, and the aquaporin RiAQPF2 . In contrast, 78% of 2,486 DEGs in AM carrot were downregulated during drought, including the symbiosis-specific genes FatM , RAM2 , and STR , which are implicated in lipid transfer from the host to the fungus and were upregulated exclusively in AM carrot during well-watered conditions. Overall, this study provides insight into the drought response of an AM fungus in relation to its host; the expression of genes related to symbiosis and nutrient exchange were downregulated in carrot but upregulated in the fungus. This study reveals that carrot and R. irregularis exhibit contrast in their regulation of gene expression during drought, with carrot reducing its apparent investment in symbiosis and the fungus increasing its apparent symbiotic efforts. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

  • Van Gelder K, Oliveira-Filho ER, Messina CD, Venado RE, Wilker J, Rajasekar S, Ané JM, Amthor JS, Hanson AD (2023) Running the numbers on plant synthetic biology solutions to global problems. Plant science : an international journal of experimental plant biology 335:111815 · Pubmed · DOI

    Synthetic biology and metabolic engineering promise to deliver sustainable solutions to global problems such as phasing out fossil fuels and replacing industrial nitrogen fixation. While this promise is real, scale matters, and so do knock-on effects of implementing solutions. Both scale and knock-on effects can be estimated by 'Fermi calculations' (aka 'back-of-envelope calculations') that use uncontroversial input data plus simple arithmetic to reach rough but reliable conclusions. Here, we illustrate how this is done and how informative it can be using two cases: oilcane (sugarcane engineered to accumulate triglycerides instead of sugar) as a source of bio-jet fuel, and nitrogen fixation by bacteria in mucilage secreted by maize aerial roots. We estimate that oilcane could meet no more than about 1% of today's U.S. jet fuel demand if grown on all current U.S. sugarcane land and that, if cane land were expanded to meet two-thirds of this demand, the fertilizer and refinery requirements would create a large carbon footprint. Conversely, we estimate that nitrogen fixation in aerial-root mucilage could replace up to 10% of the fertilizer nitrogen applied to U.S. maize, that 2% of plant carbon income used for growth would suffice to fuel the fixation, and that this extra carbon consumption would likely reduce grain yield only slightly.

  • Bittleston LS, Wolock CJ, Maeda J, Infante V, Ané JM, Pierce NE, Pringle A (2023) Carnivorous Nepenthes Pitchers with Less Acidic Fluid House Nitrogen-Fixing Bacteria. Applied and environmental microbiology 89((7)):e0081223 PMC4450248 · Pubmed · DOI

    Carnivorous pitcher plants are uniquely adapted to nitrogen limitation, using pitfall traps to acquire nutrients from insect prey. Pitcher plants in the genus Sarracenia may also use nitrogen fixed by bacteria inhabiting the aquatic microcosms of their pitchers. Here, we investigated whether species of a convergently evolved pitcher plant genus, Nepenthes , might also use bacterial nitrogen fixation as an alternative strategy for nitrogen capture. First, we constructed predicted metagenomes of pitcher organisms from three species of Singaporean Nepenthes using 16S rRNA sequence data and correlated predicted nifH abundances with metadata. Second, we used gene-specific primers to amplify and quantify the presence or absence of nifH directly from 102 environmental samples and identified potential diazotrophs with significant differential abundance in samples that also had positive nifH PCR tests. Third, we analyzed nifH in eight shotgun metagenomes from four additional Bornean Nepenthes species. Finally, we conducted an acetylene reduction assay using greenhouse-grown Nepenthes pitcher fluids to confirm nitrogen fixation is indeed possible within the pitcher habitat. Results show active acetylene reduction can occur in Nepenthes pitcher fluid. Variation in nifH from wild samples correlates with Nepenthes host species identity and pitcher fluid acidity. Nitrogen-fixing bacteria are associated with more neutral fluid pH, while endogenous Nepenthes digestive enzymes are most active at low fluid pH. We hypothesize Nepenthes species experience a trade-off in nitrogen acquisition; when fluids are acidic, nitrogen is primarily acquired via plant enzymatic degradation of insects, but when fluids are neutral, Nepenthes plants take up more nitrogen via bacterial nitrogen fixation. IMPORTANCE Plants use different strategies to obtain the nutrients that they need to grow. Some plants access their nitrogen directly from the soil, while others rely on microbes to access the nitrogen for them. Carnivorous pitcher plants generally trap and digest insect prey, using plant-derived enzymes to break down insect proteins and generate a large portion of the nitrogen that they subsequently absorb. In this study, we present results suggesting that bacteria living in the fluids formed by Nepenthes pitcher plants can fix nitrogen directly from the atmosphere, providing an alternative pathway for plants to access nitrogen. These nitrogen-fixing bacteria are only likely to be present when pitcher plant fluids are not strongly acidic. Interestingly, the plant's enzymes are known to be more active under strongly acidic conditions. We propose a potential trade-off where pitcher plants sometimes access nitrogen using their own enzymes to digest prey and at other times take advantage of bacterial nitrogen fixation.

  • Adak A, Murray SC, Calderón CI, Infante V, Wilker J, Varela JI, Subramanian N, Isakeit T, Ané JM, Wallace J, de Leon N, Stull MA, Brun M, Hill J, Johnson CD (2023) Genetic mapping and prediction for novel lesion mimic in maize demonstrates quantitative effects from genetic background, environment and epistasis. TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik 136((7)):155 · Pubmed · DOI

    A novel locus was discovered on chromosome 7 associated with a lesion mimic in maize; this lesion mimic had a quantitative and heritable phenotype and was predicted better via subset genomic markers than whole genome markers across diverse environments. Lesion mimics are a phenotype of leaf micro-spotting in maize (Zea mays L.), which can be early signs of biotic or abiotic stresses. Dissecting its inheritance is helpful to understand how these loci behave across different genetic backgrounds. Here, 538 maize recombinant inbred lines (RILs) segregating for a novel lesion mimic were quantitatively phenotyped in Georgia, Texas, and Wisconsin. These RILs were derived from three bi-parental crosses using a tropical pollinator (Tx773) as the common parent crossed with three inbreds (LH195, LH82, and PB80). While this lesion mimic was heritable across three environments based on phenotypic ([Formula: see text] = 0.68) and genomic ([Formula: see text] = 0.91) data, transgressive segregation was observed. A genome-wide association study identified a single novel locus on chromosome 7 (at 70.6 Mb) also covered by a quantitative trait locus interval (69.3-71.0 Mb), explaining 11-15% of the variation, depending on the environment. One candidate gene identified in this region, Zm00001eb308070, is related to the abscisic acid pathway involving in cell death. Genomic predictions were applied to genome-wide markers (39,611 markers) contrasted with a marker subset (51 markers). Population structure explained more variation than environment in genomic prediction, but other substantial genetic background effects were additionally detected. Subset markers explained substantially less genetic variation (24.9%) for the lesion mimic than whole genome markers (55.4%) in the model, yet predicted the lesion mimic better (0.56-0.66 vs. 0.26-0.29). These results indicate this lesion mimic phenotype was less affected by environment than by epistasis and genetic background effects, which explain its transgressive segregation.

  • Garcia K, Cloghessy K, Cooney DR, Shelley B, Chakraborty S, Kafle A, Busidan A, Sonawala U, Collier R, Jayaraman D, Ané JM, Pilot G (2023) The putative transporter MtUMAMIT14 participates in nodule formation in Medicago truncatula. Scientific reports 13((1)):804 PMC3901275 · Pubmed · DOI

    Transport systems are crucial in many plant processes, including plant-microbe interactions. Nodule formation and function in legumes involve the expression and regulation of multiple transport proteins, and many are still uncharacterized, particularly for nitrogen transport. Amino acids originating from the nitrogen-fixing process are an essential form of nitrogen for legumes. This work evaluates the role of MtN21 (henceforth MtUMAMIT14), a putative transport system from the MtN21/EamA-like/UMAMIT family, in nodule formation and nitrogen fixation in Medicago truncatula. To dissect this transporter's role, we assessed the expression of MtUMAMIT14 using GUS staining, localized the corresponding protein in M. truncatula root and tobacco leaf cells, and investigated two independent MtUMAMIT14 mutant lines. Our results indicate that MtUMAMIT14 is localized in endosomal structures and is expressed in both the infection zone and interzone of nodules. Comparison of mutant and wild-type M. truncatula indicates MtUMAMIT14, the expression of which is dependent on the presence of NIN, DNF1, and DNF2, plays a role in nodule formation and nitrogen-fixation. While the function of the transporter is still unclear, our results connect root nodule nitrogen fixation in legumes with the UMAMIT family.

  • Rush TA, Tannous J, Lane MJ, Gopalakrishnan Meena M, Carrell AA, Golan JJ, Drott MT, Cottaz S, Fort S, Ané JM, Keller NP, Pelletier DA, Jacobson DA, Kainer D, Abraham PE, Giannone RJ, Labbé JL (2022) Lipo-Chitooligosaccharides Induce Specialized Fungal Metabolite Profiles That Modulate Bacterial Growth. mSystems 7((6)):e0105222 PMC1472692 · Pubmed · DOI

    Lipo-chitooligosaccharides (LCOs) are historically known for their role as microbial-derived signaling molecules that shape plant symbiosis with beneficial rhizobia or mycorrhizal fungi. Recent studies showing that LCOs are widespread across the fungal kingdom have raised questions about the ecological function of these compounds in organisms that do not form symbiotic relationships with plants. To elucidate the ecological function of these compounds, we investigate the metabolomic response of the ubiquitous human pathogen Aspergillus fumigatus to LCOs. Our metabolomics data revealed that exogenous application of various types of LCOs to A. fumigatus resulted in significant shifts in the fungal metabolic profile, with marked changes in the production of specialized metabolites known to mediate ecological interactions. Using network analyses, we identify specific types of LCOs with the most significant effect on the abundance of known metabolites. Extracts of several LCO-induced metabolic profiles significantly impact the growth rates of diverse bacterial species. These findings suggest that LCOs may play an important role in the competitive dynamics of non-plant-symbiotic fungi and bacteria. This study identifies specific metabolomic profiles induced by these ubiquitously produced chemicals and creates a foundation for future studies into the potential roles of LCOs as modulators of interkingdom competition. IMPORTANCE The activation of silent biosynthetic gene clusters (BGC) for the identification and characterization of novel fungal secondary metabolites is a perpetual motion in natural product discoveries. Here, we demonstrated that one of the best-studied symbiosis signaling compounds, lipo-chitooligosaccharides (LCOs), play a role in activating some of these BGCs, resulting in the production of known, putative, and unknown metabolites with biological activities. This collection of metabolites induced by LCOs differentially modulate bacterial growth, while the LCO standards do not convey the same effect. These findings create a paradigm shift showing that LCOs have a more prominent role outside of host recognition of symbiotic microbes. Importantly, our work demonstrates that fungi use LCOs to produce a variety of metabolites with biological activity, which can be a potential source of bio-stimulants, pesticides, or pharmaceuticals.

  • Knaack SA, Conde D, Chakraborty S, Balmant KM, Irving TB, Maia LGS, Triozzi PM, Dervinis C, Pereira WJ, Maeda J, Schmidt HW, Ané JM, Kirst M, Roy S (2022) Temporal change in chromatin accessibility predicts regulators of nodulation in Medicago truncatula. BMC biology 20((1)):252 PMC321926 · Pubmed · DOI

    Symbiotic associations between bacteria and leguminous plants lead to the formation of root nodules that fix nitrogen needed for sustainable agricultural systems. Symbiosis triggers extensive genome and transcriptome remodeling in the plant, yet an integrated understanding of the extent of chromatin changes and transcriptional networks that functionally regulate gene expression associated with symbiosis remains poorly understood. In particular, analyses of early temporal events driving this symbiosis have only captured correlative relationships between regulators and targets at mRNA level. Here, we characterize changes in transcriptome and chromatin accessibility in the model legume Medicago truncatula, in response to rhizobial signals that trigger the formation of root nodules.

  • Pankievicz VCS, Delaux PM, Infante V, Hirsch HH, Rajasekar S, Zamora P, Jayaraman D, Calderon CI, Bennett A, Ané JM (2022) Nitrogen fixation and mucilage production on maize aerial roots is controlled by aerial root development and border cell functions. Frontiers in plant science 13:977056 PMC6080747 · Pubmed · DOI

    Exploring natural diversity for biological nitrogen fixation in maize and its progenitors is a promising approach to reducing our dependence on synthetic fertilizer and enhancing the sustainability of our cropping systems. We have shown previously that maize accessions from the Sierra Mixe can support a nitrogen-fixing community in the mucilage produced by their abundant aerial roots and obtain a significant fraction of their nitrogen from the air through these associations. In this study, we demonstrate that mucilage production depends on root cap and border cells sensing water, as observed in underground roots. The diameter of aerial roots correlates with the volume of mucilage produced and the nitrogenase activity supported by each root. Young aerial roots produce more mucilage than older ones, probably due to their root cap's integrity and their ability to produce border cells. Transcriptome analysis on aerial roots at two different growth stages before and after mucilage production confirmed the expression of genes involved in polysaccharide synthesis and degradation. Genes related to nitrogen uptake and assimilation were up-regulated upon water exposure. Altogether, our findings suggest that in addition to the number of nodes with aerial roots reported previously, the diameter of aerial roots and abundance of border cells, polysaccharide synthesis and degradation, and nitrogen uptake are critical factors to ensure efficient nitrogen fixation in maize aerial roots.

  • Irving TB, Chakraborty S, Maia LGS, Knaack S, Conde D, Schmidt HW, Triozzi PM, Simmons CH, Roy S, Kirst M, Ané JM (2022) An LCO-responsive homolog of NODULE INCEPTION positively regulates lateral root formation in Populus sp. Plant physiology 190((3)):1699-1714 PMC3142859 · Pubmed · DOI

    The transcription factor NODULE INCEPTION (NIN) has been studied extensively for its multiple roles in root nodule symbiosis within plants of the nitrogen-fixing clade (NFC) that associate with soil bacteria, such as rhizobia and Frankia. However, NIN homologs are present in plants outside the NFC, suggesting a role in other developmental processes. Here, we show that the biofuel crop Populus sp., which is not part of the NFC, contains eight copies of NIN with diversified protein sequence and expression patterns. Lipo-chitooligosaccharides (LCOs) are produced by rhizobia and a wide range of fungi, including mycorrhizal ones, and act as symbiotic signals that promote lateral root formation. RNAseq analysis of Populus sp. treated with purified LCO showed induction of the PtNIN2 subfamily. Moreover, the expression of PtNIN2b correlated with the formation of lateral roots and was suppressed by cytokinin treatment. Constitutive expression of PtNIN2b overcame the inhibition of lateral root development by cytokinin under high nitrate conditions. Lateral root induction in response to LCOs likely represents an ancestral function of NIN retained and repurposed in nodulating plants, as we demonstrate that the role of NIN in LCO-induced root branching is conserved in both Populus sp. and legumes. We further established a visual marker of LCO perception in Populus sp. roots, the putative sulfotransferase PtSS1 that can be used to study symbiotic interactions with the bacterial and fungal symbionts of Populus sp.

  • Chakraborty S, Valdés-López O, Stonoha-Arther C, Ané JM (2022) Transcription Factors Controlling the Rhizobium-Legume Symbiosis: Integrating Infection, Organogenesis and the Abiotic Environment. Plant & cell physiology 63((10)):1326-1343 · Pubmed · DOI

    Legume roots engage in a symbiotic relationship with rhizobia, leading to the development of nitrogen-fixing nodules. Nodule development is a sophisticated process and is under the tight regulation of the plant. The symbiosis initiates with a signal exchange between the two partners, followed by the development of a new organ colonized by rhizobia. Over two decades of study have shed light on the transcriptional regulation of rhizobium-legume symbiosis. A large number of transcription factors (TFs) have been implicated in one or more stages of this symbiosis. Legumes must monitor nodule development amidst a dynamic physical environment. Some environmental factors are conducive to nodulation, whereas others are stressful. The modulation of rhizobium-legume symbiosis by the abiotic environment adds another layer of complexity and is also transcriptionally regulated. Several symbiotic TFs act as integrators between symbiosis and the response to the abiotic environment. In this review, we trace the role of various TFs involved in rhizobium-legume symbiosis along its developmental route and highlight the ones that also act as communicators between this symbiosis and the response to the abiotic environment. Finally, we discuss contemporary approaches to study TF-target interactions in plants and probe their potential utility in the field of rhizobium-legume symbiosis.

  • Wen A, Havens KL, Bloch SE, Shah N, Higgins DA, Davis-Richardson AG, Sharon J, Rezaei F, Mohiti-Asli M, Johnson A, Abud G, Ane JM, Maeda J, Infante V, Gottlieb SS, Lorigan JG, Williams L, Horton A, McKellar M, Soriano D, Caron Z, Elzinga H, Graham A, Clark R, Mak SM, Stupin L, Robinson A, Hubbard N, Broglie R, Tamsir A, Temme K (2022) Correction to "Enabling Biological Nitrogen Fixation for Cereal Crops in Fertilized Fields". ACS synthetic biology 11((4)):1706-1707 · Pubmed · DOI

    No abstract available.

  • Kleist TJ, Bortolazzo A, Keyser ZP, Perera AM, Irving TB, Venkateshwaran M, Atanjaoui F, Tang RJ, Maeda J, Cartwright HN, Christianson ML, Lemaux PG, Luan S, Ané JM (2022) Stress-associated developmental reprogramming in moss protonemata by synthetic activation of the common symbiosis pathway. iScience 25((2)):103754 PMC8819110 · Pubmed · DOI

    Symbioses between angiosperms and rhizobia or arbuscular mycorrhizal fungi are controlled through a conserved signaling pathway. Microbe-derived, chitin-based elicitors activate plant cell surface receptors and trigger nuclear calcium oscillations, which are decoded by a calcium/calmodulin-dependent protein kinase (CCaMK) and its target transcription factor interacting protein of DMI3 (IPD3). Genes encoding CCaMK and IPD3 have been lost in multiple non-mycorrhizal plant lineages yet retained among non-mycorrhizal mosses. Here, we demonstrated that the moss Physcomitrium is equipped with a bona fide CCaMK that can functionally complement a Medicago loss-of-function mutant. Conservation of regulatory phosphosites allowed us to generate predicted hyperactive forms of Physcomitrium CCaMK and IPD3. Overexpression of synthetically activated CCaMK or IPD3 in Physcomitrium led to abscisic acid (ABA) accumulation and ectopic development of brood cells, which are asexual propagules that facilitate escape from local abiotic stresses. We therefore propose a functional role for Physcomitrium CCaMK-IPD3 in stress-associated developmental reprogramming.

  • Mus F, Khokhani D, MacIntyre AM, Rugoli E, Dixon R, Ané JM, Peters JW (2022) Genetic Determinants of Ammonium Excretion in nifL Mutants of Azotobacter vinelandii. Applied and environmental microbiology 88((6)):e0187621 PMC8939361 · Pubmed · DOI

    The ubiquitous diazotrophic soil bacterium Azotobacter vinelandii has been extensively studied as a model organism for biological nitrogen fixation (BNF). In A. vinelandii, BNF is regulated by the NifL-NifA two-component system, where NifL acts as an antiactivator that tightly controls the activity of the nitrogen fixation-specific transcriptional activator NifA in response to redox, nitrogen, and carbon status. While several studies reported that mutations in A. vinelandii nifL resulted in the deregulation of nitrogenase expression and the release of large quantities of ammonium, knowledge about the specific determinants for this ammonium-excreting phenotype is lacking. In this work, we report that only specific disruptions of nifL lead to large quantities of ammonium accumulated in liquid culture (∼12 mM). The ammonium excretion phenotype is associated solely with deletions of NifL domains combined with the insertion of a promoter sequence in the orientation opposite that of nifLA transcription. We further demonstrated that the strength of the inserted promoter could influence the amounts of ammonium excreted by affecting rnf1 gene expression as an additional requirement for ammonium excretion. These ammonium-excreting nifL mutants significantly stimulate the transfer of fixed nitrogen to rice. This work defines discrete determinants that bring about A. vinelandii ammonium excretion and demonstrates that strains can be generated through simple gene editing to provide promising biofertilizers capable of transferring nitrogen to crops. IMPORTANCE There is considerable interest in the engineering of ammonium-excreting bacteria for use in agriculture to promote the growth of plants under fixed-nitrogen-limiting conditions. This work defines discrete determinants that bring about A. vinelandii ammonium excretion and demonstrates that strains can be generated through simple gene editing to provide promising biofertilizers capable of transferring nitrogen to crops.

  • Pereira WJ, Knaack S, Chakraborty S, Conde D, Folk RA, Triozzi PM, Balmant KM, Dervinis C, Schmidt HW, Ané JM, Roy S, Kirst M (2022) Functional and comparative genomics reveals conserved noncoding sequences in the nitrogen-fixing clade. The New phytologist 234((2)):634-649 PMC9302667 · Pubmed · DOI

    Nitrogen is one of the most inaccessible plant nutrients, but certain species have overcome this limitation by establishing symbiotic interactions with nitrogen-fixing bacteria in the root nodule. This root-nodule symbiosis (RNS) is restricted to species within a single clade of angiosperms, suggesting a critical, but undetermined, evolutionary event at the base of this clade. To identify putative regulatory sequences implicated in the evolution of RNS, we evaluated the genomes of 25 species capable of nodulation and identified 3091 conserved noncoding sequences (CNS) in the nitrogen-fixing clade (NFC). We show that the chromatin accessibility of 452 CNS correlates significantly with the regulation of genes responding to lipochitooligosaccharides in Medicago truncatula. These included 38 CNS in proximity to 19 known genes involved in RNS. Five such regions are upstream of MtCRE1, Cytokinin Response Element 1, required to activate a suite of downstream transcription factors necessary for nodulation in M. truncatula. Genetic complementation of an Mtcre1 mutant showed a significant decrease of nodulation in the absence of the five CNS, when they are driving the expression of a functional copy of MtCRE1. CNS identified in the NFC may harbor elements required for the regulation of genes controlling RNS in M. truncatula.

  • Irving TB, Chakraborty S, Ivanov S, Schultze M, Mysore KS, Harrison MJ, Ané JM (2022) KIN3 impacts arbuscular mycorrhizal symbiosis and promotes fungal colonisation in Medicago truncatula. The Plant journal : for cell and molecular biology 110((2)):513-528 · Pubmed · DOI

    Arbuscular mycorrhizal fungi help their host plant in the acquisition of nutrients, and this association is itself impacted by soil nutrient levels. High phosphorus levels inhibit the symbiosis, whereas high nitrogen levels enhance it. The genetic mechanisms regulating the symbiosis in response to soil nutrients are poorly understood. Here, we characterised the symbiotic phenotypes in four Medicago truncatula Tnt1-insertion mutants affected in arbuscular mycorrhizal colonisation. We located their Tnt1 insertions and identified alleles for two genes known to be involved in mycorrhization, RAM1 and KIN3. We compared the effects of the kin3-2 and ram1-4 mutations on gene expression, revealing that the two genes alter the expression of overlapping but not identical gene sets, suggesting that RAM1 acts upstream of KIN3. Additionally, KIN3 appears to be involved in the suppression of plant defences in response to the fungal symbiont. KIN3 is located on the endoplasmic reticulum of arbuscule-containing cortical cells, and kin3-2 mutants plants hosted significantly fewer arbuscules than the wild type. KIN3 plays an essential role in the symbiotic response to soil nitrogen levels, as, contrary to wild-type plants, the kin3-2 mutant did not exhibit increased root colonisation under high nitrogen.

  • Wen A, Havens KL, Bloch SE, Shah N, Higgins DA, Davis-Richardson AG, Sharon J, Rezaei F, Mohiti-Asli M, Johnson A, Abud G, Ane JM, Maeda J, Infante V, Gottlieb SS, Lorigan JG, Williams L, Horton A, McKellar M, Soriano D, Caron Z, Elzinga H, Graham A, Clark R, Mak SM, Stupin L, Robinson A, Hubbard N, Broglie R, Tamsir A, Temme K (2021) Enabling Biological Nitrogen Fixation for Cereal Crops in Fertilized Fields. ACS synthetic biology 10((12)):3264-3277 · Pubmed · DOI

    Agricultural productivity relies on synthetic nitrogen fertilizers, yet half of that reactive nitrogen is lost to the environment. There is an urgent need for alternative nitrogen solutions to reduce the water pollution, ozone depletion, atmospheric particulate formation, and global greenhouse gas emissions associated with synthetic nitrogen fertilizer use. One such solution is biological nitrogen fixation (BNF), a component of the complex natural nitrogen cycle. BNF application to commercial agriculture is currently limited by fertilizer use and plant type. This paper describes the identification, development, and deployment of the first microbial product optimized using synthetic biology tools to enable BNF for corn ( Zea mays ) in fertilized fields, demonstrating the successful, safe commercialization of root-associated diazotrophs and realizing the potential of BNF to replace and reduce synthetic nitrogen fertilizer use in production agriculture. Derived from a wild nitrogen-fixing microbe isolated from agricultural soils, Klebsiella variicola 137-1036 ("Kv137-1036") retains the capacity of the parent strain to colonize corn roots while increasing nitrogen fixation activity 122-fold in nitrogen-rich environments. This technical milestone was then commercialized in less than half of the time of a traditional biological product, with robust biosafety evaluations and product formulations contributing to consumer confidence and ease of use. Tested in multi-year, multi-site field trial experiments throughout the U.S. Corn Belt, fields grown with Kv137-1036 exhibited both higher yields (0.35 ± 0.092 t/ha ± SE or 5.2 ± 1.4 bushels/acre ± SE) and reduced within-field yield variance by 25% in 2018 and 8% in 2019 compared to fields fertilized with synthetic nitrogen fertilizers alone. These results demonstrate the capacity of a broad-acre BNF product to fix nitrogen for corn in field conditions with reliable agronomic benefits.

  • Khokhani D, Carrera Carriel C, Vayla S, Irving TB, Stonoha-Arther C, Keller NP, Ané JM (2021) Deciphering the Chitin Code in Plant Symbiosis, Defense, and Microbial Networks. Annual review of microbiology 75:583-607 · Pubmed · DOI

    Chitin is a structural polymer in many eukaryotes. Many organisms can degrade chitin to defend against chitinous pathogens or use chitin oligomers as food. Beneficial microorganisms like nitrogen-fixing symbiotic rhizobia and mycorrhizal fungi produce chitin-based signal molecules called lipo-chitooligosaccharides (LCOs) and short chitin oligomers to initiate a symbiotic relationship with their compatible hosts and exchange nutrients. A recent study revealed that a broad range of fungi produce LCOs and chitooligosaccharides (COs), suggesting that these signaling molecules are not limited to beneficial microbes. The fungal LCOs also affect fungal growth and development, indicating that the roles of LCOs beyond symbiosis and LCO production may predate mycorrhizal symbiosis. This review describes the diverse structures of chitin; their perception by eukaryotes and prokaryotes; and their roles in symbiotic interactions, defense, and microbe-microbe interactions. We also discuss potential strategies of fungi to synthesize LCOs and their roles in fungi with different lifestyles.

  • Triozzi PM, Irving TB, Schmidt HW, Keyser ZP, Chakraborty S, Balmant K, Pereira WJ, Dervinis C, Mysore KS, Wen J, Ané JM, Kirst M, Conde D (2021) Spatiotemporal cytokinin response imaging and ISOPENTENYLTRANSFERASE 3 function in Medicago nodule development. Plant physiology 188((1)):560-575 PMC8774767 · Pubmed · DOI

    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.

  • Irving TB, Alptekin B, Kleven B, Ané JM (2021) A critical review of 25 years of glomalin research: a better mechanical understanding and robust quantification techniques are required. The New phytologist 232((4)):1572-1581 · Pubmed · DOI

    Arbuscular mycorrhizal fungi (AMF) are important contributors to both plant and soil health. Twenty-five years ago, researchers discovered 'glomalin', a soil component potentially produced by AMF, which was unconventionally extracted from soil and bound by a monoclonal antibody raised against Rhizophagus irregularis spores. 'Glomalin' can resist boiling, strong acids and bases, and protease treatment. Researchers proposed that 'glomalin' is a 60 kDa heat shock protein produced by AMF, while others suggested that it is a mixture of soil organic materials that are not unique to AMF. Despite disagreements on the nature of 'glomalin', it has been consistently associated with a long list of plant and soil health benefits, including soil aggregation, soil carbon storage and enhancing growth under abiotic stress. The benefits attributed to 'glomalin' have caused much excitement in the plant and soil health community; however, the mechanism(s) for these benefits have yet to be established. This review provides insights into the current understanding of the identity of 'glomalin', 'glomalin' quantification, and the associated benefits of 'glomalin'. We invite the community to think more critically about how glomalin-associated benefits are generated. We suggest a series of experiments to test hypotheses regarding the nature of 'glomalin' and associated health benefits.

  • R Cope K, B Irving T, Chakraborty S, Ané JM (2021) Perception of lipo-chitooligosaccharides by the bioenergy crop Populus . Plant signaling & behavior 16((6)):1903758 PMC8143229 · Pubmed · DOI

    Populus sp. is a developing feedstock for second-generation biofuel production. To ensure its success as a sustainable biofuel source, it is essential to capitalize on the ability of Populus sp. to associate with beneficial plant-associated microbes ( e.g ., mycorrhizal fungi) and engineer Populus sp. to associate with non-native symbionts ( e.g ., rhizobia). Here, we review recent research into the molecular mechanisms that control ectomycorrhizal associations in Populus sp. with particular emphasis on the discovery that ectomycorrhizal fungi produce lipochitooligosaccharides capable of activating the common symbiosis pathway. We also present new evidence that lipo-chitooligosaccharides produced by both ectomycorrhizal fungi and various species of rhizobia that do not associate with Populus sp. can induce nuclear calcium spiking in the roots of Populus sp. Thus, we argue Populus sp. already possesses the molecular machinery necessary for perceiving rhizobia, and the next step in engineering symbiosis with rhizobia should be focused on inducing bacterial accommodation and nodule organogenesis. The gene Nodule INception is central to these processes, and several putative orthologs are present in Populus sp. Manipulating the promoters of these genes to match that of plants in the nitrogen-fixing clade may be sufficient to introduce nodulation in Populus sp.

  • Pankievicz VCS, do Amaral FP, Ané JM, Stacey G (2021) Diazotrophic Bacteria and Their Mechanisms to Interact and Benefit Cereals. Molecular plant-microbe interactions : MPMI 34((5)):491-498 · Pubmed · DOI

    Plant-growth-promoting bacteria (PGPB) stimulate plant growth through diverse mechanisms. In addition to biological nitrogen fixation, diazotrophic PGPB can improve nutrient uptake efficiency from the soil, produce and release phytohormones to the host, and confer resistance against pathogens. The genetic determinants that drive the success of biological nitrogen fixation in nonlegume plants are understudied. These determinants include recognition and signaling pathways, bacterial colonization, and genotype specificity between host and bacteria. This review presents recent discoveries of how nitrogen-fixing PGPB interact with cereals and promote plant growth. We suggest adopting an experimental model system, such as the Setaria -diazotrophic bacteria association, as a reliable way to better understand the associated mechanisms and, ultimately, increase the use of PGPB inoculants for sustainable agriculture.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

  • Shin J, Marx H, Richards A, Vaneechoutte D, Jayaraman D, Maeda J, Chakraborty S, Sussman M, Vandepoele K, Ané JM, Coon J, Roy S (2020) A network-based comparative framework to study conservation and divergence of proteomes in plant phylogenies. Nucleic acids research 49((1)):e3 PMC7797074 · Pubmed · DOI

    Comparative functional genomics offers a powerful approach to study species evolution. To date, the majority of these studies have focused on the transcriptome in mammalian and yeast phylogenies. Here, we present a novel multi-species proteomic dataset and a computational pipeline to systematically compare the protein levels across multiple plant species. Globally we find that protein levels diverge according to phylogenetic distance but is more constrained than the mRNA level. Module-level comparative analysis of groups of proteins shows that proteins that are more highly expressed tend to be more conserved. To interpret the evolutionary patterns of conservation and divergence, we develop a novel network-based integrative analysis pipeline that combines publicly available transcriptomic datasets to define co-expression modules. Our analysis pipeline can be used to relate the changes in protein levels to different species-specific phenotypic traits. We present a case study with the rhizobia-legume symbiosis process that supports the role of autophagy in this symbiotic association.

  • Rush TA, Puech-Pagès V, Bascaules A, Jargeat P, Maillet F, Haouy A, Maës AQ, Carriel CC, Khokhani D, Keller-Pearson M, Tannous J, Cope KR, Garcia K, Maeda J, Johnson C, Kleven B, Choudhury QJ, Labbé J, Swift C, O'Malley MA, Bok JW, Cottaz S, Fort S, Poinsot V, Sussman MR, Lefort C, Nett J, Keller NP, Bécard G, Ané JM (2020) Lipo-chitooligosaccharides as regulatory signals of fungal growth and development. Nature communications 11((1)):3897 PMC7403392 · Pubmed · DOI

    Lipo-chitooligosaccharides (LCOs) are signaling molecules produced by rhizobial bacteria that trigger the nodulation process in legumes, and by some fungi that also establish symbiotic relationships with plants, notably the arbuscular and ecto mycorrhizal fungi. Here, we show that many other fungi also produce LCOs. We tested 59 species representing most fungal phyla, and found that 53 species produce LCOs that can be detected by functional assays and/or by mass spectroscopy. LCO treatment affects spore germination, branching of hyphae, pseudohyphal growth, and transcription in non-symbiotic fungi from the Ascomycete and Basidiomycete phyla. Our findings suggest that LCO production is common among fungi, and LCOs may function as signals regulating fungal growth and development.

  • Schenck CA, Westphal J, Jayaraman D, Garcia K, Wen J, Mysore KS, Ané JM, Sumner LW, Maeda HA (2020) Role of cytosolic, tyrosine-insensitive prephenate dehydrogenase in Medicago truncatula . Plant direct 4((5)):e00218 PMC7196213 · Pubmed · DOI

    l-Tyrosine (Tyr) is an aromatic amino acid synthesized de novo in plants and microbes downstream of the shikimate pathway. In plants, Tyr and a Tyr pathway intermediate, 4-hydroxyphenylpyruvate (HPP), are precursors to numerous specialized metabolites, which are crucial for plant and human health. Tyr is synthesized in the plastids by a TyrA family enzyme, arogenate dehydrogenase (ADH/TyrA), which is feedback inhibited by Tyr. Additionally, many legumes possess prephenate dehydrogenases (PDH/TyrA), which are insensitive to Tyr and localized to the cytosol. Yet the role of PDH enzymes in legumes is currently unknown. This study isolated and characterized Tnt1 -transposon mutants of MtPDH1 ( pdh1 ) in Medicago truncatula to investigate PDH function. The  pdh1 mutants lacked PDH transcript and PDH activity, and displayed little aberrant morphological phenotypes under standard growth conditions, providing genetic evidence that MtPDH1 is responsible for the PDH activity detected in M. truncatula . Though plant PDH enzymes and activity have been specifically found in legumes, nodule number and nitrogenase activity of pdh1  mutants were not significantly reduced compared with wild-type (Wt) during symbiosis with nitrogen-fixing bacteria. Although Tyr levels were not significantly different between Wt and mutants under standard conditions, when carbon flux was increased by shikimate precursor feeding, mutants accumulated significantly less Tyr than Wt. These data suggest that MtPDH1 is involved in Tyr biosynthesis when the shikimate pathway is stimulated and possibly linked to unidentified legume-specific specialized metabolism.

  • Paudel D, Liu F, Wang L, Crook M, Maya S, Peng Z, Kelley K, Ané JM, Wang J (2020) Isolation, Characterization, and Complete Genome Sequence of a Bradyrhizobium Strain Lb8 From Nodules of Peanut Utilizing Crack Entry Infection. Frontiers in microbiology 11:93 PMC7020250 · Pubmed · DOI

    In many legumes, the colonization of roots by rhizobia is via "root hair entry" and its molecular mechanisms have been extensively studied. However, the nodulation of peanuts ( Arachis hypogaea L.) by Bradyrhizobium strains requires an intercellular colonization process called "crack entry," which is understudied. To understand the intercellular crack entry process, it is critical to develop the tools and resources related to the rhizobium in addition to focus on investigating the mechanisms of the plant host. In this study, we isolated a Bradyrhizobium sp. strain, Lb8 from peanut root nodules and sequenced it using PacBio long reads. The complete genome sequence was a circular chromosome of 8,718,147 base-pair (bp) with an average GC content of 63.14%. No plasmid sequence was detected in the sequenced DNA sample. A total of 8,433 potential protein-encoding genes, one rRNA cluster, and 51 tRNA genes were annotated. Fifty-eight percent of the predicted genes showed similarity to genes of known functions and were classified into 27 subsystems representing various biological processes. The genome shared 92% of the gene families with B. diazoefficens USDA 110. A presumptive symbiosis island of 778 Kb was detected, which included two clusters of nif and nod genes. A total of 711 putative protein-encoding genes were in this region, among which 455 genes have potential functions related to symbiotic nitrogen fixation and DNA transmission. Of 21 genes annotated as transposase, 16 were located in the symbiosis island. Lb8 possessed both Type III and Type IV protein secretion systems, and our work elucidated the association of flagellar Type III secretion systems in bradyrhizobia. These observations suggested that complex rearrangement, such as horizontal transfer and insertion of different DNA elements, might be responsible for the plasticity of the Bradyrhizobium genome.

  • Bennett AB, Pankievicz VCS, Ané JM (2020) A Model for Nitrogen Fixation in Cereal Crops. Trends in plant science 25((3)):226-235 · Pubmed · DOI

    Nitrogen-fixing microbial associations with cereals have been of intense interest for more than a century (Roesch et al., Plant Soil 2008;302:91-104; Triplett, Plant Soil 1996;186:29-38; Mus et al., Appl. Environ. Microbiol. 2016;82:3698-3710; Beatty and Good, Science 2011;333:416-417). A recent report demonstrated that an indigenous Sierra Mixe maize landrace, characterized by an extensive development of aerial roots that secrete large amounts of mucilage, can acquire 28-82% of its nitrogen from atmospheric dinitrogen (Van Deynze et al., PLoS Biol. 2018;16:e2006352). Although the Sierra Mixe maize landrace is unique in the large quantity of mucilage produced, other cereal crops secrete mucilage from underground and aerial roots and we hypothesize that this may represent a general mechanism for cereals to support associations with microbial diazotrophs. We propose a model for the association of nitrogen-fixing microbes with maize mucilage and identify the four main functionalities for such a productive diazotrophic association.

  • Ryu MH, Zhang J, Toth T, Khokhani D, Geddes BA, Mus F, Garcia-Costas A, Peters JW, Poole PS, Ané JM, Voigt CA (2019) Control of nitrogen fixation in bacteria that associate with cereals. Nature microbiology 5((2)):314-330 PMC8634771 · Pubmed · DOI

    Legumes obtain nitrogen from air through rhizobia residing in root nodules. Some species of rhizobia can colonize cereals but do not fix nitrogen on them. Disabling native regulation can turn on nitrogenase expression, even in the presence of nitrogenous fertilizer and low oxygen, but continuous nitrogenase production confers an energy burden. Here, we engineer inducible nitrogenase activity in two cereal endophytes (Azorhizobium caulinodans ORS571 and Rhizobium sp. IRBG74) and the well-characterized plant epiphyte Pseudomonas protegens Pf-5, a maize seed inoculant. For each organism, different strategies were taken to eliminate ammonium repression and place nitrogenase expression under the control of agriculturally relevant signals, including root exudates, biocontrol agents and phytohormones. We demonstrate that R. sp. IRBG74 can be engineered to result in nitrogenase activity under free-living conditions by transferring a nif cluster from either Rhodobacter sphaeroides or Klebsiella oxytoca. For P. protegens Pf-5, the transfer of an inducible cluster from Pseudomonas stutzeri and Azotobacter vinelandii yields ammonium tolerance and higher oxygen tolerance of nitrogenase activity than that from K. oxytoca. Collectively, the data from the transfer of 12 nif gene clusters between 15 diverse species (including Escherichia coli and 12 rhizobia) help identify the barriers that must be overcome to engineer a bacterium to deliver a high nitrogen flux to a cereal crop.

  • Pankievicz VCS, Irving TB, Maia LGS, Ané JM (2019) Are we there yet? The long walk towards the development of efficient symbiotic associations between nitrogen-fixing bacteria and non-leguminous crops. BMC biology 17((1)):99 PMC6889567 · Pubmed · DOI

    Nitrogen is an essential element of life, and nitrogen availability often limits crop yields. Since the Green Revolution, massive amounts of synthetic nitrogen fertilizers have been produced from atmospheric nitrogen and natural gas, threatening the sustainability of global food production and degrading the environment. There is a need for alternative means of bringing nitrogen to crops, and taking greater advantage of biological nitrogen fixation seems a logical option. Legumes are used in most cropping systems around the world because of the nitrogen-fixing symbiosis with rhizobia. However, the world's three major cereal crops-rice, wheat, and maize-do not associate with rhizobia. In this review, we will survey how genetic approaches in rhizobia and their legume hosts allowed tremendous progress in understanding the molecular mechanisms controlling root nodule symbioses, and how this knowledge paves the way for engineering such associations in non-legume crops. We will also discuss challenges in bringing these systems into the field and how they can be surmounted by interdisciplinary collaborations between synthetic biologists, microbiologists, plant biologists, breeders, agronomists, and policymakers.

  • Kim S, Zeng W, Bernard S, Liao J, Venkateshwaran M, Ane JM, Jiang Y (2019) Publisher Correction: Ca-regulated Ca channels with an RCK gating ring control plant symbiotic associations. Nature communications 10((1)):4607 PMC6779904 · Pubmed · DOI

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

  • Kim S, Zeng W, Bernard S, Liao J, Venkateshwaran M, Ane JM, Jiang Y (2019) Ca-regulated Ca channels with an RCK gating ring control plant symbiotic associations. Nature communications 10((1)):3703 PMC6697748 · Pubmed · DOI

    A family of plant nuclear ion channels, including DMI1 (Does not Make Infections 1) and its homologs CASTOR and POLLUX, are required for the establishment of legume-microbe symbioses by generating nuclear and perinuclear Ca spiking. Here we show that CASTOR from Lotus japonicus is a highly selective Ca channel whose activation requires cytosolic/nucleosolic Ca, contrary to the previous suggestion of it being a K channel. Structurally, the cytosolic/nucleosolic ligand-binding soluble region of CASTOR contains two tandem RCK (Regulator of Conductance for K) domains, and four subunits assemble into the gating ring architecture, similar to that of large conductance, Ca-gated K (BK) channels despite the lack of sequence similarity. Multiple ion binding sites are clustered at two locations within each subunit, and three of them are identified to be Ca sites. Our in vitro and in vivo assays also demonstrate the importance of these gating-ring Ca binding sites to the physiological function of CASTOR as well as DMI1.

  • Cope KR, Bascaules A, Irving TB, Venkateshwaran M, Maeda J, Garcia K, Rush TA, Ma C, Labbé J, Jawdy S, Steigerwald E, Setzke J, Fung E, Schnell KG, Wang Y, Schlief N, Bücking H, Strauss SH, Maillet F, Jargeat P, Bécard G, Puech-Pagès V, Ané JM (2019) The Ectomycorrhizal Fungus Laccaria bicolor Produces Lipochitooligosaccharides and Uses the Common Symbiosis Pathway to Colonize Populus Roots. The Plant cell 31((10)):2386-2410 PMC6790088 · Pubmed · DOI

    Mycorrhizal fungi form mutualistic associations with the roots of most land plants and provide them with mineral nutrients from the soil in exchange for fixed carbon derived from photosynthesis. The common symbiosis pathway (CSP) is a conserved molecular signaling pathway in all plants capable of associating with arbuscular mycorrhizal fungi. It is required not only for arbuscular mycorrhizal symbiosis but also for rhizobia-legume and actinorhizal symbioses. Given its role in such diverse symbiotic associations, we hypothesized that the CSP also plays a role in ectomycorrhizal associations. We showed that the ectomycorrhizal fungus Laccaria bicolor produces an array of lipochitooligosaccharides (LCOs) that can trigger both root hair branching in legumes and, most importantly, calcium spiking in the host plant Populus in a CASTOR/POLLUX -dependent manner. Nonsulfated LCOs enhanced lateral root development in Populus in a calcium/calmodulin-dependent protein kinase ( CCaMK )-dependent manner, and sulfated LCOs enhanced the colonization of Populus by L. bicolor Compared with the wild-type Populus , the colonization of CASTOR / POLLUX and CCaMK RNA interference lines by L. bicolor was reduced. Our work demonstrates that similar to other root symbioses, L. bicolor uses the CSP for the full establishment of its mutualistic association with Populus .

  • Labbé J, Muchero W, Czarnecki O, Wang J, Wang X, Bryan AC, Zheng K, Yang Y, Xie M, Zhang J, Wang D, Meidl P, Wang H, Morrell-Falvey JL, Cope KR, Maia LGS, Ané JM, Mewalal R, Jawdy SS, Gunter LE, Schackwitz W, Martin J, Le Tacon F, Li T, Zhang Z, Ranjan P, Lindquist E, Yang X, Jacobson DA, Tschaplinski TJ, Barry K, Schmutz J, Chen JG, Tuskan GA (2019) Mediation of plant-mycorrhizal interaction by a lectin receptor-like kinase. Nature plants 5((7)):676-680 · Pubmed · DOI

    The molecular mechanisms underlying mycorrhizal symbioses, the most ubiquitous and impactful mutualistic plant-microbial interaction in nature, are largely unknown. Through genetic mapping, resequencing and molecular validation, we demonstrate that a G-type lectin receptor-like kinase (lecRLK) mediates the symbiotic interaction between Populus and the ectomycorrhizal fungus Laccaria bicolor. This finding uncovers an important molecular step in the establishment of symbiotic plant-fungal associations and provides a molecular target for engineering beneficial mycorrhizal relationships.

  • Wahlig TA, Bixler BJ, Valdés-López O, Mysore KS, Wen J, Ané JM, Kaspar CW (2019) Salmonella enterica serovar Typhimurium ATCC 14028S is tolerant to plant defenses triggered by the flagellin receptor FLS2. FEMS microbiology letters 366((4)): PMC6420342 · Pubmed · DOI

    Salmonellosis outbreaks associated with sprouted legumes have been a food safety concern for over two decades. Despite evidence that Salmonella enterica triggers biotic plant defense pathways, it has remained unclear how plant defenses impact Salmonella growth on sprouted legumes. We used Medicago truncatula mutants in which the gene for the flagellin receptor FLS2 was disrupted to demonstrate that plant defenses triggered by FLS2 elicitation do not impact the growth of Salmonella enterica serovar Typhimurium ATCC 14028S. As a control, we tested the growth of Salmonella enterica serovar Typhimurium LT2, which has a defect in rpoS that increases its sensitivity to reactive oxygen species. LT2 displayed enhanced growth on M. truncatula FLS2 mutants in comparison to wild-type M. truncatula. We hypothesize that these growth differences are primarily due to differences in 14028S and LT2 reactive oxygen species sensitivity. Results from this study show that FLS2-mediated plant defenses are ineffective in inhibiting growth of Salmonella entrica 14028S.

  • Valdés-López O, Jayaraman D, Maeda J, Delaux PM, Venkateshwaran M, Isidra-Arellano MC, Reyero-Saavedra MR, Sánchez-Correa MS, Verastegui-Vidal MA, Delgado-Buenrostro N, Van Ness L, Mysore KS, Wen J, Sussman MR, Ané JM (2018) A Novel Positive Regulator of the Early Stages of Root Nodule Symbiosis Identified by Phosphoproteomics. Plant & cell physiology 60((3)):575-586 · Pubmed · DOI

    Signals and signaling pathways underlying the symbiosis between legumes and rhizobia have been studied extensively over the past decades. In a previous phosphoproteomic study on the Medicago truncatula-Sinorhizobium meliloti symbiosis, we identified plant proteins that are differentially phosphorylated upon the perception of rhizobial signals, called Nod factors. In this study, we provide experimental evidence that one of these proteins, Early Phosphorylated Protein 1 (EPP1), is required for the initiation of this symbiosis. Upon inoculation with rhizobia, MtEPP1 expression was induced in curled root hairs. Down-regulation of MtEPP1 in M. truncatula roots almost abolished calcium spiking, reduced the expression of essential symbiosis-related genes (MtNIN, MtNF-YB1, MtERN1 and MtENOD40) and strongly decreased nodule development. Phylogenetic analyses revealed that orthologs of MtEPP1 are present in legumes and specifically in plant species able to host arbuscular mycorrhizal fungi, suggesting a possible role in this association too. Short chitin oligomers induced the phosphorylation of MtEPP1 like Nod factors. However, the down-regulation of MtEPP1 affected the colonization of M. truncatula roots by arbuscular mycorrhizal fungi only moderately. Altogether, these findings indicate that MtEPP1 is essential for the establishment of the legume-rhizobia symbiosis but might plays a limited role in the arbuscular mycorrhizal symbiosis.

  • Keller C, Maeda J, Jayaraman D, Chakraborty S, Sussman MR, Harris JM, Ané JM, Li L (2018) Comparison of Vacuum MALDI and AP-MALDI Platforms for the Mass Spectrometry Imaging of Metabolites Involved in Salt Stress in Medicago truncatula . Frontiers in plant science 9:1238 PMC6121006 · Pubmed · DOI

    No abstract available.

  • Van Deynze A, Zamora P, Delaux PM, Heitmann C, Jayaraman D, Rajasekar S, Graham D, Maeda J, Gibson D, Schwartz KD, Berry AM, Bhatnagar S, Jospin G, Darling A, Jeannotte R, Lopez J, Weimer BC, Eisen JA, Shapiro HY, Ané JM, Bennett AB (2018) Nitrogen fixation in a landrace of maize is supported by a mucilage-associated diazotrophic microbiota. PLoS biology 16((8)):e2006352 PMC6080747 · Pubmed · DOI

    No abstract available.

  • Griesmann M, Chang Y, Liu X, Song Y, Haberer G, Crook MB, Billault-Penneteau B, Lauressergues D, Keller J, Imanishi L, Roswanjaya YP, Kohlen W, Pujic P, Battenberg K, Alloisio N, Liang Y, Hilhorst H, Salgado MG, Hocher V, Gherbi H, Svistoonoff S, Doyle JJ, He S, Xu Y, Xu S, Qu J, Gao Q, Fang X, Fu Y, Normand P, Berry AM, Wall LG, Ané JM, Pawlowski K, Xu X, Yang H, Spannagl M, Mayer KFX, Wong GK, Parniske M, Delaux PM, Cheng S (2018) Phylogenomics reveals multiple losses of nitrogen-fixing root nodule symbiosis. Science (New York, N.Y.) 361((6398)): · Pubmed · DOI

    No abstract available.

  • Ranjan A, Jayaraman D, Grau C, Hill JH, Whitham SA, Ané JM, Smith DL, Kabbage M (2017) The pathogenic development of Sclerotinia sclerotiorum in soybean requires specific host NADPH oxidases. Molecular plant pathology 19((3)):700-714 PMC6638103 · Pubmed · DOI

    No abstract available.

  • Garcia K, Ané JM (2017) Polymorphic responses of Medicago truncatula accessions to potassium deprivation. Plant signaling & behavior 12((4)):e1307494 PMC5437819 · Pubmed · DOI

    No abstract available.

  • Jayaraman D, Richards AL, Westphall MS, Coon JJ, Ané JM (2017) Identification of the phosphorylation targets of symbiotic receptor-like kinases using a high-throughput multiplexed assay for kinase specificity. The Plant journal : for cell and molecular biology 90((6)):1196-1207 PMC5461195 · Pubmed · DOI

    No abstract available.

  • Garcia K, Chasman D, Roy S, Ané JM (2017) Physiological Responses and Gene Co-Expression Network of Mycorrhizal Roots under K Deprivation. Plant physiology 173((3)):1811-1823 PMC5338680 · Pubmed · DOI

    No abstract available.

  • Garcia K, Ané JM (2016) Comparative Analysis of Secretomes from Ectomycorrhizal Fungi with an Emphasis on Small-Secreted Proteins. Front Microbiol 7:1734 (PMC5089978) · Pubmed

    No abstract available.

  • Marx H, Minogue CE, Jayaraman D, Richards AL, Kwiecien NW, Siahpirani AF, Rajasekar S, Maeda J, Garcia K, Del Valle-Echevarria AR, Volkening JD, Westphall MS, Roy S, Sussman MR, Ané JM, Coon JJ (2016) A proteomic atlas of the legume Medicago truncatula and its nitrogen-fixing endosymbiont Sinorhizobium meliloti. Nat. Biotechnol. 34(11):1198-1205 · Pubmed

    Legumes are essential components of agricultural systems because they enrich the soil in nitrogen and require little environmentally deleterious fertilizers. A complex symbiotic association between legumes and nitrogen-fixing soil bacteria called rhizobia culminates in the development of root nodules, where rhizobia fix atmospheric nitrogen and transfer it to their plant host. Here we describe a quantitative proteomic atlas of the model legume Medicago truncatula and its rhizobial symbiont Sinorhizobium meliloti, which includes more than 23,000 proteins, 20,000 phosphorylation sites, and 700 lysine acetylation sites. Our analysis provides insight into mechanisms regulating symbiosis. We identify a calmodulin-binding protein as a key regulator in the host and assign putative roles and targets to host factors (bioactive peptides) that control gene expression in the symbiont. Further mining of this proteomic resource may enable engineering of crops and their microbial partners to increase agricultural productivity and sustainability.

  • Kamel L, Keller-Pearson M, Roux C, Ané JM (2016) Biology and evolution of arbuscular mycorrhizal symbiosis in the light of genomics. The New phytologist 213((2)):531-536 · Pubmed · DOI

    No abstract available.

  • Poinsot V, Crook MB, Erdn S, Maillet F, Bascaules A, Ané JM (2016) New insights into Nod factor biosynthesis: Analyses of chitooligomers and lipo-chitooligomers of Rhizobium sp. IRBG74 mutants. Carbohydr. Res. 434:83-93 (PMC5080398) · Pubmed

    Soil-dwelling, nitrogen-fixing rhizobia signal their presence to legume hosts by secreting lipo-chitooligomers (LCOs) that are decorated with a variety of chemical substituents. It has long been assumed, but never empirically shown, that the LCO backbone is synthesized first by NodC, NodB, and NodA, followed by addition of one or more substituents by other Nod proteins. By analyzing a collection of in-frame deletion mutants of key nod genes in the bacterium Rhizobium sp. IRBG74 by mass spectrometry, we were able to shed light on the possible substitution order of LCO decorations, and we discovered that the prevailing view is probably erroneous. We found that most substituents could be transferred to a short chitin backbone prior to acylation by NodA, which is probably one of the last steps in LCO biosynthesis. The existence of substituted, short chitin oligomers offers new insights into symbiotic plant-microbe signaling.

  • Palmer AG, Mukherjee A, Stacy DM, Lazar S, Ané JM, Blackwell HE (2016) Interkingdom Responses to Bacterial Quorum Sensing Signals Regulate Frequency and Rate of Nodulation in Legume-Rhizobia Symbiosis. Chembiochem 17(22):2199-2205 · Pubmed

    Density-dependent phenotypic switching in bacteria, the phenomenon of quorum sensing (QS), is instrumental in many pathogenic and mutualistic behaviors. In many Gram-negative bacteria, QS is regulated by N-acylated-l-homoserine lactones (AHLs). Synthetic analogues of these AHLs hold significant promise for regulating QS at the host-symbiont interface. Regulation depends on refined temporal and spatial models of quorums under native conditions. Critical to this is an understanding of how the presence of these signals may affect a prospective host. We screened a library of AHL analogues for their ability to regulate the legume-rhizobia mutualistic symbiosis (nodulation) between Medicago truncatula and Sinorhizobium meliloti. Using an established QS-reporter line of S. meliloti and nodulation assays with wild-type bacteria, we identified compounds capable of increasing either the rate of nodule formation or total nodule number. Most importantly, we identified compounds with activity exclusive to either host or pathogen, underscoring the potential to generate QS modulators selective to bacteria with limited effects on a prospective host.

  • Gemperline E, Keller C, Jayaraman D, Maeda J, Sussman MR, Ané JM, Li L (2016) Examination of Endogenous Peptides in Medicago truncatula Using Mass Spectrometry Imaging. J. Proteome Res. 15(12):4403-4411 · Pubmed

    Plant science is an important, rapidly developing area of study. Within plant science, one area of study that has grown tremendously with recent technological advances, such as mass spectrometry, is the field of plant-omics; however, plant peptidomics is relatively underdeveloped in comparison with proteomics and metabolomics. Endogenous plant peptides can act as signaling molecules and have been shown to affect cell division, development, nodulation, reproduction, symbiotic associations, and defense reactions. There is a growing need to uncover the role of endogenous peptides on a molecular level. Mass spectrometric imaging (MSI) is a valuable tool for biological analyses as it allows for the detection of thousands of analytes in a single experiment and also displays spatial information for the detected analytes. Despite the prediction of a large number of plant peptides, their detection and imaging with spatial localization and chemical specificity is currently lacking. Here we analyzed the endogenous peptides and proteins in Medicago truncatula using matrix-assisted laser desorption/ionization (MALDI)-MSI. Hundreds of endogenous peptides and protein fragments were imaged, with interesting peptide spatial distribution changes observed between plants in different developmental stages.

  • Mitra S, Mukherjee A, Wiley-Kalil A, Das S, Owen H, Reddy PM, Ané JM, James EK, Gyaneshwar P (2016) A rhamnose-deficient lipopolysaccharide mutant of Rhizobium sp. IRBG74 is defective in root colonization and beneficial interactions with its flooding-tolerant hosts Sesbania cannabina and wetland rice. J. Exp. Bot. 67(19):5869-5884 · Pubmed

    Rhizobium sp. IRBG74 develops a classical nitrogen-fixing symbiosis with the aquatic legume Sesbania cannabina (Retz.). It also promotes the growth of wetland rice (Oryza sativa L.), but little is known about the rhizobial determinants important for these interactions. In this study, we analyzed the colonization of S. cannabina and rice using a strain of Rhizobium sp. IRBG74 dually marked with β-glucuronidase and the green fluorescent protein. This bacterium colonized S. cannabina by crack entry and through root hair infection under flooded and non-flooded conditions, respectively. Rhizobium sp. IRBG74 colonized the surfaces of wetland rice roots, but also entered them at the base of lateral roots. It became endophytically established within intercellular spaces in the rice cortex, and intracellularly within epidermal and hypodermal cells. A mutant of Rhizobium sp. IRBG74 altered in the synthesis of the rhamnose-containing O-antigen exhibited significant defects, not only in nodulation and symbiotic nitrogen fixation with S. cannabina, but also in rice colonization and plant growth promotion. Supplementation with purified lipopolysaccharides from the wild-type strain, but not from the mutant, restored the beneficial colonization of rice roots, but not fully effective nodulation of S. cannabina Commonalities and differences in the rhizobial colonization of the roots of wetland legume and rice hosts are discussed.

  • Van Ness LK, Jayaraman D, Maeda J, Barrett-Wilt GA, Sussman MR, Ané JM (2016) Mass Spectrometric-Based Selected Reaction Monitoring of Protein Phosphorylation during Symbiotic Signaling in the Model Legume, Medicago truncatula. PLoS ONE 11(5):e0155460 (PMC4874550) · Pubmed

    Unlike the major cereal crops corn, rice, and wheat, leguminous plants such as soybean and alfalfa can meet their nitrogen requirement via endosymbiotic associations with soil bacteria. The establishment of this symbiosis is a complex process playing out over several weeks and is facilitated by the exchange of chemical signals between these partners from different kingdoms. Several plant components that are involved in this signaling pathway have been identified, but there is still a great deal of uncertainty regarding the early events in symbiotic signaling, i.e., within the first minutes and hours after the rhizobial signals (Nod factors) are perceived at the plant plasma membrane. The presence of several protein kinases in this pathway suggests a mechanism of signal transduction via posttranslational modification of proteins in which phosphate is added to the hydroxyl groups of serine, threonine and tyrosine amino acid side chains. To monitor the phosphorylation dynamics and complement our previous untargeted 'discovery' approach, we report here the results of experiments using a targeted mass spectrometric technique, Selected Reaction Monitoring (SRM) that enables the quantification of phosphorylation targets with great sensitivity and precision. Using this approach, we confirm a rapid change in the level of phosphorylation in 4 phosphosites of at least 4 plant phosphoproteins that have not been previously characterized. This detailed analysis reveals aspects of the symbiotic signaling mechanism in legumes that, in the long term, will inform efforts to engineer this nitrogen-fixing symbiosis in important non-legume crops such as rice, wheat and corn.

  • Mus F, Crook MB, Garcia K, Garcia Costas A, Geddes BA, Kouri ED, Paramasivan P, Ryu MH, Oldroyd GE, Poole PS, Udvardi MK, Voigt CA, Ané JM, Peters JW (2016) Symbiotic Nitrogen Fixation and the Challenges to Its Extension to Nonlegumes. Appl. Environ. Microbiol. 82(13):3698-710 (PMC4907175) · Pubmed

    Access to fixed or available forms of nitrogen limits the productivity of crop plants and thus food production. Nitrogenous fertilizer production currently represents a significant expense for the efficient growth of various crops in the developed world. There are significant potential gains to be had from reducing dependence on nitrogenous fertilizers in agriculture in the developed world and in developing countries, and there is significant interest in research on biological nitrogen fixation and prospects for increasing its importance in an agricultural setting. Biological nitrogen fixation is the conversion of atmospheric N2 to NH3, a form that can be used by plants. However, the process is restricted to bacteria and archaea and does not occur in eukaryotes. Symbiotic nitrogen fixation is part of a mutualistic relationship in which plants provide a niche and fixed carbon to bacteria in exchange for fixed nitrogen. This process is restricted mainly to legumes in agricultural systems, and there is considerable interest in exploring whether similar symbioses can be developed in nonlegumes, which produce the bulk of human food. We are at a juncture at which the fundamental understanding of biological nitrogen fixation has matured to a level that we can think about engineering symbiotic relationships using synthetic biology approaches. This minireview highlights the fundamental advances in our understanding of biological nitrogen fixation in the context of a blueprint for expanding symbiotic nitrogen fixation to a greater diversity of crop plants through synthetic biology.

  • Patron NJ, Orzaez D, Marillonnet S, Warzecha H, Matthewman C, Youles M, Raitskin O, Leveau A, Farré G, Rogers C, Smith A, Hibberd J, Webb AA, Locke J, Schornack S, Ajioka J, Baulcombe DC, Zipfel C, Kamoun S, Jones JD, Kuhn H, Robatzek S, Van Esse HP, Sanders D, Oldroyd G, Martin C, Field R, O'Connor S, Fox S, Wulff B, Miller B, Breakspear A, Radhakrishnan G, Delaux PM, Loqué D, Granell A, Tissier A, Shih P, Brutnell TP, Quick WP, Rischer H, Fraser PD, Aharoni A, Raines C, South PF, Ané JM, Hamberger BR, Langdale J, Stougaard J, Bouwmeester H, Udvardi M, Murray JA, Ntoukakis V, Schäfer P, Denby K, Edwards KJ, Osbourn A, Haseloff J (2015) Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytol. 208(1):13-9 · Pubmed

    Inventors in the field of mechanical and electronic engineering can access multitudes of components and, thanks to standardization, parts from different manufacturers can be used in combination with each other. The introduction of BioBrick standards for the assembly of characterized DNA sequences was a landmark in microbial engineering, shaping the field of synthetic biology. Here, we describe a standard for Type IIS restriction endonuclease-mediated assembly, defining a common syntax of 12 fusion sites to enable the facile assembly of eukaryotic transcriptional units. This standard has been developed and agreed by representatives and leaders of the international plant science and synthetic biology communities, including inventors, developers and adopters of Type IIS cloning methods. Our vision is of an extensive catalogue of standardized, characterized DNA parts that will accelerate plant bioengineering.

  • Garcia K, Delaux PM, Cope KR, Ané JM (2015) Molecular signals required for the establishment and maintenance of ectomycorrhizal symbioses. New Phytol. 208(1):79-87 · Pubmed

    Ectomycorrhizal (ECM) symbioses are among the most widespread associations between roots of woody plants and soil fungi in forest ecosystems. These associations contribute significantly to the sustainability and sustainagility of these ecosystems through nutrient cycling and carbon sequestration. Unfortunately, the molecular mechanisms controlling the mutual recognition between both partners are still poorly understood. Elegant work has demonstrated that effector proteins from ECM and arbuscular mycorrhizal (AM) fungi regulate host defenses by manipulating plant hormonal pathways. In parallel, genetic and evolutionary studies in legumes showed that a 'common symbiosis pathway' is required for the establishment of the ancient AM symbiosis and has been recruited for the rhizobia-legume association. Given that genes of this pathway are present in many angiosperm trees that develop ectomycorrhizas, we propose their potential involvement in some but not all ECM associations. The maintenance of a successful long-term relationship seems strongly regulated by resource allocation between symbiotic partners, suggesting that nutrients themselves may serve as signals. This review summarizes our current knowledge on the early and late signal exchanges between woody plants and ECM fungi, and we suggest future directions for decoding the molecular basis of the underground dance between trees and their favorite fungal partners.

  • Delaux PM, Radhakrishnan GV, Jayaraman D, Cheema J, Malbreil M, Volkening JD, Sekimoto H, Nishiyama T, Melkonian M, Pokorny L, Rothfels CJ, Sederoff HW, Stevenson DW, Surek B, Zhang Y, Sussman MR, Dunand C, Morris RJ, Roux C, Wong GK, Oldroyd GE, Ané JM (2015) Algal ancestor of land plants was preadapted for symbiosis. Proc. Natl. Acad. Sci. U.S.A. 112(43):13390-5 (PMC4629359) · Pubmed

    Colonization of land by plants was a major transition on Earth, but the developmental and genetic innovations required for this transition remain unknown. Physiological studies and the fossil record strongly suggest that the ability of the first land plants to form symbiotic associations with beneficial fungi was one of these critical innovations. In angiosperms, genes required for the perception and transduction of diffusible fungal signals for root colonization and for nutrient exchange have been characterized. However, the origin of these genes and their potential correlation with land colonization remain elusive. A comprehensive phylogenetic analysis of 259 transcriptomes and 10 green algal and basal land plant genomes, coupled with the characterization of the evolutionary path leading to the appearance of a key regulator, a calcium- and calmodulin-dependent protein kinase, showed that the symbiotic signaling pathway predated the first land plants. In contrast, downstream genes required for root colonization and their specific expression pattern probably appeared subsequent to the colonization of land. We conclude that the most recent common ancestor of extant land plants and green algae was preadapted for symbiotic associations. Subsequent improvement of this precursor stage in early land plants through rounds of gene duplication led to the acquisition of additional pathways and the ability to form a fully functional arbuscular mycorrhizal symbiosis.

  • Venkateshwaran M, Jayaraman D, Chabaud M, Genre A, Balloon AJ, Maeda J, Forshey K, den Os D, Kwiecien NW, Coon JJ, Barker DG, Ané JM (2015) A role for the mevalonate pathway in early plant symbiotic signaling. Proc. Natl. Acad. Sci. U.S.A. 112(31):9781-6 (PMC4534228) · Pubmed

    Rhizobia and arbuscular mycorrhizal fungi produce signals that are perceived by host legume receptors at the plasma membrane and trigger sustained oscillations of the nuclear and perinuclear Ca(2+) concentration (Ca(2+) spiking), which in turn leads to gene expression and downstream symbiotic responses. The activation of Ca(2+) spiking requires the plasma membrane-localized receptor-like kinase Does not Make Infections 2 (DMI2) as well as the nuclear cation channel DMI1. A key enzyme regulating the mevalonate (MVA) pathway, 3-Hydroxy-3-Methylglutaryl CoA Reductase 1 (HMGR1), interacts with DMI2 and is required for the legume-rhizobium symbiosis. Here, we show that HMGR1 is required to initiate Ca(2+) spiking and symbiotic gene expression in Medicago truncatula roots in response to rhizobial and arbuscular mycorrhizal fungal signals. Furthermore, MVA, the direct product of HMGR1 activity, is sufficient to induce nuclear-associated Ca(2+) spiking and symbiotic gene expression in both wild-type plants and dmi2 mutants, but interestingly not in dmi1 mutants. Finally, MVA induced Ca(2+) spiking in Human Embryonic Kidney 293 cells expressing DMI1. This demonstrates that the nuclear cation channel DMI1 is sufficient to support MVA-induced Ca(2+) spiking in this heterologous system.

  • Nguyen TT, Volkening JD, Rose CM, Venkateshwaran M, Westphall MS, Coon JJ, Ané JM, Sussman MR (2015) Potential regulatory phosphorylation sites in a Medicago truncatula plasma membrane proton pump implicated during early symbiotic signaling in roots. FEBS Lett. 589(17):2186-93 · Pubmed

    In plants and fungi the plasma membrane proton pump generates a large proton-motive force that performs essential functions in many processes, including solute transport and the control of cell elongation. Previous studies in yeast and higher plants have indicated that phosphorylation of an auto-inhibitory domain is involved in regulating pump activity. In this report we examine the Medicago truncatula plasma membrane proton pump gene family, and in particular MtAHA5. Yeast complementation assays with phosphomimetic mutations at six candidate sites support a phosphoregulatory role for two residues, suggesting a molecular model to explain early Nod factor-induced changes in the plasma membrane proton-motive force of legume root cells.

  • Sun J, Miller JB, Granqvist E, Wiley-Kalil A, Gobbato E, Maillet F, Cottaz S, Samain E, Venkateshwaran M, Fort S, Morris RJ, Ané JM, Dénarié J, Oldroyd GE (2015) Activation of symbiosis signaling by arbuscular mycorrhizal fungi in legumes and rice. Plant Cell 27(3):823-38 (PMC4558648) · Pubmed

    Establishment of arbuscular mycorrhizal interactions involves plant recognition of diffusible signals from the fungus, including lipochitooligosaccharides (LCOs) and chitooligosaccharides (COs). Nitrogen-fixing rhizobial bacteria that associate with leguminous plants also signal to their hosts via LCOs, the so-called Nod factors. Here, we have assessed the induction of symbiotic signaling by the arbuscular mycorrhizal (Myc) fungal-produced LCOs and COs in legumes and rice (Oryza sativa). We show that Myc-LCOs and tetra-acetyl chitotetraose (CO4) activate the common symbiosis signaling pathway, with resultant calcium oscillations in root epidermal cells of Medicago truncatula and Lotus japonicus. The nature of the calcium oscillations is similar for LCOs produced by rhizobial bacteria and by mycorrhizal fungi; however, Myc-LCOs activate distinct gene expression. Calcium oscillations were activated in rice atrichoblasts by CO4, but not the Myc-LCOs, whereas a mix of CO4 and Myc-LCOs activated calcium oscillations in rice trichoblasts. In contrast, stimulation of lateral root emergence occurred following treatment with Myc-LCOs, but not CO4, in M. truncatula, whereas both Myc-LCOs and CO4 were active in rice. Our work indicates that legumes and non-legumes differ in their perception of Myc-LCO and CO signals, suggesting that different plant species respond to different components in the mix of signals produced by arbuscular mycorrhizal fungi.

  • Gemperline E, Jayaraman D, Maeda J, Ané JM, Li L (2015) Multifaceted investigation of metabolites during nitrogen fixation in Medicago via high resolution MALDI-MS imaging and ESI-MS. J. Am. Soc. Mass Spectrom. 26(1):149-58 (PMC4286419) · Pubmed

    Legumes have developed the unique ability to establish a symbiotic relationship with soil bacteria known as rhizobia. This interaction results in the formation of root nodules in which rhizobia thrive and reduce atmospheric dinitrogen into plant-usable ammonium through biological nitrogen fixation (BNF). Owing to the availability of genetic information for both of the symbiotic partners, the Medicago truncatula-Sinorhizobium meliloti association is an excellent model for examining the BNF process. Although metabolites are important in this symbiotic association, few studies have investigated the array of metabolites that influence this process. Of these studies, most target only a few specific metabolites, the roles of which are either well known or are part of a well-characterized metabolic pathway. Here, we used a multifaceted mass spectrometric (MS) approach to detect and identify the key metabolites that are present during BNF using the Medicago truncatula-Sinorhizobium meliloti association as the model system. High mass accuracy and high resolution matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) Orbitrap instruments were used in this study and provide complementary results for more in-depth characterization of the nitrogen-fixation process. We used well-characterized plant and bacterial mutants to highlight differences between the metabolites that are present in functional versus nonfunctional nodules. Our study highlights the benefits of using a combination of mass spectrometric techniques to detect differences in metabolite composition and the distributions of these metabolites in plant biology.

  • Jayaraman D, Gilroy S, Ané JM (2014) Staying in touch: mechanical signals in plant-microbe interactions. Curr. Opin. Plant Biol. 20:104-9 · Pubmed

    Mechanical stimulations play a significant role in the day to day existence of plants. Plants exhibit varied responses depending on the nature and intensity of these stimuli. In this review, we present recent literature on the responses of plants to mechanical stimuli, focusing primarily on those exerted during plant-microbe interactions. We discuss how microbes are able to apply mechanical stimuli on plants and how some plant responses to pathogenic and symbiotic microbes present striking similarities with responses to mechanical stimuli applied, for instance, using micro-needles. We hypothesize that appropriate responses of plants to pathogenic and symbiotic microbes may require a tight integration of both chemical and mechanical stimulations exerted by these microbes.

  • Palmer AG, Senechal AC, Mukherjee A, Ané JM, Blackwell HE (2014) Plant responses to bacterial N-acyl L-homoserine lactones are dependent on enzymatic degradation to L-homoserine. ACS Chem. Biol. 9(8):1834-45 (PMC4136694) · Pubmed

    Many bacteria use quorum sensing (QS) to regulate phenotypes that ultimately benefit the bacterial population at high cell densities. These QS-dependent phenotypes are diverse and can have significant impacts on the bacterial host, including virulence factor production, motility, biofilm formation, bioluminescence, and root nodulation. As bacteria and their eukaryotic hosts have coevolved over millions of years, it is not surprising that certain hosts appear to be able to sense QS signals, potentially allowing them to alter QS outcomes. Recent experiments have established that eukaryotes have marked responses to the N-acyl L-homoserine lactone (AHL) signals used by Gram-negative bacteria for QS, and the responses of plants to AHLs have received considerable scrutiny to date. However, the molecular mechanisms by which plants, and eukaryotes in general, sense bacterial AHLs remain unclear. Herein, we report a systematic analysis of the responses of the model plants Arabidopsis thaliana and Medicago truncatula to a series of native AHLs and byproducts thereof. Our results establish that AHLs can significantly alter seedling growth in an acyl-chain length dependent manner. Based upon A. thaliana knockout studies and in vitro biochemical assays, we conclude that the observed growth effects are dependent upon AHL amidolysis by a plant-derived fatty acid amide hydrolase (FAAH) to yield L-homoserine. The accumulation of l-homoserine appears to encourage plant growth at low concentrations by stimulating transpiration, while higher concentrations inhibit growth by stimulating ethylene production. These results offer new insights into the mechanisms by which plant hosts can respond to QS signals and the potential role of QS in interkingdom associations.

  • Delaux PM, Varala K, Edger PP, Coruzzi GM, Pires JC, Ané JM (2014) Comparative phylogenomics uncovers the impact of symbiotic associations on host genome evolution. PLoS Genet. 10(7):e1004487 (PMC4102449) · Pubmed

    Mutualistic symbioses between eukaryotes and beneficial microorganisms of their microbiome play an essential role in nutrition, protection against disease, and development of the host. However, the impact of beneficial symbionts on the evolution of host genomes remains poorly characterized. Here we used the independent loss of the most widespread plant-microbe symbiosis, arbuscular mycorrhization (AM), as a model to address this question. Using a large phenotypic approach and phylogenetic analyses, we present evidence that loss of AM symbiosis correlates with the loss of many symbiotic genes in the Arabidopsis lineage (Brassicales). Then, by analyzing the genome and/or transcriptomes of nine other phylogenetically divergent non-host plants, we show that this correlation occurred in a convergent manner in four additional plant lineages, demonstrating the existence of an evolutionary pattern specific to symbiotic genes. Finally, we use a global comparative phylogenomic approach to track this evolutionary pattern among land plants. Based on this approach, we identify a set of 174 highly conserved genes and demonstrate enrichment in symbiosis-related genes. Our findings are consistent with the hypothesis that beneficial symbionts maintain purifying selection on host gene networks during the evolution of entire lineages.

  • Jayaraman D, Valdés-López O, Kaspar CW, Ané JM (2014) Response of Medicago truncatula seedlings to colonization by Salmonella enterica and Escherichia coli O157:H7. PLoS ONE 9(2):e87970 (PMC3925098) · Pubmed

    Disease outbreaks due to the consumption of legume seedlings contaminated with human enteric bacterial pathogens like Escherichia coli O157:H7 and Salmonella enterica are reported every year. Besides contaminations occurring during food processing, pathogens present on the surface or interior of plant tissues are also responsible for such outbreaks. In the present study, surface and internal colonization of Medicago truncatula, a close relative of alfalfa, by Salmonella enterica and Escherichia coli O157:H7 were observed even with inoculum levels as low as two bacteria per plant. Furthermore, expression analyses revealed that approximately 30% of Medicago truncatula genes were commonly regulated in response to both of these enteric pathogens. This study highlights that very low inoculum doses trigger responses from the host plant and that both of these human enteric pathogens may in part use similar mechanisms to colonize legume seedlings.

  • Crook MB, Mitra S, Ané JM, Sadowsky MJ, Gyaneshwar P (2013) Complete Genome Sequence of the Sesbania Symbiont and Rice Growth-Promoting Endophyte Rhizobium sp. Strain IRBG74. Genome Announc 1(6): (PMC3837170) · Pubmed

    Rhizobium sp. strain IRBG74 is the first known nitrogen-fixing symbiont in the Agrobacterium/Rhizobium clade that nodulates the aquatic legume Sesbania sp. and is also a growth-promoting endophyte of wetland rice. Here, we present the sequence of the IRBG74 genome, which is composed of a circular chromosome, a linear chromosome, and a symbiotic plasmid, pIRBG74a.

  • Ye H, Gemperline E, Venkateshwaran M, Chen R, Delaux PM, Howes-Podoll M, Ané JM, Li L (2013) MALDI mass spectrometry-assisted molecular imaging of metabolites during nitrogen fixation in the Medicago truncatula-Sinorhizobium meliloti symbiosis. Plant J. 75(1):130-45 · Pubmed

    Symbiotic associations between leguminous plants and nitrogen-fixing rhizobia culminate in the formation of specialized organs called root nodules, in which the rhizobia fix atmospheric nitrogen and transfer it to the plant. Efficient biological nitrogen fixation depends on metabolites produced by and exchanged between both partners. The Medicago truncatula-Sinorhizobium meliloti association is an excellent model for dissecting this nitrogen-fixing symbiosis because of the availability of genetic information for both symbiotic partners. Here, we employed a powerful imaging technique - matrix-assisted laser desorption/ionization (MALDI)/mass spectrometric imaging (MSI) - to study metabolite distribution in roots and root nodules of M. truncatula during nitrogen fixation. The combination of an efficient, novel MALDI matrix [1,8-bis(dimethyl-amino) naphthalene, DMAN] with a conventional matrix 2,5-dihydroxybenzoic acid (DHB) allowed detection of a large array of organic acids, amino acids, sugars, lipids, flavonoids and their conjugates with improved coverage. Ion density maps of representative metabolites are presented and correlated with the nitrogen fixation process. We demonstrate differences in metabolite distribution between roots and nodules, and also between fixing and non-fixing nodules produced by plant and bacterial mutants. Our study highlights the benefits of using MSI for detecting differences in metabolite distributions in plant biology.

  • Delaux PM, Séjalon-Delmas N, Bécard G, Ané JM (2013) Evolution of the plant-microbe symbiotic 'toolkit'. Trends Plant Sci. 18(6):298-304 · Pubmed

    Beneficial associations between plants and arbuscular mycorrhizal fungi play a major role in terrestrial environments and in the sustainability of agroecosystems. Proteins, microRNAs, and small molecules have been identified in model angiosperms as required for the establishment of arbuscular mycorrhizal associations and define a symbiotic 'toolkit' used for other interactions such as the rhizobia-legume symbiosis. Based on recent studies, we propose an evolutionary framework for this toolkit. Some components appeared recently in angiosperms, whereas others are highly conserved even in land plants unable to form arbuscular mycorrhizal associations. The exciting finding that some components pre-date the appearance of arbuscular mycorrhizal fungi suggests the existence of unknown roles for this toolkit and even the possibility of symbiotic associations in charophyte green algae.

  • Venkateshwaran M, Volkening JD, Sussman MR, Ané JM (2013) Symbiosis and the social network of higher plants. Curr. Opin. Plant Biol. 16(1):118-27 · Pubmed

    In the Internet era, communicating with friends and colleagues via social networks constitutes a significant proportion of our daily activities. Similarly animals and plants also interact with many organisms, some of which are pathogens and do no good for the plant, while others are beneficial symbionts. Almost all plants indulge in developing social networks with microbes, in particular with arbuscular mycorrhizal fungi, and emerging evidence indicates that most employ an ancient and widespread central 'social media' pathway made of signaling molecules within what is called the SYM pathway. Some plants, like legumes, are particularly active recruiters of friends, as they have established very sophisticated and beneficial interactions with nitrogen-fixing bacteria, also via the SYM pathway. Interestingly, many members of the Brassicaceae, including the model plant Arabidopsis thaliana, seem to have removed themselves from this ancestral social network and lost the ability to engage in mutually favorable interactions with arbuscular mycorrhizal fungi. Despite these generalizations, recent studies exploring the root microbiota of A. thaliana have found that in natural conditions, A. thaliana roots are colonized by many different bacterial species and therefore may be using different and probably more recent 'social media' for these interactions. In general, recent advances in the understanding of such molecular machinery required for plant-symbiont associations are being obtained using high throughput genomic profiling strategies including transcriptomics, proteomics and metabolomics. The crucial mechanistic understanding that such data reveal may provide the infrastructure for future efforts to genetically manipulate crop social networks for our own food and fiber needs.

  • Volkening JD, Bailey DJ, Rose CM, Grimsrud PA, Howes-Podoll M, Venkateshwaran M, Westphall MS, Ané JM, Coon JJ, Sussman MR (2012) A proteogenomic survey of the Medicago truncatula genome. Mol. Cell Proteomics 11(10):933-44 (PMC3494139) · Pubmed

    Peptide sequencing by computational assignment of tandem mass spectra to a database of putative protein sequences provides an independent approach to confirming or refuting protein predictions based on large-scale DNA and RNA sequencing efforts. This use of mass spectrometrically-derived sequence data for testing and refining predicted gene models has been termed proteogenomics. We report herein the application of proteogenomic methodology to a database of 10.9 million tandem mass spectra collected over a period of two years from proteolytically generated peptides isolated from the model legume Medicago truncatula. These spectra were searched against a database of predicted M. truncatula protein sequences generated from public databases, in silico gene model predictions, and a whole-genome six-frame translation. This search identified 78,647 distinct peptide sequences, and a comparison with the publicly available proteome from the recently published M. truncatula genome supported translation of 9,843 existing gene models and identified 1,568 novel peptides suggesting corrections or additions to the current annotations. Each supporting and novel peptide was independently validated using mRNA-derived deep sequencing coverage and an overall correlation of 93% between the two data types was observed. We have additionally highlighted examples of several aspects of structural annotation for which tandem MS provides unique evidence not easily obtainable through typical DNA or RNA sequencing. Proteogenomic analysis is a valuable and unique source of information for the structural annotation of genomes and should be included in such efforts to ensure that the genome models used by biologists mirror as accurately as possible what is present in the cell.

  • Zhang N, Venkateshwaran M, Boersma M, Harms A, Howes-Podoll M, den Os D, Ané JM, Sussman MR (2012) Metabolomic profiling reveals suppression of oxylipin biosynthesis during the early stages of legume-rhizobia symbiosis. FEBS Lett. 586(19):3150-8 · Pubmed

    The establishment of symbiosis between leguminous plants and rhizobial bacteria requires rapid metabolic changes in both partners. We utilized untargeted quantitative mass spectrometry to perform metabolomic profiling of small molecules in extracts of the model legume Medicago truncatula treated with rhizobial Nod factors. One metabolite closely resembling the 9(R)-HODE class of oxylipins reproducibly showed a decrease in concentration within the first hour of in planta nod factor treatment. Oxylipins are precursors of the jasmonic acid biosynthetic pathway and we showed that both this metabolite and jasmonic acid inhibit Nod factor signaling. Since, oxylipins have been implicated as antimicrobial compounds produced by plants, these observations suggest that the oxylipin pathway may play multiple roles in facilitating Nod factor signaling during the early stages of symbiosis.

  • Rose CM, Venkateshwaran M, Volkening JD, Grimsrud PA, Maeda J, Bailey DJ, Park K, Howes-Podoll M, den Os D, Yeun LH, Westphall MS, Sussman MR, Ané JM, Coon JJ (2012) Rapid phosphoproteomic and transcriptomic changes in the rhizobia-legume symbiosis. Mol. Cell Proteomics 11(9):724-44 (PMC3434772) · Pubmed

    Symbiotic associations between legumes and rhizobia usually commence with the perception of bacterial lipochitooligosaccharides, known as Nod factors (NF), which triggers rapid cellular and molecular responses in host plants. We report here deep untargeted tandem mass spectrometry-based measurements of rapid NF-induced changes in the phosphorylation status of 13,506 phosphosites in 7739 proteins from the model legume Medicago truncatula. To place these phosphorylation changes within a biological context, quantitative phosphoproteomic and RNA measurements in wild-type plants were compared with those observed in mutants, one defective in NF perception (nfp) and one defective in downstream signal transduction events (dmi3). Our study quantified the early phosphorylation and transcription dynamics that are specifically associated with NF-signaling, confirmed a dmi3-mediated feedback loop in the pathway, and suggested "cryptic" NF-signaling pathways, some of them being also involved in the response to symbiotic arbuscular mycorrhizal fungi.

  • Venkateshwaran M, Cosme A, Han L, Banba M, Satyshur KA, Schleiff E, Parniske M, Imaizumi-Anraku H, Ané JM (2012) The recent evolution of a symbiotic ion channel in the legume family altered ion conductance and improved functionality in calcium signaling. Plant Cell 24(6):2528-45 (PMC3406897) · Pubmed

    Arbuscular mycorrhiza and the rhizobia-legume symbiosis are two major root endosymbioses that facilitate plant nutrition. In Lotus japonicus, two symbiotic cation channels, CASTOR and POLLUX, are indispensable for the induction of nuclear calcium spiking, one of the earliest plant responses to symbiotic partner recognition. During recent evolution, a single amino acid substitution in DOES NOT MAKE INFECTIONS1 (DMI1), the POLLUX putative ortholog in the closely related Medicago truncatula, rendered the channel solo sufficient for symbiosis; castor, pollux, and castor pollux double mutants of L. japonicus were rescued by DMI1 alone, while both Lj-CASTOR and Lj-POLLUX were required for rescuing a dmi1 mutant of M. truncatula. Experimental replacement of the critical serine by an alanine in the selectivity filter of Lj-POLLUX conferred a symbiotic performance indistinguishable from DMI1. Electrophysiological characterization of DMI1 and Lj-CASTOR (wild-type and mutants) by planar lipid bilayer experiments combined with calcium imaging in Human Embryonic Kidney-293 cells expressing DMI1 (the wild type and mutants) suggest that the serine-to-alanine substitution conferred reduced conductance with a long open state to DMI1 and improved its efficiency in mediating calcium oscillations. We propose that this single amino acid replacement in the selectivity filter made DMI1 solo sufficient for symbiosis, thus explaining the selective advantage of this allele at the mechanistic level.

  • Rose CM, Venkateshwaran M, Grimsrud PA, Westphall MS, Sussman MR, Coon JJ, Ané JM (2012) Medicago PhosphoProtein Database: a repository for Medicago truncatula phosphoprotein data. Front Plant Sci 3:122 (PMC3371616) · Pubmed

    The ability of legume crops to fix atmospheric nitrogen via a symbiotic association with soil rhizobia makes them an essential component of many agricultural systems. Initiation of this symbiosis requires protein phosphorylation-mediated signaling in response to rhizobial signals named Nod factors. Medicago truncatula (Medicago) is the model system for studying legume biology, making the study of its phosphoproteome essential. Here, we describe the Medicago PhosphoProtein Database (MPPD; http://phospho.medicago.wisc.edu), a repository built to house phosphoprotein, phosphopeptide, and phosphosite data specific to Medicago. Currently, the MPPD holds 3,457 unique phosphopeptides that contain 3,404 non-redundant sites of phosphorylation on 829 proteins. Through the web-based interface, users are allowed to browse identified proteins or search for proteins of interest. Furthermore, we allow users to conduct BLAST searches of the database using both peptide sequences and phosphorylation motifs as queries. The data contained within the database are available for download to be investigated at the user's discretion. The MPPD will be updated continually with novel phosphoprotein and phosphopeptide identifications, with the intent of constructing an unparalleled compendium of large-scale Medicago phosphorylation data.

  • Jayaraman D, Forshey KL, Grimsrud PA, Ané JM (2012) Leveraging proteomics to understand plant-microbe interactions. Front Plant Sci 3:44 (PMC3355735) · Pubmed

    Understanding the interactions of plants with beneficial and pathogenic microbes is a promising avenue to improve crop productivity and agriculture sustainability. Proteomic techniques provide a unique angle to describe these intricate interactions and test hypotheses. The various approaches for proteomic analysis generally include protein/peptide separation and identification, but can also provide quantification and the characterization of post-translational modifications. In this review, we discuss how these techniques have been applied to the study of plant-microbe interactions. We also present some areas where this field of study would benefit from the utilization of newly developed methods that overcome previous limitations. Finally, we reinforce the need for expanding, integrating, and curating protein databases, as well as the benefits of combining protein-level datasets with those from genetic analyses and other high-throughput large-scale approaches for a systems-level view of plant-microbe interactions.

  • Horváth B, Yeun LH, Domonkos A, Halász G, Gobbato E, Ayaydin F, Miró K, Hirsch S, Sun J, Tadege M, Ratet P, Mysore KS, Ané JM, Oldroyd GE, Kaló P (2011) Medicago truncatula IPD3 is a member of the common symbiotic signaling pathway required for rhizobial and mycorrhizal symbioses. Mol. Plant Microbe Interact. 24(11):1345-58 · Pubmed

    Legumes form endosymbiotic associations with nitrogen-fixing bacteria and arbuscular mycorrhizal (AM) fungi which facilitate nutrient uptake. Both symbiotic interactions require a molecular signal exchange between the plant and the symbiont, and this involves a conserved symbiosis (Sym) signaling pathway. In order to identify plant genes required for intracellular accommodation of nitrogen-fixing bacteria and AM fungi, we characterized Medicago truncatula symbiotic mutants defective for rhizobial infection of nodule cells and colonization of root cells by AM hyphae. Here, we describe mutants impaired in the interacting protein of DMI3 (IPD3) gene, which has been identified earlier as an interacting partner of the calcium/calmodulin-dependent protein, a member of the Sym pathway. The ipd3 mutants are impaired in both rhizobial and mycorrhizal colonization and we show that IPD3 is necessary for appropriate Nod-factor-induced gene expression. This indicates that IPD3 is a member of the common Sym pathway. We observed differences in the severity of ipd3 mutants that appear to be the result of the genetic background. This supports the hypothesis that IPD3 function is partially redundant and, thus, additional genetic components must exist that have analogous functions to IPD3. This explains why mutations in an essential component of the Sym pathway have defects at late stages of the symbiotic interactions.

  • Capoen W, Sun J, Wysham D, Otegui MS, Venkateshwaran M, Hirsch S, Miwa H, Downie JA, Morris RJ, Ané JM, Oldroyd GE (2011) Nuclear membranes control symbiotic calcium signaling of legumes. Proc. Natl. Acad. Sci. U.S.A. 108(34):14348-53 (PMC3161518) · Pubmed

    Nuclear-associated oscillations in calcium act as a secondary messenger in the symbiotic signaling pathway of legumes. These are decoded by a nuclear-localized calcium and calmodulin-dependent protein kinase, the activation of which is sufficient to drive downstream responses. This implies that the calcium oscillations within the nucleus are the predominant signals for legume symbiosis. However, the mechanisms that allow targeted release of calcium in the nuclear region have not been defined. Here we show that symbiosis-induced calcium changes occur in both the nucleoplasm and the perinuclear cytoplasm and seem to originate from the nuclear membranes. Reaction diffusion simulations suggest that spike generation within the nucleoplasm is not possible through transmission of a calcium wave from the cytoplasm alone and that calcium is likely to be released across the inner nuclear membrane to allow nuclear calcium changes. In agreement with this, we found that the cation channel DMI1, which is essential for symbiotic calcium oscillations, is preferentially located on the inner nuclear membrane, implying an essential function for the inner nuclear membrane in symbiotic calcium signaling. Furthermore, a sarco/endoplasmic reticulum calcium ATPase (SERCA) essential for symbiotic calcium oscillations is targeted to the inner nuclear membrane, as well as the outer nuclear membrane and endoplasmic reticulum (ER). We propose that release of calcium across the inner nuclear membrane allows targeted release of the ER calcium store, and efficient reloading of this calcium store necessitates the capture of calcium from the nucleoplasm and nuclear-associated cytoplasm.

  • Mukherjee A, Ané JM (2011) Germinating spore exudates from arbuscular mycorrhizal fungi: molecular and developmental responses in plants and their regulation by ethylene. Mol. Plant Microbe Interact. 24(2):260-70 · Pubmed

    Arbuscular mycorrhizal (AM) fungi stimulate root development and induce expression of mycorrhization-specific genes in both eudicots and monocots. Diffusible factors released by AM fungi have been shown to elicit similar responses in Medicago truncatula. Colonization of roots by AM fungi is inhibited by ethylene. We compared the effects of germinating spore exudates (GSE) from Glomus intraradices in monocots and in eudicots, their genetic control, and their regulation by ethylene. GSE modify root architecture and induce symbiotic gene expression in both monocots and eudicots. The genetic regulation of root architecture and gene expression was analyzed using M. truncatula and rice symbiotic mutants. These responses are dependent on the common symbiotic pathway as well as another uncharacterized pathway. Significant differences between monocots and eudicots were observed in the genetic control of plant responses to GSE. However, ethylene inhibits GSE-induced symbiotic gene expression and root development in both groups. Our results indicate that GSE signaling shares similarities and differences in monocots versus eudicots, that only a subset of AM signaling pathways has been co-opted in legumes for the establishment of root nodulation with rhizobia, and that regulation of these pathways by ethylene is a feature conserved across higher land plants.

  • Riely BK, He H, Venkateshwaran M, Sarma B, Schraiber J, Ané JM, Cook DR (2011) Identification of legume RopGEF gene families and characterization of a Medicago truncatula RopGEF mediating polar growth of root hairs. Plant J. 65(2):230-43 · Pubmed

    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.

  • Wang B, Yeun LH, Xue JY, Liu Y, Ané JM, Qiu YL (2010) Presence of three mycorrhizal genes in the common ancestor of land plants suggests a key role of mycorrhizas in the colonization of land by plants. New Phytol. 186(2):514-25 · Pubmed

    *The colonization of land by plants fundamentally altered environmental conditions on earth. Plant-mycorrhizal fungus symbiosis likely played a key role in this process by assisting plants to absorb water and nutrients from soil. *Here, in a diverse set of land plants, we investigated the evolutionary histories and functional conservation of three genes required for mycorrhiza formation in legumes and rice (Oryza sativa), DMI1, DMI3 and IPD3. *The genes were isolated from nearly all major plant lineages. Phylogenetic analyses showed that they had been vertically inherited since the origin of land plants. Further, cross-species mutant rescue experiments demonstrated that DMI3 genes from liverworts and hornworts could rescue Medicago truncatula dmi3 mutants for mycorrhiza formation. Yeast two-hybrid assays also showed that bryophyte DMI3 proteins could bind to downstream-acting M. trunculata IPD3 protein. Finally, molecular evolutionary analyses revealed that these genes were under purifying selection for maintenance of their ancestral functions in all mycorrhizal plant lineages. *These results indicate that the mycorrhizal genes were present in the common ancestor of land plants, and that their functions were largely conserved during land plant evolution. The evidence presented here strongly suggests that plant-mycorrhizal fungus symbiosis was one of the key processes that contributed to the origin of land flora.

  • Grimsrud PA, den Os D, Wenger CD, Swaney DL, Schwartz D, Sussman MR, Ané JM, Coon JJ (2010) Large-scale phosphoprotein analysis in Medicago truncatula roots provides insight into in vivo kinase activity in legumes. Plant Physiol. 152(1):19-28 (PMC2799343) · Pubmed

    Nitrogen fixation in legumes requires the development of root organs called nodules and their infection by symbiotic rhizobia. Over the last decade, Medicago truncatula has emerged as a major model plant for the analysis of plant-microbe symbioses and for addressing questions pertaining to legume biology. While the initiation of symbiosis and the development of nitrogen-fixing root nodules depend on the activation of a protein phosphorylation-mediated signal transduction cascade in response to symbiotic signals produced by the rhizobia, few sites of in vivo phosphorylation have previously been identified in M. truncatula. We have characterized sites of phosphorylation on proteins from M. truncatula roots, from both whole cell lysates and membrane-enriched fractions, using immobilized metal affinity chromatography and tandem mass spectrometry. Here, we report 3,457 unique phosphopeptides spanning 3,404 nonredundant sites of in vivo phosphorylation on 829 proteins in M. truncatula Jemalong A17 roots, identified using the complementary tandem mass spectrometry fragmentation methods electron transfer dissociation and collision-activated dissociation. With this being, to our knowledge, the first large-scale plant phosphoproteomic study to utilize electron transfer dissociation, analysis of the identified phosphorylation sites revealed phosphorylation motifs not previously observed in plants. Furthermore, several of the phosphorylation motifs, including LxKxxs and RxxSxxxs, have yet to be reported as kinase specificities for in vivo substrates in any species, to our knowledge. Multiple sites of phosphorylation were identified on several key proteins involved in initiating rhizobial symbiosis, including SICKLE, NUCLEOPORIN133, and INTERACTING PROTEIN OF DMI3. Finally, we used these data to create an open-access online database for M. truncatula phosphoproteomic data.

  • Bhaskar PB, Venkateshwaran M, Wu L, Ané JM, Jiang J (2009) Agrobacterium-mediated transient gene expression and silencing: a rapid tool for functional gene assay in potato. PLoS ONE 4(6):e5812 (PMC2686102) · Pubmed

    Potato is the third most important food crop worldwide. However, genetic and genomic research of potato has lagged behind other major crops due to the autopolyploidy and highly heterozygous nature associated with the potato genome. Reliable and technically undemanding techniques are not available for functional gene assays in potato. Here we report the development of a transient gene expression and silencing system in potato. Gene expression or RNAi-based gene silencing constructs were delivered into potato leaf cells using Agrobacterium-mediated infiltration. Agroinfiltration of various gene constructs consistently resulted in potato cell transformation and spread of the transgenic cells around infiltration zones. The efficiency of agroinfiltration was affected by potato genotypes, concentration of Agrobacterium, and plant growth conditions. We demonstrated that the agroinfiltration-based transient gene expression can be used to detect potato proteins in sub-cellular compartments in living cells. We established a double agroinfiltration procedure that allows to test whether a specific gene is associated with potato late blight resistance pathway mediated by the resistance gene RB. This procedure provides a powerful approach for high throughput functional assay for a large number of candidate genes in potato late blight resistance.

  • Chen C, Ané JM, Zhu H (2009) OsIPD3, an ortholog of the Medicago truncatula DMI3 interacting protein IPD3, is required for mycorrhizal symbiosis in rice. New Phytol. 180(2):311-5 · Pubmed

    Medicago truncatula IPD3 (MtIPD3) is an interacting protein of DMI3 (does not make infections 3), a Ca(2+)/calmodulin-dependent protein kinase (CCaMK) essential for both arbuscular mycorrhizal (AM) and rhizobial symbioses. However, the function of MtIPD3 in root symbioses has not been demonstrated in M. truncatula, because of a lack of knockout mutants for functional analysis. In this study, the availability of IPD3 knockout mutants in rice (Oryza sativa) was exploited to test the function of OsIPD3 in AM symbiosis. Three independent retrotransposon Tos17 insertion lines of OsIPD3 were selected and the phenotypes characterized upon inoculation with the AM fungus Glomus intraradices. Phenotypic and genetic analyses revealed that the Osipd3 mutants were unable to establish a symbiotic association with G. intraradices. In conclusion, IPD3 represents a novel gene required for root symbiosis with AM fungi in plants.

  • Ané JM, Zhu H, Frugoli J (2008) Recent Advances in Medicago truncatula Genomics. Int J Plant Genomics 2008:256597 (PMC2216067) · Pubmed

    Legume rotation has allowed a consistent increase in crop yield and consequently in human population since the antiquity. Legumes will also be instrumental in our ability to maintain the sustainability of our agriculture while facing the challenges of increasing food and biofuel demand. Medicago truncatula and Lotus japonicus have emerged during the last decade as two major model systems for legume biology. Initially developed to dissect plant-microbe symbiotic interactions and especially legume nodulation, these two models are now widely used in a variety of biological fields from plant physiology and development to population genetics and structural genomics. This review highlights the genetic and genomic tools available to the M. truncatula community. Comparative genomic approaches to transfer biological information between model systems and legume crops are also discussed.

  • Kevei Z, Lougnon G, Mergaert P, Horváth GV, Kereszt A, Jayaraman D, Zaman N, Marcel F, Regulski K, Kiss GB, Kondorosi A, Endre G, Kondorosi E, Ané JM (2007) 3-hydroxy-3-methylglutaryl coenzyme a reductase 1 interacts with NORK and is crucial for nodulation in Medicago truncatula. Plant Cell 19(12):3974-89 (PMC2217646) · Pubmed

    NORK in legumes encodes a receptor-like kinase that is required for Nod factor signaling and root nodule development. Using Medicago truncatula NORK as bait in a yeast two-hybrid assay, we identified 3-hydroxy-3-methylglutaryl CoA reductase 1 (Mt HMGR1) as a NORK interacting partner. HMGR1 belongs to a multigene family in M. truncatula, and different HMGR isoforms are key enzymes in the mevalonate biosynthetic pathway leading to the production of a diverse array of isoprenoid compounds. Testing other HMGR members revealed a specific interaction between NORK and HMGR1. Mutagenesis and deletion analysis showed that this interaction requires the cytosolic active kinase domain of NORK and the cytosolic catalytic domain of HMGR1. NORK homologs from Lotus japonicus and Sesbania rostrata also interacted with Mt HMGR1, but homologous nonsymbiotic kinases of M. truncatula did not. Pharmacological inhibition of HMGR activities decreased nodule number and delayed nodulation, supporting the importance of the mevalonate pathway in symbiotic development. Decreasing HMGR1 expression in M. truncatula transgenic roots by RNA interference led to a dramatic decrease in nodulation, confirming that HMGR1 is essential for nodule development. Recruitment of HMGR1 by NORK could be required for production of specific isoprenoid compounds, such as cytokinins, phytosteroids, or isoprenoid moieties involved in modification of signaling proteins.

  • Peiter E, Sun J, Heckmann AB, Venkateshwaran M, Riely BK, Otegui MS, Edwards A, Freshour G, Hahn MG, Cook DR, Sanders D, Oldroyd GE, Downie JA, Ané JM (2007) The Medicago truncatula DMI1 protein modulates cytosolic calcium signaling. Plant Physiol. 145(1):192-203 (PMC1976572) · Pubmed

    In addition to establishing symbiotic relationships with arbuscular mycorrhizal fungi, legumes also enter into a nitrogen-fixing symbiosis with rhizobial bacteria that results in the formation of root nodules. Several genes involved in the development of both arbuscular mycorrhiza and legume nodulation have been cloned in model legumes. Among them, Medicago truncatula DMI1 (DOESN'T MAKE INFECTIONS1) is required for the generation of nucleus-associated calcium spikes in response to the rhizobial signaling molecule Nod factor. DMI1 encodes a membrane protein with striking similarities to the Methanobacterium thermoautotrophicum potassium channel (MthK). The cytosolic C terminus of DMI1 contains a RCK (regulator of the conductance of K(+)) domain that in MthK acts as a calcium-regulated gating ring controlling the activity of the channel. Here we show that a dmi1 mutant lacking the entire C terminus acts as a dominant-negative allele interfering with the formation of nitrogen-fixing nodules and abolishing the induction of calcium spikes by the G-protein agonist Mastoparan. Using both the full-length DMI1 and this dominant-negative mutant protein we show that DMI1 increases the sensitivity of a sodium- and lithium-hypersensitive yeast (Saccharomyces cerevisiae) mutant toward those ions and that the C-terminal domain plays a central role in regulating this response. We also show that DMI1 greatly reduces the release of calcium from internal stores in yeast, while the dominant-negative allele appears to have the opposite effect. This work suggests that DMI1 is not directly responsible for Nod factor-induced calcium changes, but does have the capacity to regulate calcium channels in both yeast and plants.

  • Messinese E, Mun JH, Yeun LH, Jayaraman D, Rougé P, Barre A, Lougnon G, Schornack S, Bono JJ, Cook DR, Ané JM (2007) A novel nuclear protein interacts with the symbiotic DMI3 calcium- and calmodulin-dependent protein kinase of Medicago truncatula. Mol. Plant Microbe Interact. 20(8):912-21 · Pubmed

    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.

  • Riely BK, Lougnon G, Ané JM, Cook DR (2007) The symbiotic ion channel homolog DMI1 is localized in the nuclear membrane of Medicago truncatula roots. Plant J. 49(2):208-16 · Pubmed

    Legumes utilize a common signaling pathway to form symbiotic associations both with rhizobial bacteria and arbuscular mycorrhizal fungi. The perception of microbial signals is believed to take place at the plasma membrane, activating a cascade that converges on the nucleus where transcriptional reprogramming facilitates the symbioses. Forward genetic strategies have identified genes in this signaling pathway including Medicago truncatula DMI1 (Doesn't Make Infections 1) that encodes a putative ion channel. Although the DMI1 homologs from Lotus japonicus, CASTOR and POLLUX, were recently reported to be localized in plastids, we report here that a functional DMI1::GFP fusion is localized to the nuclear envelope in M. truncatula roots when expressed both from a constitutive 35S promoter and from a native DMI1 promoter. Localization may be mediated in part by sequences located within the amino-terminus of DMI1. This region of DMI1 is required for symbiotic signal transduction, and its replacement with a bona fide plastid transit peptide from the glutamine synthetase 2 gene does not restore DMI1 function. These new data place DMI1 in the nuclear envelope in close proximity to the origin of Nod-factor-induced calcium spiking.

  • Riely BK, Mun JH, Ané JM (2006) Unravelling the molecular basis for symbiotic signal transduction in legumes. Mol. Plant Pathol. 7(3):197-207 · Pubmed

    No abstract available.

  • Zhu H, Riely BK, Burns NJ, Ané JM (2006) Tracing nonlegume orthologs of legume genes required for nodulation and arbuscular mycorrhizal symbioses. Genetics 172(4):2491-9 (PMC1456400) · Pubmed

    Most land plants can form a root symbiosis with arbuscular mycorrhizal (AM) fungi for assimilation of inorganic phosphate from the soil. In contrast, the nitrogen-fixing root nodule symbiosis is almost completely restricted to the legumes. The finding that the two symbioses share common signaling components in legumes suggests that the evolutionarily younger nitrogen-fixing symbiosis has recruited functions from the more ancient AM symbiosis. The recent advances in cloning of the genes required for nodulation and AM symbioses from the two model legumes, Medicago truncatula and Lotus japonicus, provide a unique opportunity to address biological questions pertaining to the evolution of root symbioses in plants. Here, we report that nearly all cloned legume genes required for nodulation and AM symbioses have their putative orthologs in nonlegumes. The orthologous relationship can be clearly defined on the basis of both sequence similarity and microsyntenic relationship. The results presented here serve as a prelude to the comparative analysis of orthologous gene function between legumes and nonlegumes and facilitate our understanding of how gene functions and signaling pathways have evolved to generate species- or family-specific phenotypes.

  • Riely BK, Ané JM, Penmetsa RV, Cook DR (2004) Genetic and genomic analysis in model legumes bring Nod-factor signaling to center stage. Curr. Opin. Plant Biol. 7(4):408-13 · Pubmed

    The control of host-specificity in the Rhizobium-legume symbiosis has been a topic of long-standing interest to plant biologists. By the early 1990s, biologists had deciphered the chemical signals that trigger early symbiotic responses. Flavonoids from the plant root trigger bacterial gene expression and the production of lipo-chitooligosaccharide signals (called Nod factors) that are recognized by the plant host. Genetic differences between bacterial strains modify the oligosaccharide backbone, for example by the addition of sulfate, acetate or fucose, and simultaneously alter the host-specificity of the purified Nod factor and the bacterium. Recent studies have begun to reveal the genetic and molecular basis of Nod-factor perception in legumes, a signaling system that also controls plant interactions with mycorrhizal fungi.