Dhileepkumar Jayaraman

3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase1 Interacts with NORK and Is Crucial for Nodulation in Medicago truncatula

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.

Algal ancestor of land plants was preadapted for symbiosis

Significance Colonization of land by plants was a critical event for the emergence of extant ecosystems. The innovations that allowed the algal ancestor of land plants to succeed in such a transition remain unknown. Beneficial interaction with symbiotic fungi has been proposed as one of these innovations. Here we show that the genes required for this interaction appeared in a stepwise manner: Some evolved before the colonization of land by plants and others first appeared in land plants. We thus propose that the algal ancestor of land plants was preadapted for interaction with beneficial fungi and employed these gene networks to colonize land successfully. 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.

A role for the mevalonate pathway in early plant symbiotic signaling

Significance Metabolites of the mevalonate (MVA) pathway play essential roles in the regulation of growth and development in many organisms. In this study, we demonstrate that a key regulatory enzyme of the MVA pathway is directly involved in the signaling pathway that transduces endosymbiotic microbial signals in Medicago truncatula. Furthermore, we show that exogenous MVA application is sufficient to activate this transduction pathway. The use of mutants in the signaling pathway and a heterologous expression system provides evidence that the MVA pathway is a missing link between the initial perception of microbial signals at the host plasma membrane and the regulation of symbiotic gene expression in the nucleus. 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 Ca2+ concentration (Ca2+ spiking), which in turn leads to gene expression and downstream symbiotic responses. The activation of Ca2+ 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 Ca2+ 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 Ca2+ spiking and symbiotic gene expression in both wild-type plants and dmi2 mutants, but interestingly not in dmi1 mutants. Finally, MVA induced Ca2+ spiking in Human Embryonic Kidney 293 cells expressing DMI1. This demonstrates that the nuclear cation channel DMI1 is sufficient to support MVA-induced Ca2+ spiking in this heterologous system.

A proteomic atlas of the legume Medicago truncatula and its nitrogen-fixing endosymbiont Sinorhizobium meliloti

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.

Multifaceted investigation of metabolites during nitrogen fixation in Medicago via high resolution MALDI-MS imaging and ESI-MS.

AbstractLegumes 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. Graphical Abstractᅟ

Staying in touch: mechanical signals in plant-microbe interactions

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.

Leveraging Proteomics to Understand Plant–Microbe Interactions

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.

Response of Medicago truncatula Seedlings to Colonization by Salmonella enterica and Escherichia coli O157:H7

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.

Nitrogen fixation in a landrace of maize is supported by a mucilage-associated diazotrophic microbiota

Plants are associated with a complex microbiota that contributes to nutrient acquisition, plant growth, and plant defense. Nitrogen-fixing microbial associations are efficient and well characterized in legumes but are limited in cereals, including maize. We studied an indigenous landrace of maize grown in nitrogen-depleted soils in the Sierra Mixe region of Oaxaca, Mexico. This landrace is characterized by the extensive development of aerial roots that secrete a carbohydrate-rich mucilage. Analysis of the mucilage microbiota indicated that it was enriched in taxa for which many known species are diazotrophic, was enriched for homologs of genes encoding nitrogenase subunits, and harbored active nitrogenase activity as assessed by acetylene reduction and 15N2 incorporation assays. Field experiments in Sierra Mixe using 15N natural abundance or 15N-enrichment assessments over 5 years indicated that atmospheric nitrogen fixation contributed 29%–82% of the nitrogen nutrition of Sierra Mixe maize.

Comparison of Vacuum MALDI and AP-MALDI Platforms for the Mass Spectrometry Imaging of Metabolites Involved in Salt Stress in Medicago truncatula

Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is routinely used to determine the spatial distributions of various biomolecules in tissues. Recently, there has been an increased interest in creating higher resolution images using sources with more focused beams. One such source, an atmospheric pressure (AP) MALDI source from MassTech, has a laser capable of reaching spatial resolutions of 10 μm. Here, the AP-MALDI source coupled with a Q Exactive HF Orbitrap platform is compared to the commercial MALDI LTQ Orbitrap XL system using Medicago truncatula root nodules. AP-MALDI parameters, such as the S-lens value, capillary temperature, and spray voltage, were optimized on the Q Exactive-HF platform for optimal detection of plant metabolites. The performance of the two systems was evaluated for sensitivity, spatial resolution, and overall ability to detect plant metabolites. The commercial MALDI LTQ Orbitrap XL was superior regarding the number of compounds detected, as at least two times more m/z were detected compared to the AP-MALDI system. However, although the AP-MALDI source requires a spatial resolution higher than 10 μm to get the best signal, the spatial resolution at 30 μm is still superior compared to the 75 μm spatial resolution achieved on the MALDI platform. The AP-MALDI system was also used to investigate the metabolites present in M. truncatula roots and root nodules under high salt and low salt conditions. A discriminative analysis with SCiLS software revealed m/z ions specific to the control and salt conditions. This analysis revealed 44 m/z ions present at relatively higher abundances in the control samples, and 77 m/z enriched in the salt samples. Liquid chromatography-tandem MS was performed to determine the putative molecular identities of some of the mass ions enriched in each sample, including, asparagine, adenosine, and nicotianamine in the control samples, and arginine and soyasaponin I in the salt treated samples.