Evolution of endosymbiosis-mediated nuclear calcium signaling in land plants.

The ability of fungi to establish mycorrhizal associations with plants and enhance the acquisition of mineral nutrients stands out as a key feature of terrestrial life. Evidence indicates that arbuscular mycorrhizal (AM) association is a trait present in the common ancestor of land plants,1,2,3,4 suggesting that AM symbiosis was an important adaptation for plants in terrestrial environments.5 The activation of nuclear calcium signaling in roots is essential for AM within flowering plants.6 Given that the earliest land plants lacked roots, whether nuclear calcium signals are required for AM in non-flowering plants is unknown. To address this question, we explored the functional conservation of symbiont-induced nuclear calcium signals between the liverwort Marchantia paleacea and the legume Medicago truncatula. In M. paleacea, AM fungi penetrate the rhizoids and form arbuscules in the thalli.7 Here, we demonstrate that AM germinating spore exudate (GSE) activates nuclear calcium signals in the rhizoids of M. paleacea and that this activation is dependent on the nuclear-localized ion channel DOES NOT MAKE INFECTIONS 1 (MpaDMI1). However, unlike flowering plants, MpaDMI1-mediated calcium signaling is only required for the thalli colonization but not for the AM penetration within rhizoids. We further demonstrate that the mechanism of regulation of DMI1 has diverged between M. paleacea and M. truncatula, including a key amino acid residue essential to sustain DMI1 in an inactive state. Our study reveals functional evolution of nuclear calcium signaling between liverworts and flowering plants and opens new avenues of research into the mechanism of endosymbiosis signaling.

A highly diversified NLR cluster in melon contains homologs that confer powdery mildew and aphid resistance.

Podosphaera xanthii is the main causal agent of powdery mildew (PM) on Cucurbitaceae. In Cucumis melo, the Pm-w resistance gene, which confers resistance to P. xanthii, is located on chromosome 5 in a cluster of nucleotide-binding leucine-rich repeat receptors (NLRs). We used positional cloning and transgenesis, to isolate the Pm-wWMR 29 gene encoding a coiled-coil NLR (CC-NLR). Pm-wWMR 29 conferred high level of resistance to race 1 of PM and intermediate level of resistance to race 3 of PM. Pm-wWMR 29 turned out to be a homolog of the Aphis gossypii resistance gene Vat-1PI 161375. We confirmed that Pm-wWMR 29 did not confer resistance to aphids, while Vat-1PI 161375 did not confer resistance to PM. We showed that both homologs were included in a highly diversified cluster of NLRs, the Vat cluster. Specific Vat-1PI 161375 and Pm-wWMR 29 markers were present in 10% to 13% of 678 accessions representative of wild and cultivated melon types worldwide. Phylogenic reconstruction of 34 protein homologs of Vat-1PI 161375 and Pm-wWMR 29 identified in 24 melon accessions revealed an ancestor with four R65aa-a specific motif in the LRR domain, evolved towards aphid and virus resistance, while an ancestor with five R65aa evolved towards PM resistance. The complexity of the cluster comprising the Vat/Pm-w genes and its diversity in melon suggest that Vat homologs may contribute to the recognition of a broad range of yet to be identified pests and pathogens.

A novel mutant allele of AtCNGC15 reveals a dual function of nuclear calcium release in the root meristem.

Calcium release to the nucleoplasm of root meristem cells was demonstrated to modulate root development. The calcium channel encoded by cyclic nucleotide-gated channel (CNGC) 15 localizes at the nuclear envelope in young Arabidopsis seedlings. In contrast, at later stages of root growth, overexpression analysis showed that AtCNGC15 can relocalize to the plasma membrane to mediate primary nitrate-induced gene expression. This raises the question as to whether nuclear localized AtCNGC15 is required for root apical meristem development in young Arabidopsis seedlings, and whether nitrate signalling occurs independently of nuclear localized AtCNGC15 at this developmental stage. In this study, we characterize a novel mutant allele of AtCNGC15 and demonstrate that the mutation of a highly conserved aspartic acid in the C-linker domain is sufficient to impair the gating of AtCNCG15. We demonstrate that AtCNGC15 mediates the nuclear calcium release that modulates root apical meristem development and nitrate-induced LBD39 expression. We also show that, in the presence of nitrate, the relocalization of AtCNGC15 at the plasma membrane occurs specifically in the columella cells. Our results further suggest that the induction of LBD37, LBD38, and LBD39 in the presence of nitrate is modulated by different inputs of cytoplasmic or nuclear calcium release.

Engineered CaM2 modulates nuclear calcium oscillation and enhances legume root nodule symbiosis.

SignificanceOscillations in intracellular calcium concentration play an essential role in the regulation of multiple cellular processes. In plants capable of root endosymbiosis with nitrogen-fixing bacteria and/or arbuscular mycorrhizal fungi, nuclear localized calcium oscillations are essential to transduce the microbial signal. Although the ion channels required to generate the nuclear localized calcium oscillations have been identified, their mechanisms of regulation are unknown. Here, we combined proteomics and engineering approaches to demonstrate that the calcium-bound form of the calmodulin 2 (CaM2) associates with CYCLIC NUCLEOTIDE GATED CHANNEL 15 (CNGC15s), closing the channels and providing the negative feedback to sustain the oscillatory mechanism. We further unraveled that the engineered CaM2 accelerates early endosymbioses and enhanced root nodule symbiosis but not arbuscular mycorrhization.

Publisher Correction: The calcium-permeable channel OSCA1.3 regulates plant stomatal immunity.

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

The calcium-permeable channel OSCA1.3 regulates plant stomatal immunity.

Perception of biotic and abiotic stresses often leads to stomatal closure in plants1,2. Rapid influx of calcium ions (Ca2+) across the plasma membrane has an important role in this response, but the identity of the Ca2+ channels involved has remained elusive3,4. Here we report that the Arabidopsis thaliana Ca2+-permeable channel OSCA1.3 controls stomatal closure during immune signalling. OSCA1.3 is rapidly phosphorylated upon perception of pathogen-associated molecular patterns (PAMPs). Biochemical and quantitative phosphoproteomics analyses reveal that the immune receptor-associated cytosolic kinase BIK1 interacts with and phosphorylates the N-terminal cytosolic loop of OSCA1.3 within minutes of treatment with the peptidic PAMP flg22, which is derived from bacterial flagellin. Genetic and electrophysiological data reveal that OSCA1.3 is permeable to Ca2+, and that BIK1-mediated phosphorylation on its N terminus increases this channel activity. Notably, OSCA1.3 and its phosphorylation by BIK1 are critical for stomatal closure during immune signalling, and OSCA1.3 does not regulate stomatal closure upon perception of abscisic acid-a plant hormone associated with abiotic stresses. This study thus identifies a plant Ca2+ channel and its activation mechanisms underlying stomatal closure during immune signalling, and suggests specificity in Ca2+ influx mechanisms in response to different stresses.

Mildew Locus O facilitates colonization by arbuscular mycorrhizal fungi in angiosperms.

Loss of barley Mildew Resistance Locus O (MLO) is known to confer durable and robust resistance to powdery mildew (Blumeria graminis), a biotrophic fungal leaf pathogen. Based on the increased expression of MLO in mycorrhizal roots and its presence in a clade of the MLO family that is specific to mycorrhizal-host species, we investigated the potential role of MLO in arbuscular mycorrhizal interactions. Using mutants from barley (Hordeum vulgare), wheat (Triticum aestivum), and Medicago truncatula, we demonstrate a role for MLO in colonization by the arbuscular mycorrhizal fungus Rhizophagus irregularis. Early mycorrhizal colonization was reduced in mlo mutants of barley, wheat, and M. truncatula, and this was accompanied by a pronounced decrease in the expression of many of the key genes required for intracellular accommodation of arbuscular mycorrhizal fungi. These findings show that clade IV MLOs are involved in the establishment of symbiotic associations with beneficial fungi, a role that has been appropriated by powdery mildew.

Nuclear calcium signatures are associated with root development.

In plants, nuclear Ca2+ releases are essential to the establishment of nitrogen-fixing and phosphate-delivering arbuscular mycorrhizal endosymbioses. In the legume Medicago truncatula, these nuclear Ca2+ signals are generated by a complex of nuclear membrane-localised ion channels including the DOES NOT MAKE INFECTIONS 1 (DMI1) and the cyclic nucleotide-gated channels (CNGC) 15s. DMI1 and CNCG15s are conserved among land plants, suggesting roles for nuclear Ca2+ signalling that extend beyond symbioses. Here we show that nuclear Ca2+ signalling initiates in the nucleus of Arabidopsis root cells and that these signals are correlated with primary root development, including meristem development and auxin homeostasis. In addition, we demonstrate that altering genetically AtDMI1 is sufficient to modulate the nuclear Ca2+ signatures, and primary root development. This finding supports the postulate that stimulus-specific information can be encoded in the frequency and duration of a Ca2+ signal and thereby regulate cellular function.

Medicago LINC Complexes Function in Nuclear Morphology, Nuclear Movement, and Root Nodule Symbiosis.

Nuclear movement is involved in cellular and developmental processes across eukaryotic life, often driven by Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes, which bridge the nuclear envelope (NE) via the interaction of Klarsicht/ANC-1/Syne-1 Homology (KASH) and Sad1/UNC-84 (SUN) proteins. Arabidopsis (Arabidopsis thaliana) LINC complexes are involved in nuclear movement and positioning in several cell types. Observations since the 1950s have described targeted nuclear movement and positioning during symbiosis initiation between legumes and rhizobia, but it has not been established whether these movements are functional or incidental. Here, we identify and characterize LINC complexes in the model legume Medicago truncatula We show that LINC complex characteristics such as NE localization, dependence of KASH proteins on SUN protein binding for NE enrichment, and direct SUN-KASH binding are conserved between plant species. Using a SUN dominant-negative strategy, we demonstrate that LINC complexes are necessary for proper nuclear shaping and movement in Medicago root hairs, and are important for infection thread initiation and nodulation.

Calcium Signals in the Plant Nucleus: Origin and Function.

The universality of calcium as an intracellular messenger depends on the dynamics of its spatial and temporal release from calcium stores. Accumulating evidence over the past two decades supports an essential role for nuclear calcium signalling in the transduction of specific stimuli into cellular responses. This review focusses on mechanisms underpinning changes in nuclear calcium concentrations and discusses what is known so far, about the origin of the nuclear calcium signals identified, primarily in the context of microbial symbioses and abiotic stresses.

Dual Color Sensors for Simultaneous Analysis of Calcium Signal Dynamics in the Nuclear and Cytoplasmic Compartments of Plant Cells.

Spatiotemporal changes in cellular calcium (Ca2+) concentrations are essential for signal transduction in a wide range of plant cellular processes. In legumes, nuclear and perinuclear-localized Ca2+ oscillations have emerged as key signatures preceding downstream symbiotic signaling responses. Förster resonance energy transfer (FRET) yellow-based Ca2+ cameleon probes have been successfully exploited to measure the spatiotemporal dynamics of symbiotic Ca2+ signaling in legumes. Although providing cellular resolution, these sensors were restricted to measuring Ca2+ changes in single subcellular compartments. In this study, we have explored the potential of single fluorescent protein-based Ca2+ sensors, the GECOs, for multicolor and simultaneous imaging of the spatiotemporal dynamics of cytoplasmic and nuclear Ca2+ signaling in root cells. Single and dual fluorescence nuclear and cytoplasmic-localized GECOs expressed in transgenic Medicago truncatula roots and Arabidopsis thaliana were used to successfully monitor Ca2+ responses to microbial biotic and abiotic elicitors. In M. truncatula, we demonstrate that GECOs detect symbiosis-related Ca2+ spiking variations with higher sensitivity than the yellow FRET-based sensors previously used. Additionally, in both M. truncatula and A. thaliana, the dual sensor is now able to resolve in a single root cell the coordinated spatiotemporal dynamics of nuclear and cytoplasmic Ca2+ signaling in vivo. The GECO-based sensors presented here therefore represent powerful tools to monitor Ca2+ signaling dynamics in vivo in response to different stimuli in multi-subcellular compartments of plant cells.

Highlight on the dynamic organization of the nucleus.

The last decade has seen rapid advances in our understanding of the proteins of the nuclear envelope, which have multiple roles including positioning the nucleus, maintaining its structural organization, and in events ranging from mitosis and meiosis to chromatin positioning and gene expression. Diverse new and stimulating results relating to nuclear organization and genome function from across kingdoms were presented in a session stream entitled "Dynamic Organization of the Nucleus" at this year's Society of Experimental Biology (SEB) meeting in Brighton, UK (July 2016). This was the first session stream run by the Nuclear Dynamics Special Interest Group, which was organized by David Evans, Katja Graumann (both Oxford Brookes University, UK) and Iris Meier (Ohio State University, USA). The session featured presentations on areas relating to nuclear organization across kingdoms including the nuclear envelope, chromatin organization, and genome function.

Nuclear-localized cyclic nucleotide-gated channels mediate symbiotic calcium oscillations.

Nuclear-associated Ca(2+) oscillations mediate plant responses to beneficial microbial partners--namely, nitrogen-fixing rhizobial bacteria that colonize roots of legumes and arbuscular mycorrhizal fungi that colonize roots of the majority of plant species. A potassium-permeable channel is known to be required for symbiotic Ca(2+) oscillations, but the calcium channels themselves have been unknown until now. We show that three cyclic nucleotide-gated channels in Medicago truncatula are required for nuclear Ca(2+) oscillations and subsequent symbiotic responses. These cyclic nucleotide-gated channels are located at the nuclear envelope and are permeable to Ca(2+) We demonstrate that the cyclic nucleotide-gated channels form a complex with the postassium-permeable channel, which modulates nuclear Ca(2+) release. These channels, like their counterparts in animal cells, might regulate multiple nuclear Ca(2+) responses to developmental and environmental conditions.

Abscisic acid promotion of arbuscular mycorrhizal colonization requires a component of the PROTEIN PHOSPHATASE 2A complex.

Legumes can establish intracellular interactions with symbiotic microbes to enhance their fitness, including the interaction with arbuscular mycorrhizal (AM) fungi. AM fungi colonize root epidermal cells to gain access to the root cortex, and this requires the recognition by the host plant of fungus-made mycorrhizal factors. Genetic dissection has revealed the symbiosis signaling pathway that allows the recognition of AM fungi, but the downstream processes that are required to promote fungal infection are poorly understood. Abscisic acid (ABA) has been shown to promote arbuscule formation in tomato (Solanum lycopersicum). Here, we show that ABA modulates the establishment of the AM symbiosis in Medicago truncatula by promoting fungal colonization at low concentrations and impairing it at high concentrations. We show that the positive regulation of AM colonization via ABA requires a PROTEIN PHOSPHATASE 2A (PP2A) holoenzyme subunit, PP2AB'1. Mutations in PP2AB'1 cause reduced levels of AM colonization that cannot be rescued with permissive ABA application. The action of PP2AB'1 in response to ABA is unlinked to the generation of calcium oscillations, as the pp2aB'1 mutant displays a normal calcium response. This contrasts with the application of high concentrations of ABA that impairs mycorrhizal factor-induced calcium oscillations, suggesting different modes of action of ABA on the AM symbiosis. Our work reveals that ABA functions at multiple levels to regulate the AM symbiosis and that a PP2A phosphatase is required for the ABA promotion of AM colonization.

The role of DMI1 in establishing Ca (2+) oscillations in legume symbioses.

Calcium (Ca (2+)) is a key secondary messenger in many plant signaling pathways. One such pathway is the SYM pathway, required in the establishment of both arbuscular mycorrhizal and rhizobial root symbioses with legume host plants. (1) When the host plant has perceived the diffusible signals from the microbial symbionts, one of the earliest physiological responses are Ca (2+) oscillations in and around the nucleus. (2) These oscillations are essential for activating downstream gene expression, but the precise mechanisms of encoding and decoding the Ca (2+) signals are unclear and still under intense investigation. Here we put forward a hypothesis for the mechanism of the cation channel DMI1.

Two microRNAs linked to nodule infection and nitrogen-fixing ability in the legume Lotus japonicus.

Legumes overcome nitrogen shortage by developing root nodules in which symbiotic bacteria fix atmospheric nitrogen in exchange for host-derived carbohydrates and mineral nutrients. Nodule development involves the distinct processes of nodule organogenesis, bacterial infection, and the onset of nitrogen fixation. These entail profound, dynamic gene expression changes, notably contributed to by microRNAs (miRNAs). Here, we used deep-sequencing, candidate-based expression studies and a selection of Lotus japonicus mutants uncoupling different symbiosis stages to identify miRNAs involved in symbiotic nitrogen fixation. Induction of a noncanonical miR171 isoform, which targets the key nodulation transcription factor Nodulation Signaling Pathway2, correlates with bacterial infection in nodules. A second candidate, miR397, is systemically induced in the presence of active, nitrogen-fixing nodules but not in that of noninfected or inactive nodule organs. It is involved in nitrogen fixation-related copper homeostasis and targets a member of the laccase copper protein family. These findings thus identify two miRNAs specifically responding to symbiotic infection and nodule function in legumes.

Buffering capacity explains signal variation in symbiotic calcium oscillations.

Legumes form symbioses with rhizobial bacteria and arbuscular mycorrhizal fungi that aid plant nutrition. A critical component in the establishment of these symbioses is nuclear-localized calcium (Ca(2+)) oscillations. Different components on the nuclear envelope have been identified as being required for the generation of the Ca(2+) oscillations. Among these an ion channel, Doesn't Make Infections1, is preferentially localized on the inner nuclear envelope and a Ca(2+) ATPase is localized on both the inner and outer nuclear envelopes. Doesn't Make Infections1 is conserved across plants and has a weak but broad similarity to bacterial potassium channels. A possible role for this cation channel could be hyperpolarization of the nuclear envelope to counterbalance the charge caused by the influx of Ca(2+) into the nucleus. Ca(2+) channels and Ca(2+) pumps are needed for the release and reuptake of Ca(2+) from the internal store, which is hypothesized to be the nuclear envelope lumen and endoplasmic reticulum, but the release mechanism of Ca(2+) remains to be identified and characterized. Here, we develop a mathematical model based on these components to describe the observed symbiotic Ca(2+) oscillations. This model can recapitulate Ca(2+) oscillations, and with the inclusion of Ca(2+)-binding proteins it offers a simple explanation for several previously unexplained phenomena. These include long periods of frequency variation, changes in spike shape, and the initiation and termination of oscillations. The model also predicts that an increase in buffering capacity in the nucleoplasm would cause a period of rapid oscillations. This phenomenon was observed experimentally by adding more of the inducing signal.

Vapyrin, a gene essential for intracellular progression of arbuscular mycorrhizal symbiosis, is also essential for infection by rhizobia in the nodule symbiosis of Medicago truncatula.

Intracellular invasion of root cells is required for the establishment of successful endosymbioses in legumes of both arbuscular mycorrhizal (AM) fungi and rhizobial bacteria. In both interactions a requirement for successful entry is the activation of a common signalling pathway that includes five genes required to generate calcium oscillations and two genes required for the perception of the calcium response. Recently, it has been discovered that in Medicago truncatula, the Vapyrin (VPY) gene is essential for the establishment of the arbuscular mycorrhizal symbiosis. Here, we show by analyses of mutants that the same gene is also required for rhizobial colonization and nodulation. VPY encodes a protein featuring a Major Sperm Protein domain, typically featured on proteins involved in membrane trafficking and biogenesis, and a series of ankyrin repeats. Plants mutated in this gene have abnormal rhizobial infection threads and fewer nodules, and in the case of interactions with AM fungi, epidermal penetration defects and aborted arbuscule formation. Calcium spiking in root hairs in response to supplied Nod factors is intact in the vpy-1 mutant. This, and the elevation of VPY transcripts upon application of Nod factors which we show to be dependent on NFP, DMI1, and DMI3, indicates that VPY acts downstream of the common signalling pathway.

How close are we to nitrogen-fixing cereals?

Engineering nitrogen-fixing cereals is essential for sustainable food production for the projected global population of 9 billion people in 2050. This process will require engineering cereals for nodule organogenesis and infection by nitrogen-fixing bacteria. The symbiosis signalling pathway is essential to establish both bacterial infection and nodule organogenesis in legumes and is also necessary for the establishment of mycorrhizal colonisation. Hence this signalling pathway is also present in cereals and it should be feasible to engineer this signalling pathway for cereal recognition of nitrogen-fixing bacteria. However, establishing a fully function nitrogen-fixing symbiosis in cereals will probably require additional genetic engineering for bacterial colonisation and nodule organogenesis.