Medicago LINC complexes function in nuclear morphology, nuclear movement, and root nodule symbiosis 1[OPEN]

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.National Science Foundation NSF-1440019, NSF-1613501Biotechnology and Biological Sciences Research Council BB/P007112/1European Molecular Biology Organization 699

Functional Characterisation of Two Channels Proteins Involved in Leguminous Symbiosis

Legume-rhizobial symbiosis results in the formation of a new organ, the nitrogen-fixing root nodule. A chemical communication between both partners accompanies the invasion of plant host cells by bacteria and the development of the root nodule. In response to plant-released flavonoids, rhizobia produce lipo-chito-oligosaccharide signalling molecules, so called Nod factors. Early signal transduction in legumes such as Lotus japonicus and Medicago truncatula, is associated with a succession of tightly orchestrated ion fluxes across different membrane systems of the host cell. The Nod factor perception at the plasma membrane triggers Ca2+ oscillations that are associated with the nucleus. CASTOR and POLLUX are required for Ca2+ spiking. Homology modeling suggested CASTOR and POLLUX might be ion channels. However, experimental confirmation was lacking. Therefore we performed biochemical and electrophysiological analysis to define their role. Here we show that CASTOR and POLLUX form two independent homocomplexes in the nuclear rim in planta. We reconstituted CASTOR in planar lipid bilayers and electrophysiological measurements revealed that CASTOR is a cation channel preferentially permeable to potassium. The permeability of the sequence-related POLLUX for cation could be as well demonstrated through expression of POLLUX in different yeast mutants. Furthermore, we demonstrate that a voltage-dependent magnesium blocking mechanism contributes to reduce the conductance of CASTOR at negative membrane potential. By screening a L. japonicus roots cDNA library using yeast-two-hybrid system, a SNF7 protein interacting with CASTOR was found which acts as positive regulator in the nodulation pathway. Collectively the data demonstrate that both CASTOR and POLLUX are nuclear localized cation channels. Therefore, we propose that CASTOR and POLLUX may act as counter ion channels to facilitate a rapid efflux of charge associated with the calcium efflux. Alternatively and not mutually exclusive, they may catalyze a nuclear membrane depolarization leading to the activation of calcium channels responsible for calcium spikin

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

A physical map covering the nsv locus that confers resistance to Melon necrotic spot virus in melon (Cucumis melo L.)

Melon necrotic spot virus (MNSV) is a member of the genus Carmovirus, which produces severe yield losses in melon and cucumber crops. The nsv gene is the only known natural source of resistance against MNSV in melon, and confers protection against all widespread strains of this virus. nsv has been previously mapped in melon linkage group 11, in a region spanning 5.9 cM, saturated with RAPD and AFLP markers. To identify the nsv gene by positional cloning, we started construction of a high-resolution map for this locus. On the basis of the two mapping populations, F2 and BC1, which share the same resistant parent PI 161375 (nsv/nsv), and using more than 3,000 offspring, a high-resolution genetic map has been constructed in the region around the nsv locus, spanning 3.2 cM between CAPS markers M29 and M132. The availability of two melon BAC libraries allowed for screening and the identification of new markers closer to the resistance gene, by means of BAC-end sequencing and mapping. We constructed a BAC contig in this region and identified the marker 52K20sp6, which co-segregates with nsv in 408 F2 and 2.727 BC1 individuals in both mapping populations. We also identified a single 100 kb BAC that physically contains the resistance gene and covers a genetic distance of 0.73 cM between both BAC ends. These are the basis for the isolation of the nsv recessive-resistance gene.This work was partly funded by grants GEN2003-20237-C06-02 and AGL2003-02739 from the Spanish ‘Ministerio de Ciencia y Tecnología’.Peer reviewe

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

Perception of biotic and abiotic stresses often leads to stomatal closure in plants 1,2. Rapid influx of calcium ions (Ca 2+) across the plasma membrane has an important role in this response, but the identity of the Ca 2+ channels involved has remained elusive 3,4. Here we report that the Arabidopsis thaliana Ca 2+-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 Ca 2+, 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 Ca 2+ channel and its activation mechanisms underlying stomatal closure during immune signalling, and suggests specificity in Ca 2+ influx mechanisms in response to different stresses

Le sujet en langue des signes française

Impersonal constructions are a highly debated issue in the studies of spoken languages, especially regarding the description of the Romance and Slavic languages. Impersonal constructions are linguistically distinguished from personal constructions, i. e. verbal forms associated with a referential argument in the subject position. The present study is devoted to impersonal constructions with a non-referential subject, while taking into account a very specific language : French Sign Language (LSF). While spoken languages are said to be visual-gestural and audio-vocal languages, signed languages are characterized by their exclusive visual-gestural modality, involving different features of their linguistic system, such as iconicity or – what is at issue here – the referential use of space. In sign linguistics, spatialization refers to the signer’s ability to introduce the reference to an entity linked with a specific location in space. For example, this referential strategy allows him or her to point at a location to refer back to the argument that was linked to it earlier in the discourse (Lillo-Martin, 1986 ; Bahan et al., 2000 ; Meurant, 2008 ; Risler, 2013). Therefore, we aim to study how a loss of referentiality can be expressed in a visual-gestural language. In the recent past, linguistic descriptions of sign languages have provided evidence of agent-defocusing structures, such as the passive (Janzen et al., 2001 ; Guitteny, 2005) or the impersonal constructions (Barberà & Quer, 2013 ; Kimmelman, 2016). Hence, on the basis of previous findings on impersonal structures in spoken languages, and taking into account studies describing the syntactic functioning of signed languages, this article analyses personal and impersonal structures in LSF. Different items lead our path of reflexion. Based on identification features of the subject in spoken languages, we define in this article the properties of the subject in LSF. With regard to the literature, we distinguish primarily two paths for analysing the subject in sign language. On the one hand, French studies reveal that thematic roles are predominant in the analysis of LSF, whereas the syntactic functions of the phrasal constituents are rarely set out. However, the interpreted sentences illustrate in general the choice of an active perspective : the agent corresponds to the syntactic function of subject. We can conclude from these analyses that, with agreement verbs (Padden, 1988), the starting point of the verbal movement is analysed as marking the agent-subject position. On the other hand, the study of Meir et al. (2007) offers different analysis possibilities, by assigning the syntactic function of the subject to the signer’s body, regardless of its thematic role. In light of these studies, our article highlights possible interpretations of personal and agent-defocusing constructions in French Sign Language, and raises the question whether the subject function is marked on a specific location or not. In order to conduct this study, we have chosen to work on empirical data, extracted from semi-spontaneous and narrative corpora, since they favor referential structures and anaphora. The analysis consists of two stages. The first part of this study is devoted to the analysis of basic sentences in LSF, in which the personal subject is specified, in order to define the features of the subject in this language. This analysis raises a syntactic specificity of sign language : like other signed languages (Lillo-Martin, 1986 ; Bahan et al., 2000 ; Kimmelman, 2017), LSF accepts the argument position of the subject, and/or the object, to be lexically empty. In the context of this study, we focus on the subject argument and note that the subject position is not systematically filled by a lexical component. Our analysis suggests that the arguments are mentioned by the expression of anaphora, in the predicative form : the syntactic mark of the subject argument on a spatial location allows the spatial instantiation of the argument in the inflection. Moreover, the observation of personal references allows us to note the role the body plays in the argumental structure, encoding the properties of the subject argument. Following Meir et al. (2007), we analyse this corporal locus as marking the syntactical function of the subject. However, we suggest that the syntactic function of the subject is marked by the corporal locus, irrespective of the type of the verb. The second part of this study is devoted to structures with an unspecified human subject and compares them to the personal constructions analysed in the first part. The comparative approach does not show syntactic distinctions between these personal and impersonal structures. Indeed, we observe both lexical and empty subject constructions with personal as with non-specified subjects. Finally, with respect to the role of the body, this comparative study allows us to observe that the body encodes the properties of the first argument regardless of its referentiality. Then, in the absence of syntactic differences between personal and impersonal references, only the semantic level allows us to notice the distinction. In personal as well as in agent-demoting constructions, this assignation of the subject function to the body calls into question the interpretation of the starting point of agreement verbs’ movement. Indeed, if the end point of the verbal movement – which represents the patient – is on the signer’s body, and is assigned the function of subject, it may consequently be a structure defocusing the agent. This assumption is corroborated by the observation of the use of specific spatial markings, on a high and lateral space, emphasizing the process by not referring to any specified agent. Similar spatial markings have been studied for Catalan Sign Language (LSC) (Barberà & Quer, 2013), giving us an interesting perspective to guide our analysis of this phenomenon. Moreover, previous French studies on the role of space in the actantial distribution in LSF describe the use of a high lateral space, on each side of the signer’s head, for the reference to an indefinite agent (Millet, 2004, 2006).These considerations in relation to the assignation of the subject syntactical function to the signer’s body or to the starting point of the verbal movement led us in our questioning of the passive or the active perspective. Though it is too soon to settle the question, this analysis suggests that a distinction between impersonal and passive constructions could be drawn. Thus, the questioning raised by this study will be the basis for further research on defocusing agent strategies and their semantic tendencies in LSF, with the aim of distinguishing impersonal and passive constructions in this language

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