PLENTY, a hydroxyproline O-arabinosyltransferase, negatively regulates root nodule symbiosis in Lotus japonicus.
ABSTRACT: Legumes can survive in nitrogen-deficient environments by forming root-nodule symbioses with rhizobial bacteria; however, forming nodules consumes energy, and nodule numbers must thus be strictly controlled. Previous studies identified major negative regulators of nodulation in Lotus japonicus, including the small peptides CLAVATA3/ESR (CLE)-RELATED-ROOT SIGNAL1 (CLE-RS1), CLE-RS2, and CLE-RS3, and their putative major receptor HYPERNODULATION AND ABERRANT ROOT FORMATION1 (HAR1). CLE-RS2 is known to be expressed in rhizobia-inoculated roots, and is predicted to be post-translationally arabinosylated, a modification essential for its activity. Moreover, all three CLE-RSs suppress nodulation in a HAR1-dependent manner. Here, we identified PLENTY as a gene responsible for the previously isolated hypernodulation mutant plenty. PLENTY encoded a hydroxyproline O-arabinosyltransferase orthologous to ROOT DETERMINED NODULATION1 in Medicago truncatula. PLENTY was localized to the Golgi, and an in vitro analysis of the recombinant protein demonstrated its arabinosylation activity, indicating that CLE-RS1/2/3 may be substrates for PLENTY. The constitutive expression experiments showed that CLE-RS3 was the major candidate substrate for PLENTY, suggesting the substrate preference of PLENTY for individual CLE-RS peptides. Furthermore, a genetic analysis of the plenty har1 double mutant indicated the existence of another PLENTY-dependent and HAR1-independent pathway negatively regulating nodulation.
Project description:Autoregulatory negative-feedback loops play important roles in fine-balancing tissue and organ development. Such loops are composed of short-range intercellular signaling pathways via cell-cell communications. On the other hand, leguminous plants use a long-distance negative-feedback system involving root-shoot communication to control the number of root nodules, root lateral organs that harbor symbiotic nitrogen-fixing bacteria known as rhizobia. This feedback system, known as autoregulation of nodulation (AON), consists of two long-distance mobile signals: root-derived and shoot-derived signals. Two Lotus japonicus CLAVATA3/endosperm surrounding region (CLE)-related small peptides, CLE root signal1 (CLE-RS1) and CLE-RS2, function as root-derived signals and are perceived by a shoot-acting AON factor, the hypernodulation aberrant root formation1 (HAR1) receptor protein, an ortholog of Arabidopsis CLAVATA1, which is responsible for shoot apical meristem homeostasis. This peptide-receptor interaction is necessary for systemic suppression of nodulation. How the onset of nodulation activates AON and how optimal nodule numbers are maintained remain unknown, however. Here we show that an RWP-RK-containing transcription factor, nodule inception (NIN), which induces nodule-like structures without rhizobial infection when expressed ectopically, directly targets CLE-RS1 and CLE-RS2. Roots constitutively expressing NIN systemically repress activation of endogenous NIN expression in untransformed roots of the same plant in a HAR1-dependent manner, leading to systemic suppression of nodulation and down-regulation of CLE expression. Our findings provide, to our knowledge, the first molecular evidence of a long-distance autoregulatory negative-feedback loop that homeostatically regulates nodule organ formation.
Project description:Legumes utilize a shoot-mediated signaling system to maintain a mutualistic relationship with nitrogen-fixing bacteria in root nodules. In Lotus japonicus, shoot-to-root transfer of microRNA miR2111 that targets TOO MUCH LOVE, a nodulation suppressor in roots, has been proposed to explain the mechanism underlying nodulation control from shoots. However, the role of shoot-accumulating miR2111s for the systemic regulation of nodulation was not clearly shown. Here, we find L. japonicus has seven miR2111 loci, including those mapped through RNA-seq. MIR2111-5 expression in leaves is the highest among miR2111 loci and repressed after rhizobial infection depending on a shoot-acting HYPERNODULATION ABERRANT ROOT FORMATION1 (HAR1) receptor. MIR2111-5 knockout mutants show significantly decreased nodule numbers and miR2111 levels. Furthermore, grafting experiments using transformants demonstrate scions with altered miR2111 levels influence nodule numbers in rootstocks in a dose-dependent manner. Therefore, miR2111 accumulation in leaves through MIR2111-5 expression is required for HAR1-dependent systemic optimization of nodule number.
Project description:Dynein motors and regulatory complexes repeat every 96 nm along the length of motile cilia. Each repeat contains three radial spokes, RS1, RS2, and RS3, which transduct signals between the central microtubules and dynein arms. Each radial spoke has a distinct structure, but little is known about the mechanisms of assembly and function of the individual radial spokes. In Chlamydomonas, calmodulin and spoke-associated complex (CSC) is composed of FAP61, FAP91, and FAP251 and has been linked to the base of RS2 and RS3. We show that in Tetrahymena, loss of either FAP61 or FAP251 reduces cell swimming and affects the ciliary waveform and that RS3 is either missing or incomplete, whereas RS1 and RS2 are unaffected. Specifically, FAP251-null cilia lack an arch-like density at the RS3 base, whereas FAP61-null cilia lack an adjacent portion of the RS3 stem region. This suggests that the CSC proteins are crucial for stable and functional assembly of RS3 and that RS3 and the CSC are important for ciliary motility.
Project description:Legumes and rhizobia establish symbiosis in root nodules. To balance the gains and costs associated with the symbiosis, plants have developed two strategies for adapting to nitrogen availability in the soil: plants can regulate nodule number and/or stop the development or function of nodules. Although the former is accounted for by autoregulation of nodulation, a form of systemic long-range signaling, the latter strategy remains largely enigmatic. Here, we show that the Lotus japonicus NITRATE UNRESPONSIVE SYMBIOSIS 1 (NRSYM1) gene encoding a NIN-LIKE PROTEIN transcription factor acts as a key regulator in the nitrate-induced pleiotropic control of root nodule symbiosis. NRSYM1 accumulates in the nucleus in response to nitrate and directly regulates the production of CLE-RS2, a root-derived mobile peptide that acts as a negative regulator of nodule number. Our data provide the genetic basis for how plants respond to the nitrogen environment and control symbiosis to achieve proper plant growth.
Project description:Motile cilia and flagella are highly conserved organelles that play important roles in human health and development. We recently discovered a calmodulin- and spoke-associ-ated complex (CSC) that is required for wild-type motility and for the stable assembly of a subset of radial spokes. Using cryo-electron tomography, we present the first structure-based localization model of the CSC. Chlamydomonas flagella have two full-length radial spokes, RS1 and RS2, and a shorter RS3 homologue, the RS3 stand-in (RS3S). Using newly developed techniques for analyzing samples with structural heterogeneity, we demonstrate that the CSC connects three major axonemal complexes involved in dynein regulation: RS2, the nexin-dynein regulatory complex (N-DRC), and RS3S. These results provide insights into how signals from the radial spokes may be transmitted to the N-DRC and ultimately to the dynein motors. Our results also indicate that although structurally very similar, RS1 and RS2 likely serve different functions in regulating flagellar motility.
Project description:Radial spokes (RSs) are ubiquitous components of motile cilia and flagella and play an essential role in transmitting signals that regulate the activity of the dynein motors, and thus ciliary and flagellar motility. In some organisms, the 96 nm axonemal repeat unit contains only a pair of spokes, RS1 and RS2, while most organisms have spoke triplets with an additional spoke RS3. The spoke pairs in Chlamydomonas flagella have been well characterized, while spoke triplets have received less attention. Here, we used cryoelectron tomography and subtomogram averaging to visualize the three-dimensional structure of spoke triplets in Strongylocentrotus purpuratus (sea urchin) sperm flagella in unprecedented detail. Only small differences were observed between RS1 and RS2, but the structure of RS3 was surprisingly unique and structurally different from the other two spokes. We observed novel doublet specific features that connect RS2, RS3, and the nexin-dynein regulatory complex, three key ciliary and flagellar structures. The distribution of these doublet specific structures suggests that they could be important for establishing the asymmetry of dynein activity required for the oscillatory movement of cilia and flagella. Surprisingly, a comparison with other organisms demonstrated both that this considerable RS heterogeneity is conserved and that organisms with RS pairs contain the basal part of RS3. This conserved RS heterogeneity may also reflect functional differences between the spokes and their involvement in regulating ciliary and flagellar motility.
Project description:Peptides are signaling molecules regulating various aspects of plant development, including the balance between cell division and differentiation in different meristems. Among those, CLAVATA3/Embryo Surrounding Region-related (CLE-ESR) peptide activity depends on leucine-rich-repeat receptor-like-kinases (LRR-RLK) belonging to the subclass XI. In legume plants, such as the Medicago truncatula model, specific CLE peptides were shown to regulate root symbiotic nodulation depending on the LRR-RLK SUNN (Super Numeric Nodules). Amongst the ten M. truncatula LRR-RLK most closely related to SUNN, only one showed a nodule-induced expression, and was so-called MtNRLK1 (Nodule-induced Receptor-Like Kinase 1). MtNRLK1 expression is associated to root and nodule vasculature as well as to the proximal meristem and rhizobial infection zone in the nodule apex. Except for the root vasculature, the MtNRLK1 symbiotic expression pattern is different than the one of MtSUNN. Functional analyses either based on RNA interference, insertional mutagenesis, and overexpression of MtNRLK1 however failed to identify a significant nodulation phenotype, either regarding the number, size, organization or nitrogen fixation capacity of the symbiotic organs formed.
Project description:Because of the developmental similarities between root nodules induced by symbiotic rhizobia and root galls formed by parasitic nematodes, we investigated the involvement of nodulation genes in the infection of Medicago truncatula by the root knot nematode (RKN), Meloidogyne javanica. We found that gall formation, including giant cell formation, pericycle and cortical cell division, as well as egg laying, occurred successfully in the non-nodulating mutants nfp1 (nod factor perception1), nin1 (nodule inception1) and nsp2 (nodulation signaling pathway2) and the cytokinin perception mutant cre1 (cytokinin receptor1). Gall and egg formation were significantly reduced in the ethylene insensitive, hypernodulating mutant skl (sickle), and to a lesser extent, in the low nodulation, abscisic acid insensitive mutant latd/nip (lateral root-organ defective/numerous infections and polyphenolics). Despite its supernodulation phenotype, the sunn4 (super numeric nodules4) mutant, which has lost the ability to autoregulate nodule numbers, did not form excessive numbers of galls. Co-inoculation of roots with nematodes and rhizobia significantly reduced nodule numbers compared to rhizobia-only inoculated roots, but only in the hypernodulation mutant skl. Thus, this effect is likely to be influenced by ethylene signaling, but is not likely explained by resource competition between galls and nodules. Co-inoculation with rhizobia also reduced gall numbers compared to nematode-only infected roots, but only in the wild type. Therefore, the protective effect of rhizobia on nematode infection does not clearly depend on nodule number or on Nod factor signaling. Our study demonstrates that early nodulation genes that are essential for successful nodule development are not necessary for nematode-induced gall formation, that gall formation is not under autoregulation of nodulation control, and that ethylene signaling plays a positive role in successful RKN parasitism in M. truncatula.
Project description:Legumes control the nitrogen-fixing root nodule symbiosis in response to external and internal stimuli, such as nitrate, and via systemic autoregulation of nodulation (AON). Overexpression of the CLV3/ESR-related (CLE) pre-propeptide-encoding genes GmNIC1 (nitrate-induced and acting locally) and GmRIC1 (Bradyrhizobium-induced and acting systemically) suppresses soybean nodulation dependent on the activity of the nodulation autoregulation receptor kinase (GmNARK). This nodule inhibition response was used to assess the relative importance of key structural components within and around the CLE domain sequences of these genes. Using a site-directed mutagenesis approach, mutants were produced at each amino acid within the CLE domain (RLAPEGPDPHHN) of GmRIC1. This approach identified the Arg1, Ala3, Pro4, Gly6, Pro7, Asp8, His11, and Asn12 residues as critical to GmRIC1 nodulation suppression activity (NSA). In contrast, none of the mutations in conserved residues outside of the CLE domain showed compromised NSA. Chimeric genes derived from combinations of GmRIC1 and GmNIC1 domains were used to determine the role of each pre-propeptide domain in NSA differences that exist between the two peptides. It was found that the transit peptide and CLE peptide regions of GmRIC1 significantly enhanced activity of GmNIC1. In contrast, the comparable GmNIC1 domains reduced the NSA of GmRIC1. Identification of these critical residues and domains provides a better understanding of how these hormone-like peptides function in plant development and regulation.
Project description:Radial spokes are conserved macromolecular complexes that are essential for ciliary motility. A triplet of three radial spokes, RS1, RS2, and RS3, repeats every 96 nm along the doublet microtubules. Each spoke has a distinct base that docks to the doublet and is linked to different inner dynein arms. Little is known about the assembly and functions of individual radial spokes. A knockout of the conserved ciliary protein FAP206 in the ciliate Tetrahymena resulted in slow cell motility. Cryo-electron tomography showed that in the absence of FAP206, the 96-nm repeats lacked RS2 and dynein c. Occasionally, RS2 assembled but lacked both the front prong of its microtubule base and dynein c, whose tail is attached to the front prong. Overexpressed GFP-FAP206 decorated nonciliary microtubules in vivo. Thus FAP206 is likely part of the front prong and docks RS2 and dynein c to the microtubule.