RNA Interference highlights the role of CCaMK in dissemination of endosymbionts in the aeschynomeneae legume arachis

In legume–rhizobia symbiosis, Ca2+/Calmodulin-dependent Protein Kinase (CCaMK) is essential for rhizobial invasion through infection threads in the epidermis and nodule organogenesis in the cortex. Though CCaMK is actively transcribed in the infected zone of nodules, its role in the later stages of nodule development remain elusive because of the epidermal arrest of “loss-of-function” mutants. In Aeschynomeneae legumes such as Arachis hypogea, rhizobia directly access the cortex, where nodule organogenesis as well as endosymbiont dissemination take place by multiplication of infected cortical cells. We characterized CCaMK (GI:195542474) from A. hypogea and downregulated the kinase through RNA interference (RNAi) to understand its role during organogenesis of its characteristic aeschynomenoid nodules. In CCaMK downregulated plants, the inception of nodules was delayed by approximately 4 weeks and nodulation capacity was decreased (>90%). The infected zones of the RNA interference nodules were scattered with uninfected or binucleated cells as opposed to the homogeneous infection zone in empty-vector-transformed nodules. Symbiosomes in RNAi nodules were pleomorphic with diverse geometrical shapes or arrested during division in the final stages of their fission as opposed to uniform-sized, spherical symbiosomes in empty-vector-transformed nodules. Together, our results reveal CCaMK to be essential for development of functional aeschynomenoid nodules, with a critical role in rhizobial dissemination during nodule organogenesis

Characterization of the regulatory domain of the γ-subunit of phosphorylase kinase

Phosphorylase kinase is a multimeric protein kinase (α4β4γ4δ4) whose enzymatic activity is conferred by its γ-subunit. A library of 18 overlapping synthetic peptides spanning residues 277-386 of the γ-subunit has been prepared to use in identifying important regulatory structures in the protein. In the present study, the library was screened to identify regions that might function as autoinhibitory domains. Peptides from two distinct regions were found to inhibit the Ca2+-activated holoenzyme. The same regions were previously found to bind calmodulin (i.e. the δ-subunit; Dasgupta, M. Honeycutt, T. and Blumenthal, D. K.(1989) J. Biol. Chem. 264, 17156-17163). The most potent substrate antagonist peptides were PhK13 (residues 302-326; Ki = 300 nM) and PhK5 (residues 342-366; Ki = 20 μM). Both peptides inhibited the holoenzyme competitively with respect to phosphorylase b and noncompetitively with respect to Mg•ATP. When the pattern of inhibition with both peptides present was analyzed, inhibition was observed to be synergistic and modestly cooperative indicating that the two peptides can simultaneously occupy the protein substrate-binding site(s). These data are consistent with a model in which the regions of the γ-subunit represented by PhK5 and PhK13 work in concert as regulatory subdomains that transduce Ca2+-induced conformational changes in the δ-subunit to the catalytic γ-subunit through a pseudosubstrate autoinhibitory mechanism

IntGeom: a server for the calculation of the interaction geometry between planar groups in proteins

IntGeom is a server for the calculation of the relative orientation between any two planar groups in protein side chains. IntGeom1 considers ten planar groups, while IntGeom2 is meant for studying the contact between a S-containing group and an aromatic residue. When the interaction is between two aromatic residues or involving an aromatic ring with Pro or Arg or an amide side chain, the occurrence of any C-H···π (N-H···π) interaction is also studied. All contacts between any two of the above types of residues juxtaposed on the protein structure can be displayed. The software is available at: http://www.boseinst.ernet.in/resources/bioinfo/stag.html

Activation and inhibition of phosphorylase kinase by monospecific antibodies raised against peptides from the regulatory domain of the γ-subunit

The C terminus of the catalytic γ-subunit of phosphorylase kinase comprises a regulatory domain that contains regions important for subunit interactions and autoinhibitory functions. Monospecific antibodies raised against four synthetic peptides from this region, PhK1 (362-386), PhK5 (342-366), PhK9 (322-346) and PhK13 (302-326), were found to have significant effects on the catalytic activities of phosphorylase kinase holoenzyme and the γ•δ complex. Antibodies raised against the very C terminus of the γ-subunit, anti-PhK1 and anti-PhK5, markedly activated both holoenzyme and the γ•δ complex, in the presence and absence of Ca2+. In the presence of Ca2+ at pH 8.2, anti-PhK1 activated the holoenzyme more than 11-fold and activated the γ•δ complex 2.5-fold. Activation of the holoenzyme and the γ•δ complex by anti-PhK5 was 50-70 % of that observed with anti-PhK1. Prior phosphorylation of the holoenzyme by the cAMP-dependent protein kinase blocked activation by both anti-PhK1 and anti-PhK5. Antibodies raised against the peptides from the N terminus of the regulatory domain, anti-PhK9 and anti-PhK13, were inhibitory, with their greatest effects on the γ•δ complex. These data demonstrate that the binding of antibodies to specific regions within the regulatory domain of the γ-subunit can augment or inhibit structural changes and subunit interactions important in regulating phosphorylase kinase activity

A phylogenetic framework of the legume genus Aeschynomene for comparative genetic analysis of the Nod-dependent and Nod-independent symbioses

Background : Among semi-aquatic species of the legume genus Aeschynomene, some have the property of being nodulated by photosynthetic Bradyrhizobium lacking the nodABC genes necessary for the synthesis of Nod factors. Knowledge of the specificities underlying this Nod-independent symbiosis has been gained from the model legume Aeschynomene evenia but our understanding remains limited due to the lack of comparative genetics with related taxa using a Nod factor-dependent process. To fill this gap, we combined different approaches to perform a thorough comparative analysis in the genus Aeschynomene. Results: This study significantly broadened previous taxon sampling, including in allied genera, in order to construct a comprehensive phylogeny. In the phylogenetic tree, five main lineages were delineated, including a novel lineage, the Nod-independent clade and another one containing a polytomy that comprised several Aeschynomene groups and all the allied genera. This phylogeny was matched with data on chromosome number, genome size and low-copy nuclear gene sequences to reveal the diploid species and a polytomy containing mostly polyploid taxa. For these taxa, a single allopolyploid origin was inferred and the putative parental lineages were identified. Finally, nodulation tests with different Bradyrhizobium strains revealed new nodulation behaviours and the diploid species outside of the Nod-independent clade were compared for their experimental tractability and genetic diversity. Conclusions: The extended knowledge of the genetics and biology of the different lineages sheds new light of the evolutionary history of the genus Aeschynomene and they provide a solid framework to exploit efficiently the diversity encountered in Aeschynomene legumes. Notably, our backbone tree contains all the species that are diploid and it clarifies the genetic relationships between the Nod-independent clade and the Nod-dependent lineages. This study enabled the identification of A. americana and A. patula as the most suitable species to undertake a comparative genetic study of the Nod-independent and Nod-dependent symbioses

Phosphorylation−dephosphorylation of light-harvesting complex II as a response to variation in irradiance is thiol sensitive and thylakoid sufficient: modulation of the sensitivity of the phenomenon by a peripheral component

Downregulation of phosphorylation of chlorophyll a/b-binding proteins (LHCII) of the photosystem II at high irradiance could only be demonstrated with leaf discs but not in isolated thylakoids. The present view suggests this phenomenon to be regulated by stromal thioredoxin. Here, we show that high-light inactivation of LHCII phosphorylation can be reproduced in isolated thylakoids and have explained the apparent absence of inactivation in vitro to be due to the derepressed activity of a peripheral kinase. We investigated this phenomenon with Arachis hypogea thylakoids prepared with (Th:A) or without (Th:B) tricine, where tricine is known for removing peripheral proteins from thylakoids. While LHCII remained phosphorylated at high irradiance in Th:B, the response of Th:A mimicked Arachis leaflets where LHCII was transiently phosphorylated with irradiance. LHCII phosphorylation in Th:A was sensitive to thiol reducing conditions, but in Th:B, the phenomenon became insensitive to thiol reduction following illumination. Washing Th:B with tricine made them resemble Th:A and conversely, Th:A reconstituted with the Tricine extract resembled Th:B with respect to both irradiance response and thiol sensitivity. In vitro phosphorylation reactions indicated a thiol insensitive kinase activity to be present in the Tricine extract that was capable of phosphorylating histone H1 as well as purified LHCII. This peripherally associated kinase activity explained the sustenance of LHCII phosphorylation as well as its thiol insensitivity at high irradiance in Th:B thylakoids. Contrary to the current view, our results clearly show that irradiance dependent phosphorylation and dephosphorylation of LHCII is a thylakoid sufficient phenomenon, although it remained open to regulation by thiol redox state modulation

Gamma-subunit of skeletal muscle phosphorylase kinase contains two noncontiguous domains that act in concert to bind calmodulin.

Journal ArticlePhosphorylase kinase is a Ca2+-regulated, multisubunit enzyme that contains calmodulin as an integral subunit (termed the delta-subunit). Ca2+-dependent activity of the enzyme is thought to be regulated by direct interaction of the delta-subunit with the catalytic subunit (the gamma-subunit) in the holoenzyme complex. In order to systematically search for putative calmodulin (delta-subunit)-binding domain(s) in the gamma-subunit of phosphorylase kinase, a series of 18 overlapping peptides corresponding to the C terminus of the gamma-subunit was chemically synthesized using a tea bag method. The calmodulin-binding activity of each peptide was tested for its ability to inhibit Ca2+/calmodulin-dependent activation of myosin light chain kinase. Data were obtained indicating that two distinct regions in the gamma-subunit, one spanning residues 287-331 (termed domain-N) and the other residues 332-371 (domain-C), are capable of binding calmodulin with nanomolar affinity. Peptides from both of these two domains also inhibited calmodulin-dependent reactivation of denatured gamma-subunit. The interactions of peptides from both domain-N and domain-C with calmodulin were found to be Ca2+-dependent. Dixon plots obtained using mixtures of peptides from domain-N and domain-C indicate that these two domains can bind simultaneously to a single molecule of calmodulin. Multiple contacts between the gamma-subunit and calmodulin (delta-subunit), as indicated by our data, may help to explain why strongly denaturing conditions are required to dissociate these two subunits, whereas complexes of calmodulin with most other target enzymes can be readily dissociated by merely lowering Ca2+ to submicromolar concentrations. Comparison of the sequences of the two calmodulin-binding domains in the gamma-subunit of phosphorylase kinase with corresponding regions in troponin I indicates similarities that may have functional and evolutionary significance

Does SUNN-SYMRK crosstalk occur in medicago truncatulafor regulating nodule organogenesis

Recently we reported that overexpression of intracellular kinase domain of Symbiosis Receptor Kinase (SYMRK-kd) hyperactivated spontaneous nodulation in Medicago truncatula indicating the importance of SYMRK ectodomain in restricting nodule number. To clarify whether sunn and sickle pathways were overcome by SYMRK-kd for hyperactivation of nodule organogenesis, we overexpressed SYMRK-kd in these mutants and analyzed for spontaneous nodulation in absence of rhizobia. Spontaneous nodulation in skl/SYMRK-kd roots was 2-fold higher than A17/SYMRK-kd roots indicating nodule organogenesis induced by SYMRK-kd to be ethylene sensitive. Intriguingly, sunn/SYMRK-kd roots failed to generate any spontaneous nodule which directly indicate the LRR-RLK SUNN to have a role in SYMRK-kd mediated nodule development under non-symbiotic conditions. We hypothesize a crosstalk between SUNN and SYMRK receptors for activation as well as restriction of nodule development

Transformed Hairy Roots of Arachis hypogea: A Tool for Studying Root Nodule Symbiosis in a Non–Infection Thread Legume of the Aeschynomeneae Tribe

Arachis hypogea is a non–“infection thread” (IT) legume where rhizobial entry or dissemination in the nodules never involves IT. Rhizobia invade through epidermal “cracks” and directly access the cortical cells to develop the characteristic aeschynomenoid nodules. For investigating these nonclassical nodulation features in Arachis spp., we developed an efficient procedure for Agrobacterium rhizogenes R1000-mediated transformation of this plant. In this study, we optimized the induction of hairy roots and nodulation of composite Arachis hypogea plants in the presence of Bradyrhizobium sp. (Arachis) strain NC92. 35S promoterdriven green fluorescent protein and β-glucuronidase expression indicated transformation frequency to be above 80%. The transformed roots had the characteristic rosettetype root hairs and had normal level of expression of symbiosis- related genes SymRK and CCaMK. The transgenic nodules resembled the wild-type nodules with an exception of 2 to 3%, where they structurally deviated from the wildtype nodules to form nodular roots. A 16S rRNA profile of an infected-zone metagenome indicated that identical populations of bradyrhizobia invaded both composite wild-type plants grown in natural soil. Our results demonstrate that Arachis hairy root is an attractive system for undertaking investigations of the nonclassical features associated with its nitrogen-fixing symbiotic interaction