A mobile loop near the active site acts as a switch between the dual activities of a viral protease/deubiquitinase.

The positive-strand RNA virus Turnip yellow mosaic virus (TYMV) encodes an ovarian tumor (OTU)-like protease/deubiquitinase (PRO/DUB) protein domain involved both in proteolytic processing of the viral polyprotein through its PRO activity, and in removal of ubiquitin chains from ubiquitylated substrates through its DUB activity. Here, the crystal structures of TYMV PRO/DUB mutants and molecular dynamics simulations reveal that an idiosyncratic mobile loop participates in reversibly constricting its unusual catalytic site by adopting "open", "intermediate" or "closed" conformations. The two cis-prolines of the loop form a rigid flap that in the most closed conformation zips up against the other side of the catalytic cleft. The intermediate and closed conformations also correlate with a reordering of the TYMV PRO/DUB catalytic dyad, that then assumes a classical, yet still unusually mobile, OTU DUB alignment. Further structure-based mutants designed to interfere with the loop's mobility were assessed for enzymatic activity in vitro and in vivo, and were shown to display reduced DUB activity while retaining PRO activity. This indicates that control of the switching between the dual PRO/DUB activities resides prominently within this loop next to the active site. Introduction of mutations into the viral genome revealed that the DUB activity contributes to the extent of viral RNA accumulation both in single cells and in whole plants. In addition, the conformation of the mobile flap was also found to influence symptoms severity in planta. Such mutants now provide powerful tools with which to study the specific roles of reversible ubiquitylation in viral infection

Identification and Molecular Characterization of the Chloroplast Targeting Domain of Turnip yellow mosaic virus Replication Proteins

Turnip yellow mosaic virus (TYMV) is a positive-strand RNA virus infecting plants. The TYMV 140K replication protein is a key organizer of viral replication complex (VRC) assembly, being responsible for recruitment of the viral polymerase and for targeting the VRCs to the chloroplast envelope where viral replication takes place. However, the structural requirements determining the subcellular localization and membrane association of this essential viral protein have not yet been defined. In this study, we investigated determinants for the in vivo chloroplast targeting of the TYMV 140K replication protein. Subcellular localization studies of deletion mutants identified a 41-residue internal sequence as the chloroplast targeting domain (CTD) of TYMV 140K; this sequence is sufficient to target GFP to the chloroplast envelope. The CTD appears to be located in the C-terminal extension of the methyltransferase domain—a region shared by 140K and its mature cleavage product 98K, which behaves as an integral membrane protein during infection. We predicted the CTD to fold into two amphipathic α-helices—a folding that was confirmed in vitro by circular dichroism spectroscopy analyses of a synthetic peptide. The importance for subcellular localization of the integrity of these amphipathic helices, and the function of 140K/98K, was demonstrated by performing amino acid substitutions that affected chloroplast targeting, membrane association and viral replication. These results establish a short internal α-helical peptide as an unusual signal for targeting proteins to the chloroplast envelope membrane, and provide new insights into membrane targeting of viral replication proteins—a universal feature of positive-strand RNA viruses

CML8, an Arabidopsis Calmodulin-Like Protein, Plays a Role in Pseudomonas syringae Plant Immunity

International audienceCalcium is a universal second messenger involved in various cellular processes including plant development and stress responses. Its conversion into biological responses requires the presence of calcium sensor relays such as calmodulin (CaM) and calmodulin-like (CML) proteins. While the role of CaM is well described, the functions CML proteins remain largely uncharacterized. Here, we show that Arabidopsis CML8 expression is strongly and transiently induced by Pseudomonas syringae, and reverse genetic approaches indicated that the overexpression of CML8 confers on plants a better resistance to pathogenic bacteria compared with wild-type, knock-down and knock-out lines, indicating that CML8 participates as a positive regulator in plant immunity. However, this difference disappeared when inoculations were performed using bacteria unable to inject effectors into a plant host cell or deficient for some effectors known to target the salicylic acid (SA) signaling pathway. SA content and PR1 protein accumulation were altered in CML8 transgenic lines, supporting a role for CML8 in SA-dependent processes. Pathogen-associated molecular pattern (PAMP) treatments with flagellin and elf18 peptides have no effects on CML8 gene expression and do not modify root growth of CML8 knock-down and overexpressing lines compared with wild-type plants. Collectively, our results support a role for CML8 in plant immunity against P. syringae

Primers used for RTqPCR experiments.

<p>Primers used for RTqPCR experiments.</p

Layouts of the two sides of the active site clefts in viral OTU PRO/DUBs and cellular OTU DUBs.

<p>For each enzyme, the top panel is the view from the P side with residues bridging the hydrophobic constriction as sticks and labeled. The bottom panel is the view from the P' side with residues known to contribute to catalysis as sticks and labeled. For these DUBs for which structures are available in both free and Ub-bound forms (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006714#ppat.1006714.s005" target="_blank">S5</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006714#ppat.1006714.s006" target="_blank">S6</a> Figs), the free enzyme is displayed on the left, and the complex on the right. In complexes, Ub is in grey with its C-terminal residues 73-LRGG-76 as sticks. L73 and R74 are labeled in underlined italics in one view, and the C-terminus is labeled with an italicized and underlined '76' in the other view. (A) TYMV PRO/DUB. A snapshot at t = 24 ns from the molecular dynamics simulation of the wild type molecule was used here. This snapshot represents the typical equilibrium conformation where the GPP flap is in the intermediate position (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006714#ppat.1006714.g002" target="_blank">Fig 2A</a>, top and Fig 8A, top) and the catalytic dyad realigned (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006714#ppat.1006714.g002" target="_blank">Fig 2A</a>, bottom and Fig 8A, bottom). For clarity, hydrogens added for the simulation are omitted. (B) The cellular OTU DUB domain OTUD2 in free (PDB 4BOQ) and Ub-bound (PDB 4BOS) forms. The latter is a catalytic cysteine mutant C160A and its catalytic histidine displays static disorder with alternate canonical and swung out conformations. (C) The arterivirus PRO/DUB in complex with Ub (PDB 4IUM). Despite its divergence from other OTU DUBs, this enzyme displays a canonical OTU DUB active site cleft both on the P and P' sides. The only exception is the third catalytic residue (here N263) that comes from a neighbouring strand.</p

<i>In vitro</i> DUB activity of structure-guided mutants of TYMV PRO/DUB.

<p><i>In vitro</i> DUB activity of structure-guided mutants of TYMV PRO/DUB.</p

Impact of DUB activity and mutations of the GPP flap on viral infectivity and symptoms appearance in planta.

<p>Arabidopsis plants were mechanically inoculated with water, or <i>in vitro</i> transcripts as indicated. (A) Symptoms development were monitored and representative pictures of the symptoms observed on the inoculated leaves (upper panel) at 14 dpi and on the systemic leaves (lower panel) at 17 dpi are shown for each inoculum. The same type and severity of symptoms were consistently observed in at least four independent experiments involving n ≥ 26 plants. (B) The ability of the transcripts to multiply in systemic leaves was assessed by extracting total RNAs and quantifying viral genomic RNA by RTqPCR at 17 dpi. Viral RNA levels were calibrated and normalized to <i>EF1α</i> and <i>18S</i> rRNA reference genes RNAs. The relative accumulation of viral mutant RNAs as compared to the WT TYMV RNA is represented as the mean +/- SD. Mean and SD values, as well as the number of samples (n) analyzed in at least three independent experiments are indicated below panel (B).</p

Crystal structure of TYMV PRO/DUB mutant ΔC5.

<p>(A) Superposition of PRO/DUB wild type (in pink) and ΔC5 ('A' and 'B', in light cyan and purple-blue, respectively). The displacement of loop 864–868 (865-GPP-867 flap) is indicated with a blue arrow following P866. (B) Superposition of the two most distant conformations (wild type and ΔC5 'B' conformation) seen from the P' side of the catalytic cleft. Residues forming the hydrophobic zipper, as well as the catalytic dyad C783-H869, are displayed as sticks and labeled. The distances between the Sγ of C783 and the Nδ1 of H869 are indicated in black and the distances between the Cαs of P867 and L820 in gray. The displacement of P866 in the zipper is indicated by a blue arrow as in panel (A). (C) Same superposition as in (B) seen from the other side of the GPP flap. Note that D843 follows the movement of the neighbouring GPP loop but that some of its interactions with the base of the loop break.</p

Molecular dynamics simulations for TYMV PRO/DUB and structure-based flap mutants.

<p>25-ns simulations were performed in identical conditions for wild type PRO/DUB (A) and mutants D843A (B), G865A (C) and P866G/P867G (D). Top, evolution along the simulations of the distance between Cα carbons of P867 and L820. Middle, snapshot taken from the end of each simulation (t = 25 ns), that shows a representative view of the GPP flap for all systems. Mutated residues' side chains are displayed as sticks, as well as L820. For L820 and for residues that are glycines or were mutated to glycines, Cα carbons are also shown as spheres. For clarity, hydrogens added for the simulations are omitted. Bottom, evolution along the simulations of the distance between the Sγ of C783 and the Nδ1 of H869. The dotted-and-broken line signals a 3.2 Å value below which the activating hydrogen bond between H869 and C783 is formed. (A) The three P867-L820 distances found in the crystal structures are labeled "Open" (dotted line, for a molecule taken from the PRO:PRO complex, the conformation from which the simulations were started), "Interm." (solid line, value found in the ΔC5 'A' conformation) and "Closed" (broken line, value found in the ΔC5 'B' conformation).</p

Phosphoribulokinase abundance is not limiting the Calvin-Benson-Bassham cycle in Chlamydomonas reinhardtii

Improving photosynthetic efficiency in plants and microalgae is of utmost importance to support the growing world population and to enable the bioproduction of energy and chemicals. Limitations in photosynthetic light conversion efficiency can be directly attributed to kinetic bottlenecks within the Calvin-Benson-Bassham cycle (CBBC) responsible for carbon fixation. A better understanding of these bottlenecks in vivo is crucial to overcome these limiting factors through bio-engineering. The present study is focused on the analysis of phosphoribulokinase (PRK) in the unicellular green alga Chlamydomonas reinhardtii. We have characterized a PRK knock-out mutant strain and showed that in the absence of PRK, Chlamydomonas cannot grow photoautotrophically while functional complementation with a synthetic construct allowed restoration of photoautotrophy. Nevertheless, using standard genetic elements, the expression of PRK was limited to 40% of the reference level in complemented strains and could not restore normal growth in photoautotrophic conditions suggesting that the CBBC is limited. We were subsequently able to overcome this initial limitation by improving the design of the transcriptional unit expressing PRK using diverse combinations of DNA parts including PRK endogenous promoter and introns. This enabled us to obtain strains with PRK levels comparable to the reference strain and even overexpressing strains. A collection of strains with PRK levels between 16% and 250% of WT PRK levels was generated and characterized. Immunoblot and growth assays revealed that a PRK content of ≈86% is sufficient to fully restore photoautotrophic growth. This result suggests that PRK is present in moderate excess in Chlamydomonas. Consistently, the overexpression of PRK did not increase photosynthetic growth indicating that that the endogenous level of PRK in Chlamydomonas is not limiting the Calvin-Benson-Bassham cycle under optimal conditions