Marc Bergdoll

Argonaute quenching and global changes in Dicer homeostasis caused by a pathogen-encoded GW repeat protein

In plants and invertebrates, viral-derived siRNAs processed by the RNaseIII Dicer guide Argonaute (AGO) proteins as part of antiviral RNA-induced silencing complexes (RISC). As a counterdefense, viruses produce suppressor proteins (VSRs) that inhibit the host silencing machinery, but their mechanisms of action and cellular targets remain largely unknown. Here, we show that the Turnip crinckle virus (TCV) capsid, the P38 protein, acts as a homodimer, or multiples thereof, to mimic host-encoded glycine/tryptophane (GW)-containing proteins normally required for RISC assembly/function in diverse organisms. The P38 GW residues bind directly and specifically to Arabidopsis AGO1, which, in addition to its role in endogenous microRNA-mediated silencing, is identified as a major effector of TCV-derived siRNAs. Point mutations in the P38 GW residues are sufficient to abolish TCV virulence, which is restored in Arabidopsis ago1 hypomorphic mutants, uncovering both physical and genetic interactions between the two proteins. We further show how AGO1 quenching by P38 profoundly impacts the cellular availability of the four Arabidopsis Dicers, uncovering an AGO1-dependent, homeostatic network that functionally connects these factors together. The likely widespread occurrence and expected consequences of GW protein mimicry on host silencing pathways are discussed in the context of innate and adaptive immunity in plants and metazoans.

Metabolic engineering of monoterpene synthesis in yeast

Terpenoids are one of the largest and most diverse families of natural compounds. They are heavily used in industry, and the trend is toward engineering modified microorganisms that produce high levels of specific terpenoids. Most studies have focused on creating specific heterologous pathways for sesquiterpenes in Escherichia coli or yeast. We subjected the Saccharomyces cerevisiae ERG20 gene (encoding farnesyl diphosphate synthase) to a set of amino acid mutations in the catalytic site at position K197. Mutated strains have been shown to exhibit various growth rate, sterol amount, and monoterpenol‐producing capacities. These results are discussed in the context of the potential use of these mutated strains for heterologous expression of monoterpenoid synthases, which was investigated using Ocimum basilicum geraniol synthase. The results obtained with up to 5 mg/L geraniol suggest a major improvement compared with previous available expression systems like Escherichia coli or yeast strains with an unmodified ERG20 gene that respectively delivered amounts in the 10 and 500 µg/L range or even a previously characterized K197E mutation that delivered amounts in the 1 mg/L range. Biotechnol. Bioeng. 2011; 108:1883–1892.

The Plant TPX2 Protein Regulates Prospindle Assembly before Nuclear Envelope Breakdown

The Targeting Protein for Xklp2 (TPX2) is a central regulator of spindle assembly in vertebrate cells. The absence or excess of TPX2 inhibits spindle formation. We have defined a TPX2 signature motif that is present once in vertebrate sequences but twice in plants. Plant TPX2 is predominantly nuclear during interphase and is actively exported before nuclear envelope breakdown to initiate prospindle assembly. It localizes to the spindle microtubules but not to the interdigitating polar microtubules during anaphase or to the phragmoplast as it is rapidly degraded during telophase. We characterized the Arabidopsis thaliana TPX2-targeting domains and show that the protein is able to rescue microtubule assembly in TPX2-depleted Xenopus laevis egg extracts. Injection of antibodies to TPX2 into living plant cells inhibits the onset of mitosis. These results demonstrate that plant TPX2 already functions before nuclear envelope breakdown. Thus, plants have adapted nuclear–cytoplasmic shuttling of TPX2 to maintain proper spindle assembly without centrosomes.

Selective proteolysis sets the tempo of the cell cycle

Ubiquitin-mediated proteolysis is one of the key mechanisms underlying cell cycle control in all eukaryotes. This is achieved by the action of ubiquitin ligases (E3s), which remove both negative and positive regulators of the cell cycle. Though our current understanding of the plant cell cycle has improved a lot these recent years, the identity of the E3s regulating it and their mode of action is still in its infancy. Nevertheless, recent research in Arabidopsis revealed some novel findings in this area. Thus the anaphase promoting complex/cyclosome (APC/C) not only controls mitotic events, but is also important in post-mitotic cells for normal plant development and cell differentiation. Moreover conserved and novel E3s were identified that target cyclin-dependent kinase inhibitors at different plant developmental stages. Finally, environmental constrains and stress hormones negatively impact on the cell cycle by processes that also include E3s.

Arabidopsis tRNA Adenosine Deaminase Arginine Edits the Wobble Nucleotide of Chloroplast tRNAArg(ACG) and Is Essential for Efficient Chloroplast Translation

RNA editing changes the coding/decoding information relayed by transcripts via nucleotide insertion, deletion, or conversion. Editing of tRNA anticodons by deamination of adenine to inosine is used both by eukaryotes and prokaryotes to expand the decoding capacity of individual tRNAs. This limits the number of tRNA species required for codon-anticodon recognition. We have identified the Arabidopsis thaliana gene that codes for tRNA adenosine deaminase arginine (TADA), a chloroplast tRNA editing protein specifically required for deamination of chloroplast (cp)-tRNAArg(ACG) to cp-tRNAArg(ICG). Land plant TADAs have a C-terminal domain similar in sequence and predicted structure to prokaryotic tRNA deaminases and also have very long N-terminal extensions of unknown origin and function. Biochemical and mutant complementation studies showed that the C-terminal domain is sufficient for cognate tRNA deamination both in vitro and in planta. Disruption of TADA has profound effects on chloroplast translation efficiency, leading to reduced yields of chloroplast-encoded proteins and impaired photosynthetic function. By contrast, chloroplast transcripts accumulate to levels significantly above those of wild-type plants. Nevertheless, absence of cp-tRNAArg(ICG) is compatible with plant survival, implying that two out of three CGN codon recognition occurs in chloroplasts, though this mechanism is less efficient than wobble pairing.

Effect of vanillic acid on COQ6 mutants identified in patients with coenzyme Q10 deficiency.

Human COQ6 encodes a monooxygenase which is responsible for the C5-hydroxylation of the quinone ring of coenzyme Q (CoQ). Mutations in COQ6 cause primary CoQ deficiency, a condition responsive to oral CoQ10 supplementation. Treatment is however still problematic given the poor bioavailability of CoQ10. We employed S. cerevisiae lacking the orthologous gene to characterize the two different human COQ6 isoforms and the mutations found in patients. COQ6 isoform a can partially complement the defective yeast, while isoform b, which lacks part of the FAD-binding domain, is inactive but partially stable, and could have a regulatory/inhibitory function in CoQ10 biosynthesis. Most mutations identified in patients, including the frameshift Q461fs478X mutation, retain residual enzymatic activity, and all patients carry at least one hypomorphic allele, confirming that the complete block of CoQ biosynthesis is lethal. These mutants are also partially stable and allow the assembly of the CoQ biosynthetic complex. In fact treatment with two hydroxylated analogues of 4-hydroxybenzoic acid, namely, vanillic acid or 3-4-hydroxybenzoic acid, restored the respiratory growth of yeast Δcoq6 cells expressing the mutant huCOQ6-isoa proteins. These compounds, and particularly vanillic acid, could therefore represent an interesting therapeutic option for COQ6 patients.

Structural Insights Into Viral Determinants of Nematode Mediated Grapevine Fanleaf Virus Transmission.

Many animal and plant viruses rely on vectors for their transmission from host to host. Grapevine fanleaf virus (GFLV), a picorna-like virus from plants, is transmitted specifically by the ectoparasitic nematode Xiphinema index. The icosahedral capsid of GFLV, which consists of 60 identical coat protein subunits (CP), carries the determinants of this specificity. Here, we provide novel insight into GFLV transmission by nematodes through a comparative structural and functional analysis of two GFLV variants. We isolated a mutant GFLV strain (GFLV-TD) poorly transmissible by nematodes, and showed that the transmission defect is due to a glycine to aspartate mutation at position 297 (Gly297Asp) in the CP. We next determined the crystal structures of the wild-type GFLV strain F13 at 3.0 Å and of GFLV-TD at 2.7 Å resolution. The Gly297Asp mutation mapped to an exposed loop at the outer surface of the capsid and did not affect the conformation of the assembled capsid, nor of individual CP molecules. The loop is part of a positively charged pocket that includes a previously identified determinant of transmission. We propose that this pocket is a ligand-binding site with essential function in GFLV transmission by X. index. Our data suggest that perturbation of the electrostatic landscape of this pocket affects the interaction of the virion with specific receptors of the nematodes feeding apparatus, and thereby severely diminishes its transmission efficiency. These data provide a first structural insight into the interactions between a plant virus and a nematode vector.

A Stretch of 11 Amino Acids in the βB-βC Loop of the Coat Protein of Grapevine Fanleaf Virus Is Essential for Transmission by the Nematode Xiphinema index

ABSTRACT Grapevine fanleaf virus (GFLV) and Arabis mosaic virus (ArMV) from the genus Nepovirus, family Secoviridae, cause a severe degeneration of grapevines. GFLV and ArMV have a bipartite RNA genome and are transmitted specifically by the ectoparasitic nematodes Xiphinema index and Xiphinema diversicaudatum, respectively. The transmission specificity of both viruses maps to their respective RNA2-encoded coat protein (CP). To further delineate the GFLV CP determinants of transmission specificity, three-dimensional (3D) homology structure models of virions and CP subunits were constructed based on the crystal structure of Tobacco ringspot virus, the type member of the genus Nepovirus. The 3D models were examined to predict amino acids that are exposed at the external virion surface, highly conserved among GFLV isolates but divergent between GFLV and ArMV. Five short amino acid stretches that matched these topographical and sequence conservation criteria were selected and substituted in single and multiple combinations by their ArMV counterparts in a GFLV RNA2 cDNA clone. Among the 21 chimeric RNA2 molecules engineered, transcripts of only three of them induced systemic plant infection in the presence of GFLV RNA1. Nematode transmission assays of the three viable recombinant viruses showed that swapping a stretch of (i) 11 residues in the βB-βC loop near the icosahedral 3-fold axis abolished transmission by X. index but was insufficient to restore transmission by X. diversicaudatum and (ii) 7 residues in the βE-αB loop did not interfere with transmission by the two Xiphinema species. This study provides new insights into GFLV CP determinants of nematode transmission.

A novel pollen-specific α-tubulin in sunflower: structure and characterization

We describe here a new α-tubulin isoform from sunflower we named απ-tubulin. απ-tubulin is the most divergent higher-plant α-tubulin described so far, having an unusual deletion in the H1/B2 loop and a glutamine-rich C-terminus. We constructed a three-dimensional model and discuss its implications. Using specific antibodies, we show that απ-tubulin expression is restricted to the male gametophyte. απ-tubulin mRNA represents 90% of α-tubulin mRNA and a small percentage of total pollen mRNA. Among the plants tested, απ-tubulin was only detected in sunflower and in Cosmos. Since both plants are Asteraceae, we propose that απ-tubulin is specific to this family. Our results suggest that απ-tubulin can inhibit tubulin assembly in pollen. This hypothesis is reinforced by the fact that απ-tubulin is found in a complex with β-tubulin in mature sunflower pollen.

Evolutionary and functional aspects of C-to-U editing at position 28 of tRNA(Cys)(GCA) in plant mitochondria.

In plant mitochondria, editing of messenger RNA by C-to-U conversions is essential for correct gene expression as it usually improves the protein-sequence conservation between different species or sometimes affects the reading frames (for a review, see Maier et al+, 1996)+ Editing sites have been identified in mitochondrial (mt) RNA of all major groups of land plants, including Bryophytes, Pteridophytes, Prespermaphytes, and Spermaphytes (Hiesel et al+, 1994a,b;Malek et al+, 1996)+ Editing mainly affects messenger RNA, but editing sites have also been identified in three transfer RNAs+ In dicot mitochondria a C-to-U editing event corrects a C:A mismatch into a U:A base pair in the acceptor stem of tRNAPhe(GAA) (Marechal-Drouard et al+, 1993; Binder et al+, 1994)+ In the gymnosperm Larix leptoeuropaea, three C-to-U conversions restore a U:A base pair in the acceptor stem, D stem, and anticodon stem of tRNAHis(GUG), respectively (Marechal-Drouard et al+, 1996b)+ The third example described is the Oenothera berteriana mt tRNACys(GCA), where a C28:U42 mismatch is converted into a U28:U42 noncanonical base pair (Binder et al+, 1994)+ In the case of both tRNAPhe and tRNAHis, editing of precursors is a prerequisite for 59 and 39 processing to generate a mature tRNA (Marchfelder et al+, 1996; Marechal-Drouard et al+, 1996a, 1996b; Kunzmann et al+, 1998)+ The role of editing in the case of tRNACys has not been studied so far, although it has been shown that it occurs at the precursor level (Binder et al+, 1994)+ In this letter, we report an evolutionary and functional study of mt tRNACys(GCA) editing in plant mitochondria+ The cloverleaf structure of the mt tRNACys(GCA) deduced from the sequence of the single Solanum tuberosum mt trnC gene (EMBL Accession Number X93575) is identical to its counterpart in O. berteriana and reveals a weak anticodon stem with a U27:G43 noncanonical interaction, and a C28:U42 mismatch+ By analyzing RT-PCR amplified cDNAs of S. tuberosum mt tRNACys precursors (362 nt in length), we found that 7 out of 11 independent clones contained a T at position 28+ The ratio of edited versus nonedited mature tRNACys was determined by RT-mini-sequencing+ When total S. tuberosum mt tRNAs were used as template, only dATP was incorporated, demonstrating that the mature tRNACys is fully edited in vivo (Fig+ 1B)+ From an evolutionary point of view, the comparison of the S. tuberosum mt trnC gene with its counterpart in Marchantia polymorpha shows in particular two differences in the anticodon stem (Fig+ 1A)+ In M. polymorpha, an A residue at position 43 allows a T27:A43 base pairing, and a T residue is present at position 28+ Considering that this sequence is more closely related to the ancestral sequence, we postulated that the C-to-U editing site found in dicot mitochondria restores this ancestral sequence+ To confirm this hypothesis, we first tried to determine when, during the evolution of land plants, the mt trnC gene acquired a C at position 28 and when the C28-to-U28 editing event occurred+ To do so, the internal sequence of trnC (from position 25 to 52) was PCR-amplified, cloned, and sequenced in several species that belong to different groups of land plants+ A single difference could be observed in this region between the different plants tested: a T residue was present at position 28 of mt trnC in the Pteridophyte Pteris nephrolepis (Filicales order) and in the Reprint requests to: Laurence Marechal-Drouard, Institut de Biologie Moleculaire des Plantes, Centre National de la Recherche Scientifique, Universite Louis Pasteur, 12 rue du General Zimmer, F-67084 Strasbourg Cedex, France; e-mail: laurence+drouard@ibmpulp+u-strasbg+fr 2Present address: Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, New York 14853-1801, USA 3Present address: Department of Molecular Biophysics and Biochemistry, Yale University, School of Medicine, New Haven, Connecticut 06520-8024, USA RNA (2000), 6:470–474+ Cambridge University Press+ Printed in the USA+ Copyright