Enzyme Promiscuity in Enolase Superfamily. Theoretical Study of o-Succinylbenzoate Synthase Using QM/MM Methods

The promiscuous activity of the enzyme o-succinylbenzoate synthase (OSBS) from the actinobacteria Amycolatopsis is investigated by means of QM/MM methods, using both density functional theory and semiempirical Hamiltonians. This enzyme catalyzes not only the dehydration of 2-succinyl-6R-hydroxy-2,4-cyclohexadiene-1R-carboxylate but also catalyzes racemization of different acylamino acids, with N-succinyl-R-phenylglycine being the best substrate. We investigated the molecular mechanisms for both reactions exploring the potential energy surface. Then, molecular dynamics simulations were performed to obtain the free energy profiles and the averaged interaction energies of enzymatic residues with the reacting system. Our results confirm the plausibility of the reaction mechanisms proposed in the literature, with a good agreement between theoretical and experimentally derived activation free energies. Our simulations unravel the role played by the different residues in each of the two possible reactions. The presence of flexible loops in the active site and the selection of structural modifications in the substrate seem to be key elements to promote the promiscuity of this enzyme.This work was supported by the Spanish Ministerio de Economia y Competitividad project CTQ2012-36253-C03-03 ́ and FEDER funds. K.S. thanks the Polish National Science Center (NCN) for Grant 2011/02/A/ST4/00246. The authors acknowledge computational facilities of the Servei d’Informatica ̀ de la Universitat de Valencia in the ̀ “Tirant” supercomputer, which is part of the Spanish Supercomputing Network

Molecular and Physiological Properties Associated with Zebra Complex Disease in Potatoes and Its Relation with Candidatus Liberibacter Contents in Psyllid Vectors

Zebra complex (ZC) disease on potatoes is associated with Candidatus Liberibacter solanacearum (CLs), an α-proteobacterium that resides in the plant phloem and is transmitted by the potato psyllid Bactericera cockerelli (Šulc). The name ZC originates from the brown striping in fried chips of infected tubers, but the whole plants also exhibit a variety of morphological features and symptoms for which the physiological or molecular basis are not understood. We determined that compared to healthy plants, stems of ZC-plants accumulate starch and more than three-fold total protein, including gene expression regulatory factors (e.g. cyclophilin) and tuber storage proteins (e.g., patatins), indicating that ZC-affected stems are reprogrammed to exhibit tuber-like physiological properties. Furthermore, the total phenolic content in ZC potato stems was elevated two-fold, and amounts of polyphenol oxidase enzyme were also high, both serving to explain the ZC-hallmark rapid brown discoloration of air-exposed damaged tissue. Newly developed quantitative and/or conventional PCR demonstrated that the percentage of psyllids in laboratory colonies containing detectable levels of CLs and its titer could fluctuate over time with effects on colony prolificacy, but presumed reproduction-associated primary endosymbiont levels remained stable. Potato plants exposed in the laboratory to psyllid populations with relatively low-CLs content survived while exposure of plants to high-CLs psyllids rapidly culminated in a lethal collapse. In conclusion, we identified plant physiological biomarkers associated with the presence of ZC and/or CLs in the vegetative potato plant tissue and determined that the titer of CLs in the psyllid population directly affects the rate of disease development in plants

Distinct Effects of p19 RNA Silencing Suppressor on Small RNA Mediated Pathways in Plants

RNA silencing is one of the main defense mechanisms employed by plants to fight viruses. In change, viruses have evolved silencing suppressor proteins to neutralize antiviral silencing. Since the endogenous and antiviral functions of RNA silencing pathway rely on common components, it was suggested that viral suppressors interfere with endogenous silencing pathway contributing to viral symptom development. In this work, we aimed to understand the effects of the tombusviral p19 suppressor on endogenous and antiviral silencing during genuine virus infection. We showed that ectopically expressed p19 sequesters endogenous small RNAs (sRNAs) in the absence, but not in the presence of virus infection. Our presented data question the generalized model in which the sequestration of endogenous sRNAs by the viral suppressor contributes to the viral symptom development. We further showed that p19 preferentially binds the perfectly paired ds-viral small interfering RNAs (vsiRNAs) but does not select based on their sequence or the type of the 5’ nucleotide. Finally, co-immunoprecipitation of sRNAs with AGO1 or AGO2 from virus-infected plants revealed that p19 specifically impairs vsiRNA loading into AGO1 but not AGO2. Our findings, coupled with the fact that p19-expressing wild type Cymbidium ringspot virus (CymRSV) overcomes the Nicotiana benthamiana silencing based defense killing the host, suggest that AGO1 is the main effector of antiviral silencing in this host-virus combination

Plant ARGONAUTEs: Features, Functions and Unknowns

ARGONAUTEs (AGOs) are the effector proteins in eukaryotic small RNA(sRNA)– based gene silencing pathways controlling gene expression and transposon activity. In plants, AGOs regulate key biological processes such as development, response to stress, genome structure and integrity, and pathogen defense. Canonical functions of plant AGO–sRNA complexes include the endonucleolytic cleavage or translational inhibition of target RNAs, and the methylation of target DNAs. Here, I provide a brief update on the major features, molecular functions and biological roles of plant AGOs. A special focus is given to the more recent discoveries related to emerging molecular or biological functions of plant AGOs, as well as to the major unknowns in the plant AGO field.This work was supported by an Individual Fellowship from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 655841 to A.C.Carbonell Olivares, A. (2017). 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Antiviral Silencing and Suppression of Gene Silencing in Plants

RNA silencing is an evolutionary conserved sequence-specific gene inactivation mechanism that contributes to the control of development, maintains heterochromatin, acts in stress responses, DNA repair and defends against invading nucleic acids like transposons and viruses. In plants RNA silencing functions as one of the main immune systems. RNA silencing process involves the small RNAs and trans factor components like Dicers, Argonautes and RNA-dependent RNA poly- merases. To deal with host antiviral silencing responses viruses evolved mecha- nisms to avoid or counteract this, most notably through expression of viral suppressors of RNA silencing. Due to the overlap between endogenous and antiviral silencing pathways while blocking antiviral pathways viruses also impact endogenous silencing processes. Here we provide an overview of antiviral silencing pathway, host factors implicated in it and the crosstalk between antiviral and endogenous branches of silencing. We summarize the current status of knowledge about the viral counter-defense strategies acting at various steps during virus infection in plants with the focus on representative, well studied silencing suppres- sor proteins. Finally we discuss future challenges of the antiviral silencing and counter-defense research field