@Article{IPB-597, author = {Serra, P. and Carbonell, A. and Navarro, B. and Gago-Zachert, S. and Li, S. and Di Serio, F. and Flores, R. and}, title = {{Symptomatic plant viroid infections in phytopathogenic fungi: A request for a critical reassessment}}, year = {2020}, pages = {10126-10128}, journal = {Proc. Natl. Acad. Sci. U.S.A.}, doi = {10.1073/pnas.1922249117}, volume = {117}, } @Article{IPB-565, author = {Kuhn, J. H. and Adkins, S. and Alioto, D. and Gago-Zachert, S. and et al., . and}, title = {{2020 taxonomic update for phylum Negarnaviricota (Riboviria: Orthornavirae), including the large orders Bunyavirales and Mononegavirales}}, year = {2020}, pages = {3023–3072}, journal = {Arch. Virol.}, doi = {10.1007/s00705-020-04731-2}, url = {https://doi.org/10.1007/s00705-020-04731-2}, volume = {165}, abstract = {In March 2020, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. At the genus rank, 20 new genera were added, two were deleted, one was moved, and three were renamed. At the species rank, 160 species were added, four were deleted, ten were moved and renamed, and 30 species were renamed. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.} } @Article{IPB-667, author = {Gago-Zachert, S. and Schuck, J. and Weinholdt, C. and Knoblich, M. and Pantaleo, V. and Grosse, I. and Gursinsky, T. and Behrens, S.-E. and}, title = {{Highly efficacious antiviral protection of plants by small interfering RNAs identified in vitro}}, year = {2019}, pages = {9343-9357}, journal = {Nucleic Acids Res.}, doi = {10.1093/nar/gkz678}, volume = {47}, abstract = {In response to a viral infection, the plant’s RNA silencing machinery processes viral RNAs into a huge number of small interfering RNAs (siRNAs). However, a very few of these siRNAs actually interfere with viral replication. A reliable approach to identify these immunologically effective siRNAs (esiRNAs) and to define the characteristics underlying their activity has not been available so far. Here, we develop a novel screening approach that enables a rapid functional identification of antiviral esiRNAs. Tests on the efficacy of such identified esiRNAs of a model virus achieved a virtual full protection of plants against a massive subsequent infection in transient applications. We find that the functionality of esiRNAs depends crucially on two properties: the binding affinity to Argonaute proteins and the ability to access the target RNA. The ability to rapidly identify functional esiRNAs could be of great benefit for all RNA silencing-based plant protection measures against viruses and other pathogens.} } @Article{IPB-785, author = {García, M. L. and Bó, E. D. and da Graça, J. V. and Gago-Zachert, S. and Hammond, J. and Moreno, P. and Natsuaki, T. and Pallás, V. and Navarro, J. A. and Reyes, C. A. and Luna, G. R. and Sasaya, T. and Tzanetakis, I. E. and Vaira, A. M. and Verbeek, M. and ICTV Report Consortium, . and}, title = {{Corrigendum: ICTV Virus Taxonomy Profile: Ophioviridae}}, year = {2018}, pages = {949-949}, journal = {J. Gen. Virol.}, doi = {10.1099/jgv.0.001093}, volume = {99}, } @Article{IPB-908, author = {López-Carrasco, A. and Ballesteros, C. and Sentandreu, V. and Delgado, S. and Gago-Zachert, S. and Flores, R. and Sanjuán, R. and}, title = {{Different rates of spontaneous mutation of chloroplastic and nuclear viroids as determined by high-fidelity ultra-deep sequencing}}, year = {2017}, pages = {e1006547}, journal = {PLOS Pathog.}, doi = {10.1371/journal.ppat.1006547}, volume = {13}, abstract = {Mutation rates vary by orders of magnitude across biological systems, being higher for simpler genomes. The simplest known genomes correspond to viroids, subviral plant replicons constituted by circular non-coding RNAs of few hundred bases. Previous work has revealed an extremely high mutation rate for chrysanthemum chlorotic mottle viroid, a chloroplast-replicating viroid. However, whether this is a general feature of viroids remains unclear. Here, we have used high-fidelity ultra-deep sequencing to determine the mutation rate in a common host (eggplant) of two viroids, each representative of one family: the chloroplastic eggplant latent viroid (ELVd, Avsunviroidae) and the nuclear potato spindle tuber viroid (PSTVd, Pospiviroidae). This revealed higher mutation frequencies in ELVd than in PSTVd, as well as marked differences in the types of mutations produced. Rates of spontaneous mutation, quantified in vivo using the lethal mutation method, ranged from 1/1000 to 1/800 for ELVd and from 1/7000 to 1/3800 for PSTVd depending on sequencing run. These results suggest that extremely high mutability is a common feature of chloroplastic viroids, whereas the mutation rates of PSTVd and potentially other nuclear viroids appear significantly lower and closer to those of some RNA viruses.} } @Article{IPB-884, author = {García, M. L. and Bó, E. D. and da Graça, J. V. and Gago-Zachert, S. and Hammond, J. and Moreno, P. and Natsuaki, T. and Pallás, V. and Navarro, J. A. and Reyes, C. A. and Luna, G. R. and Sasaya, T. and Tzanetakis, I. E. and Vaira, A. M. and Verbeek, M. and ICTV Report Consortium, . and}, title = {{ICTV Virus Taxonomy Profile: Ophioviridae}}, year = {2017}, pages = {1161-1162}, journal = {J. Gen. Virol.}, doi = {10.1099/jgv.0.000836}, volume = {98}, abstract = {The Ophioviridae is a family of filamentous plant viruses, with single-stranded negative, and possibly ambisense, RNA genomes of 11.3–12.5 kb divided into 3–4 segments, each encapsidated separately. Virions are naked filamentous nucleocapsids, forming kinked circles of at least two different contour lengths. The sole genus, Ophiovirus, includes seven species. Four ophioviruses are soil-transmitted and their natural hosts include trees, shrubs, vegetables and bulbous or corm-forming ornamentals, both monocots and dicots. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Ophioviridae, which is available at http://www.ictv.global/report/ophioviridae.} } @INBOOK{IPB-39, author = {Flores, R. and Gago-Zachert, S. and Serra, P. and De la Peña, M. and Navarro, B. and}, title = {{Viroids and Satellites}}, year = {2017}, pages = {331-338}, chapter = {{Chrysanthemum Chlorotic Mottle Viroid}}, editor = {Hadidi, A., et al., eds.}, doi = {10.1016/B978-0-12-801498-1.00031-0}, abstract = {Chrysanthemum chlorotic mottle viroid (CChMVd) (398–401 nt) belongs to genus Pelamoviroid, family Avsunviroidae and, like other members of this family, replicates in plastids through a rolling-circle mechanism involving hammerhead ribozymes. CChMVd RNA adopts a branched conformation stabilized by a kissing-loop interaction, resembling peach latent mosaic viroid in this respect. Chrysanthemum is the only natural and experimental host for CChMVd, which in the most sensitive varieties induces leaf mottling and chlorosis, delay in flowering, and dwarfing. The viroid has been found in major chrysanthemum growing areas including Europe and Asia. There are natural variants in which the change (UUUC→GAAA) mapping at a tetraloop in the CChMVd branched conformation is sufficient to change the symptomatic phenotype into a nonsymptomatic one without altering the viroid titer. Preinfection with nonsymptomatic variants prevents challenge inoculation with symptomatic ones. Moreover, experimental coinoculation with symptomatic and nonsymptomatic CChMVd variants results in symptomless phenotypes only when the latter is in vast excess, thus indicating its lower fitness.} } @Article{IPB-1017, author = {López-Carrasco, A. and Gago-Zachert, S. and Mileti, G. and Minoia, S. and Flores, R. and Delgado, S. and}, title = {{The transcription initiation sites of eggplant latent viroid strands map within distinct motifs in their in vivo RNA conformations}}, year = {2016}, pages = {83-97}, journal = {RNA Biol.}, doi = {10.1080/15476286.2015.1119365}, volume = {13}, abstract = {Eggplant latent viroid (ELVd), like other members of family Avsunviroidae, replicates in plastids through a symmetric rolling-circle mechanism in which elongation of RNA strands is most likely catalyzed by a nuclear-encoded polymerase (NEP) translocated to plastids. Here we have addressed where NEP initiates transcription of viroid strands. Because this step is presumably directed by sequence/structural motifs, we have previously determined the conformation of the monomeric linear (\+) and (−) RNAs of ELVd resulting from hammerhead-mediated self-cleavage. In silico predictions with 3 softwares led to similar bifurcated conformations for both ELVd strands. In vitro examination by non-denaturing PAGE showed that they migrate as prominent single bands, with the ELVd (\+) RNA displaying a more compact conformation as revealed by its faster electrophoretic mobility. In vitro SHAPE analysis corroborated the ELVd conformations derived from thermodynamics-based predictions in silico. Moreover, sequence analysis of 94 full-length natural ELVd variants disclosed co-variations, and mutations converting canonical into wobble pairs or vice versa, which confirmed in vivo most of the stems predicted in silico and in vitro, and additionally helped to introduce minor structural refinements. Therefore, results from the 3 experimental approaches were essentially consistent among themselves. Application to RNA preparations from ELVd-infected tissue of RNA ligase-mediated rapid amplification of cDNA ends, combined with pretreatments to modify the 5′ ends of viroid strands, mapped the transcription initiation sites of ELVd (\+) and (−) strands in vivo at different sequence/structural motifs, in contrast with the situation previously observed in 2 other members of the family Avsunviroidae.} } @Article{IPB-997, author = {Gharsallah, C. and Fakhfakh, H. and Grubb, D. and Gorsane, F. and}, title = {{Effect of salt stress on ion concentration, proline content, antioxidant enzyme activities and gene expression in tomato cultivars}}, year = {2016}, pages = {plw055}, journal = {AoB PLANTS}, doi = {10.1093/aobpla/plw055}, volume = {8}, abstract = {Salinity is a constraint limiting plant growth and productivity of crops throughout the world. Understanding the mechanism underlying plant response to salinity provides new insights into the improvement of salt tolerance-crops of importance. In the present study, we report on the responses of twenty cultivars of tomato. We have clustered genotypes into scale classes according to their response to increased NaCl levels. Three local tomato genotypes, representative of different saline scale classes, were selected for further investigation. During early (0 h, 6 h and 12 h) and later (7 days) stages of the response to salt treatment, ion concentrations (Na \+ , K \+  and Ca 2\+ ), proline content, enzyme activities (catalase, ascorbate peroxidase and guiacol peroxidase) were recorded. qPCR analysis of candidate genes WRKY (8, 31and 39), ERF (9, 16 and 80), LeNHX (1, 3 and 4) and HKT (class I) were performed. A high K \+ , Ca 2 \+ and proline accumulation as well as a decrease of Na \+  concentration-mediated salt tolerance. Concomitant with a pattern of high-antioxidant enzyme activities, tolerant genotypes also displayed differential patterns of gene expression during the response to salt stress.} } @Article{IPB-994, author = {Gago-Zachert, S. and}, title = {{Viroids, infectious long non-coding RNAs with autonomous replication}}, year = {2016}, pages = {12-24}, journal = {Virus Res.}, doi = {10.1016/j.virusres.2015.08.018}, volume = {212}, abstract = {Transcriptome deep-sequencing studies performed during the last years confirmed that the vast majority of the RNAs transcribed in higher organisms correspond to several types of non-coding RNAs including long non-coding RNAs (lncRNAs). The study of lncRNAs and the identification of their functions, is still an emerging field in plants but the characterization of some of them indicate that they play an important role in crucial regulatory processes like flowering regulation, and responses to abiotic stress and plant hormones. A second group of lncRNAs present in plants is formed by viroids, exogenous infectious subviral plant pathogens well known since many years. Viroids are composed of circular RNA genomes without protein-coding capacity and subvert enzymatic activities of their hosts to complete its own biological cycle. Different aspects of viroid biology and viroid-host interactions have been elucidated in the last years and some of them are the main topic of this review together with the analysis of the state-of-the-art about the growing field of endogenous lncRNAs in plants.} } @Article{IPB-1108, author = {Hamdi, I. and Elleuch, A. and Bessaies, N. and Grubb, C. D. and Fakhfakh, H. and}, title = {{First report of Citrus viroid V in North Africa}}, year = {2015}, pages = {87-91}, journal = {J. Gen. Plant Pathol.}, doi = {10.1007/s10327-014-0556-9}, volume = {81}, abstract = {We tested citrus samples from Tunisia using reverse transcription-polymerase chain reaction (RT-PCR), and for the first time, Citrus viroid V (CVd-V) was reported in North Africa. Fourteen of 38 tested citrus trees were infected by CVd-V including the majority of varieties grown in Tunisia. Some RT-PCR results were also supported by biological indexing. After sequencing the RT-PCR products, three new CVd-V variants were identified, showing 80–91 % nucleotide sequence identity with those reported previously. Based on phylogenetic analysis using all CVd-V sequences in GenBank, two main CVd-V groups were identified. Furthermore, construction of a genetic network of the detected haplotypes using the same sequences shows a clear geographical structuring of Tunisian CVd-V variants.} } @Article{IPB-1176, author = {Zayneb, C. and Bassem, K. and Zeineb, K. and Grubb, C. D. and Noureddine, D. and Hafedh, M. and Amine, E. and}, title = {{Physiological responses of fenugreek seedlings and plants treated with cadmium}}, year = {2015}, pages = {10679-10689}, journal = {Environ. Sci. Pollut. Res.}, doi = {10.1007/s11356-015-4270-8}, volume = {22}, abstract = {The bioaccumulation efficiency of cadmium (Cd) by fenugreek (Trigonella foenum-graecum) was examined using different concentrations of CdCl2. The germination rate was similar to control except at 10 mM Cd. However, early seedling growth was quite sensitive to the metal from the lowest Cd level. Accordingly, amylase activity was reduced substantially on treatment of seeds with 0.5, 1, and 10 mM Cd. Cadmium also affected various other plant growth parameters. Its accumulation was markedly lower in shoots as compared to roots, reducing root biomass by almost 50 %. Plants treated with 1 and 5 mM Cd presented chlorosis due to a significant reduction in chlorophyll b especially. Furthermore, at Cd concentrations greater than 0.1 mM, plants showed several signs of oxidative stress; an enhancement in root hydrogen peroxide (H2O2) level and in shoot malondialdehyde (MDA) content was observed. Conversely, antioxidant enzyme activities (superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT)) increased in various plant parts. Likewise, total phenolic and flavonoid contents reached their highest values in the 0.5 mM Cd treatment, consistent with their roles in quenching low concentrations of reactive oxygen species (ROS). Consequently, maintaining oxidant and antioxidant balance may permit fenugreek to hyperaccumulate Cd and allow it to be employed in extremely Cd polluted soils for detoxification purposes.} } @Article{IPB-1175, author = {Zayneb, C. and Lamia, K. and Olfa, E. and Naïma, J. and Grubb, C. D. and Bassem, K. and Hafedh, M. and Amine, E. and}, title = {{Morphological, Physiological and Biochemical Impact of Ink Industry Effluent on Germination of Maize (Zea mays), Barley (Hordeum vulgare) and Sorghum (Sorghum bicolor)}}, year = {2015}, pages = {687-693}, journal = {Bull. Environ. Contam. Toxicol.}, doi = {10.1007/s00128-015-1600-y}, volume = {95}, abstract = {The present study focuses on effects of untreated and treated ink industry wastewater on germination of maize, barley and sorghum. Wastewater had a high chemical oxygen demand (COD) and metal content compared to treated effluent. Germination decreased with increasing COD concentration. Speed of germination also followed the same trend, except for maize seeds exposed to untreated effluent (E), which germinated slightly faster than controls. These alterations of seedling development were mirrored by changes in soluble protein content. E exerted a positive effect on soluble protein content and maximum levels occurred after 10 days with treated effluent using coagulation/flocculation (TEc/f) process and treated effluent using combined process (coagulation/flocculation/biosorption) (TEc/f/b). Likewise, activity of α-amylase was influenced by effluent composition. Its expression depended on the species, exposure time and applied treatment. Nevertheless, current results indicated TEc/f/b had no observable toxic effects on germination and could be a beneficial alternative resource to irrigation water.} } @Article{IPB-1147, author = {Rekik, I. and Drira, N. and Grubb, C. D. and Elleuch, A. and}, title = {{Molecular characterization and evolution studies of a SERK like gene transcriptionally induced during somatic embryogenesis in Phoenix Dactylifera L v Deglet Nour}}, year = {2015}, pages = {323-337}, journal = {Genetika}, doi = {10.2298/GENSR1501323R}, volume = {47}, abstract = {A somatic embryogenesis receptor kinase like (SERKL) cDNA, designated PhSERKL, was isolated from date palm (Phoenix Dactylifera L) using RACE PCR. PhSERKL protein shared all the characteristic domains of the SERK family, including five leucine-rich repeats, one proline-rich region motif, a transmembrane domain, and kinase domains. Phylogenetic analyses using PHYLIP and Notung 2.7 programs suggest that the SERK proteins of some plant species resulted from relatively ancient duplication events. We predict an ancestor protein of monocots and dicots SERK using FASTML program. Somatic embryogenic cultures of date palm were established following transfer of callus cultures to medium containing 2, 4-dichlorophenoxyacetic acid. The role of PhSERKL gene during establishment of somatic embryogenesis in culture was investigated using quantitative real-time PCR. PhSERKL gene was highly expressed during embryogenic competence acquisition and globular embryo formation in culture. Overall, levels of expression of PhSERKL gene were lower in nonembryogenic tissues and organs than in embryogenic callus.} } @Article{IPB-1146, author = {Rekik, I. and Chaâbene, Z. and Grubb, C. D. and Drira, N. and Cheour, F. and Elleuch, A. and}, title = {{In silico characterization and Molecular modeling of double-strand break repair protein MRE11 from Phoenix dactylifera v deglet nour}}, year = {2015}, pages = {23}, journal = {Theor. Biol. Med. Model.}, doi = {10.1186/s12976-015-0013-2}, volume = {12}, abstract = {BackgroundDNA double-strand breaks (DSBs) are highly cytotoxic and mutagenic. MRE11 plays an essential role in repairing DNA by cleaving broken ends through its 3′ to 5′ exonuclease and single-stranded DNA endonuclease activities.MethodsThe present study aimed to in silico characterization and molecular modeling of MRE11 from Phoenix dactylifera L cv deglet nour (DnMRE11) by various bioinformatic approaches. To identify DnMRE11 cDNA, assembled contigs from our cDNA libraries were analysed using the Blast2GO2.8 program.ResultsThe DnMRE11 protein length was 726 amino acids. The results of HUMMER show that DnMRE11 is formed by three domains: the N-terminal core domain containing the nuclease and capping domains, the C-terminal half containing the DNA binding and coiled coil region. The structure of DnMRE11 is predicted using the Swiss-Model server, which contains the nuclease and capping domains. The obtained model was verified with the structure validation programs such as ProSA and QMEAN servers for reliability. Ligand binding studies using COACH indicated the interaction of DnMRE11 protein with two Mn2\+ ions and dAMP. The ConSurf server predicted that residues of the active site and Nbs binding site have high conservation scores between plant species.ConclusionsA model structure of DnMRE11 was constructed and validated with various bioinformatics programs which suggested the predicted model to be satisfactory. Further validation studies were conducted by COACH analysis for active site ligand prediction, and revealed the presence of six ligands binding sites and two ligands (2 Mn2\+ and dAMP).} } @Article{IPB-1281, author = {Ziegler, J. and Qwegwer, J. and Schubert, M. and Erickson, J. L. and Schattat, M. and Bürstenbinder, K. and Grubb, C. D. and Abel, S. and}, title = {{Simultaneous analysis of apolar phytohormones and 1-aminocyclopropan-1-carboxylic acid by high performance liquid chromatography/electrospray negative ion tandem mass spectrometry via 9-fluorenylmethoxycarbonyl chloride derivatization}}, year = {2014}, pages = {102-109}, journal = {J. Chromatogr. A}, doi = {10.1016/j.chroma.2014.08.029}, volume = {1362}, abstract = {A strategy to detect and quantify the polar ethylene precursor 1-aminocyclopropan-1-carboxylic acid (ACC) along with the more apolar phytohormones abscisic acid (ABA), indole-3-acetic acid (IAA), jasmonic acid (JA), jasmonic acid-isoleucine conjugate (JA-Ile), 12-oxo-phytodienoic acid (OPDA), trans-zeatin, and trans-zeatin 9-riboside using a single extraction is presented. Solid phase resins commonly employed for extraction of phytohormones do not allow the recovery of ACC. We circumvent this problem by attaching an apolar group to ACC via derivatization with the amino group specific reagent 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl). Derivatization in the methanolic crude extract does not modify other phytohormones. The derivatized ACC could be purified and detected together with the more apolar phytohormones using common solid phase extraction resins and reverse phase HPLC/electrospray negative ion tandem mass spectrometry. The limit of detection was in the low nanomolar range for all phytohormones, a sensitivity sufficient to accurately determine the phytohormone levels from less than 50 mg (fresh weight) of Arabidopsis thaliana and Nicotiana benthamiana tissues. Comparison with previously published phytohormone levels and the reported changes in phytohormone levels after stress treatments confirmed the accuracy of the method.} } @Article{IPB-1234, author = {Maldonado-Bonilla, L. D. and Eschen-Lippold, L. and Gago-Zachert, S. and Tabassum, N. and Bauer, N. and Scheel, D. and Lee, J. and}, title = {{The Arabidopsis Tandem Zinc Finger 9 Protein Binds RNA and Mediates Pathogen-Associated Molecular Pattern-Triggered Immune Responses}}, year = {2014}, pages = {412-425}, journal = {Plant Cell Physiol.}, doi = {10.1093/pcp/pct175}, volume = {55}, abstract = {Recognition of pathogen-associated molecular patterns (PAMPs) induces multiple defense mechanisms to limit pathogen growth. Here, we show that the Arabidopsis thaliana tandem zinc finger protein 9 (TZF9) is phosphorylated by PAMP-responsive mitogen-activated protein kinases (MAPKs) and is required to trigger a full PAMP-triggered immune response. Analysis of a tzf9 mutant revealed attenuation in specific PAMP-triggered reactions such as reactive oxygen species accumulation, MAPK activation and, partially, the expression of several PAMP-responsive genes. In accordance with these weaker PAMP-triggered responses, tzf9 mutant plants exhibit enhanced susceptibility to virulent Pseudomonas syringae pv. tomato DC3000. Visualization of TZF9 localization by fusion to green fluorescent protein revealed cytoplasmic foci that co-localize with marker proteins of processing bodies (P-bodies). This localization pattern is affected by inhibitor treatments that limit mRNA availability (such as cycloheximide or actinomycin D) or block nuclear export (leptomycin B). Coupled with its ability to bind the ribohomopolymers poly(rU) and poly(rG), these results suggest involvement of TZF9 in post-transcriptional regulation, such as mRNA processing or storage pathways, to regulate plant innate immunity.} } @Article{IPB-1219, author = {Grubb, C. D. and Zipp, B. J. and Kopycki, J. and Schubert, M. and Quint, M. and Lim, E.-K. and Bowles, D. J. and Pedras, M. S. C. and Abel, S. and}, title = {{Comparative analysis of Arabidopsis UGT74 glucosyltransferases reveals a special role of UGT74C1 in glucosinolate biosynthesis}}, year = {2014}, pages = {92-105}, journal = {Plant J.}, doi = {10.1111/tpj.12541}, volume = {79}, abstract = {The study of glucosinolates and their regulation has provided a powerful framework for the exploration of fundamental questions about the function, evolution, and ecological significance of plant natural products, but uncertainties about their metabolism remain. Previous work has identified one thiohydroximate S‐glucosyltransferase, UGT74B1, with an important role in the core pathway, but also made clear that this enzyme functions redundantly and cannot be the sole UDP‐glucose dependent glucosyltransferase (UGT) in glucosinolate synthesis. Here, we present the results of a nearly comprehensive in vitro activity screen of recombinant Arabidopsis Family 1 UGTs, which implicate other members of the UGT74 clade as candidate glucosinolate biosynthetic enzymes. Systematic genetic analysis of this clade indicates that UGT74C1 plays a special role in the synthesis of aliphatic glucosinolates, a conclusion strongly supported by phylogenetic and gene expression analyses. Finally, the ability of UGT74C1 to complement phenotypes and chemotypes of the ugt74b1‐2 knockout mutant and to express thiohydroximate UGT activity in planta provides conclusive evidence for UGT74C1 being an accessory enzyme in glucosinolate biosynthesis with a potential function during plant adaptation to environmental challenge.} } @Article{IPB-1213, author = {Flores, R. and Gago-Zachert, S. and Serra, P. and Sanjuán, R. and Elena, S. F. and}, title = {{Viroids: Survivors from the RNA World?}}, year = {2014}, pages = {395-414}, journal = {Annu. Rev. Microbiol.}, doi = {10.1146/annurev-micro-091313-103416}, volume = {68}, abstract = {Because RNA can be a carrier of genetic information and a biocatalyst, there is a consensus that it emerged before DNA and proteins, which eventually assumed these roles and relegated RNA to intermediate functions. If such a scenario—the so-called RNA world—existed, we might hope to find its relics in our present world. The properties of viroids that make them candidates for being survivors of the RNA world include those expected for primitive RNA replicons: (a) small size imposed by error-prone replication, (b) high G \+ C content to increase replication fidelity, (c) circular structure for assuring complete replication without genomic tags, (d) structural periodicity for modular assembly into enlarged genomes, (e) lack of protein-coding ability consistent with a ribosome-free habitat, and (f) replication mediated in some by ribozymes, the fingerprint of the RNA world. With the advent of DNA and proteins, those protoviroids lost some abilities and became the plant parasites we now know.} } @Article{IPB-1326, author = {Kopycki, J. and Wieduwild, E. and Kohlschmidt, J. and Brandt, W. and Stepanova, A. and Alonso, J. and Pedras, M. S. and Abel, S. and Grubb, C. D. and}, title = {{Kinetic analysis of Arabidopsis glucosyltransferase UGT74B1 illustrates a general mechanism by which enzymes can escape product inhibition}}, year = {2013}, pages = {37-46}, journal = {Biochem. J.}, doi = {10.1042/BJ20121403}, volume = {450}, abstract = {Plant genomes encode numerous small molecule glycosyltransferases which modulate the solubility, activity, immunogenicity and/or reactivity of hormones, xenobiotics and natural products. The products of these enzymes can accumulate to very high concentrations, yet somehow avoid inhibiting their own biosynthesis. Glucosyltransferase UGT74B1 (UDP-glycosyltransferase 74B1) catalyses the penultimate step in the core biosynthetic pathway of glucosinolates, a group of natural products with important functions in plant defence against pests and pathogens. We found that mutation of the highly conserved Ser284 to leucine [wei9-1 (weak ethylene insensitive)] caused only very mild morphological and metabolic phenotypes, in dramatic contrast with knockout mutants, indicating that steady state glucosinolate levels are actively regulated even in unchallenged plants. Analysis of the effects of the mutation via a structural modelling approach indicated that the affected serine interacts directly with UDP-glucose, but also predicted alterations in acceptor substrate affinity and the kcat value, sparking an interest in the kinetic behaviour of the wild-type enzyme. Initial velocity and inhibition studies revealed that UGT74B1 is not inhibited by its glycoside product. Together with the effects of the missense mutation, these findings are most consistent with a partial rapid equilibrium ordered mechanism. This model explains the lack of product inhibition observed both in vitro and in vivo, illustrating a general mechanism whereby enzymes can continue to function even at very high product/precursor ratios.} } @Article{IPB-1301, author = {Elleuch, A. and Chaâbene, Z. and Grubb, D. C. and Drira, N. and Mejdoub, H. and Khemakhem, B. and}, title = {{Morphological and biochemical behavior of fenugreek (Trigonella foenum-graecum) under copper stress}}, year = {2013}, pages = {46-53}, journal = {Ecotoxicol. Environ. Saf.}, doi = {10.1016/j.ecoenv.2013.09.028}, volume = {98}, abstract = {The effects of copper on germination and growth of fenugreek (Trigonella foenum-graecum) was investigated separately using different concentrations of CuSO4. The germination percentage and radical length had different responses to cupric ions: the root growth increased with increasing copper concentration up to 1 mM and Cu2\+ was inhibited thereafter. In contrast, the germination percentage was largely unaffected by concentrations of copper below 10 mM.The reduction in root growth may have been due to inhibition of hydrolytic enzymes such as amylase. Indeed, the average total amylolytic activity decreased from the first day of treatment with [Cu2\+] greater than 1 mM. Furthermore, copper affected various plant growth parameters. Copper accumulation was markedly higher in roots as compared to shoots. While both showed a gradual decrease in growth, this was more pronounced in roots than in leaves and in stems. Excess copper induced an increase in the rate of hydrogen peroxide (H2O2) production and lipid peroxidation in all plant parts, indicating oxidative stress. This redox stress affected leaf chlorophyll and carotenoid content which decreased in response to augmented Cu levels. Additionally, the activities of proteins involved in reactive oxygen species (ROS) detoxification were affected. Cu stress elevated the ascorbate peroxidase (APX) activity more than two times at 10 mM CuSO4. In contrast, superoxide dismutase (SOD) and catalase (CAT) levels showed only minor variations, only at 1 mM Cu2\+. Likewise, total phenol and flavonoid contents were strongly induced by low concentrations of copper, consistent with the role of these potent antioxidants in scavenging ROS such as H2O2, but returned to control levels or below at high [Cu2\+]. Taken together, these results indicate a fundamental shift in the plant response to copper toxicity at low versus high concentrations.} } @INBOOK{IPB-89, author = {Vaira, A. M. and Gago-Zachert, S. and Garcia, M. L. and Guerri, J. and Hammond, J. and Milne, R. G. and Moreno, P. and Morikawa, T. and Natsuaki, T. and Navarro, J. A. and Pallas, V. and Torok, V. and Verbeek, M. and Vetten, H. J. and}, title = {{Virus Taxonomy: Ninth Report of the International Committee on Taxonomy of Viruses}}, year = {2012}, pages = {743-748}, chapter = {{Family - Ophioviridae}}, editor = {King, A. M. Q., et al., eds.}, doi = {10.1016/B978-0-12-384684-6.00060-4}, abstract = {This chapter focuses on Ophioviridae family whose sole member genus is Ophiovirus. The member species of the genus include Citrus psorosis virus (CPsV), Freesia sneak virus(FreSV), Lettuce ring necrosis virus (LRNV), and Mirafiori lettuce big-vein virus (MiLBVV).The single stranded negative/possibly ambisense RNA genome is divided into 3–4 segments, each of which is encapsidated in a single coat protein (43–50 kDa) forming filamentous virions of about 3 nm in diameter, in shape of kinked or probably internally coiled circles of at least two different contour lengths. Ophioviruses can be mechanically transmitted to a limited range of test plants, inducing local lesions and systemic mottle. The natural hosts of CPsV, ranunculus white mottle virus (RWMV), MiLBVV, and LRNV are dicotyledonous plants of widely differing taxonomy. CPsV has a wide geographical distribution in citrus in the Americas, in the Mediterranean and in New Zealand. FreSV has been reported in two species of the family Ranunculacae from Northern Italy, and in lettuce in France and Germany. Tulip mild mottle mosaic virus (TMMMV) has been reported in tulips in Japan. LRNV is closely associated with lettuce ring necrosis disease in The Netherlands, Belgium, and France, and FreSV has been reported in Europe, Africa, North America and New Zealand.} } @INBOOK{IPB-86, author = {Carbonell, A. and Flores, R. and Gago, S. and}, title = {{From Nucleic Acids Sequences to Molecular Medicine}}, year = {2012}, pages = {411-427}, chapter = {{Hammerhead Ribozymes Against Virus and Viroid RNAs}}, journal = {RNA Technologies}, editor = {Erdmann, V. A. \& Barciszewski, J., eds.}, doi = {10.1007/978-3-642-27426-8_16}, abstract = {The hammerhead ribozyme, a small catalytic motif that promotes self-cleavage of the RNAs in which it is found naturally embedded, can be manipulated to recognize and cleave specifically in trans other RNAs in the presence of Mg2\+. To be really effective, hammerheads need to operate at the low concentration of Mg2\+ existing in vivo. Evidence has been gathered along the last years showing that tertiary stabilizing motifs (TSMs), particularly interactions between peripheral loops, are critical for the catalytic activity of hammerheads at physiological levels of Mg2\+. These TSMs, in two alternative formats, have been incorporated into a new generation of more efficient trans-cleaving hammerheads, some of which are active in vitro and in planta when targeted against the highly structured RNA of a viroid (a small plant pathogen). This strategy has potential to confer protection against other RNA replicons, like RNA viruses infecting plants and animals.} } @Article{IPB-1499, author = {Kopycki, J. and Schmidt, J. and Abel, S. and Grubb, C. D. and}, title = {{Chemoenzymatic synthesis of diverse thiohydroximates from glucosinolate-utilizing enzymes from Helix pomatia and Caldicellulosiruptor saccharolyticus}}, year = {2011}, pages = {1039-1046}, journal = {Biotechnol. Lett.}, doi = {10.1007/s10529-011-0530-y}, volume = {33}, abstract = {Thiohydroximates comprise a diverse class of compounds important in both biological and industrial chemistry. Their syntheses are generally limited to simple alkyl and aryl compounds with few stereocenters and a narrow range of functional groups. We hypothesized that sequential action of two recombinant enzymes, a sulfatase from Helix pomatia and a β-O-glucosidase from Caldicellulosiruptor saccharolyticus, on glucosinolates would allow synthesis of thiohydroximates from a structurally broad array of abundant precursors. We report successful synthesis of thiohydroximates of varied chemical classes, including from homochiral compounds of demonstrated biological activity. The chemoenzymatic synthetic route reported here should allow access to many, if not all, of the thiohydroximate core structures of the ~200 known naturally occurring glucosinolates. The enrichment of this group for compounds with possible pharmacological potential is discussed.} } @Article{IPB-1484, author = {Flores, R. and Grubb, D. and Elleuch, A. and Nohales, M.-?. and Delgado, S. and Gago, S. and}, title = {{Rolling-circle replication of viroids, viroid-like satellite RNAs and hepatitis delta virus: Variations on a theme}}, year = {2011}, pages = {200-206}, journal = {RNA Biol.}, doi = {10.4161/rna.8.2.14238}, volume = {8}, abstract = {Viroids and viroid-like satellite RNAs from plants, and the human hepatitis delta virus (HDV) RNA share some properties that include small size, circularity and replication through a rolling-circle mechanism. Replication occurs in different cell compartments (nucleus, chloroplast and membrane-associated cytoplasmatic vesicles) and has three steps: RNA polymerization, cleavage and ligation. The first step generates oligomeric RNAs that result from the reiterative transcription of the circular templates of one or both polarities, and is catalyzed by either the RNA-dependent RNA polymerase of the helper virus on which viroid-like satellite RNAs are functionally dependent, or by host DNA-dependent RNA polymerases that, remarkably, viroids and HDV redirect to transcribe RNA templates. Cleavage is mediated by host enzymes in certain viroids and viroid-like satellite RNAs, while in others and in HDV is mediated by cis-acting ribozymes of three classes. Ligation appears to be catalyzed mainly by host enzymes. Replication most likely also involves many other non-catalytic proteins of host origin and, in HDV, the single virus-encoded protein.} } @Article{IPB-1476, author = {Carbonell, A. and Flores, R. and Gago, S. and}, title = {{Trans-cleaving hammerhead ribozymes with tertiary stabilizing motifs: in vitro and in vivo activity against a structured viroid RNA}}, year = {2011}, pages = {2432-2444}, journal = {Nucleic Acids Res.}, doi = {10.1093/nar/gkq1051}, volume = {39}, abstract = {Trans -cleaving hammerheads with discontinuous or extended stem I and with tertiary stabilizing motifs (TSMs) have been tested previously against short RNA substrates in vitro at low Mg 2\+ concentration. However, the potential of these ribozymes for targeting longer and structured RNAs in vitro and in vivo has not been examined. Here, we report the in vitro cleavage of short RNAs and of a 464-nt highly structured RNA from potato spindle tuber viroid (PSTVd) by hammerheads with discontinuous and extended formats at submillimolar Mg 2\+ . Under these conditions, hammerheads derived from eggplant latent viroid and peach latent mosaic viroid (PLMVd) with discontinuous and extended formats, respectively, where the most active. Furthermore, a PLMVd-derived hammerhead with natural TSMs showed activity in vivo against the same long substrate and interfered with systemic PSTVd infection, thus reinforcing the idea that this class of ribozymes has potential to control pathogenic RNA replicons.} } @Article{IPB-1577, author = {Renovell, ?. and Gago, S. and Ruiz-Ruiz, S. and Velázquez, K. and Navarro, L. and Moreno, P. and Vives, M. C. and Guerri, J. and}, title = {{Mapping the subgenomic RNA promoter of the Citrus leaf blotch virus coat protein gene by Agrobacterium-mediated inoculation}}, year = {2010}, pages = {360-369}, journal = {Virology}, doi = {10.1016/j.virol.2010.07.034}, volume = {406}, abstract = {Citrus leaf blotch virus has a single-stranded positive-sense genomic RNA (gRNA) of 8747 nt organized in three open reading frames (ORFs). The ORF1, encoding a polyprotein involved in replication, is translated directly from the gRNA, whereas ORFs encoding the movement (MP) and coat (CP) proteins are expressed via 3\' coterminal subgenomic RNAs (sgRNAs). We characterized the minimal promoter region critical for the CP-sgRNA expression in infected cells by deletion analyses using Agrobacterium-mediated infection of Nicotiana benthamiana plants. The minimal CP-sgRNA promoter was mapped between nucleotides −67 and \+ 50 nt around the transcription start site. Surprisingly, larger deletions in the region between the CP-sgRNA transcription start site and the CP translation initiation codon resulted in increased CP-sgRNA accumulation, suggesting that this sequence could modulate the CP-sgRNA transcription. Site-specific mutational analysis of the transcription start site revealed that the \+ 1 guanylate and the \+ 2 adenylate are important for CP-sgRNA synthesis.} } @Article{IPB-1628, author = {Gago, S. and Elena, S. F. and Flores, R. and Sanjuan, R. and}, title = {{Extremely High Mutation Rate of a Hammerhead Viroid}}, year = {2009}, pages = {1308-1308}, journal = {Science}, doi = {10.1126/science.1169202}, volume = {323}, abstract = {The mutation rates of viroids, plant pathogens with minimal non-protein-coding RNA genomes, are unknown. Their replication is mediated by host RNA polymerases and, in some cases, by hammerhead ribozymes, small self-cleaving motifs embedded in the viroid. By using the principle that the population frequency of nonviable genotypes equals the mutation rate, we screened for changes that inactivated the hammerheads of Chrysanthemum chlorotic mottle viroid. We obtained a mutation rate of 1/400 per site, the highest reported for any biological entity. Such error-prone replication can only be tolerated by extremely simple genomes such as those of viroids and, presumably, the primitive replicons of the RNA world. Our results suggest that the emergence of replication fidelity was critical for the evolution of complexity in the early history of life.} } @Article{IPB-1625, author = {Flores, R. and Gas, M.-E. and Molina-Serrano, D. and Nohales, M.-?. and Carbonell, A. and Gago, S. and De la Peña, M. and Daròs, J.-A. and}, title = {{Viroid Replication: Rolling-Circles, Enzymes and Ribozymes}}, year = {2009}, pages = {317-334}, journal = {Viruses}, doi = {10.3390/v1020317}, volume = {1}, abstract = {Viroids, due to their small size and lack of protein-coding capacity, must rely essentially on their hosts for replication. Intriguingly, viroids have evolved the ability to replicate in two cellular organella, the nucleus (family Pospiviroidae) and the chloroplast (family Avsunviroidae). Viroid replication proceeds through an RNA-based rolling-circle mechanism with three steps that, with some variations, operate in both polarity strands: i) synthesis of longer-than-unit strands catalyzed by either the nuclear RNA polymerase II or a nuclear-encoded chloroplastic RNA polymerase, in both instances redirected to transcribe RNA templates, ii) cleavage to unit-length, which in the family Avsunviroidae is mediated by hammerhead ribozymes embedded in both polarity strands, while in the family Pospiviroidae the oligomeric RNAs provide the proper conformation but not the catalytic activity, and iii) circularization. The host RNA polymerases, most likely assisted by additional host proteins, start transcription from specific sites, thus implying the existence of viroid promoters. Cleavage and ligation in the family Pospiviroidae is probably catalyzed by an RNase III-like enzyme and an RNA ligase able to circularize the resulting 5’ and 3’ termini. Whether a chloroplastic RNA ligase mediates circularization in the family Avsunviroidae, or this reaction is autocatalytic, remains an open issue.} } @Article{IPB-1618, author = {Dufour, D. and De la Peña, M. and Gago, S. and Flores, R. and Gallego, J. and}, title = {{Structure–function analysis of the ribozymes of chrysanthemum chlorotic mottle viroid: a loop–loop interaction motif conserved in most natural hammerheads}}, year = {2009}, pages = {368-381}, journal = {Nucleic Acids Res.}, doi = {10.1093/nar/gkn918}, volume = {37}, abstract = {Loop–loop tertiary interactions play a key role in the folding and catalytic activity of natural hammerhead ribozymes. Using a combination of NMR spectroscopy, site-directed mutagenesis and kinetic and infectivity analyses, we have examined the structure and function of loops 1 and 2 of the (\+) and (–) hammerheads of chrysanthemum chlorotic mottle viroid RNA. In both hammerheads, loop 1 is a heptanucleotide hairpin loop containing an exposed U at its 5′ side and an extrahelical U at its 3′-side critical for the catalytic activity of the ribozyme in vitro and for viroid infectivity in vivo , whereas loop 2 has a key opened A at its 3′-side. These structural features promote a specific loop–loop interaction motif across the major groove. The essential features of this tertiary structure element, base pairing between the 5′ U of loop 1 and the 3′ A of loop 2, and interaction of the extrahelical pyrimidine of loop 1 with loop 2, are likely shared by a significant fraction of natural hammerheads.} } @Article{IPB-1657, author = {Serra, P. and BANI HASHEMIAN, S. M. and PENSABENE-BELLAVIA, G. and Gago, S. and DURAN-VILA, N. and}, title = {{An artificial chimeric derivative of Citrus viroid V involves the terminal left domain in pathogenicity}}, year = {2009}, pages = {515-522}, journal = {Mol. Plant Pathol.}, doi = {10.1111/j.1364-3703.2009.00553.x}, volume = {10}, abstract = {The recently described Citrus viroid V (CVd‐V) induces, in Etrog citron, mild stunting and very small necrotic lesions and cracks, sometimes filled with gum. As Etrog citron plants co‐infected with Citrus dwarfing viroid (CDVd) and CVd‐V show synergistic interactions, these host–viroid combinations provide a convenient model to identify the pathogenicity determinant(s). The biological effects of replacing limited portions of the rod‐like structure of CVd‐V with the corresponding portions of CDVd are reported. Chimeric constructs were synthesized using a novel polymerase chain reaction‐based approach, much more flexible than those based on restriction enzymes used in previous studies. Of the seven chimeras (Ch) tested, only one (Ch5) proved to be infectious. Plants infected with Ch5 showed no symptoms and, although this novel chimera was able to replicate to relatively high titres in singly infected plants, it was rapidly displaced by either CVd‐V or CDVd in doubly infected plants. The results demonstrate that direct interaction(s) between structural elements in the viroid RNA (in this case, the terminal left domain) and as yet unidentified host factors play an important role in modulating viroid pathogenicity. This is the first pathogenic determinant mapped in species of the genus Apscaviroid.} } @Article{IPB-1704, author = {Iglesias, N. G. and Gago-Zachert, S. P. and Robledo, G. and Costa, N. and Plata, M. I. and Vera, O. and Grau, O. and Semorile, L. C. and}, title = {{Population structure of Citrus tristeza virus from field Argentinean isolates}}, year = {2008}, pages = {199-207}, journal = {Virus Genes}, doi = {10.1007/s11262-007-0169-x}, volume = {36}, abstract = {We studied the genetic variability of three genomic regions (p23, p25 and p27 genes) from 11 field Citrus tristeza virus isolates from the two main citrus growing areas of Argentina, a country where the most efficient vector of the virus, Toxoptera citricida, is present for decades. The pathogenicity of the isolates was determinated by biological indexing, single-strand conformation polymorphism analysis showed that most isolates contained high intra-isolate variability. Divergent sequence variants were detected in some isolates, suggesting re-infections of the field plants. Phylogenetic analysis of the predominant sequence variants of each isolate revealed similar grouping of isolates for genes p25 and p27. The analysis of p23 showed two groups contained the severe isolates. Our results showed a high intra-isolate sequence variability suggesting that re-infections could contribute to the observed variability and that the host can play an important role in the selection of the sequence variants present in these isolates.} } @Article{IPB-1688, author = {Carbonell, A. and Martínez de Alba, A.-E. and Flores, R. and Gago, S. and}, title = {{Double-stranded RNA interferes in a sequence-specific manner with the infection of representative members of the two viroid families}}, year = {2008}, pages = {44-53}, journal = {Virology}, doi = {10.1016/j.virol.2007.09.031}, volume = {371}, abstract = {Infection by viroids, non-protein-coding circular RNAs, occurs with the accumulation of 21–24 nt viroid-derived small RNAs (vd-sRNAs) with characteristic properties of small interfering RNAs (siRNAs) associated to RNA silencing. The vd-sRNAs most likely derive from dicer-like (DCL) enzymes acting on viroid-specific dsRNA, the key elicitor of RNA silencing, or on the highly structured genomic RNA. Previously, viral dsRNAs delivered mechanically or agroinoculated have been shown to interfere with virus infection in a sequence-specific manner. Here, we report similar results with members of the two families of nuclear- and chloroplast-replicating viroids. Moreover, homologous vd-sRNAs co-delivered mechanically also interfered with one of the viroids examined. The interference was sequence-specific, temperature-dependent and, in some cases, also dependent on the dose of the co-inoculated dsRNA or vd-sRNAs. The sequence-specific nature of these effects suggests the involvement of the RNA induced silencing complex (RISC), which provides sequence specificity to RNA silencing machinery. Therefore, viroid titer in natural infections might be regulated by the concerted action of DCL and RISC. Viroids could have evolved their secondary structure as a compromise between resistance to DCL and RISC, which act preferentially against RNAs with compact and relaxed secondary structures, respectively. In addition, compartmentation, association with proteins or active replication might also help viroids to elude their host RNA silencing machinery.} } @Article{IPB-1736, author = {Serra, P. and Gago, S. and Duran-Vila, N. and}, title = {{A single nucleotide change in Hop stunt viroid modulates citrus cachexia symptoms}}, year = {2008}, pages = {130-134}, journal = {Virus Res.}, doi = {10.1016/j.virusres.2008.08.003}, volume = {138}, abstract = {Cachexia disease of citrus is caused by Hop stunt viroid (HSVd). In citrus, pathogenic and non-pathogenic strains differ by a “cachexia expression motif” of five to six nucleotides located in the variable domain of the proposed rod-like secondary structure. Here, site-directed mutants were generated to investigate if all these nucleotides were required for infectivity and/or symptom expression. Specifically an artificial cachexia inducing mutant M0 was generated by introducing the six nucleotides changes of the “cachexia expression motif” into a non-pathogenic sequence variant and M0 was used as a template to systematically restore some of the introduced changes. The resulting mutants in which specific changes introduced to generate M0, were restored presented a variety of responses: (i) M1, obtained by introducing two insertions forming a base-pair, was infectious but non-pathogenic; (ii) M2, obtained by introducing an insertion and restoring a substitution, presented low infectivity and the resulting progeny reverted to M0; (iii) M3, obtained by restoring a single substitution in the lower strand of the viroid secondary structure, was infectious but induced only mild cachexia symptoms; (iv) M4, obtained by restoring a single susbtitution in the upper strand of the viroid secondary structure, was non-infectious. These results confirm that the “cachexia expression motif” plays a major role in inciting cachexia symptoms, and that subtle changes within this motif affect symptom severity and may even suppress symptom expression.} } @INBOOK{IPB-108, author = {Flores, R. and Carbonell, A. and Gago, S. and Martínez de Alba, A.-E. and Delgado, S. and Rodio, M.-E. and Di Serio, F. and}, year = {2008}, pages = {1-9}, chapter = {{Viroid-host interactions: A molecular dialogue between two uneven partners}}, journal = {Biology of Plant-Microbe Interactions}, volume = {6}, } @Article{IPB-1783, author = {Flores, R. and Navarro, B. and Gago, S. and De la Peña, M. and}, title = {{Chrysanthemum Chlorotic Mottle Viroid: a System for Reverse Genetics in the Family Avsunviroidae (Hammerhead Viroids)}}, year = {2007}, pages = {27-32}, journal = {Plant Viruses}, url = {http://www.globalsciencebooks.info/Online/GSBOnline/OnlinePV_1_1.html}, volume = {1}, abstract = {Viroids are small single-stranded circular RNAs able to infect plants. Chrysanthemum chlorotic mottle was one of the first viroid diseases reported, but identification and characterization of the causing RNA was delayed by its low accumulation in vivo. Chrysanthemum chlorotic mottle viroid (CChMVd) (398-401 nt) adopts a branched conformation instead of the rod-like secondary structure characteristic of most viroids. The natural sequence variability and the effects of artificial mutants support that the branched conformation is physiologically relevant and additionally stabilized by a kissing-loop interaction critical for RNA in vitro folding and in vivo viability. CChMVd shares structural similarities with peach latent mosaic viroid, with which forms the genus Pelamoviroid within the family Avsunviroidae. CChMVd adopts hammerhead structures that catalyze self-cleavage of the oligomeric strands of both polarities resulting from replication through a symmetric rolling-circle mechanism. The two CChMVd hammerheads display peculiarities: the plus has an extra A close to the central conserved core, and the minus an unsually long helix II. There are non-symptomatic strains (CChMVd-NS) that protect against challenge inoculation with severe strains (CChMVd-S). Introduction by site-directed mutagenesis of one of the CChMVd-NS specific mutations (UUUC?GAAA) is sufficient to change the symptomatic phenotype into non-symptomatic without altering the viroid titer. This pathogenicity determinant maps at a tetraloop of the CChMVd branched conformation. Co-inoculations with typical CChMVd-S and -NS variants showed that the infected plants remain symptomless only when the latter was in more than a 100-fold excess, indicating the higher fitness of the S variant. RNA silencing could mediate the observed cross-protection.} } @Article{IPB-1766, author = {Biondi, E. and Branciamore, S. and Fusi, L. and Gago, S. and Gallori, E. and}, title = {{Catalytic activity of hammerhead ribozymes in a clay mineral environment: Implications for the RNA world}}, year = {2007}, pages = {10-18}, journal = {Gene}, doi = {10.1016/j.gene.2006.09.002}, volume = {389}, abstract = {The hypothesized RNA-based world would have required the presence of a protected environment in which RNA, or an RNA-like molecule, could originate and express its biological activity.Recent studies have indicated that RNA molecules adsorbed/bound on clay minerals are able to persist in the presence of degrading agents, to interact with surrounding molecules, and to transmit the information contained in their nucleotide sequences.In this study, we assessed the ability of RNA molecules with catalytic activity to perform a specific reaction in a mineral environment. For this purpose, we investigated the self-cleavage reaction of the hammerhead ribozyme of the Avocado Sun Blotch Viroid (ASBVd), both in the monomeric and in dimeric forms. The monomeric transcript was tightly bound on the clay mineral montmorillonite to form a stable complex, while the behaviour of the dimeric transcript was studied in the presence of the clay particles in the reaction mixture.The results indicated that the hammerhead ribozyme was still active when the monomeric transcript was adsorbed on the clay surface, even though its efficiency was reduced to about 20% of that in solution. Moreover, the self-cleavage of clay-adsorbed molecule was significantly enhanced (∼ four times) by the presence of the 5′ reaction product.The self-cleavage reaction of the dimeric transcript in the presence of montmorillonite indicated that the mineral particles protected the RNA molecules against aspecific degradation and increased the rate of cleavage kinetics by about one order of magnitude.These findings corroborate the hypothesis that clay-rich environments would have been a good habitat in which RNA or RNA-like molecules could originate, accumulate and undergo Darwinian evolutionary processes, leading to the first living cells on Earth.} } @INBOOK{IPB-112, author = {Flores, R. and Carbonell, A. and De la Peña, M. and Gago, S. and}, title = {{Herramientas Biotecnológicas en Fitopatología}}, year = {2007}, pages = {407-425}, chapter = {{RNAs Autocatalíticos: Ribozimas de Cabeza de Martillo}}, } @Article{IPB-1869, author = {Grubb, C. D. and Abel, S. and}, title = {{Glucosinolate metabolism and its control}}, year = {2006}, pages = {89-100}, journal = {Trends Plant Sci.}, doi = {10.1016/j.tplants.2005.12.006}, volume = {11}, abstract = {Glucosinolates and their associated degradation products have long been recognized for their distinctive benefits to human nutrition and plant defense. Because most of the structural genes of glucosinolate metabolism have been identified and functionally characterized in Arabidopsis thaliana, current research increasingly focuses on questions related to the regulation of glucosinolate synthesis, distribution and degradation as well as to the feasibility of engineering customized glucosinolate profiles. Here, we highlight recent progress in glucosinolate research, with particular emphasis on the biosynthetic pathway and its metabolic relationships to auxin homeostasis. We further discuss emerging insight into the signaling networks and regulatory proteins that control glucosinolate accumulation during plant development and in response to environmental challenge.} } @Article{IPB-1853, author = {Carbonell, A. and De la Peña, M. and Flores, R. and Gago, S. and}, title = {{Effects of the trinucleotide preceding the self-cleavage site on eggplant latent viroid hammerheads: differences in co- and post-transcriptional self-cleavage may explain the lack of trinucleotide AUC in most natural hammerheads}}, year = {2006}, pages = {5613-5622}, journal = {Nucleic Acids Res.}, doi = {10.1093/nar/gkl717}, volume = {34}, abstract = {Eggplant latent viroid (ELVd) can form stable hammerhead structures in its (\+) and (−) strands. These ribozymes have the longest helices I reported in natural hammerheads, with that of the ELVd (\+) hammerhead being particularly stable (5/7 bp are G-C). Moreover, the trinucleotide preceding the self-cleavage site of this hammerhead is AUA, which together with GUA also found in some natural hammerheads, deviate from the GUC present in most natural hammerheads including the ELVd (−) hammerhead. When the AUA trinucleotide preceding the self-cleavage site of the ELVd (\+) hammerhead was substituted by GUA and GUC, as well as by AUC (essentially absent in natural hammerheads), the values of the self-cleavage rate constants at low magnesium of the purified hammerheads were: ELVd-(\+)-AUC≈ELVd-(\+)-GUC\>ELVd-(\+)-GUA\> ELVd-(\+)-AUA. However, the ELVd-(\+)-AUC hammerhead was the catalytically less efficient during in vitro transcription, most likely because of the transient adoption of catalytically-inactive metastable structures. These results suggest that natural hammerheads have been evolutionary selected to function co-transcriptionally, and provide a model explaining the lack of trinucleotide AUC preceding the self-cleavage site of most natural hammerheads. Comparisons with other natural hammerheads showed that the ELVd-(\+)-GUC and ELVd-(\+)-AUC hammerheads are the catalytically most active in a post-transcriptional context with low magnesium.} } @Article{IPB-1950, author = {Gago, S. and De la Peña, M. and Flores, R. and}, title = {{A kissing-loop interaction in a hammerhead viroid RNA critical for its in vitro folding and in vivo viability}}, year = {2005}, pages = {1073-1083}, journal = {RNA}, doi = {10.1261/rna.2230605}, volume = {11}, abstract = {Chrysanthemum chlorotic mottle viroid (CChMVd) RNA (398–401 nucleotides) can form hammerhead ribozymes that play a functional role in its replication through a rolling-circle mechanism. In contrast to most other viroids, which adopt rod-like or quasi-rod-like secondary structures of minimal free energy, the computer-predicted conformations of CChMVd and Peach latent mosaic viroid (PLMVd) RNAs are branched. Moreover, the covariations found in a number of natural CChMVd variants support that the same or a closely related conformation exists in vivo. Here we report that the CChMVd natural variability also supports that the branched conformation is additionally stabilized by a kissing-loop interaction resembling another one proposed in PLMVd from in vitro assays. Moreover, site-directed mutagenesis combined with bioassays and progeny analysis showed that: (1) single CChMVd mutants affecting the kissing loops had low or no infectivity at all, whereas infectivity was recovered in double mutants restoring the interaction; (2) mutations affecting the structure of the regions adjacent to the kissing loops reverted to wild type or led to rearranged stems, also supporting their interaction; and (3) the interchange between 4 nucleotides of each of the two kissing loops generated a viable CChMVd variant with eight mutations. PAGE analysis under denaturing and nondenaturing conditions revealed that the kissing-loop interaction determines proper in vitro folding of CChMVd RNA. Preservation of a similar kissing-loop interaction in two hammerhead viroids with an overall low sequence similarity suggests that it facilitates in vivo the adoption and stabilization of a compact folding critical for viroid viability.} } @INBOOK{IPB-132, author = {Vaira, A. M. and Acotto, G. P. and Gago-Zachert, S. and Garcia, M. L. and Grau, O. and Milne, R. G. and Morikawa, T. and Natsuaki, T. and Torov, V. and Verbeek, M. and Vetten, H. J. and}, title = {{Virus Taxonomy}}, year = {2005}, pages = {673-679}, chapter = {{Genus Ophiovirus}}, doi = {10.1016/B978-0-12-249951-7.50014-6}, } @Article{IPB-2019, author = {Grubb, C. D. and Zipp, B. J. and Ludwig-Müller, J. and Masuno, M. N. and Molinski, T. F. and Abel, S. and}, title = {{Arabidopsis glucosyltransferase UGT74B1 functions in glucosinolate biosynthesis and auxin homeostasis}}, year = {2004}, pages = {893-908}, journal = {Plant J.}, doi = {10.1111/j.1365-313X.2004.02261.x}, volume = {40}, abstract = {Glucosinolates are a class of secondary metabolites with important roles in plant defense and human nutrition. Here, we characterize a putative UDP‐glucose:thiohydroximate S‐glucosyltransferase, UGT74B1, to determine its role in the Arabidopsis glucosinolate pathway. Biochemical analyses demonstrate that recombinant UGT74B1 specifically glucosylates the thiohydroximate functional group. Low K m values for phenylacetothiohydroximic acid (approximately 6 μ m ) and UDP‐glucose (approximately 50 μm ) strongly suggest that thiohydroximates are in vivo substrates of UGT74B1. Insertional loss‐of‐function ugt74b1 mutants exhibit significantly decreased, but not abolished, glucosinolate accumulation. In addition, ugt74b1 mutants display phenotypes reminiscent of auxin overproduction, such as epinastic cotyledons, elongated hypocotyls in light‐grown plants, excess adventitious rooting and incomplete leaf vascularization. Indeed, during early plant development, mutant ugt74b1 seedlings accumulate nearly threefold more indole‐3‐acetic acid than the wild type. Other phenotypes, however, such as chlorosis along the leaf veins, are likely caused by thiohydroximate toxicity. Analysis of UGT74B1 promoter activity during plant development reveals expression patterns consistent with glucosinolate metabolism and induction by auxin treatment. The results are discussed in the context of known mutations in glucosinolate pathway genes and their effects on auxin homeostasis. Taken together, our work provides complementary in vitro and in vivo evidence for a primary role of UGT74B1 in glucosinolate biosynthesis.} } @Article{IPB-2012, author = {Flores, R. and Delgado, S. and Gas, M.-E. and Carbonell, A. and Molina, D. and Gago, S. and De la Peña, M. and}, title = {{Viroids: the minimal non-coding RNAs with autonomous replication}}, year = {2004}, pages = {42-48}, journal = {FEBS Lett.}, doi = {10.1016/j.febslet.2004.03.118}, volume = {567}, abstract = {Viroids are small (246–401 nucleotides), non‐coding, circular RNAs able to replicate autonomously in certain plants. Viroids are classified into the families Pospiviroidae and Avsunviroidae , whose members replicate in the nucleus and chloroplast, respectively. Replication occurs by an RNA‐based rolling‐circle mechanism in three steps: (1) synthesis of longer‐than‐unit strands catalyzed by host DNA‐dependent RNA polymerases forced to transcribe RNA templates, (2) processing to unit‐length, which in family Avsunviroidae is mediated by hammerhead ribozymes, and (3) circularization either through an RNA ligase or autocatalytically. Disease induction might result from the accumulation of viroid‐specific small interfering RNAs that, via RNA silencing, could interfere with normal developmental pathways.} } @Article{IPB-2104, author = {Naum-Onganı́a, G. and Gago-Zachert, S. and Peña, E. and Grau, O. and Laura Garcia, M. and}, title = {{Citrus psorosis virus RNA 1 is of negative polarity and potentially encodes in its complementary strand a 24K protein of unknown function and 280K putative RNA dependent RNA polymerase}}, year = {2003}, pages = {49-61}, journal = {Virus Res.}, doi = {10.1016/S0168-1702(03)00172-2}, volume = {96}, abstract = {Citrus psorosis virus (CPsV), the type member of genus Ophiovirus, has three genomic RNAs. Complete sequencing of CPsV RNA 1 revealed a size of 8184 nucleotides and Northern blot hybridization with chain specific probes showed that its non-coding strand is preferentially encapsidated. The complementary strand of RNA 1 contains two open reading frames (ORFs) separated by a 109-nt intergenic region, one located near the 5′-end potentially encoding a 24K protein of unknown function, and another of 280K containing the core polymerase motifs characteristic of viral RNA-dependent RNA polymerases (RdRp). Comparison of the core RdRp motifs of negative-stranded RNA viruses, supports grouping CPsV, Ranunculus white mottle virus (RWMV) and Mirafiori lettuce virus (MiLV) within the same genus (Ophiovirus), constituting a monophyletic group separated from all other negative-stranded RNA viruses. Furthermore, RNAs 1 of MiLV, CPsV and RWMV are similar in size and those of MiLV and CPsV also in genomic organization and sequence.} } @Article{IPB-2079, author = {De la Peña, M. and Gago, S. and Flores, R. and}, title = {{Peripheral regions of natural hammerhead ribozymes greatly increase their self-cleavage activity}}, year = {2003}, pages = {5561-5570}, journal = {EMBO J.}, doi = {10.1093/emboj/cdg530}, volume = {22}, abstract = {Natural hammerhead ribozymes are mostly found in some viroid and viroid‐like RNAs and catalyze their cis cleavage during replication. Hammerheads have been manipulated to act in trans and assumed to have a similar catalytic behavior in this artificial context. However, we show here that two natural cis‐acting hammerheads self‐cleave much faster than trans‐acting derivatives and other reported artificial hammerheads. Moreover, modifications of the peripheral loops 1 and 2 of one of these natural hammerheads induced a \>100‐fold reduction of the self‐cleavage constant, whereas engineering a trans‐acting artificial hammerhead into a cis derivative by introducing a loop 1 had no effect. These data show that regions external to the central conserved core of natural hammerheads play a role in catalysis, and suggest the existence of tertiary interactions between these peripheral regions. The interactions, determined by the sequence and size of loops 1 and 2 and most likely of helices I and II, must result from natural selection and should be studied in order to better understand the hammerhead requirements in vivo.} } @Article{IPB-2195, author = {Wang, Q. and Grubb, C. D. and Abel, S. and}, title = {{Direct analysis of single leaf disks for chemopreventive glucosinolates}}, year = {2002}, pages = {152-157}, journal = {Phytochem. Anal.}, doi = {10.1002/pca.636}, volume = {13}, abstract = {Natural isothiocyanates, produced during plant tissue damage from methionine‐derived glucosinolates, are potent inducers of mammalian phase 2 detoxification enzymes such as quinone reductase (QR). A greatly simplified bioassay for glucosinolates based on induction and colorimetric detection of QR activity in murine hepatoma cells is described. It is demonstrated that excised leaf disks of Arabidopsis thaliana (ecotype Columbia) can directly and reproducibly substitute for cell‐free leaf extracts as inducers of murine QR, which reduces sample preparation to a minimum and maximizes throughput. A comparison of 1 and 3 mm diameter leaf disks indicated that QR inducer potency was proportional to disk circumference (extent of tissue damage) rather than to area. When compared to the QR inducer potency of the corresponding amount of extract, 1 mm leaf disks were equally effective, whereas 3 mm disks were 70% as potent. The QR inducer potency of leaf disks correlated positively with the content of methionine‐derived glucosinolates, as shown by the analysis of wild‐type plants and mutant lines with lower or higher glucosinolate content. Thus, the microtitre plate‐based assay of single leaf disks provides a robust and inexpensive visual method for rapidly screening large numbers of plants in mapping populations or mutant collections and may be applicable to other glucosinolate‐producing species.} } @Article{IPB-2162, author = {Grubb, C. D. and Gross, H. B. and Chen, D. L. and Abel, S. and}, title = {{Identification of Arabidopsis mutants with altered glucosinolate profiles based on isothiocyanate bioactivity}}, year = {2002}, pages = {143-152}, journal = {Plant Sci.}, doi = {10.1016/S0168-9452(01)00550-7}, volume = {162}, abstract = {Glucosinolates are a diverse class of nitrogen- and sulfur-containing secondary metabolites. They are rapidly hydrolyzed on tissue disruption to a number of biologically active compounds that are increasingly attracting interest as anticarcinogenic phytochemicals and crop protectants. Several glucosinolate-derived isothiocyanates are potent chemopreventive agents that favorably modulate carcinogen metabolism in mammals. Methylsulfinylalkyl isothiocyanates, in particular the 4-methylsulfinylbutyl derivative, are selective and potent inducers of mammalian detoxification enzymes such as quinone reductase (QR). Cruciferous plants including Arabidopsis thaliana (L.) Heyhn, synthesize methylsulfinylalkyl glucosinolates, which are derived from methionine. Using a colorimetric assay for QR activity in murine hepatoma cells and high performance liquid chromatography (HPLC) analysis of desulfoglucosinolates, we have demonstrated a strong positive correlation between leaf QR inducer potency and leaf content of methionine-derived glucosinolates in various A. thaliana ecotypes and available glucosinolate mutants. In a molecular genetic approach to glucosinolate biosynthesis, we screened 3000 chemically mutagenized M2 plants of the Columbia ecotype for altered leaf QR inducer potency. Subsequent HPLC analysis of progeny of putative mutants identified six lines with significant and heritable changes in leaf glucosinolate content and composition.} } @Article{IPB-2255, author = {Gross, H. B. and Dalebout, T. and Grubb, C. D. and Abel, S. and}, title = {{Functional detection of chemopreventive glucosinolates in Arabidopsis thaliana}}, year = {2000}, pages = {265-272}, journal = {Plant Sci.}, doi = {10.1016/S0168-9452(00)00354-X}, volume = {159}, abstract = {Natural isothiocyanates, derived from glucosinolates by myrosinase-catalyzed hydrolysis, are potent chemopreventive agents that favorably modify carcinogen metabolism in mammals by inhibiting metabolic activation of carcinogens and/or by inducing carcinogen-detoxifying enzymes. Methylsulfinylalkyl isothiocyanates are potent selective inducers of mammalian Phase 2 detoxification enzymes such as quinone reductase [NADP(H):quinone-acceptor oxidoreductase, EC 1.6.99.2]. Members of the Cruciferae family, including the model plant species Arabidopsis thaliana (L.) Heyhn, synthesize methylsulfinylalkyl glucosinolates. We have adapted a colorimetric bioassay for quinone reductase activity in Hepa 1c1c7 murine hepatoma cells as a versatile tool to rapidly monitor methylsulfinylalkyl glucosinolate content in A. thaliana leaf extracts. Using wild type plants and mutant plants defective in the synthesis of 4-methylsulfinylbutyl glucosinolate (glucoraphanin), we have demonstrated that A. thaliana (ecotype Columbia) is a rich source of Phase 2 enzyme inducers and that methylsulfinylalkyl glucosinolates, predominantly glucoraphanin, account for about 80% of the quinone reductase inducer potency of Columbia leaf extracts. We have optimized leaf extraction conditions and the quinone reductase bioassay to allow for screening of large numbers of plant extracts in a molecular genetic approach to dissecting glucosinolate biosynthesis in A. thaliana.} } @Article{IPB-2305, author = {Gago-Zachert, S. and Costa, N. and Semorile, L. and Grau, O. and}, title = {{Sequence variability in p27 gene of Citrus Tristeza Virus (CTV) revealed by SSCP analysis}}, year = {1999}, pages = {41-50}, journal = {Electron. J. Biotechnol.}, doi = {10.2225/vol2-issue1-fulltext-3}, volume = {2}, abstract = {Citrus tristeza closterovirus (CTV), is a phloem-limited virus transmitted by aphids in a semipersistent manner. The genome of CTV is composed of a ssRNA with two capsid proteins: CP, covering about 95% of the particle length, and a diverged coat protein (dCP), present only in one end of the particle, forming a rattlesnake structure. dCP is the product of p27 gene for which it is also postulated a function in the transmissibility by aphid vectors. Hybridization analysis showed a p27 gene region, which exhibits different patterns with two probes derived from two biological distinct CTV isolates. In an attempt to screen whether that gene region differs in mild and severe strains, six CTV isolates belonging to different biogroups were compared for variations in their p27 gene by analysis of single-strand conformation polymorphism (SSCP). The p27 gene was reverse transcribed and amplified by PCR and thirty clones of each isolate were obtained. From each clone, two fragments of the gene were amplified by PCR: fragment (a), 459 bp long, and fragment (b), 281 bp long. Sequence variations in both gene fragments were studied by SSCP analysis. A variety of SSCP patterns was obtained from each isolate, being isolates belonging to the groups II-IV and III those with the higher and lower number of them. Moreover, SSCP analysis provided a rapid procedure to screen the genetic heterogeneity of the viral isolates reducing considerably the amount of nucleic acid sequenciation necessary to gain that knowledge.} }