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Publikation

López-Carrasco, A.; Ballesteros, C.; Sentandreu, V.; Delgado, S.; Gago-Zachert, S.; Flores, R.; Sanjuán, R.; Different rates of spontaneous mutation of chloroplastic and nuclear viroids as determined by high-fidelity ultra-deep sequencing PLOS Pathog. 13, e1006547, (2017) DOI: 10.1371/journal.ppat.1006547

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.
Publikation

López-Carrasco, A.; Gago-Zachert, S.; Mileti, G.; Minoia, S.; Flores, R.; Delgado, S.; The transcription initiation sites of eggplant latent viroid strands map within distinct motifs in their in vivo RNA conformations RNA Biol. 13, 83-97, (2016) DOI: 10.1080/15476286.2015.1119365

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.
Publikation

Flores, R.; Grubb, D.; Elleuch, A.; Nohales, M.-?.; Delgado, S.; Gago, S.; Rolling-circle replication of viroids, viroid-like satellite RNAs and hepatitis delta virus: Variations on a theme RNA Biol. 8, 200-206, (2011) DOI: 10.4161/rna.8.2.14238

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.
Bücher und Buchkapitel

Flores, R.; Carbonell, A.; Gago, S.; Martínez de Alba, A.-E.; Delgado, S.; Rodio, M.-E.; Di Serio, F.; Viroid-host interactions: A molecular dialogue between two uneven partners Biology of Plant-Microbe Interactions 6, 1-9, (2008)

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Publikation

Pedranzani, H.; Sierra-de-Grado, R.; Vigliocco, A.; Miersch, O.; Abdala, G.; Cold and water stresses produce changes in endogenous jasmonates in two populations of Pinus pinaster Ait Plant Growth Regul. 52, 111-116, (2007) DOI: 10.1007/s10725-007-9166-2

There is considerable evidence suggesting that jasmonates (JAs) play a role in plant resistance against abiotic stress. It is well known that in Angiosperms JAs are involved in the defense response, however there is little information about their role in Gymnosperms. Our proposal was to study the involvement of JAs in Pinus pinaster Ait. reaction to cold and water stress, and to compare the response of two populations of different provenances (Gredos and Bajo Tiétar) to these stresses. We detected 12-oxo-phytodienoic acid (OPDA), jasmonic acid (JA), and the hydroxylates 11-hydroxyjasmonate and 12-hydroxyjasmonate in foliage and shoots of P. pinaster plants. The response of the Gredos population to cold stress differed from that of Bajo Tiétar. Gredos plants showed a lower JA-basal level than Bajo Tiétar; under cold stress JA increased twofold at 72 h, while it decreased in Bajo Tiétar plants. The hydroxylates slightly increased in both populations due to cold stress treatment. Under water stress, plants from Gredos showed a remarkable JA-increase; thus the JA-response was much more prominent under water stress than under cold stress. In contrast, no change was found in JA-level in Bajo Tiétar plants under water stress. The level of JA-precursor, OPDA, was very low in control plants from Gredos and Bajo Tiétar. Under water stress OPDA increased only in plants from Bajo Tiétar. Therefore, we inform here of a different JAs-accumulation pattern after the stress treatment in P. pinaster from two provenances, and suggest a possible correlation with adaptations to diverse ecological conditions.
Publikation

Vigliocco, A.; Alemano, S.; Miersch, O.; Alvarez, D.; Abdala, G.; Endogenous jasmonates in dry and imbibed sunflower seeds from plants grown at different soil moisture contents Seed Sci. Res. 17, 91-98, (2007) DOI: 10.1017/S0960258507708371

In this study, we characterized two sunflower (Helianthus annuus L.) lines with differential sensitivity to drought, the sensitive line B59 and the tolerant line B71. Using both lines, we compared the content of endogenous jasmonates (JAs) in dry and imbibed seeds from plants grown under irrigation and drought. Jasmonic acid (JA), 12-oxo-phytodienoic acid (OPDA), 11-hydroxyjasmonate (11-OH-JA) and 12-hydroxyjasmonate (12-OH-JA) were detected in dry and imbibed sunflower seeds. Seeds from plants grown under drought had a lower content of total JAs and exhibited higher germination percentages than seeds from irrigated plants, demonstrating that environmental conditions have a strong influence on the progeny. OPDA and 12-OH-JA were the main compounds found in dry seeds of both lines. Imbibed seeds showed an enhanced amount of total JAs with respect to dry seeds produced by plants grown in both soil moisture conditions. Imbibition triggered a dramatic OPDA increase in the embryo, suggesting a role of this compound in germination. We conclude that JAs patterns vary during sunflower germination and that the environmental conditions experienced by the mother plant modify the hormonal content of the seed progeny.
Publikation

Andrade, A.; Vigliocco, A.; Alemano, S.; Miersch, O.; Botella, M. A.; Abdala, G.; Endogenous jasmonates and octadecanoids in hypersensitive tomato mutants during germination and seedling development in response to abiotic stress Seed Sci. Res. 15, 309-318, (2005) DOI: 10.1079/SSR2005219

Although jasmonates (JAs) are involved in germination and seedling development, the regulatory mechanism of JAs, and their relation with endogenous level modifications in these processes, is not well understood. We report here the detection of 12-oxo-phytodienoic acid (OPDA), jasmonic acid (JA), 11-hydroxyjasmonate (11-OH-JA), 12-hydroxyjasmonate (12-OH-JA) and methyljasmonate (JAME) in unimbibed seeds and seedlings of tomato Lycopersicon esculentum Mill cv. Moneymaker (wild type) and tss1, tss2, tos1 mutants. The main compounds in wild-type and tss1, tss2, tos1 seeds were the hydroxylate-JAs; 12-OH-JA was the major component in dry seeds of the wild type and in tss2 and tos1. The amounts of these derivatives were higher in seeds than in seedlings. Changes in JAs during wild-type and tss1 imbibition were analysed in seeds and the imbibition water. In wild-type imbibed seeds, 11-OH-JA content was higher than in tss1. 12-OH-JA showed a different tendency with respect to 11-OH-JA, with high levels in the wild type at early imbibition. In tss1, levels of 12-OH-JA rose from 24 to 48 h of imbibition. At 72 h of imbibition, when radicles had emerged, the amounts of both hydroxylates in wild-type and tss1 seeds were minimal. An important release of the hydroxylate forms was observed in the imbibition water. 11-OH-JA decreased in the imbibition water of wild-type seeds at 48 h. On the contrary, a high and sustained liberation of this compound was observed in tss1 after 24 h. 12-OH-JA increased in wild-type as well in tss1 until 24 h. Thereafter, a substantial reduction in the content of this compound was registered. NaCl-treated wild-type seedlings increased their 12-OH-JA, but tss1 seedlings increased their JA in response to salt treatment. In tss2 seedlings, NaCl caused a slight decrease in 11-OH-JA and JAME, whereas tos1 seedlings showed a dramatic OPDA and 12-OH-JA decrease in response to salt treatment. Under salt stress the mutant seedlings showed different patterns of JAs according to their differential hypersensitivity to abiotic stress. The JA-hydroxylate forms found, and the differential accumulation of JAs during germination, imbibition and seedling development, as well as their response to NaCl stress, provide new evidence about the control of many developmental processes by JA.
Publikation

Flores, R.; Delgado, S.; Gas, M.-E.; Carbonell, A.; Molina, D.; Gago, S.; De la Peña, M.; Viroids: the minimal non-coding RNAs with autonomous replication FEBS Lett. 567, 42-48, (2004) DOI: 10.1016/j.febslet.2004.03.118

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.
Publikation

Abdala, G.; Miersch, O.; Kramell, R.; Vigliocco, A.; Agostini, E.; Forchetti, G.; Alemano, S.; Jasmonate and octadecanoid occurrence in tomato hairy roots. Endogenous level changes in response to NaCl Plant Growth Regul. 40, 21-27, (2003) DOI: 10.1023/A:1023016412454

Jasmonic acid biosynthesis occurs in leaves and there is also evidence of a similar pathway in roots. The expression of lipoxygenase, allene oxide cyclase and low amounts of transcripts of allene oxide synthase in tomato roots indicates that some steps of the jasmonate synthesis may occur in these organs. Thus, the aim of the present work was to study the jasmonate and octadecanoid occurrence in tomato roots using isolated cultures of hairy roots. These were obtained by the transformation of cv. Pera roots with Agrobacterium rhyzogenes. Also we investigated the effect of NaCl stress on the endogenous levels of these compounds. Jasmonic acid, 12-oxophytodienoic acid and their methylated derivatives, as well as a jasmonate-isoleucine conjugate, were present in control hairy roots of 30 d of culture. The 12-oxophytodienoic acid and its methylated derivative showed higher levels than jasmonic acid and its methylated form, although the content of the conjugate was the same as that of jasmonic acid. After salinization of hairy roots for 14, 20 and 30 d, free jasmonates and octadecanoids were measured. Fourteen days after salt treatment, increased levels of these compounds were found, jasmonic acid and 12-oxophytodienoic acid showed the most remarkable rise. 11-OH-jasmonic acid was found at 14 d of culture in control and salt-treated hairy roots; whereas the 12-OH- form of jasmonic acid was only detected in the salt-treated hairy roots. Agrobacterium rhizogenes cultures did not produce jasmonates and/or octadecanoids.
Publikation

Vigliocco, A.; Bonamico, B.; Alemano, S.; Miersch, O.; Abdala, G.; Stimulation of jasmonic acid production in Zea Mays L. infected by the maize rough dwarf virus - Río Cuarto. Reversion of symptoms by salicylic acid Biocell 26, 369-374, (2002)

In the present paper we study the possible biological relevance of endogenous jasmonic acid (JA) and exogenous salicylic acid (SA) in a plant-microbial system maize-virus. The virus disease "Mal de Río Cuarto" is caused by the maize rough dwarf virus - Río Cuarto. The characteristic symptoms are the appearance of galls or "enations" in leaves, shortening of the stem internodes, poor radical system and general stunting. Changes in JA and protein pattern in maize control and infected plants of a virus-tolerant cultivar were investigated. Healthy and infected-leaf discs were collected for JA measurement at different post-infection times (20, 40, 60 and 68 days). JA was also measured in roots on day 60 after infection. For SDS-PAGE protein analysis, leaf discs were also harvested on day 60 after infection. Infected leaves showed higher levels of JA than healthy leaves, and the rise in endogenous JA coincided with the enation formation. The soluble protein amount did not show differences between infected and healthy leaves; moreover, no difference in the expression of soluble protein was revealed by SDS-PAGE. Our results show that the octadecanoid pathway was stimulated in leaves and roots of the tolerant maize cultivar when infected by this virus. This finding, together with fewer plants with the disease symptoms, suggest that higher foliar and roots JA content may be related to disease tolerance. SA exogenous treatment caused the reversion of the dwarfism symptom.
  • Die Leibniz-Gemeinschaft
  • Digitalisierung in der Landwirtschaft
  • Martin-Luther Universität Halle
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