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Publikationen - Molekulare Signalverarbeitung

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Publikation

Gasperini, D.; Acosta, I. F.; Farmer, E. E.; Cotyledon Wounding of Arabidopsis Seedlings Bio Protoc. 6, e1712, (2016) DOI: 10.21769/BioProtoc.1712

Damage to plant organs through both biotic and abiotic injury is very common in nature. Arabidopsis thaliana 5-day-old (5-do) seedlings represent an excellent system in which to study plant responses to mechanical wounding, both at the site of the damage and in distal unharmed tissues. Seedlings of wild type, transgenic or mutant lines subjected to single or repetitive cotyledon wounding can be used to quantify morphological alterations (e.g., root length, Gasperini et al., 2015), analyze the dynamics of reporter genes in vivo (Larrieu et al., 2015; Gasperini et al., 2015), follow transcriptional changes by quantitative RT-PCR (Acosta et al., 2013; Gasperini et al., 2015) or examine additional aspects of the wound response with a plethora of downstream procedures. Here we illustrate how to rapidly and reliably wound cotyledons of young seedlings, and show the behavior of two promoters driving the expression of β-glucuronidase (GUS) in entire seedlings and in the primary root meristem, following single or repetitive cotyledon wounding respectively. We describe two procedures that can be easily adapted to specific experimental needs.
Publikation

Gasperini, D.; Chételat, A.; Acosta, I. F.; Goossens, J.; Pauwels, L.; Goossens, A.; Dreos, R.; Alfonso, E.; Farmer, E. E.; Multilayered Organization of Jasmonate Signalling in the Regulation of Root Growth PLOS Genet. 11, e1005300, (2015) DOI: 10.1371/journal.pgen.1005300

Physical damage can strongly affect plant growth, reducing the biomass of developing organs situated at a distance from wounds. These effects, previously studied in leaves, require the activation of jasmonate (JA) signalling. Using a novel assay involving repetitive cotyledon wounding in Arabidopsis seedlings, we uncovered a function of JA in suppressing cell division and elongation in roots. Regulatory JA signalling components were then manipulated to delineate their relative impacts on root growth. The new transcription factor mutant myc2-322B was isolated. In vitro transcription assays and whole-plant approaches revealed that myc2-322B is a dosage-dependent gain-of-function mutant that can amplify JA growth responses. Moreover, myc2-322B displayed extreme hypersensitivity to JA that totally suppressed root elongation. The mutation weakly reduced root growth in undamaged plants but, when the upstream negative regulator NINJA was genetically removed, myc2-322B powerfully repressed root growth through its effects on cell division and cell elongation. Furthermore, in a JA-deficient mutant background, ninja1 myc2-322B still repressed root elongation, indicating that it is possible to generate JA-responses in the absence of JA. We show that NINJA forms a broadly expressed regulatory layer that is required to inhibit JA signalling in the apex of roots grown under basal conditions. By contrast, MYC2, MYC3 and MYC4 displayed cell layer-specific localisations and MYC3 and MYC4 were expressed in mutually exclusive regions. In nature, growing roots are likely subjected to constant mechanical stress during soil penetration that could lead to JA production and subsequent detrimental effects on growth. Our data reveal how distinct negative regulatory layers, including both NINJA-dependent and -independent mechanisms, restrain JA responses to allow normal root growth. Mechanistic insights from this work underline the importance of mapping JA signalling components to specific cell types in order to understand and potentially engineer the growth reduction that follows physical damage.
Publikation

Gasperini, D.; Chauvin, A.; Acosta, I. F.; Kurenda, A.; Stolz, S.; Chételat, A.; Wolfender, J.-L.; Farmer, E. E.; Axial and Radial Oxylipin Transport Plant Physiol. 169, 2244-2254, (2015) DOI: 10.1104/pp.15.01104

Jasmonates are oxygenated lipids (oxylipins) that control defense gene expression in response to cell damage in plants. How mobile are these potent mediators within tissues? Exploiting a series of 13-lipoxygenase (13-lox) mutants in Arabidopsis (Arabidopsis thaliana) that displays impaired jasmonic acid (JA) synthesis in specific cell types and using JA-inducible reporters, we mapped the extent of the transport of endogenous jasmonates across the plant vegetative growth phase. In seedlings, we found that jasmonate (or JA precursors) could translocate axially from wounded shoots to unwounded roots in a LOX2-dependent manner. Grafting experiments with the wild type and JA-deficient mutants confirmed shoot-to-root oxylipin transport. Next, we used rosettes to investigate radial cell-to-cell transport of jasmonates. After finding that the LOX6 protein localized to xylem contact cells was not wound inducible, we used the lox234 triple mutant to genetically isolate LOX6 as the only JA precursor-producing LOX in the plant. When a leaf of this mutant was wounded, the JA reporter gene was expressed in distal leaves. Leaf sectioning showed that JA reporter expression extended from contact cells throughout the vascular bundle and into extravascular cells, revealing a radial movement of jasmonates. Our results add a crucial element to a growing picture of how the distal wound response is regulated in rosettes, showing that both axial (shoot-to-root) and radial (cell-to-cell) transport of oxylipins plays a major role in the wound response. The strategies developed herein provide unique tools with which to identify intercellular jasmonate transport routes.
Publikation

Farmer, E. E.; Gasperini, D.; Acosta, I. F.; The squeeze cell hypothesis for the activation of jasmonate synthesis in response to wounding New Phytol. 204, 282-288, (2014) DOI: 10.1111/nph.12897

Jasmonates are lipid mediators that control defence gene expression in response to wounding and other environmental stresses. These small molecules can accumulate at distances up to several cm from sites of damage and this is likely to involve cell‐to‐cell jasmonate transport. Also, and independently of jasmonate synthesis, transport and perception, different long‐distance wound signals that stimulate distal jasmonate synthesis are propagated at apparent speeds of several cm min–1 to tissues distal to wounds in a mechanism that involves clade 3 GLUTAMATE RECEPTOR‐LIKE (GLR) genes. A search for jasmonate synthesis enzymes that might decode these signals revealed LOX6, a lipoxygenase that is necessary for much of the rapid accumulation of jasmonic acid at sites distal to wounds. Intriguingly, the LOX6 promoter is expressed in a distinct niche of cells that are adjacent to mature xylem vessels, a location that would make these contact cells sensitive to the release of xylem water column tension upon wounding. We propose a model in which rapid axial changes in xylem hydrostatic pressure caused by wounding travel through the vasculature and lead to slower, radially dispersed pressure changes that act in a clade 3 GLR‐dependent mechanism to promote distal jasmonate synthesis.
Publikation

Acosta, I. F.; Gasperini, D.; Chételat, A.; Stolz, S.; Santuari, L.; Farmer, E. E.; Role of NINJA in root jasmonate signaling Proc. Natl. Acad. Sci. U.S.A. 110, 15473-15478, (2013) DOI: 10.1073/pnas.1307910110

Wound responses in plants have to be coordinated between organs so that locally reduced growth in a wounded tissue is balanced by appropriate growth elsewhere in the body. We used a JASMONATE ZIM DOMAIN 10 (JAZ10) reporter to screen for mutants affected in the organ-specific activation of jasmonate (JA) signaling in Arabidopsis thaliana seedlings. Wounding one cotyledon activated the reporter in both aerial and root tissues, and this was either disrupted or restricted to certain organs in mutant alleles of core components of the JA pathway including COI1, OPR3, and JAR1. In contrast, three other mutants showed constitutive activation of the reporter in the roots and hypocotyls of unwounded seedlings. All three lines harbored mutations in Novel Interactor of JAZ (NINJA), which encodes part of a repressor complex that negatively regulates JA signaling. These ninja mutants displayed shorter roots mimicking JA-mediated growth inhibition, and this was due to reduced cell elongation. Remarkably, this phenotype and the constitutive JAZ10 expression were still observed in backgrounds lacking the ability to synthesize JA or the key transcriptional activator MYC2. Therefore, JA-like responses can be recapitulated in specific tissues without changing a plant’s ability to make or perceive JA, and MYC2 either has no role or is not the only derepressed transcription factor in ninja mutants. Our results show that the role of NINJA in the root is to repress JA signaling and allow normal cell elongation. Furthermore, the regulation of the JA pathway differs between roots and aerial tissues at all levels, from JA biosynthesis to transcriptional activation.
Bücher und Buchkapitel

Carbonell, A.; Flores, R.; Gago, S.; Hammerhead Ribozymes Against Virus and Viroid RNAs (Erdmann, V. A. & Barciszewski, J., eds.). RNA Technologies 411-427, (2012) ISBN: 978-3-642-27426-8 DOI: 10.1007/978-3-642-27426-8_16

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.
Publikation

Carbonell, A.; Flores, R.; Gago, S.; Trans-cleaving hammerhead ribozymes with tertiary stabilizing motifs: in vitro and in vivo activity against a structured viroid RNA Nucleic Acids Res. 39, 2432-2444, (2011) DOI: 10.1093/nar/gkq1051

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.
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.
Publikation

Renovell, ?.; Gago, S.; Ruiz-Ruiz, S.; Velázquez, K.; Navarro, L.; Moreno, P.; Vives, M. C.; Guerri, J.; Mapping the subgenomic RNA promoter of the Citrus leaf blotch virus coat protein gene by Agrobacterium-mediated inoculation Virology 406, 360-369, (2010) DOI: 10.1016/j.virol.2010.07.034

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.
Publikation

Gago, S.; Elena, S. F.; Flores, R.; Sanjuan, R.; Extremely High Mutation Rate of a Hammerhead Viroid Science 323, 1308-1308, (2009) DOI: 10.1126/science.1169202

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.
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