Instituto Universitario Mixto de Biología Molecular y Celular de Plantas

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CRISPR-Mediated Strand Displacement Logic Circuits with Toehold-Free DNA
(American Chemical Society, 2021-05-21) Montagud-Martínez, Roser; Heras-Hernández, María; Goiriz, Lucas; Rodrigo Tarrega, Guillermo; Daros Arnau, Jose Antonio; Instituto Universitario Mixto de Biología Molecular y Celular de Plantas; Agencia Estatal de Investigación; European Regional Development Fund; Ministerio de Economía y Competitividad
[EN] DNA nanotechnology, and DNA computing in particular, has grown extensively over the past decade to end with a variety of functional stable structures and dynamic circuits. However, the use as designer elements of regular DNA pieces, perfectly complementary double strands, has remained elusive. Here, we report the exploitation of CRISPR-Cas systems to engineer logic circuits based on isothermal strand displacement that perform with toehold-free double-stranded DNA. We designed and implemented molecular converters for signal detection and amplification, showing good interoperability between enzymatic and nonenzymatic processes. Overall, these results contribute to enlarge the repertoire of substrates and reactions (hardware) for DNA computing.
CRISPR-Cas12a genome editing at the whole-plant level using two compatible RNA virus vectors
(Mary Ann Liebert Inc., 2021-10-15) Uranga, Mireia; Vázquez-Vilar, Marta; Orzáez Calatayud, Diego Vicente; Daros Arnau, Jose Antonio; Instituto Universitario Mixto de Biología Molecular y Celular de Plantas; European Commission; Generalitat Valenciana; Agencia Estatal de Investigación; European Regional Development Fund; Ministerio de Ciencia e Innovación
[EN] The use of viral vectors that can replicate and move systemically through the host plant to deliver bacterial CRISPR components enables genome editing at the whole-plant level and avoids the requirement for labor-intensive stable transformation. However, this approach usually relies on previously transformed plants that stably express a CRISPR-Cas nuclease. Here, we describe successful DNA-free genome editing of Nicotiana benthamiana using two compatible RNA virus vectors derived from tobacco etch virus (TEV; genus Potyvirus) and potato virus X (PVX; genus Potexvirus), which replicate in the same cells. The TEV and PVX vectors respectively express a Cas12a nuclease and the corresponding guide RNA. This novel two-virus vector system improves the toolbox for transformation-free virus-induced genome editing in plants and will advance efforts to breed more nutritious, resistant, and productive crops.
Molecular signatures of silencing suppression degeneracy from a complex RNA virus
(Public Library of Science, 2021-06) Ambrós, Silvia; Gómez-Muñoz, Neus; Giménez-Santamarina, Silvia; Sánchez-Vicente, Javier; Navarro-López, Josep; Martínez, Fernando; Daros Arnau, Jose Antonio; Rodrigo Tarrega, Guillermo; Instituto Universitario Mixto de Biología Molecular y Celular de Plantas; Generalitat Valenciana; Agencia Estatal de Investigación; European Regional Development Fund; Ministerio de Ciencia e Innovación
[EN] As genomic architectures become more complex, they begin to accumulate degenerate and redundant elements. However, analyses of the molecular mechanisms underlying these genetic architecture features remain scarce, especially in compact but sufficiently complex genomes. In the present study, we followed a proteomic approach together with a computational network analysis to reveal molecular signatures of protein function degeneracy from a plant virus (as virus-host protein-protein interactions). We employed affinity purification coupled to mass spectrometry to detect several host factors interacting with two proteins of Citrus tristeza virus (p20 and p25) that are known to function as RNA silencing suppressors, using an experimental system of transient expression in a model plant. The study was expanded by considering two different isolates of the virus, and some key interactions were confirmed by bimolecular fluorescence complementation assays. We found that p20 and p25 target a common set of plant proteins including chloroplastic proteins and translation factors. Moreover, we noted that even specific targets of each viral protein overlap in function. Notably, we identified argonaute proteins (key players in RNA silencing) as reliable targets of p20. Furthermore, we found that these viral proteins preferentially do not target hubs in the host protein interactome, but elements that can transfer information by bridging different parts of the interactome. Overall, our results demonstrate that two distinct proteins encoded in the same viral genome that overlap in function also overlap in their interactions with the cell proteome, thereby highlighting an overlooked connection from a degenerate viral system.
Involvement of the Chloroplastic Isoform of tRNA Ligase in the Replication of Viroids Belonging to the Family Avsunviroidae
(American Society for Microbiology, 2012) Nohales Zafra, Maria Angeles; Molina Serrano, Diego; Flores Pedauye, Ricardo; Daros Arnau, Jose Antonio; Instituto Universitario Mixto de Biología Molecular y Celular de Plantas; Ministerio de Educación y Ciencia
Avocado sunblotch viroid, peach latent mosaic viroid, chrysanthemum chlorotic mottle viroid, and eggplant latent viroid (ELVd), the four recognized members of the family Avsunviroidae, replicate through the symmetric pathway of an RNA-to-RNA rolling-circle mechanism in chloroplasts of infected cells. Viroid oligomeric transcripts of both polarities contain embedded hammerhead ribozymes that, during replication, mediate their self-cleavage to monomeric-length RNAs with 5'-hydroxyl and 2',3'-phosphodiester termini that are subsequently circularized. We report that a recombinant version of the chloroplastic isoform of the tRNA ligase from eggplant (Solanum melongena L.) efficiently catalyzes in vitro circularization of the plus [(+)] and minus [(-)] monomeric linear replication intermediates from the four Avsunviroidae. We also show that while this RNA ligase specifically recognizes the genuine monomeric linear (+) ELVd replication intermediate, it does not do so with five other monomeric linear (+) ELVd RNAs with their ends mapping at different sites along the molecule, despite containing the same 5'-hydroxyl and 2',3'-phosphodiester terminal groups. Moreover, experiments involving transient expression of a dimeric (+) ELVd transcript in Nicotiana benthamiana Domin plants preinoculated with a tobacco rattle virus-derived vector to induce silencing of the plant endogenous tRNA ligase show a significant reduction of ELVd circularization. In contrast, circularization of a viroid replicating in the nucleus occurring through a different pathway is unaffected. Together, these results support the conclusion that the chloroplastic isoform of the plant tRNA ligase is the host enzyme mediating circularization of both (+) and (-) monomeric linear intermediates during replication of the viroids belonging to the family Avsunviroidae.
Strong and tunable anti-CRISPR/Cas activities in plants
(Blackwell Publishing, 2022-02) Calvache Román, Camilo Alberto; Selma, Sara; Vázquez-Vilar, Marta; Uranga, Mireia; Fernández Del Carmen, María Asunción; Daros Arnau, Jose Antonio; Orzáez Calatayud, Diego Vicente; Instituto Universitario Mixto de Biología Molecular y Celular de Plantas; European Commission; Generalitat Valenciana; Agencia Estatal de Investigación; European Regional Development Fund; Ministerio de Ciencia e Innovación; Universitat Politècnica de València; Ministerio de Economía y Competitividad
[EN] CRISPR/Cas has revolutionized genome engineering in plants. However, the use of anti-CRISPR proteins as tools to prevent CRISPR/Cas-mediated gene editing and gene activation in plants has not been explored yet. This study describes the characterization of two anti-CRISPR proteins, AcrIIA4 and AcrVA1, in Nicotiana benthamiana. Our results demonstrate that AcrIIA4 prevents site-directed mutagenesis in leaves when transiently co-expressed with CRISPR/Cas9. In a similar way, AcrVA1 is able to prevent CRISPR/Cas12a-mediated gene editing. Moreover, using a N. benthamiana line constitutively expressing Cas9, we show that the viral delivery of AcrIIA4 using Tobacco etch virus is able to completely abolish the high editing levels obtained when the guide RNA is delivered with a virus, in this case Potato virus X. We also show that AcrIIA4 and AcrVA1 repress CRISPR/dCas-based transcriptional activation of reporter genes. In the case of AcrIIA4, this repression occurs in a highly efficient, dose-dependent manner. Furthermore, the fusion of an auxin degron to AcrIIA4 results in auxin-regulated activation of a downstream reporter gene. The strong anti-Cas activity of AcrIIA4 and AcrVA1 reported here opens new possibilities for customized control of gene editing and gene expression in plants.
Dynamics of alternative modes of RNA replication for positive-sense RNA viruses
(Royal Society, The, 2012-04-07) Sardanyes Cayuela, Jose; Martinez, Fernando; Daros Arnau, Jose Antonio; Elena Fito, Santiago Fco; Instituto Universitario Mixto de Biología Molecular y Celular de Plantas; Ministerio de Ciencia e Innovación; Human Frontier Science Program Organization; National Science Foundation, EEUU
[EN] We propose and study nonlinear mathematical models describing the intracellular time dynamics of viral RNA accumulation for positive-sense single-stranded RNA viruses. Our models consider different replication modes ranging between two extremes represented by the geometric replication (GR) and the linear stamping machine replication (SMR). We first analyse a model that quantitatively reproduced experimental data for the accumulation dynamics of both polarities of turnip mosaic potyvirus RNAs. We identify a non-degenerate transcritical bifurcation governing the extinction of both strands depending on three key parameters: the mode of replication (a), the replication rate (r) and the degradation rate (d) of viral strands. Our results indicate that the bifurcation associated with a generically takes place when the replication mode is closer to the SMR, thus suggesting that GR may provide viral strands with an increased robustness against degradation. This transcritical bifurcation, which is responsible for the switching from an active to an absorbing regime, suggests a smooth (i.e. secondorder), absorbing-state phase transition. Finally, we also analyse a simplified model that only incorporates asymmetry in replication tied to differential replication modes.
Identification and Characterization of Stress-Responsive TAS3-Derived TasiRNAs in Melon
(Oxford University Press, 2019-11) Cervera-Seco, Luis; Marques, M.C.; Sanz-Carbonell, Alejandro; Márquez-Molins, Joan; Carbonell Olivares, Alberto; Daros Arnau, Jose Antonio; Gomez, Gustavo Germán; Instituto Universitario Mixto de Biología Molecular y Celular de Plantas; European Regional Development Fund; Ministerio de Economía y Competitividad; Agencia Estatal de Investigación
[EN] Small interfering RNAs (siRNA) are key regulators of gene expression that play essential roles in diverse biological processes. Trans-acting siRNAs (tasiRNAs) are a class of plant-endogenous siRNAs that lead the cleavage of nonidentical transcripts. TasiRNAs are usually involved in fine-tuning development. However, increasing evidence supports that tasiRNAs may be involved in stress response. Melon is a crop of great economic importance extensively cultivated in semiarid regions frequently exposed to changing environmental conditions that limit its productivity. However, knowledge of the precise role of siRNAs in general, and of tasiRNAs in particular, in regulating the response to adverse environmental conditions is limited. Here, we provide the first comprehensive analysis of computationally inferred melon-tasiRNAs responsive to two biotic (viroid-infection) and abiotic (cold treatment) stress conditions. We identify two TAS3-loci encoding to length (TAS3-L) and short (TAS3-S) transcripts. The TAS candidates predicted from small RNA-sequencing data were characterized according to their chromosome localization and expression pattern in response to stress. The functional activity of cmTAS genes was validated by transcript quantification and degradome assays of the tasiRNA precursors and their predicted targets. Finally, the functionality of a representative cmTAS3-derived tasiRNA (TAS3-S) was confirmed by transient assays showing the cleavage of ARF target transcripts.
Boolean Computation in Plants Using Post-translational Genetic Control and a Visual Output Signal
(American Chemical Society, 2018-10) Cordero-Cucart, María Teresa; Rosado, A.; Majer, Eszter; Jaramillo Rosales, Alfonso; Rodrigo Tarrega, Guillermo; Daros Arnau, Jose Antonio; Instituto Universitario Mixto de Biología Molecular y Celular de Plantas; European Regional Development Fund; UK Research and Innovation; Ministerio de Educación, Cultura y Deporte; Engineering and Physical Sciences Research Council, Reino Unido; Biotechnology and Biological Sciences Research Council, Reino Unido; Agencia Estatal de Investigación; Ministerio de Economía y Competitividad
[EN] Due to autotrophic growing capacity and extremely rich secondary metabolism, plants should be preferred targets of synthetic biology. However, developments in plants usually run below those in other taxonomic groups. In this work we engineered genetic circuits capable of logic YES, OR and AND Boolean computation in plant tissues with a visual output signal. The circuits, which are deployed by means of Agrobacterium tumefaciens, perform with the conditional activity of the MYB transcription factor Roseal from Antirrhinum majus inducing the accumulation of anthocyanins, plant endogenous pigments that are directly visible to the naked eye or accurately quantifiable by spectrophotometric analysis. The translational fusion of Roseal to several viral proteins, such as potyvirus NIb or fragments thereof, rendered the transcription factor inactive. However, anthocyanin accumulation could be restored by inserting protease cleavage sites between both moieties of the fusion and by coexpressing specific proteases, such as potyvirus nuclear inclusion a protease.
Efficient Cas9 multiplex editing using unspaced sgRNA arrays engineering in a Potato virus X vector
(Blackwell Publishing, 2021-04) Uranga-Ruiz De Eguino, Mireia; Aragonés Blasco, Verónica; Selma García, Sara; Vázquez-Vilar, Marta; Orzáez Calatayud, Diego Vicente; Daros Arnau, Jose Antonio; Instituto Universitario Mixto de Biología Molecular y Celular de Plantas; EU H2020; Agencia Estatal de Investigación; Ministerio de Ciencia e Innovación; Ministerio de Ciencia, Innovación y Universidades (Gobierno de España)
[EN] Systems based on the clustered, regularly interspaced, short palindromic repeat (CRISPR) and CRISPR-associated proteins (Cas) have revolutionized genome editing in many organisms, including plants. Most CRISPR-Cas strategies in plants rely on genetic transformation using Agrobacterium tumefaciens to supply the gene editing reagents, such as Cas nucleases or the synthetic guide RNA (sgRNA). While Cas nucleases are constant elements in editing approaches, sgRNAs are target-specific and a screening process is usually required to identify those most effective. Plant virus-derived vectors are an alternative for the fast and efficient delivery of sgRNAs into adult plants, due to the virus capacity for genome amplification and systemic movement, a strategy known as virus-induced genome editing. We engineered Potato virus X (PVX) to build a vector that easily expresses multiple sgRNAs in adult solanaceous plants. Using the PVX-based vector, Nicotiana benthamiana genes were efficiently targeted, producing nearly 80% indels in a transformed line that constitutively expresses Streptococcus pyogenes Cas9. Interestingly, results showed that the PVX vector allows expression of arrays of unspaced sgRNAs, achieving highly efficient multiplex editing in a few days in adult plant tissues. Moreover, virus-free edited progeny can be obtained from plants regenerated from infected tissues or infected plant seeds, which exhibit a high rate of heritable biallelic mutations. In conclusion, this new PVX vector allows easy, fast and efficient expression of sgRNA arrays for multiplex CRISPR-Cas genome editing and will be a useful tool for functional gene analysis and precision breeding across diverse plant species, particularly in Solanaceae crops.
Chrysanthemum stunt viroid in Brazil: survey, identification, biological and molecular characterization and detection methods
(Edizioni ETS, 2014-03) Gobatto, D.; Chaves, A.L.R.; Harakava, R.; Marque, J. M.; Daros Arnau, Jose Antonio; Eiras, M.; Instituto Universitario Mixto de Biología Molecular y Celular de Plantas; Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brasil; Fundação de Amparo à Pesquisa do Estado de São Paulo; Ministerio de Economía y Competitividad
[EN] In Brazil, the ornamental flowers and plants market moves in the wholesale and retail more than two billion US dollars annually, and chrysanthemum stands out as one of the most valuable commercial species. The stunting disease induced by Chrysanthemum stunt viroid (CSVd) has become a serious problem in chrysanthemum production systems worldwide. CSVd incites also colour breaking and retards flowering, but in many situations it does not induce visible symptoms, facilitating its spread in the field and passing international borders unnoticed. In Brazil there are few studies on this pathogen, with a single report of its possible occurrence in chrysanthemum in the State of Sao Paulo. In this work we aimed at surveying, identifying and characterizyng viroids in chrysanthemum crops in the State of Sao Paulo; challenging chrysanthemum varieties with a Brazilian CSVd isolate; and establishing diagnostic strategies to strengthen quarantine and indexing programs. Our survey showed that CSVd is widely disseminated in chrysanthemum crops in the State of Sao Paulo. All evaluated chrysanthemum varieties were susceptible of infection, although infection was sometimes symptomless. The complete genome sequencing of seven CSVd isolates showed high nucleotide identity when compared with other isolates from databases. Specific oligonucleotides were designed for conventional RT-PCR and RT-qPCR CSVd analysis. Results show that CSVd can be identified with a sensitivity about 100,000 times higher than PAGE. Dot-blot proved also to be highly sensitive and, consequently, most useful for large scale diagnosis. This is the first biological and molecular identification and characterization of CSVd isolates in Brazil.