Multi-targeting of viral RNAs with synthetic trans-acting small interfering RNAs enhances plant antiviral resistance

[EN] RNA interference (RNAi)-based tools are used in multiple organisms to induce antiviral resistance through the sequence-specific degradation of target RNAs by complementary small RNAs. In plants, highly specific antiviral RNAi-based tools include artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs). syn-tasiRNAs have emerged as a promising antiviral tool allowing for the multi-targeting of viral RNAs through the simultaneous expression of several syn-tasiRNAs from a single precursor. Here, we compared in tomato plants the effects of an amiRNA construct expressing a single amiRNA and a syn-tasiRNA construct expressing four different syn-tasiRNAs against Tomato spotted wilt virus (TSWV), an economically important pathogen affecting tomato crops worldwide. Most of the syn-tasiRNA lines were resistant to TSWV, whereas the majority of the amiRNA lines were susceptible and accumulated viral progenies with mutations in the amiRNA target site. Only the two amiRNA lines with higher amiRNA accumulation were resistant, whereas resistance in syn-tasiRNA lines was not exclusive of lines with high syn-tasiRNA accumulation. Collectively, these results suggest that syn-tasiRNAs induce enhanced antiviral resistance because of the combined silencing effect of each individual syn-tasiRNA, which minimizes the possibility that the virus simultaneously mutates all different target sites to fully escape each syn-tasiRNA.We thank V. Aragones and E. Moya for invaluable technical assistance. This work was supported by grants from Ministerio de Ciencia, Innovacion y Universidades (MCIU, Spain), Agencia Estatal de Investigacion (AEI, Spain) and Fondo Europeo de Desarrollo Regional (FEDER, European Union) (RTI2018-095118-A-100 and RYC-2017-21648 to A.C.; BIO2017-83184-R to J.-A.D.).Carbonell, A.; Lisón, P.; Daròs, J. (2019). Multi-targeting of viral RNAs with synthetic trans-acting small interfering RNAs enhances plant antiviral resistance. 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Cryo-EM structure and rRNA modification sites of a plant ribosome

[EN] Protein synthesis in crop plants contributes to the balance of food and fuel on our planet, which influences human metabolic activity and lifespan. Protein synthesis can be regulated with respect to changing environmental cues via the deposition of chemical modifications into rRNA. Here, we present the structure of a plant ribosome from tomato and a quantitative mass spectrometry analysis of its rRNAs. The study reveals fine features of the ribosomal proteins and 71 plant-specific rRNA modifications, and it re-annotates 30 rRNA residues in the available sequence. At the protein level, isoAsp is found in position 137 of uS11, and a zinc finger previously believed to be universal is missing from eL34, suggesting a lower effect of zinc deficiency on protein synthesis in plants. At the rRNA level, the plant ribosome differs markedly from its human counterpart with respect to the spatial distribution of modifications. Thus, it represents an additional layer of gene expression regulation, highlighting the molecular signature of a plant ribosome. The results provide a reference model of a plant ribosome for structural studies and an accurate marker for molecular ecology.This work was supported by the Swedish Foundation for Strategic Research (ARC19:0051), the Knut and Alice Wallenberg Foundation (2018.0080), the EMBO Young Investigator Program, and a NASA award (80NSSC18K1139 to A.S.P.).Cottilli, P.; Itoh, Y.; Nobe, Y.; Petrov, AS.; Lisón, P.; Taoka, M.; Amunts, A. (2022). Cryo-EM structure and rRNA modification sites of a plant ribosome. Plant communications. 3(5):1-9. https://doi.org/10.1016/j.xplc.2022.100342193

Relevance of Translational Regulation on Plant Growth and Environmental Responses

The authors acknowledge funding by MINECO BIO2015-70483-R to AF, by CAM S2013/ABI-2734 and by ERC GA260468 to MMC, by the Deutsche Forschungsgemeinschaft (DFG, grant TRR175-C05) to DL, by NSF IOS 1444561 and NSF IOS PAPM-EAGER 1650139 to AS, and by Bio4Energy, a Strategic Research Environment appointed by the Swedish government to JH.Ferrando Monleón, AR.; Castellano, M.; Lisón, P.; Leister, D.; Stepanova, AN.; Hanson, J. (2017). Relevance of Translational Regulation on Plant Growth and Environmental Responses. Frontiers in Plant Science. 8:1-2. https://doi.org/10.3389/fpls.2017.02170S128Hummel, M., Cordewener, J. H. G., de Groot, J. C. M., Smeekens, S., America, A. H. P., & Hanson, J. (2012). Dynamic protein composition of Arabidopsis thaliana cytosolic ribosomes in response to sucrose feeding as revealed by label free MSE proteomics. PROTEOMICS, 12(7), 1024-1038. doi:10.1002/pmic.201100413Vermeulen, S. J., Campbell, B. M., & Ingram, J. S. I. (2012). Climate Change and Food Systems. Annual Review of Environment and Resources, 37(1), 195-222. doi:10.1146/annurev-environ-020411-130608Vogel, C., & Marcotte, E. M. (2012). Insights into the regulation of protein abundance from proteomic and transcriptomic analyses. Nature Reviews Genetics, 13(4), 227-232. doi:10.1038/nrg318

Ethylene is Involved in Symptom Development and Ribosomal Stress of Tomato Plants upon Citrus Exocortis Viroid Infection

[EN] Citrus exocortis viroid (CEVd) is known to cause different symptoms in citrus trees, and its mechanism of infection has been studied in tomato as an experimental host, producing ribosomal stress on these plants. Some of the symptoms caused by CEVd in tomato plants resemble those produced by the phytohormone ethylene. The present study is focused on elucidating the relationship between CEVd infection and ethylene on disease development. To this purpose, the ethylene insensitive Never ripe (Nr) tomato mutants were infected with CEVd, and several aspects such as susceptibility to infection, defensive response, ethylene biosynthesis and ribosomal stress were studied. Phenotypic characterization revealed higher susceptibility to CEVd in these mutants, which correlated with higher expression levels of both defense and ethylene biosynthesis genes, as well as the ribosomal stress marker SlNAC082. In addition, Northern blotting revealed compromised ribosome biogenesis in all CEVd infected plants, particularly in Nr mutants. Our results indicate a higher ethylene biosynthesis in Nr mutants and suggest an important role of this phytohormone in disease development and ribosomal stress caused by viroid infection.Vázquez Prol, F.; López-Gresa, MP.; Rodrigo Bravo, I.; Belles Albert, JM.; Lisón, P. (2020). Ethylene is Involved in Symptom Development and Ribosomal Stress of Tomato Plants upon Citrus Exocortis Viroid Infection. Plants. 9(5):1-15. https://doi.org/10.3390/plants9050582S11595Flores, R., Hernández, C., Alba, A. E. M. de, Daròs, J.-A., & Serio, F. D. (2005). Viroids and Viroid-Host Interactions. Annual Review of Phytopathology, 43(1), 117-139. doi:10.1146/annurev.phyto.43.040204.140243Adkar‐Purushothama, C. R., & Perreault, J. (2019). Current overview on viroid–host interactions. WIREs RNA, 11(2). doi:10.1002/wrna.1570Di Serio, F., Flores, R., Verhoeven, J. T. 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Viruses, 11(5), 453. doi:10.3390/v11050453Jia, C., Zhang, L., Liu, L., Wang, J., Li, C., & Wang, Q. (2013). Multiple phytohormone signalling pathways modulate susceptibility of tomato plants to Alternaria alternata f. sp. lycopersici. Journal of Experimental Botany, 64(2), 637-650. doi:10.1093/jxb/ers360Van Loon, L. C., Geraats, B. P. J., & Linthorst, H. J. M. (2006). Ethylene as a modulator of disease resistance in plants. Trends in Plant Science, 11(4), 184-191. doi:10.1016/j.tplants.2006.02.005Bellés, J. M., & Conejero, V. (1989). Ethylene Mediation of the Viroid-Like Syndrome Induced by Ag+Ions inGynura aurantiacaDC Plants. Journal of Phytopathology, 124(4), 275-284. doi:10.1111/j.1439-0434.1989.tb04924.xDubois, M., Van den Broeck, L., & Inzé, D. (2018). The Pivotal Role of Ethylene in Plant Growth. Trends in Plant Science, 23(4), 311-323. doi:10.1016/j.tplants.2018.01.003Yang, S. F., & Hoffman, N. E. (1984). Ethylene Biosynthesis and its Regulation in Higher Plants. 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Salicylic Acid Is Involved in the Basal Resistance of Tomato Plants to Citrus Exocortis Viroid and Tomato Spotted Wilt Virus

[EN] Tomato plants expressing the NahG transgene, which prevents accumulation of endogenous salicylic acid (SA), were used to study the importance of the SA signalling pathway in basal defence against Citrus Exocortis Viroid (CEVd) or Tomato Spotted Wilt Virus (TSWV). The lack of SA accumulation in the CEVd- or TSWV-infected NahG tomato plants led to an early and dramatic disease phenotype, as compared to that observed in the corresponding parental Money Maker. Addition of acibenzolar-S-methyl, a benzothiadiazole (BTH), which activates the systemic acquired resistance pathway downstream of SA signalling, improves resistance of NahG tomato plants to CEVd and TSWV. CEVd and TSWV inoculation induced the accumulation of the hydroxycinnamic amides p-coumaroyltyramine, feruloyltyramine, caffeoylputrescine, and feruloylputrescine, and the defence related proteins PR1 and P23 in NahG plants earlier and with more intensity than in Money Maker plants, indicating that SA is not essential for the induction of these plant defence metabolites and proteins. In addition, NahG plants produced very high levels of ethylene upon CEVd or TSWV infection when compared with infected Money Maker plants, indicating that the absence of SA produced additional effects on other metabolic pathways. This is the first report to show that SA is an important component of basal resistance of tomato plants to both CEVd and TSWV, indicating that SA-dependent defence mechanisms play a key role in limiting the severity of symptoms in CEVd- and TSWV-infected NahG tomato plants.This work was supported by grant BIO2012-33419 from the Spanish Ministry of Economy and Competitiveness received by JMB. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.López-Gresa, MP.; Lisón, P.; Yenush, L.; Conejero Tomás, V.; Rodrigo Bravo, I.; Belles Albert, JM. (2016). Salicylic Acid Is Involved in the Basal Resistance of Tomato Plants to Citrus Exocortis Viroid and Tomato Spotted Wilt Virus. PLoS ONE. 11(11). https://doi.org/10.1371/journal.pone.0166938S111

(Z)-3-Hexenyl Butyrate induces stomata closure and ripening in Vitis vinifera

[EN] Agronomy solutions for modifying pre-harvest grape ripening are needed for a more sustainable viticulture. Field experiments were performed inVitis viniferaL. vines to study the effect of the previously described stomata-closing compound (Z)-3-hexenyl butyrate (HB). Exogenous treatments at different doses were periodically carried out using a randomized block design. Firstly, we observed that HB was able to induce stomatal closure in grapevine plants. Under field conditions, the application of HB around veraison induced a higher color intensity in berries, and vines treated at higher doses reached this stage earlier than the un-treated controls. There was also a clear increase in both grape anthocyanin concentration and total soluble solids without having a negative impact on total yield. We therefore, confirm the role of HB as a universal natural stomatal closure compound and propose a new use for HB in viticulture as a ripening inducer, by accelerating anthocyanin accumulation.This research was funded by Grant INNVAL10/18/005 from the Agencia Valenciana de la Innovacio (Spain). C.P. was a recipient of a predoctoral contract of the Generalitat Valenciana (ACIF/2019/187). D.S.I. is supported by AEI-FEDER grant AGL2017-83738-C3-3-R.Payá Montes, C.; López-Gresa, MP.; Intrigliolo, DS.; Rodrigo Bravo, I.; Belles Albert, JM.; Lisón, P. (2020). (Z)-3-Hexenyl Butyrate induces stomata closure and ripening in Vitis vinifera. Agronomy. 10(8):1-12. https://doi.org/10.3390/agronomy10081122S112108Zoccatelli, G., Zenoni, S., Savoi, S., Dal Santo, S., Tononi, P., Zandonà, V., … Tornielli, G. B. (2013). Skin pectin metabolism during the postharvest dehydration of berries from three distinct grapevine cultivars. 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Tomato glycosyltransferase Twi1 plays a role in flavonoid glycosylation and defence against virus

[EN] Background: Secondary metabolites play an important role in the plant defensive response. They are produced as a defence mechanism against biotic stress by providing plants with antimicrobial and antioxidant weapons. In higher plants, the majority of secondary metabolites accumulate as glycoconjugates. Glycosylation is one of the commonest modifications of secondary metabolites, and is carried out by enzymes called glycosyltransferases. Results: Here we provide evidence that the previously described tomato wound and pathogen-induced glycosyltransferase Twi1 displays in vitro activity toward the coumarins scopoletin, umbelliferone and esculetin, and the flavonoids quercetin and kaempferol, by uncovering a new role of this gene in plant glycosylation. To test its activity in vivo, Twi1-silenced transgenic tomato plants were generated and infected with Tomato spotted wilt virus. The Twi1- silenced plants showed a differential accumulation of Twi1 substrates and enhanced susceptibility to the virus. Conclusions: Biochemical in vitro assays and transgenic plants generation proved to be useful strategies to assign a role of tomato Twi1 in the plant defence response. Twi1 glycosyltransferase showed to regulate quercetin and kaempferol levels in tomato plants, affecting plant resistance to viral infection.This work was supported by grant BIO2012-33419 from the Direccion General de Programas y Transferencia de Conocimiento, Spanish Ministry of Science and Innovation, and grant AICO/2017/048 from the Valencian Local Government (Generalitat Valenciana, Spain). LC was supported by a predoctoral fellowship (ACIF/2010/231) from the Valencian Local Government (Generalitat Valenciana, Spain). 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Downregulation of Tomato STEROL GLYCOSYLTRANSFERASE 1 Perturbs Plant Development and Facilitates Viroid Infection

[EN] Potato spindle tuber viroid (PSTVd) is a plant pathogen naturally infecting economically important crops such as tomato (Solanum lycopersicum). Here, we aimed to engineer tomato plants highly resistant to PSTVd and developed several S. lycopersicum lines expressing an artificial microRNA (amiRNA) against PSTVd (amiR-PSTVd). Infectivity assays revealed that amiR-PSTVd-expressing lines were not resistant but rather hypersusceptible to the viroid. A combination of phenotypic, molecular and metabolic analyses of amiRNAexpressing lines non-inoculated with the viroid revealed that amiR-PSTVd was accidentally silencing the tomato STEROL GLYCOSYLTRANSFERASE 1 (SlSGT1) gene, which caused late developmental and reproductive defects such as leaf epinasty, dwarfism or reduced fruit size. Importantly, two independent transgenic tomato lines each expressing a different amiRNA specifically designed to target SlSGT1 were also hypersusceptible to PSTVd, thus confirming that downregulation of SlSGT1 was responsible for the viroid hypersusceptibility phenotype. Our results highlight the role of SGTs in proper plant development and indicate that the unbalance of sterol glycosylation levels favors viroid infection most likely by facilitating viroid movement.Cisneros, AE.; Lisón, P.; Campos, L.; López-Tubau, JM.; Altabella, T.; Ferrer, A.; Daròs, J.... (2022). Downregulation of Tomato STEROL GLYCOSYLTRANSFERASE 1 Perturbs Plant Development and Facilitates Viroid Infection. Journal of Experimental Botany. 1-37. https://doi.org/10.1093/jxb/erac36113

Physiological and morphological characterisation of Limonium species in their natural habitats: Insights into their abiotic stress responses

[EN] Background and aims: Morphological and biochemical traits of four halophytes of the genus Limonium were analysed in plants sampled from salt marshes in SE Spain. This work aimed to explore the mechanism(s) behind the adaptation of these species to stressful habitats, with particular emphasis on responses to drought. Methods: Plants of each species together with soil samples were collected in summer, which is the most stressful season in the Mediterranean. Soil parameters and plant morphological traits were determined, and the levels of several biochemical stress markers in plants were measured using spectrophotometric assays. A multivariate analysis was performed to correlate soil and plant data. Results: Morphological characteristics regarding the underground system topology and several biochemical traits (higher foliar Ca2+, sucrose and glucose, and lower proline, glycine-betaine and fructose) clearly separate L. santapolense individuals from plants of the other three species. Conclusions: Drought tolerance of L. santapolense in the field is mostly dependent on morphological adaptations: when growing in an arid location, plants of this species develop long taproots that can extract water from the deep, moist layers of the soil.This research was partly supported by the project AICO/2017/039 from Generalitat Valenciana. We are indebted to Dr. Inmaculada Bautista (Universitat Politècnica de Valencia, Spain) for her useful suggestions for improving the manuscript.González-Orenga, S.; Llinares Palacios, JV.; Al Hassan, M.; Fita, A.; Collado, F.; Lisón, P.; Vicente, O.... (2020). Physiological and morphological characterisation of Limonium species in their natural habitats: Insights into their abiotic stress responses. 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Avignon and its interspecific hybrid Limoniun caspia x Limonium latifolium cv. Beltlaard during salt stress. J Plant Physiol 154:795–780Allen RG, Pereira LS, Raes D, Smith M (1998) Crop evapotranspiration – guidelines for computing crop water requirements. FAO irrigation and drainage paper 56. Rome, Italy: Food and agriculture Organization of the United NationsAlvarez-Flores R, Nguyen-Thi-Truc N, Peredo-Parada S, Joffre R, Winkel T (2018) Rooting plasticity in wild and cultivated Andean Chenopodium species under soil water deficit. Plant Soil 425:479–492Álvarez-Rogel J, Hernández J, Ortiz Silla R, Alcaraz F (1997) Patterns of spatial and temporal variations in soil salinity: example of a salt marsh in a semiarid climate. Arid Soil Res Rehabil 11:315–329Álvarez-Rogel J, Alcaraz Ariza F, Ortiz Silla R (2000) Soil salinity and moisture gradients and plant zonation in Mediterranean salt marshes of Southeast Spain. 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Bacillus subtilis IAB/BS03 as a potential biological control agent

[EN] We describe the efficacy of Bacillus subtilis strain IAB/BS03 in reducing disease incidence of B. subtilis IAB/BS03 as a foliar treatment against Botrytis cinerea and Pseudomonas syringae on greenhouse-grown tomato (Solanum lycopersicon) plants. We also tested the effect of foliar treatments on lettuce (Lactuca sativa) against lettuce downy mildew caused by Bremia lactucae in multiple trials under different field conditions. All the assays indicated that B. subtilis IAB/BS03 reduced disease. To ascertain the mechanism of action, the induction of pathogenesis-related (PR) proteins, the accumulation of salicylic acid and the activation of peroxidase caused by foliar or root treatments with B. subtilis IAB/BS03 were studied in tomato. A salicylic acid-independent induction of the antifungal protein PR1 was observed after treatment with B. subtilis IAB/BS03, with the strongest induction due to root treatment compared with foliar application. A metabolic analysis of B. subtilis IAB/BS03 culture broth using Ultra Performance Liquid Chromatography coupled with ultraviolet and mass spectrometric detection determined surfactin and iturin A isomers. These compounds have been described as antifungal and antibiotic lipopeptides. The results indicated that B. subtilis IAB/BS03 could be effectively used as a biocontrol agent.This work was funded by IAB S. L. (Investigaciones y Aplicaciones Biotecnologicas, S. L.), and by grant BIO2012-33419 from the Spanish Ministry of Economy and Competitiveness. Mayte Castellano was the recipient of a research grant also funded by IAB S. L. The authors would like to thank Cristina Torres (IBMCP, UPV-CSIC) for her excellent technical assistance.Hinarejos, E.; Castellano Pérez, M.; Rodrigo Bravo, I.; Belles Albert, JM.; Conejero Tomás, V.; López-Gresa, MP.; Lisón, P. (2016). Bacillus subtilis IAB/BS03 as a potential biological control agent. 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