Seleccionar contextos relevantes y poner en acción el pensamiento crítico del alumnado y que establezca conexiones entre conocimiento del aula e interrogantes de la vida diaria fue el objeto de la unidad “Tranquilo, es solo un pinchazo”. Centrada en un dilema de interés socio-científico como el uso de vacunas frente al SARS-CoV-2, se aplicó en la materia “Ciencias Aplicadas a la Actividad Profesional” de 4º de ESO en un grupo de 19 estudiantes (15-16 años), utilizando prácticas científicas de modelización e indagación para fundamentar su toma de decisiones. Mediante diversas producciones del alumnado –informe científico, póster y prueba escrita– se analizaron sus niveles de aprendizaje, detectándose la necesidad de mejora sobre su calidad argumentativa y de modelización de los procesos. Al respecto, se proponen alternativas metodológicas y se aportan implicaciones para la educación científica y la formación del profesorado.

Ricardo Flores (1947-2020) focused his research on the identification, replication, pathogenesis, and evolution of viroids, the minimal non-protein-coding circular RNAs (250-400 nt) able to replicate and incite diseases in plants that are remarkable for being at the lowest step of the biological scale. He and his collaborators initially identified and characterized additional group members, adding six new ones to the family Pospiviroidae, and expanding the Avsunviroidae from one to four members. They showed that members of the second family "encode" ribozymes, a property that, together with others, makes them candidates for being the most primitive replicons that emerged on our planet 3500 million years ago. He also made important contributions regarding how viroids replicate, providing relevant data on the templates, enzymes, and ribozymes that mediate this process and on the mutation rate, which turned out to be the highest reported for any biological entity. More recently, he concentrated on the role that RNA silencing could play on viroid-host interactions, describing details of this process. Ricardo also worked on citrus tristeza virus, a widely different type of subcellular pathogen, and made important contributions on the structure, localization and functions of its unique p23 protein. His research has produced 170 original articles and reviews, according to Web of Science. He encouraged the scientific careers of a large number of researchers, and collaborated with many others, some of whom have recapitulated his scientific legacy in this review and contributed with other chapters in this special issue.

Knowledge on diseases caused by Citrus tristeza virus (CTV) has greatly increased in last decades after their etiology was demonstrated in the past seventies. Professor Ricardo Flores substantially contributed to these advances in topics like: i) improvement of virus purification to obtain biologically active virions, ii) sequencing mild CTV isolates for genetic comparisons with sequences of moderate or severe isolates and genetic engineering, iii) analysis of genetic variation of both CTV genomic RNA ends and features of the highly variable 5' end that allow accommodating this variation within a conserved secondary structure, iv) studies on the structure, subcellular localization and biological functions of the CTV-unique p23 protein, and v) potential use of p23 and other 3'-proximal regions of the CTV genome to develop transgenic citrus resistant to the virus. Here we review his main achievements on these topics and how they contributed to deeper understanding of CTV biology and to new potential measures for disease control.

To dissect the host RNA silencing response incited by citrus tristeza virus (CTV, genus Closterovirus), a (+) ssRNA of similar to 19300 nt, and the counter reaction deployed by the virus via its three RNA silencing suppressors (RSS), the small RNAs (sRNAs) of three virus-host combinations were deep sequenced. The subsequent analysis indicated that CTV sRNAs (1) constitute more than half of the total sRNAs in the susceptible Mexican lime and sweet orange, while only 3.5% in the restrictive sour orange; (2) are mostly of 21-22 nt, with those of (+) sense predominating slightly; and (3) derive from all the CTV genome, as evidenced by its entire recomposition from viral sRNA contigs but adopt an asymmetric pattern with a hotspot mapping at the 3'-terminal similar to 2500 nt. The citrus homologues of Arabidopsis Dicer-like (DCL) 4 and 2 most likely generate the 21 and 22 nt CTV sRNAs, respectively, by dicing the gRNA and the 3' co-terminal sgRNAs and, particularly, their double-stranded forms accumulating in infected cells. The plant sRNA profile, very similar and dominated by the 24 nt sRNAs in the three mock-inoculated controls, displayed a major reduction of the 24 nt sRNAs in Mexican lime and sweet orange, but not in sour orange. CTV infection also influences the levels of certain microRNAs. The high accumulation of CTV sRNAs in two of the citrus hosts examined suggests that it is not their synthesis, but their function, the target of the RSS encoded by CTV: p25 (intercellular), p23 (intracellular) and p20 (both). The two latter might block the loading of CTV sRNAs into the RNA silencing complex or interfere with it through alternative mechanisms. Of the three CTV RSS, p23 is the one that has been more thoroughly studied. It is a multifunctional RNA-binding protein with a putative Zn finger domain and basic motifs that (1) has no homologues in other closteroviruses, (2) accumulates in the nucleolus and plasmodesmata, (3) regulates the asymmetric balance of CTV (+) and (-) RNA strands, and (4) induces CTV syndromes and stimulates systemic infection in certain citrus species when expressed as a transgene ectopically or in phloem-associated cells.

Conventional breeding of citrus types demands a long-term effort due to their complex reproductive biology and long juvenile period. As a compelling alternative, genetic engineering of mature tissues allows the insertion of specific traits into specific elite cultivars, including well-known and widely grown varieties and rootstocks, thus reducing the time and costs involved in improving and evaluating them. Conventional breeding for resistance to CTV in citrus varieties has been largely unsuccessful as well as cloning of the genes conferring resistance to specific citrus types. RNA interference (RNAi), based on producing dsRNAs (usually using intron-hairpin constructs) highly homologous to specific CTV sequences to trigger RNA silencing, has been employed to produce virus-resistant transgenic citrus plants. The most successful construct has been an intron-hairpin vector carrying full-length, untranslatable versions of the genes p25, p20, and p23 from the virus. Using it, we have generated full resistance against CTV in Mexican lime. Moreover, this strategy is applicable to all those citrus varieties amenable to mature transformation, including sweet oranges, sour oranges, mandarins, Citrus macrophylla, and limes.

Key messageCitrus tristeza virus encodes a unique protein, p23, with multiple functional roles that include co-option of the cytoplasmic glyceraldehyde 3-phosphate dehydrogenase to facilitate the viral infectious cycle.AbstractThe genome of citrus tristeza virus (CTV), genus Closterovirus family Closteroviridae, is a single-stranded (+) RNA potentially encoding at least 17 proteins. One (p23), an RNA-binding protein of 209 amino acids with a putative Zn-finger and some basic motifs, displays singular features: (i) it has no homologues in other closteroviruses, (ii) it accumulates mainly in the nucleolus and Cajal bodies, and in plasmodesmata, and (iii) it mediates asymmetric accumulation of CTV RNA strands, intracellular suppression of RNA silencing, induction of some CTV syndromes and enhancement of systemic infection when expressed as a transgene ectopically or in phloem-associated cells in several Citrus spp. Here, a yeast two-hybrid screening of an expression library of Nicotiana benthamiana (a symptomatic experimental host for CTV), identified a transducin/WD40 domain protein and the cytosolic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as potential host interactors with p23. Bimolecular fluorescence complementation corroborated the p23-GAPDH interaction in planta and showed that p23 interacts with itself in the nucleolus, Cajal bodies and plasmodesmata, and with GAPDH in the cytoplasm (forming aggregates) and in plasmodesmata. The latter interaction was preserved in a p23 deletion mutant affecting the C-terminal domain, but not in two others affecting the Zn-finger and one internal basic motif. Virus-induced gene silencing of GAPDH mRNA resulted in a decrease of CTV titer as revealed by real-time RT-quantitative PCR and RNA gel-blot hybridization. Thus, like other viruses, CTV seems to co-opt GAPDH, via interaction with p23, to facilitate its infectious cycle.

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.

Citrus tristeza virus (CTV) induces in the field the decline and death of citrus varieties grafted on sour orange (SO) rootstock, which has forced the use of alternative decline-tolerant rootstocks in affected countries, despite the highly desirable agronomic features of the SO rootstock. Declining citrus plants display phloem necrosis below the bud union. In addition, SO is minimally susceptible to CTV compared with other citrus varieties, suggesting partial resistance of SO to CTV. Here, by silencing different citrus genes with a Citrus leaf blotch virus-based vector, we have examined the implication of the RNA silencing and salicylic acid (SA) defence pathways in the resistance of SO to CTV. Silencing of the genes RDR1, NPR1 and DCL2/DCL4, associated with these defence pathways, enhanced virus spread and accumulation in SO plants in comparison with non-silenced controls, whereas silencing of the genes NPR3/NPR4, associated with the hypersensitive response, produced a slight decrease in CTV accumulation and reduced stunting of SO grafted on CTV-infected rough lemon plants. We also found that the CTV RNA silencing suppressors p20 and p23 also suppress the SA signalling defence, with the suppressor activity being higher in the most virulent isolates.

SummaryThe long juvenile period of citrus trees (often more than 6 years) has hindered genetic improvement by traditional breeding methods and genetic studies. In this work, we have developed a biotechnology tool to promote transition from the vegetative to the reproductive phase in juvenile citrus plants by expression of the Arabidopsis thaliana or citrus FLOWERING LOCUS T (FT) genes using a Citrus leaf blotch virus‐based vector (clbvINpr‐AtFT and clbvINpr‐CiFT, respectively). Citrus plants of different genotypes graft inoculated with either of these vectors started flowering within 4–6 months, with no alteration of the plant architecture, leaf, flower or fruit morphology in comparison with noninoculated adult plants. The vector did not integrate in or recombine with the plant genome nor was it pollen or vector transmissible, albeit seed transmission at low rate was detected. The clbvINpr‐AtFT is very stable, and flowering was observed over a period of at least 5 years. Precocious flowering of juvenile citrus plants after vector infection provides a helpful and safe tool to dramatically speed up genetic studies and breeding programmes.

Citrus tristeza virus (CTV), the causal agent of the most devastating viral disease of citrus, has evolved three silencing suppressor proteins acting at intra-(p23 and p20) and inter-cellular level (p20 and p25) to overcome host antiviral defense. RNA interference (RNAi), an approach based on using dsRNA to trigger RNA silencing, has been widely used for generating transgenic plants resistant against viruses. Considering the important role of p23, p20 and p25 in CTV pathogenesis, we obtained transgenic Mexican lime plants with an intron-hairpin vector carrying full untranslatable versions of genes p25, p20, p23 and the 3'-UTR from the CTV strain T36, to attempt silencing their expression in CTV-infected cells. Complete resistance to viral infection was observed in three transgenic lines, with all their propagations remaining symptomless and virus-free after graft-inoculation with CTV-T36, either in the non-transgenic rootstock or directly in the transgenic scion. Accumulation of transgene-derived siRNAs was necessary but not sufficient for CTV resistance. When immune transformants were challenged with a dissimilar CTV strain the resistance was partially broken, stressing the importance of sequence identity in the underlying RNAi mechanism. This is the first evidence that it is possible to achieve full resistance to CTV in a highly sensitive citrus host by targeting simultaneously its three viral silencing suppressors through RNAi.

Measures to control damage caused by severe stem pitting strains (SP) of Citrus tristeza virus (CTV) include selective eradication and cross protection with mild strains (MS). Implementation of these measures needs sensitive and specific methods to discriminate between SP and MS variants in the viral populations. We developed a quantitative real-time RT-PCR method using Taqman locked nucleic acid (LNA) probes that enabled quantification of SP and MS variants in natural CTV populations. Here we used this method to monitor the evolution of the CTV population in Pineapple sweet orange plants pre-inoculated with a MS (T32) or a SP (T318) isolate, and then challenge-inoculated with budsticks infected with a SP or a MS isolate, respectively. While control plants singly inoculated with T32 or T318 only contained the cognate CTV variant in successive flushes, the plants doubly inoculated contained the two sequence variants in tissues derived from both the receptor plant and the inoculum budstick, with no strain impairing systemic invasion by the other. The relative amount of T32 and T318 variants in the co-infected plants differed between flushes. The co-infected plants showed stem pitting symptoms similar to those of plants inoculated only with T318, whereas control plants inoculated only with T32 or non-inoculated remained symptomless. This procedure enables rapid evaluation of MS for cross protecting ability.

Citrus tristeza virus (CTV) is phloem restricted in natural citrus hosts. The 23-kDa protein (p23) encoded by the virus is an RNA silencing suppressor and a pathogenicity determinant. The expression of p23, or its N-terminal 157-amino-acid fragment comprising the zinc finger and flanking basic motifs, driven by the constitutive 35S promoter of cauliflower mosaic virus, induces CTV-like symptoms and other aberrations in transgenic citrus. To better define the role of p23 in CTV pathogenesis, we compared the phenotypes of Mexican lime transformed with p23-derived transgenes from the severe T36 and mild T317 CTV isolates under the control of the phloem-specific promoter from Commelina yellow mottle virus (CoYMV) or the 35S promoter. Expression of the constructs restricted to the phloem induced a phenotype resembling CTV-specific symptoms (vein clearing and necrosis, and stem pitting), but not the non-specific aberrations (such as mature leaf epinasty and yellow pinpoints, growth cessation and apical necrosis) observed when p23 was ectopically expressed. Furthermore, vein necrosis and stem pitting in Mexican lime appeared to be specifically associated with p23 from T36. Phloem-specific accumulation of the p23Δ158-209(T36) fragment was sufficient to induce the same anomalies, indicating that the region comprising the N-terminal 157 amino acids of p23 is responsible (at least in part) for the vein clearing, stem pitting and, possibly, vein corking in this host.

Citrus vein enation (VE), a graft-transmissible disease naturally spread by several aphid species in a persistent mode, has been reported in many citrus growing areas. It causes vein enations on leaves and woody galls on trunk and branches of sensitive citrus species such as Mexican lime, rough lemon and Citrus volkameriana. The disease is currently diagnosed by biological indexing on sensitive indicator plants, an expensive and time-consuming method. In order to identify its causal agent and develop specific and reliable molecular detection methods, we analyzed small RNAs (sRNAs) from VE-infected Etrog citron plants by deep sequencing using the Illumina Solexa platform. Assembly of VE-associated sRNAs yielded several contigs that showed sequence homology with Pea enation mosaic virus 1 (PEMV-1), the type species of genus Enamovirus, family Luteoviridae. The gaps between adjacent contigs were filled by RT-PCR amplification, cloning and sequencing in order to obtain the complete genome sequence of a new virus, Citrus vein enation virus (CVEV). The CVEV genomic RNA has 5,983 nt organized in five open reading frames, resembling that of PEMV-1. Phylogenetic comparison of amino acid signatures in RNA-dependent RNA polymerases of the family Luteoviridae clearly grouped CVEV with PEMV-1. Therefore, we propose that CVEV should be included in the genus Enamovirus. A rapid and specific detection procedure was developed based on RT-PCR with CVEV-specific primers.

Citrus psorosis virus (CPsV), genus Ophiovirus, causes an important disease in many countries. In growing regions where natural disease spread occurs, damage caused by the virus can be controlled using resistant or tolerant cultivars, but sensitivity of many species and hybrids of Citrus and related genera is presently unknown. To find potential sources of CPsV resistance, we first propagated 61 species (or cultivars) and hybrids of Citrus and related genera [Citrus (37), Microcitrus (5), Fortunella (6), Eremocitrus (1), Pleiospermium (1), Atalantia (1), Severinia (1), Clausena (1), Poncirus (1) and hybrids (7)] on Rough lemon seedlings inoculated with the CPsV isolate PB 143. Out of the 61 accessions tested 54 showed symptoms and reacted by ELISA with a CPsV-specific antibody, 2 (M. inodora and F. hindsii) were symptomless but gave high ELISA values, suggesting tolerance to CPsV, 5 (C. depresa, Cleopatra mandarin, C. excavata, Carrizo citrange and CPB 4475 citrumelo) showed symptoms in the first flush but gave a negative ELISA reaction, and 1 (Poncirus trifoliata) was ELISA negative and symptomless. We then examined CPsV infection by ELISA and RT-PCR in Cleopatra mandarin, P. trifoliata and Citrumelo seedlings inoculated with CPsV isolates P 121, PB 108 and PB 143. While P 121 was detected in all Cleopatra and citrumelo plants, the other isolates were detected in only 50% of the inoculated plants, with all plants indexed as CPsV positive displaying symptoms. All P. trifoliata plants inoculated with either isolate indexed negative for CPsV and remained symptomless. Our results suggest CPsV resistance in P. trifoliata and partial isolate-specific resistance in some accessions.

Virus diseases of perennial trees and vines have characteristics not amenable to study using small model annual plants. Unique disease symptoms such as graft incompatibilities and stem pitting cause considerable crop losses. Also, viruses in these long-living plants tend to accumulate complex populations of viruses and strains. Considerable progress has been made in understanding the biology and genetics of Citrus tristeza virus (CTV) and in developing it into a tool for crop protection and improvement. The diseases in tree and vine crops have commonalities for which CTV can be used to develop a baseline. The purpose of this review is to provide a necessary background of systems and reagents developed for CTV that can be used for continued progress in this area and to point out the value of the CTV-citrus system in answering important questions on plant-virus interactions and developing new methods for controlling plant diseases.

First reported in 1896, psorosis was the first citrus disease proven to be graft transmissible and also the first for which eradication and budwood certification programs were launched to prevent its economic damage. For many years psorosis etiology remained elusive and only in 1986 it was associated with the presence of virus-like particles in infected plants. However, in the last two decades a virus with unusual morphology (Citrus psorosis virus, CPsV) was characterized and closely associated with psorosis disease as previously defined by field symptoms and by biological indexing in sensitive indicator plants. With a tripartite, negative-sense, RNA genome and a ~48 kDa coat protein, CPsV, the presumed causal agent of psorosis, is the type member of the genus Ophiovirus, within the new family Ophioviridae. Availability of the complete genomic sequence of two CPsV isolates and partial sequences of many others has enabled i) setting up rapid and sensitive RNA-based detection methods, ii) testing different citrus and relatives for resistance to CPsV, iii) identification of the two components (psorosis A and psorosis B) traditionally associated with non-scaled and scaled bark inoculum, respectively, from psorosis-infected plants and study their interactions, iv) analysis of genetic variation and evolutionary forces shaping the CPsV populations, v) preliminary studies on the interactions between virus and host factors and vi) development of transgenic citrus plants expressing variable degrees of resistance to CPsV. In summary, 120 years after the first report on psorosis we start seeing a pale light at the end of the tunnel.

Virus induced gene silencing (VIGS) is an effective technology for gene function analysis in plants. We assessed the VIGS effectiveness in Nicotiana benthamiana and citrus plants of different Citrus leaf blotch virus (CLBV)-based vectors, using insets of the phytoene desaturase (pds) gene. While in N. benthamiana the silencing phenotype was induced only by the construct carrying a 58-nt pds hairpin, in citrus plants all the constructs induced the silencing phenotype. Differences in the generation of secondary small interfering RNAs in both species are believed to be responsible for differential host–species effects. The ability of CLBV-based vectors to silence different endogenous citrus genes was further confirmed. Since CLBV-based vectors are known to be stable and induce VIGS in successive flushes for several months, these vectors provide an important genomic tool and it is expected that they will be useful to analyze gene function by reverse genetics in the long-lived citrus plants.

Analysis of four genomic regions from 37 geographically diverse isolates of broad bean wilt virus 1 (BBWV-1) showed high genetic diversity in comparison to most plant viruses. Comparison of synonymous and nonsynonymous substitutions of the small coat protein gene (SCP) revealed negative selection for most amino acid positions. Phylogenetic analysis of SCP showed that some BBWV-1 isolates from distant geographical areas were genetically close, suggesting long-distance migration. Analysis of genetic differentiation revealed high gene flow between Spanish and Near Eastern subpopulations, which were separated from North-Central and South-Eastern European subpopulations. Finally, putative recombinant and reassortant genomes were also identified.

Citrus tristeza virus (CTV) encodes a singular protein (p23, 209 amino acids) with multiple functions, including RNA silencing suppression (RSS). Confocal laser-scanning microscopy of green fluorescent protein (GFP)-p23 agroexpressed in Nicotiana benthamiana revealed its accumulation in the nucleolus, Cajal bodies, and plasmodesmata. To dissect the nucleolar localization signal (NoLS) typically associated with basic motifs, seven truncated and 10 point-mutated versions of p23 were assayed. Deletion mutants showed that regions 50 to 86 and 100 to 157 (excluding fragment 106 to 114), both with basic motifs and the first with a zinc-finger, contain the (bipartite) NoLS. Alanine substitutions delimited this signal to three cysteines of the Zn-finger and some basic amino acids. RSS activity of p23 in N. benthamiana was abolished by essentially all mutants, indicating that it involves most p23 regions. The necrotic-inducing ability of p23 when launched in N. benthamiana from Potato virus X was only retained by deletion mutant 158-209 and one substitution mutant, showing that the Zn-finger and flanking basic motifs form part of the pathogenic determinant. Ectopic expression of p23 and some deletion mutants in transgenic Mexican lime demarcated a similar determinant, suggesting that p23 affects related pathways in citrus and N. benthamiana. Both RSS activity and pathogenicity of p23 appear related to its nucleolar localization.

In nature Citrus tristeza virus (CTV), genus Closterovirus, infects only the phloem cells of species of Citrus and related genera. Finding that the CTV T36 strain replicated in Nicotiana benthamiana (NB) protoplasts and produced normal virions allowed development of the first genetic system based on protoplast transfection with RNA transcribed from a full-genome cDNA clone, a laborious and uncertain system requiring several months for each experiment. We developed a more efficient system based on agroinfiltration of NB leaves with CTV-T36-based binary plasmids, which caused systemic infection in this non-natural host within a few weeks yielding in the upper leaves enough CTV virions to readily infect citrus by slash inoculation. Stem agroinoculation of citrus and NB plants with oncogenic strains of Agrobacterium tumefaciens carrying a CTV-T36 binary vector with a GUS marker, induced GUS positive galls in both species. However, while most NB tumors were CTV positive and many plants became systemically infected, no coat protein or viral RNA was detected in citrus tumors, even though CTV cDNA was readily detected by PCR in the same galls. This finding suggests (1) strong silencing or CTV RNA processing in transformed cells impairing infection progress, and (2) the need for using NB as an intermediate host in the genetic system. To maintain CTV-T36 in NB or assay other CTV genotypes in this host, we also tried to graft-transmit the virus from infected to healthy NB, or to mechanically inoculate NB leaves with virion extracts. While these trials were mostly unsuccessful on non-treated NB plants, agroinfiltration with silencing suppressors enabled for the first time infecting NB plants by side-grafting and by mechanical inoculation with virions, indicating that previous failure to infect NB was likely due to virus silencing in early infection steps. Using NB as a CTV host provides new possibilities to study virus-host interactions with a simple and reliable system.