Virus-induced gene silencing (VIGS) is a powerful and cost-effective biotechnological tool for studying gene function in plants. It allows for rapid downregulation of gene expression without the need for tissue culture or genetic transformation. While VIGS is transient and may pose challenges for certain developmental genes, it has been successfully used to study organ abscission. Using a VIGS protocol based on Citrus leaf blotch virus (CLBV) viral vectors, we were able to characterize the regulatory role of INFLORESCENCE DEFICIENT IN ABSCISSION (IDA)-like genes and their potential HAESA (HAE)-like receptors in the process of corolla abscission in Nicotiana benthamiana flowers.

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.

Background Abscission is an active, organized, and highly coordinated cell separation process enabling the detachment of aerial organs through the modification of cell-to-cell adhesion and breakdown of cell walls at specific sites on the plant body known as abscission zones. In Arabidopsis thaliana , abscission of floral organs and cauline leaves is regulated by the interaction of the hormonal peptide INFLORESCENCE DEFICIENT IN ABSCISSION (IDA), a pair of redundant receptor-like protein kinases, HAESA (HAE) and HAESA-LIKE2 (HSL2), and SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) co-receptors. However, the functionality of this abscission signaling module has not yet been demonstrated in other plant species. Results The expression of the pair of NbenIDA1 homeologs and the receptor NbenHAE.1 was supressed at the base of the corolla tube by the inoculation of two virus-induced gene silencing (VIGS) constructs in Nicotiana benthamiana . These gene suppression events arrested corolla abscission but did not produce any obvious effect on plant growth. VIGS plants retained a higher number of corollas attached to the flowers than control plants, an observation related to a greater corolla breakstrength. The arrest of corolla abscission was associated with the preservation of the parenchyma tissue at the base of the corolla tube that, in contrast, was virtually collapsed in normal corollas. In contrast, the inoculation of a viral vector construct that increased the expression of NbenIDA1A at the base of the corolla tube negatively affected the growth of the inoculated plants accelerating the timing of both corolla senescence and abscission. However, the heterologous ectopic overexpression of citrus CitIDA3 and Arabidopsis AtIDA in N. benthamiana did not alter the standard plant phenotype suggesting that the proteolytic processing machinery was unable to yield active peptides. Conclusion Here, we demonstrate that the pair of NbenIDA1 homeologs encoding small peptides of the IDA -like family and the receptor NbenHAE.1 control cellular breakdown at the base of the corolla tube awhere an adventitious AZ should be formed and, therefore, corolla abscission in N. benthamiana flowers. Altogether, our results provide the first evidence supporting the notion that the IDA-HAE/HSL2 signaling module is conserved in angiosperms.

Damage caused by Citrus psorosis virus (CPsV) could be avoided obtaining resistant cultivars by breeding, but the sensitivity of most citrus hosts to CPsV is unknown. To find potential sources of resistance we inoculated 63 cultivars and hybrids of Citrus and related genera [ Citrus (36), Microcitrus (5), Fortunella (6), Eremocitrus (1), Pleiospermium (1), Atalantia (2), Severinia (1), Clausena (1), Swinglea (1), Afraegle (1), Poncirus (1) and hybrids (7)] with the CPsV isolate PB-143 and monitored symptoms and CPsV infection by ELISA. Microcitrus inodora and Fortunella hindsii were symptomless but gave high ELISA values, suggesting tolerance to CPsV, whereas Citrus depresa , Clausena excavata , Cleopatra mandarin, sour orange, Carrizo citrange and CPB4475 citrumelo were ELISA negative, suggesting resistance. Further examination of CPsV infection by reverse transcription quantitative real time PCR (RT-qPCR) in Cleopatra mandarin, Poncirus trifoliata and Carrizo citrange seedlings inoculated with CPsV isolates P-121, PB-102 and PB-143, revealed different infection rates and CPsV accumulation, depending on the isolate and citrus genotype. Bud propagation of these genotypes on CPsV-inoculated sweet orange plants incited a bud union disorder that hindered flushing and scion growth, suggesting that the high viral load in the rootstock induced a hypersensitive-like reaction in the partially resistant scion.

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 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.

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.

To invade systemically host plants, viruses need to replicate in the infected cells, spread to neighbouring cells through plasmodesmata and move to distal parts of the plant via sieve tubes to start new infection foci. To monitor the infection of Nicotiana benthamiana plants by Citrus leaf blotch virus (CLBV), leaves were agroinoculated with an infectious cDNA clone of the CLBV genomic RNA expressing green fluorescent protein (GFP) under the transcriptional control of a duplicate promoter of the coat protein subgenomic RNA. Fluorescent spots first appeared in agroinfiltrated leaves 11-12 days after infiltration, indicating CLBV replication. Then, after entering the phloem vascular system, CLBV was unloaded in the upper parts of the plant and invaded all tissues, including flower organs and meristems. GFP fluorescence was not visible in citrus plants infected with CLBV-GFP. Therefore, to detect CLBV in meristematic regions, Mexican lime (Citrus aurantifolia) plants were graft inoculated with CLBV, with Citrus tristeza virus (CTV), a virus readily eliminated by shoot-tip grafting in vitro, or with both simultaneously. Although CLBV was detected by hybridization and real-time reverse transcription-polymerase chain reaction (RT-PCR) in 0.2-mm shoot tips in all CLBV-inoculated plants, CTV was not detected. These results explain the difficulty in eliminating CLBV by shoot-tip grafting in vitro.

To identify the causal agent of citrus vein enation disease, we examined by deep sequencing (Solexa-Illumina) the small RNA (sRNA) fraction from infected and healthy Etrog citron plants. Our results showed that virus-derived sRNAs (vsRNAs): (i) represent about 14.21% of the total sRNA population, (ii) are predominantly of 21 and 24 nucleotides with a biased distribution of their 5' nucleotide and with a clear prevalence of those of (+) polarity, and (iii) derive from all the viral genome, although a prominent hotspot is present at a 5'-proximal region. Contigs assembled from vsRNAs showed similarity with luteovirus sequences, particularly with Pea enation mosaic virus, the type member of the genus Enamovirus. The genomic RNA (gRNA) sequence of a new virus, provisionally named Citrus vein enation virus (CVEV), was completed and characterized. The CVEV gRNA was found to be single-stranded, positive-sense, with a size of 5,983 nucleotides and five open reading frames. Phylogenetic comparisons based on amino acid signatures of the RNA polymerase and the coat protein clearly classifies CVEV within the genus Enamovirus. Dot-blot hybridization and reverse transcription-polymerase chain reaction tests were developed to detect CVEV in plants affected by vein enation disease. CVEV detection by these methods has already been adopted for use in the Spanish citrus quarantine, sanitation, and certification programs.

RNA viruses have a great potential for genetic variation, rapid evolution and adaptation. Characterization of the genetic variation of viral populations provides relevant information on the processes involved in virus evolution and epidemiology and it is crucial for designing reliable diagnostic tools and developing efficient and durable disease control strategies. Here we performed an updated analysis of sequences available in Genbank and reviewed present knowledge on the genetic variability and evolutionary processes of viruses of the family Closteroviridae. Several factors have shaped the genetic structure and diversity of closteroviruses. (I) A strong negative selection seems to be responsible for the high genetic stability in space and time for some viruses. (2) Long distance migration, probably by human transport of infected propagative plant material, have caused that genetically similar virus isolates are found in distant geographical regions. (3) Recombination between divergent sequence variants have generated new genotypes and plays an important role for the evolution of some viruses of the family Closteroviridae. (4) Interaction between virus strains or between different viruses in mixed infections may alter accumulation of certain strains. (5) Host change or virus transmission by insect vectors induced changes in the viral population structure due to positive selection of sequence variants with higher fitness for host-virus or vector-virus interaction (adaptation) or by genetic drift due to random selection of sequence variants during the population bottleneck associated to the transmission process.

Viral vectors have been used to express foreign proteins in plants or to silence endogenous genes. This methodology could be appropriate for citrus plants that have long juvenile periods and adult plants that are difficult to transform. We developed viral vectors based on Citrus leaf blotch virus (CLBV) by duplicating a minimum promoter (92 bp) either at the 3' untranslated region (clbv3'pr vector) or at the intergenic region between the movement and coat protein (CP) genes (clbvINpr vector). The duplicated fragment (-42/+50) around the transcription start site of the CP subgenomic RNA (sgRNA) had the full promoter activity and induced synthesis of a new sgRNA in infected plants. Agroinoculation with these vectors resulted in systemic infection of Nicotiana benthamiana and the resulting virions systemically infected citrus plants. A clbvINpr vector carrying the green fluorescent protein (GFP) gene expressed GFP in citrus plants and triggered gfp silencing in gfp-transgenic citrus plants, and vectors carrying fragments of the phytoene desaturase or the magnesium chelatase genes incited a silencing phenotype in citrus plants. These silenced phenotypes persisted in successive flushes. Because CLBV infections are symptomless in most citrus species, the effective silencing induced by CLBV-derived vectors will be helpful to analyze citrus gene function.

To counteract plant antiviral defense based on RNA silencing, many viruses express proteins that inhibit this mechanism at different levels. The genome of Citrus leaf blotch virus (CLBV) encodes a 227-kDa protein involved in replication, a 40-kDa movement protein (MP), and a 41-kDa coat protein (CP). To determine if any of these proteins might have RNA silencing suppressor activities, we have used Agrobacterium-mediated transient assays in the green fluorescent protein (GFP)-expressing Nicotiana benthamiana line 16c. Only CLBV MP was able to suppress intracellular GFP silencing induced by expression of either single- or double-stranded (ds) GFP RNA, but not cell-to-cell or long distance spread of the silencing signal. The MP suppressor activity was weak compared to other characterized viral suppressor proteins. Overall our data indicate that MP acts as a suppressor of local silencing probably by interfering in the silencing pathway downstream of the steps of dsRNA and small RNAs generation.

Citrus tristeza virus (CTV) naturally infects only some citrus species and relatives and within these it only invades phloem tissues. Failure to agroinfect citrus plants and the lack of an experimental herbaceous host hindered development of a workable genetic system. A full-genome cDNA of CTV isolate T36 was cloned in binary plasmids and was used to agroinfiltrate Nicotiana benthamiana leaves, with or without coinfiltration with plasmids expressing different silencing-suppressor proteins. A time course analysis in agroinfiltrated leaves indicated that CTV accumulates and moves cell-to-cell for at least three weeks postinoculation (wpi), and then, it moves systemically and infects the upper leaves with symptom expression. Silencing suppressors expedited systemic infection and often increased infectivity. In systemically infected Nicotiana benthamiana plants, CTV invaded first the phloem, but after 7 wpi, it was also found in other tissues and reached a high viral titer in upper leaves, thus allowing efficient transmission to citrus by stem-slash inoculation. Infected citrus plants showed the symptoms, virion morphology, and phloem restriction characteristic of the wild T36 isolate. Therefore, agroinfiltration of Nicotiana benthamiana provided the first experimental herbaceous host for CTV and an easy and efficient genetic system for this closterovirus.

The genetic variation and evolutionary mechanisms of broad bean wilt virus 2 (BBWV-2) were studied by nucleotide sequence analysis of four genomic regions of 30 isolates from different countries. Nucleotide diversity was high (0.198) for a plant virus. Phylogenetic and genetic structure analyses showed low population subdivision, suggesting a significant gene flow between distant geographic areas. Analysis of synonymous and nonsynonymous substitutions showed different negative selection pressures for different parts of the coding regions, but no positive selection was found. Several recombination detection methods showed that some BBWV-2 genomes might have originated from recombination or reassortment.

A real-time RT-PCR assay based on the TaqMan chemistry was developed for reliable detection and quantitation of Citrus leaf blotch virus (CLBV) in citrus plants. Detection by this method was highly specific and about one thousand times more sensitive than detection by conventional RT-PCR. An external standard curve using in vitro synthesized RNA transcripts of the selected target allowed a reproducible quantitative assay, with a wide dynamic range (seven logarithmic units of concentration) and very low variation coefficient values. This protocol enabled detection of as little as 100 copies of CLBV RNA in various tissues and citrus varieties infected with CLBV sources from different geographical origins. The new assay greatly improves current detection methods for CLBV and it has been most helpful for the Spanish citrus sanitation, quarantine and certification programs, and fitness evaluation of infectious cDNA clones of CLBV, useful potentially as viral vectors for citrus.