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Keywords = Citrus exocortis viroid (CEVd)

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16 pages, 4530 KiB  
Article
Salicylic Acid Modulates Volatile Organic Compound Profiles During CEVd Infection in Tomato Plants
by Marc Balanzá, Francisco Vázquez-Prol, Ismael Rodrigo, José María Bellés, Francisco Vera-Sirera, Maria Pilar López-Gresa and Purificación Lisón
Metabolites 2025, 15(2), 102; https://doi.org/10.3390/metabo15020102 - 6 Feb 2025
Viewed by 1252
Abstract
Background:Citrus Exocortis Viroid (CEVd) is a non-coding RNA pathogen capable of infecting a wide range of plant species, despite its lack of protein-coding ability. Viroid infections induce significant alterations in various physiological and biochemical processes, particularly impacting plant metabolism. This study shows [...] Read more.
Background:Citrus Exocortis Viroid (CEVd) is a non-coding RNA pathogen capable of infecting a wide range of plant species, despite its lack of protein-coding ability. Viroid infections induce significant alterations in various physiological and biochemical processes, particularly impacting plant metabolism. This study shows the metabolic changes upon viroid infection in tomato plants (Solanum lycopersicum var. ‘MoneyMaker’) exhibiting altered levels of salicylic acid (SA), a key signal molecule involved in the plant defence against this pathogen. Methods: Transgenic RNAi_S5H lines, which have the salicylic acid 5-hydroxylase gene silenced to promote SA accumulation, and NahG lines, which overexpress a salicylate hydroxylase to degrade SA into catechol and prevent its accumulation, were used to establish different SA levels in plants, resulting in varying degrees of resistance to viroid infection. The analysis was performed by using gas chromatography–mass spectrometry (GC-MS) to explore the role of volatile organic compounds (VOCs) in plant immunity against this pathogen. Results: Our results revealed distinct volatile profiles associated with plant immunity, where RNAi_S5H-resistant plants showed significantly enhanced production of monoterpenoids upon viroid infection. Moreover, viroid-susceptible NahG plants emitted a broad range of VOCs, whilst viroid-tolerant RNAi_S5H plants exhibited less variation in VOC emission. Conclusions: This study demonstrates that SA levels significantly influence metabolic responses and immunity in tomato plants infected by CEVd. The identification of differential emitted VOCs upon CEVd infection could allow the development of biomarkers for disease or strategies for disease control. Full article
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18 pages, 3899 KiB  
Article
The Elimination of Viroids through In Vitro Thermotherapy and a Meristem Tip Culture from a New Limonime Hybrid (Citrus x limon var. limon (L.) Burm. f. x Citrus latifolia var. latifolia)
by Virginia Sarropoulou, Katerina Grigoriadou, Varvara I. Maliogka, Chrysoula-Lito Sassalou and Vasileios Ziogas
BioTech 2024, 13(3), 37; https://doi.org/10.3390/biotech13030037 - 23 Sep 2024
Cited by 2 | Viewed by 1888
Abstract
Viruses and viroids pose a significant challenge in citriculture, and their control is crucial for plant health. This study evaluated the effectiveness of in vitro thermotherapy combined with a meristem tip culture for eliminating citrus exocortis viroid (CEVd) and hop stunt viroid (HSVd) [...] Read more.
Viruses and viroids pose a significant challenge in citriculture, and their control is crucial for plant health. This study evaluated the effectiveness of in vitro thermotherapy combined with a meristem tip culture for eliminating citrus exocortis viroid (CEVd) and hop stunt viroid (HSVd) from a new limonime hybrid (Citrus x limon var. limon x Citrus latifolia var. latifolia). The elimination success was confirmed by RT-PCR assays. The in vitro elimination rate for CEVd during the shoot proliferation stage (43%) was higher than for HSVd (21%). Accordingly, in the subsequent rooting stage, the in vitro elimination rate for CEVd (50%) was higher than for HSVd (33%). Successful CEVd and HSVd eradication at a 100% rate was confirmed in the ex vitro acclimatized plants in the greenhouse. The study also established an efficient micropropagation protocol. The optimal treatment for in vitro shoot induction was 0.5–2 mg L−1 benzyladenine (BA) + 0.5 mg L−1 gibberellic acid (GA3) + 0.25 mg L−1 naphthalene acetic acid (NAA), while for shoot elongation, it was 0.5 mg L−1 BA + 0.5 mg L−1 kinetin (KIN) + 0.5 mg L−1 GA3 + 0.25 mg L−1 NAA. Rooting was best promoted by 1 mg L−1 NAA. This study provides valuable insights for the mass production of viroid-free propagation material in this new lemon x lime hybrid, contributing to the conservation of genetic resources in citrus breeding programs through the combined application of in vitro thermotherapy and an in vitro meristem tip culture, a novel and highlighted achievement reported for the first time in this study. Full article
(This article belongs to the Section Industry, Agriculture and Food Biotechnology)
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12 pages, 8317 KiB  
Article
Loquat (Eriobotrya japonica) Is a New Natural Host of Tomato Mosaic Virus and Citrus Exocortis Viroid
by Chengyong He, Lingli Wang, Yarui Li, Kangyu Zhou, Ke Zhao, Dong Chen, Jing Li, Haiyan Song and Meiyan Tu
Plants 2024, 13(14), 1965; https://doi.org/10.3390/plants13141965 - 18 Jul 2024
Viewed by 1777
Abstract
Loquat leaves exhibiting obvious yellowing, blistering, mosaic, leaf upward cupping, crinkle, and leaf narrowing were identified in Panzhihua City, Sichuan Province, China. High-throughput sequencing (HTS) with the ribo-depleted cDNA library was employed to identify the virome in the loquat samples; only tomato mosaic [...] Read more.
Loquat leaves exhibiting obvious yellowing, blistering, mosaic, leaf upward cupping, crinkle, and leaf narrowing were identified in Panzhihua City, Sichuan Province, China. High-throughput sequencing (HTS) with the ribo-depleted cDNA library was employed to identify the virome in the loquat samples; only tomato mosaic virus (ToMV) and citrus exocortis viroid (CEVd) were identified in the transcriptome data. The complete genome sequence of ToMV and CEVd were obtained from the loquat leaves. The full-length genome of the ToMV-loquat is 6376 nt and comprises four open reading frames (ORFs) encoding 183 kDa protein, RNA-dependent RNA polymerase (RdRp), movement protein (MP), and coat protein (CP), respectively. A pairwise identity analysis showed that the complete sequence of the ToMV-loquat had a nucleotide identity between 98.5 and 99.3% with other ToMV isolates. A phylogenetic analysis indicated that ToMV-loquat was more closely related to ToMV-IFA9 (GenBank No. ON156781). A CEVd sequence with 361 nt in length was amplified based on the HTS contigs, sequence alignment indicated CEVd-loquat had the highest identity with the strain of CEVd-Balad (GenBank No. PP869624), phylogenetic analysis showed that CEVd-loquat was more closely related to CEVd-lettuce (GenBank No. ON993891). This significant discovery marks the first documentation and characterization of ToMV and CEVd infecting loquat plants, shedding light on potential threats to loquat cultivation and providing insights for disease management strategies. Full article
(This article belongs to the Section Plant Protection and Biotic Interactions)
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14 pages, 1886 KiB  
Article
Development of Multiplex RT-PCR Assay for the Simultaneous Detection of Four Systemic Diseases Infecting Citrus
by Shun-Min Yao, Meng-Ling Wu and Ting-Hsuan Hung
Agriculture 2023, 13(6), 1227; https://doi.org/10.3390/agriculture13061227 - 10 Jun 2023
Cited by 2 | Viewed by 2546
Abstract
The citrus systemic diseases, including citrus Huanglongbing (caused by Candidatus Liberibacter asiaticus (CLas)), citrus tristeza (caused by citrus tristeza virus (CTV)), citrus tatter leaf (caused by citrus tatter leaf virus (CTLV)), and citrus exocortis (caused by citrus exocortis viroid (CEVd)), are [...] Read more.
The citrus systemic diseases, including citrus Huanglongbing (caused by Candidatus Liberibacter asiaticus (CLas)), citrus tristeza (caused by citrus tristeza virus (CTV)), citrus tatter leaf (caused by citrus tatter leaf virus (CTLV)), and citrus exocortis (caused by citrus exocortis viroid (CEVd)), are threats to citrus production in Taiwan. Reliable diagnostic methods are important for the management of these systemic diseases. In this study, we developed a multiplex reverse transcription–polymerase chain reaction (RT-PCR) assay to detect four pathogens simultaneously. Herein, the specific amplicons from each pathogen (295 bp for CLas, 468 bp for CTV, 120 bp for CTLV, and 196 bp for CEVd) were successfully produced using the optimized multiplex RT-PCR described here. The sensitivity evaluation showed that low titers of pathogens could be detected using this multiplex RT-PCR. Compared with the published simplex assays, the detection of field samples using the multiplex RT-PCR developed in this study showed a better performance. The detections using multiplex RT-PCR revealed that these four citrus systemic pathogens were commonly found in fields, and 30.0% of field samples were mix-infected. To our knowledge, this is the first study of a survey of the four important citrus systemic diseases in Taiwan, and it provides insights for improving disease management. Therefore, the multiplex RT-PCR assay provides a useful method for routine disease surveying and the production of pathogen-free citrus plants. Full article
(This article belongs to the Section Crop Protection, Diseases, Pests and Weeds)
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17 pages, 2223 KiB  
Article
Incidence and Epidemiology of Citrus Viroids in Greece: Role of Host and Cultivar in Epidemiological Characteristics
by Matthaios M. Mathioudakis, Nikolaos Tektonidis, Antonia Karagianni, Louiza Mikalef, Pedro Gómez and Beata Hasiów-Jaroszewska
Viruses 2023, 15(3), 605; https://doi.org/10.3390/v15030605 - 22 Feb 2023
Cited by 8 | Viewed by 2259
Abstract
Viroids represent a threat to the citrus industry and also display an intricate matter for citrus tristeza virus (CTV) control as most of the commercial citrus rootstocks that are resistant/tolerant to CTV appear to be highly susceptible to viroid infection. Therefore, a detailed [...] Read more.
Viroids represent a threat to the citrus industry and also display an intricate matter for citrus tristeza virus (CTV) control as most of the commercial citrus rootstocks that are resistant/tolerant to CTV appear to be highly susceptible to viroid infection. Therefore, a detailed knowledge of the viroid’s incidence and distribution, along with the assessment of unexplored epidemiological factors leading to their occurrence, are necessary to further improve control measures. Herein, a large-scale epidemiological study of citrus viroids in five districts, 38 locations and 145 fields in Greece is presented, based on the analysis of 3005 samples collected from 29 cultivars of six citrus species. We monitored the occurrence of citrus exocortis (CEVd), hop stunt (HSVd), citrus dwarfing (CDVd), citrus bark cracking (CBCVd), and citrus bent leaf (CBLVd) viroids, and addressed their epidemiological patterns and factors shaping their population structure. Our results show a high frequency and wide distribution of four viroids in all areas and in almost all hosts, whereas CBLVd occurrence was restricted to Crete. Mixed infections were found in all districts in which a wide spread of viroids was observed. We identified a potential pathogens’ different preferences that could be partially explained by the host and cultivar, including the type of infection (single or mixed) and the number of viroids in the mixed infections. Overall, this work provides the first detailed epidemiological study on citrus viroids, enriching our knowledge for the implementation, production, and distribution of certified citrus propagative material, and the development of sustainable control strategies. Full article
(This article belongs to the Special Issue Plant Virus Epidemiology and Control 2022)
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12 pages, 3199 KiB  
Article
Reproducibility and Sensitivity of High-Throughput Sequencing (HTS)-Based Detection of Citrus Tristeza Virus and Three Citrus Viroids
by Rachelle Bester, Chanel Steyn, Johannes H. J. Breytenbach, Rochelle de Bruyn, Glynnis Cook and Hans J. Maree
Plants 2022, 11(15), 1939; https://doi.org/10.3390/plants11151939 - 26 Jul 2022
Cited by 5 | Viewed by 2353
Abstract
The credibility of a pathogen detection assay is measured using specific parameters including repeatability, specificity, sensitivity, and reproducibility. The use of high-throughput sequencing (HTS) as a routine detection assay for viruses and viroids in citrus was previously evaluated and, in this study, the [...] Read more.
The credibility of a pathogen detection assay is measured using specific parameters including repeatability, specificity, sensitivity, and reproducibility. The use of high-throughput sequencing (HTS) as a routine detection assay for viruses and viroids in citrus was previously evaluated and, in this study, the reproducibility and sensitivity of the HTS assay were assessed. To evaluate the reproducibility of HTS, the same plants assayed in a previous study were sampled again, one year later, and assessed in triplicate using the same analyses to construct the virome profile. The sensitivity of the HTS assay was compared to routinely used RT-PCR assays in a time course experiment, to compensate for natural pathogen accumulation in plants over time. The HTS pipeline applied in this study produced reproducible and comparable results to standard RT-PCR assays for the detection of CTV and three viroid species in citrus. Even though the limit of detection of HTS can be influenced by pathogen concentration, sample processing method and sequencing depth, detection with HTS was found to be either equivalent or more sensitive than RT-PCR in this study. Full article
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16 pages, 3747 KiB  
Article
Symptom Severity, Infection Progression and Plant Responses in Solanum Plants Caused by Three Pospiviroids Vary with the Inoculation Procedure
by Francisco Vázquez Prol, Joan Márquez-Molins, Ismael Rodrigo, María Pilar López-Gresa, José María Bellés, Gustavo Gómez, Vicente Pallás and Purificación Lisón
Int. J. Mol. Sci. 2021, 22(12), 6189; https://doi.org/10.3390/ijms22126189 - 8 Jun 2021
Cited by 12 | Viewed by 3100
Abstract
Infectious viroid clones consist of dimeric cDNAs used to generate transcripts which mimic the longer-than-unit replication intermediates. These transcripts can be either generated in vitro or produced in vivo by agro-inoculation. We have designed a new plasmid, which allows both inoculation methods, and [...] Read more.
Infectious viroid clones consist of dimeric cDNAs used to generate transcripts which mimic the longer-than-unit replication intermediates. These transcripts can be either generated in vitro or produced in vivo by agro-inoculation. We have designed a new plasmid, which allows both inoculation methods, and we have compared them by infecting Solanum lycopersicum and Solanum melongena with clones of Citrus exocortis virod (CEVd), Tomato chlorotic dwarf viroid (TCDVd), and Potato spindle tuber viroid (PSTVd). Our results showed more uniform and severe symptoms in agro-inoculated plants. Viroid accumulation and the proportion of circular and linear forms were different depending on the host and the inoculation method and did not correlate with the symptoms, which correlated with an increase in PR1 induction, accumulation of the defensive signal molecules salicylic (SA) and gentisic (GA) acids, and ribosomal stress in tomato plants. The alteration in ribosome biogenesis was evidenced by both the upregulation of the tomato ribosomal stress marker SlNAC082 and the impairment in 18S rRNA processing, pointing out ribosomal stress as a novel signature of the pathogenesis of nuclear-replicating viroids. In conclusion, this updated binary vector has turned out to be an efficient and reproducible method that will facilitate the studies of viroid–host interactions. Full article
(This article belongs to the Section Molecular Plant Sciences)
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9 pages, 1529 KiB  
Article
Effect of VIRP1 Protein on Nuclear Import of Citrus Exocortis Viroid (CEVd)
by Hyesu Seo, Kyunghee Kim and Woong June Park
Biomolecules 2021, 11(1), 95; https://doi.org/10.3390/biom11010095 - 13 Jan 2021
Cited by 9 | Viewed by 2661
Abstract
Before replicating, Pospiviroidae viroids must move into the plant nucleus. However, the mechanisms of viroid nuclear import are not entirely understood. To study the nuclear import of viroids, we established a nuclear import assay system using onion cell strips and observed the import [...] Read more.
Before replicating, Pospiviroidae viroids must move into the plant nucleus. However, the mechanisms of viroid nuclear import are not entirely understood. To study the nuclear import of viroids, we established a nuclear import assay system using onion cell strips and observed the import of Alexa Fluor-594-labeled citrus exocortis viroid (CEVd). To identify the plant factors involved in the nuclear import of viroids, we cloned the Viroid RNA-binding Protein 1 (VIRP1) gene from a tomato cultivar, Seokwang, and heterologously expressed and purified the VIRP1 protein. The newly prepared VIRP1 protein had alterations of amino acid residues at two points (H52R, A277G) compared with a reference VIRP1 protein (AJ249595). VIRP1 specifically bound to CEVd and promoted its nuclear import. However, it is still uncertain whether VIRP1 is the only factor required for the nuclear import of CEVd because CEVd entered the plant nuclei without VIRP1 in our assay system. The cause of the observed nuclear accumulation of CEVd in the absence of VIRP1 needs to be further clarified. Full article
(This article belongs to the Section Molecular Biology)
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16 pages, 3324 KiB  
Article
Ethylene is Involved in Symptom Development and Ribosomal Stress of Tomato Plants upon Citrus Exocortis Viroid Infection
by Francisco Vázquez Prol, M. Pilar López-Gresa, Ismael Rodrigo, José María Bellés and Purificación Lisón
Plants 2020, 9(5), 582; https://doi.org/10.3390/plants9050582 - 2 May 2020
Cited by 8 | Viewed by 4736
Abstract
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Plant Ribosome Biogenesis)
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17 pages, 3526 KiB  
Article
Global Transcriptomic Analysis Reveals Insights into the Response of ‘Etrog’ Citron (Citrus medica L.) to Citrus Exocortis Viroid Infection
by Yafei Wang, Jiaxing Wu, Yuanjian Qiu, Sagheer Atta, Changyong Zhou and Mengji Cao
Viruses 2019, 11(5), 453; https://doi.org/10.3390/v11050453 - 17 May 2019
Cited by 23 | Viewed by 5975
Abstract
Citrus exocortis viroid (CEVd) is the causal agent of citrus exocortis disease. We employed CEVd-infected ‘Etrog’ citron as a system to study the feedback regulation mechanism using transcriptome analysis in this study. Three months after CEVd infection, the transcriptome of fresh leaves was [...] Read more.
Citrus exocortis viroid (CEVd) is the causal agent of citrus exocortis disease. We employed CEVd-infected ‘Etrog’ citron as a system to study the feedback regulation mechanism using transcriptome analysis in this study. Three months after CEVd infection, the transcriptome of fresh leaves was analyzed, and 1530 differentially expressed genes were detected. The replication of CEVd in citron induced upregulation of genes encoding key proteins that were involved in the RNA silencing pathway such as Dicer-like 2, RNA-dependent RNA polymerase 1, argonaute 2, argonaute 7, and silencing defective 3, as well as those genes encoding proteins that are related to basic defense responses. Many genes involved in secondary metabolite biosynthesis and chitinase activity were upregulated, whereas other genes related to cell wall and phytohormone signal transduction were downregulated. Moreover, genes encoding disease resistance proteins, pathogenicity-related proteins, and heat shock cognate 70 kDa proteins were also upregulated in response to CEVd infection. These results suggest that basic defense and RNA silencing mechanisms are activated by CEVd infection, and this information improves our understanding of the pathogenesis of viroids in woody plants. Full article
(This article belongs to the Special Issue Viroid-2018: International Conference on Viroids and Viroid-Like RNAs)
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15 pages, 2893 KiB  
Article
Effect of Benzothiadiazole on the Metabolome of Tomato Plants Infected by Citrus Exocortis Viroid
by María Pilar López-Gresa, Celia Payá, Ismael Rodrigo, José María Bellés, Susana Barceló, Young Hae Choi, Robert Verpoorte and Purificación Lisón
Viruses 2019, 11(5), 437; https://doi.org/10.3390/v11050437 - 14 May 2019
Cited by 12 | Viewed by 4410
Abstract
Benzothiadiazole (BTH) is a functional analogue of the phytohormone salycilic acid (SA) involved in the plant immune response. NahG tomato plants are unable to accumulate SA, which makes them hypersusceptible to several pathogens. Treatments with BTH increase the resistance to bacterial, fungal, viroid, [...] Read more.
Benzothiadiazole (BTH) is a functional analogue of the phytohormone salycilic acid (SA) involved in the plant immune response. NahG tomato plants are unable to accumulate SA, which makes them hypersusceptible to several pathogens. Treatments with BTH increase the resistance to bacterial, fungal, viroid, or viral infections. In this study, metabolic alterations in BTH-treated Money Maker and NahG tomato plants infected by citrus exocortis viroid (CEVd) were investigated by nuclear magnetic resonance spectroscopy. Using multivariate data analysis, we have identified defence metabolites induced after viroid infection and BTH-treatment. Glycosylated phenolic compounds include gentisic and ferulic acid accumulated in CEVd-infected tomato plants, as well as phenylalanine, tyrosine, aspartate, glutamate, and asparagine. Besides, an increase of γ-aminobutyric acid (GABA), glutamine, adenosine, and trigonelline, contributed to a clear discrimination between the metabolome of BTH-treated tomato leaves and their corresponding controls. Among them, GABA was the only metabolite significantly accumulated in both genotypes after the chemical treatment. In view of these results, the addition of GABA was performed on tomato plants infected by CEVd, and a reversion of the NahG hypersusceptibility to CEVd was observed, indicating that GABA could regulate the resistance to CEVd induced by BTH. Full article
(This article belongs to the Special Issue Viroid-2018: International Conference on Viroids and Viroid-Like RNAs)
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10 pages, 5379 KiB  
Commentary
Viroids as Companions of a Professional Career
by Núria Duran-Vila
Viruses 2019, 11(3), 245; https://doi.org/10.3390/v11030245 - 12 Mar 2019
Cited by 3 | Viewed by 4408
Abstract
Since the early 1970s when “virus-like” agents were considered as the cause of two diseases (potato spindle tuber and citrus exocortis), their study and further characterization have been linked to the development and use of molecular biology tools. Sucrose density gradient centrifugation and [...] Read more.
Since the early 1970s when “virus-like” agents were considered as the cause of two diseases (potato spindle tuber and citrus exocortis), their study and further characterization have been linked to the development and use of molecular biology tools. Sucrose density gradient centrifugation and polyacrylamide gel electrophoresis (PAGE) played a critical role in the pioneering studies of PSTVd and citrus exocortis viroid (CEVd). This was later modified by using other PAGEs (sequential PAGE, return PAGE, two-dimensional PAGE), and/or different staining methods (ethidium bromide, silver nitrate, etc.). Since then, disease-causing agents suspected to be viroids were usually subjected to a number of tests to define their: (i) Molecular nature (RNA or DNA; single stranded or double stranded; circular or linear RNA); (ii) molecular weight; (iii) secondary and tertiary structure. Further biological assays are also essential to establish the relationship of a viroid with plant disease and to fulfill Koch’s postulates. Full article
(This article belongs to the Special Issue Viroid-2018: International Conference on Viroids and Viroid-Like RNAs)
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21 pages, 1526 KiB  
Article
Innate Immunity Activation and RNAi Interplay in Citrus Exocortis Viroid—Tomato Pathosystem
by Thibaut Olivier and Claude Bragard
Viruses 2018, 10(11), 587; https://doi.org/10.3390/v10110587 - 26 Oct 2018
Cited by 24 | Viewed by 4667
Abstract
Although viroids are the smallest and simplest plant pathogens known, the molecular mechanisms underlying their pathogenesis remain unclear. To unravel these mechanisms, a dual approach was implemented consisting of in silico identification of potential tomato silencing targets of pospiviroids, and the experimental validation [...] Read more.
Although viroids are the smallest and simplest plant pathogens known, the molecular mechanisms underlying their pathogenesis remain unclear. To unravel these mechanisms, a dual approach was implemented consisting of in silico identification of potential tomato silencing targets of pospiviroids, and the experimental validation of these targets through the sequencing of small RNAs and RNA ends extracted from tomatoes infected with a severe isolate of Citrus exocortis viroid (CEVd). The generated RNA ends were also used to monitor the differentially-expressed genes. These analyses showed that when CEVd symptoms are well established: (i) CEVd are degraded by at least three Dicer-like (DCL) proteins and possibly by RNA-induced silencing complex (RISC), (ii) five different mRNAs are partially degraded through post-transcriptional gene silencing (PTGS), including argonaute 2a, which is further degraded in phasiRNAs, (iii) Dicer-like 2b and 2d are both upregulated and degraded in phasiRNAs, and (iv) CEVd infection induced a significant shift in gene expression allowing to explain the usual symptoms of pospiviroids on tomato and to demonstrate the constant activation of host innate immunity and systemic acquired resistance (SAR) by these pathogenic RNAs. Finally, based on in silico analysis, potential immunity receptor candidates of viroid-derived RNAs are suggested. Full article
(This article belongs to the Special Issue Viroid-2018: International Conference on Viroids and Viroid-Like RNAs)
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