A Current Overview of Two Viroids Prevailing in Citrus Orchards: Citrus Exocortis Viroid and Hop Stunt Viroid

Citrus exocortis viroid (CEVd) and hop stunt viroid (HSVd) are the main viroids circulating in all citrus-growing areas worldwide, and causing two well-known diseases on citrus trees; exocortis and cachexia, respectively. These viroids are small, covalently closed single-stranded RNA, allocated to the Pospiviroidae family. CEVd is the first viroid being described on citrus trees in 1948 in California. It is considered the largest citrus viroid at 371 nucleotides. It causes bark scaling disorder on the rootstock of citrus trees grafted on trifoliate orange and its hybrids and can cause dwarfing of trees grown on these rootstocks. HSVd was first observed in 1945 in Florida. It consists of 299 nucleotides. Stunting, chlorosis, bark gumming, stem pitting, decline, and depressions in the wood are the main symptoms of HSVd in mandarin and its hybrids. The introduction and propagation of infected budwoods are the main causes of viroids spread in citrus orchards. These agents are mechanically sap-transmissible and spread by contaminated tools. Neither seed transmission nor vectors have been reported for both viroids. Root transmission, though possible, would be overshadowed by mechanical transmission. Rapid and sensitive molecular-based detection methods specific to both viroids are available. Both diseases are controlled by using viroids-free budwoods for new plantations, launching budwood certification programs, and establishing a quarantine system for new citrus varieties introduction. The most important achievements in CEVd and HSVd researches are outlined in this chapter. This would help to provide a clearer understanding of the diseases they cause and contribute to the development of better control strategies.

Effect of VIRP1 Protein on Nuclear Import of Citrus Exocortis Viroid (CEVd)

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.

Viróides em citros no Estado do Rio de Janeiro

RESUMO Os viróides infectam plantas de grande importância econômica como os citros. Objetivando detectar a presença de viróides através de métodos moleculares em árvores cítricas, cinco propriedades em Araruama, no Estado do Rio de Janeiro foram avaliadas. Vinte e duas amostras foram coletadas a partir de plantas com nanismo, rachadura no tronco e epinastia, sendo realizada a extração de RNA das folhas e empregado a técnica de RT-PCR com primers específicos para cinco espécies de viróide que infectam citros. O resultado da eletroforese em gel de agarose mostrou-se positivo para os viróides Citrus exocortis viroid (CEVd); Citrus bent leaf viroid (CBLVd); Hop stunt viroid (HSVd) e Citrus dwarfing viroid (CDVd), sendo o último encontrado em todas as propriedades e na combinação com outros viróides, o HSVd e o CBLVd estavam presentes em duas propriedades e o CEVd isoladamente em apenas uma propriedade. Não foi detectada a presença do Citrus viroid IV (CVd-IV) nas amostras avaliadas. Foram observadas diferenças na expressão dos sintomas associados ao CEVd o que pode ter ocorrido devido a interferências entre as espécies de viróides que infectavam uma mesma planta. A transmissão pode ter sido mecanicamente através da poda das plantas cítricas ou através de mudas infectadas com viróide. A utilização de métodos moleculares mostrou-se eficiente na identificação da presença de viróides em plantas cítricas no Estado do Rio de Janeiro.

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

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.

Citrus exocortis viroid causes ribosomal stress in tomato plants

Viroids are naked RNAs that do not code for any known protein and yet are able to infect plants causing severe diseases. Because of their RNA nature, many studies have focused on the involvement of viroids in RNA-mediated gene silencing as being their pathogenesis mechanism. Here, the alterations caused by the Citrus exocortis viroid (CEVd) on the tomato translation machinery were studied as a new aspect of viroid pathogenesis. The presence of viroids in the ribosomal fractions of infected tomato plants was detected. More precisely, CEVd and its derived viroid small RNAs were found to co-sediment with tomato ribosomes in vivo, and to provoke changes in the global polysome profiles, particularly in the 40S ribosomal subunit accumulation. Additionally, the viroid caused alterations in ribosome biogenesis in the infected tomato plants, affecting the 18S rRNA maturation process. A higher expression level of the ribosomal stress mediator NAC082 was also detected in the CEVd-infected tomato leaves. Both the alterations in the rRNA processing and the induction of NAC082 correlate with the degree of viroid symptomatology. Taken together, these results suggest that CEVd is responsible for defective ribosome biogenesis in tomato, thereby interfering with the translation machinery and, therefore, causing ribosomal stress.

Global Transcriptomic Analysis Reveals Insights into the Response of ‘Etrog’ Citron (Citrus medica L.) to Citrus Exocortis Viroid Infection

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.

Effect of Benzothiadiazole on the Metabolome of Tomato Plants Infected by Citrus Exocortis Viroid

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.