Citrus Exocortis Research Articles

Viability and Pathogenicity of Intersubgroup Viroid Chimeras Suggest Possible Involvement of the Terminal Right Region in Replication

To investigate the structural determinants regulating viroid replication and pathogenicity, we have examined the biological properties of four chimeric viroids containing sequences derived from hop stunt (HSVd) and citrus exocortis (CEVd) viroids. The viability of each chimera — CEHS (CEVd left half + HSVd right half), HSCE (HSVd left half + CEVd right half), CE/HS-TR (CEVd + HSVd right terminal loop), and HS/CE-TR (HSVd + CEVd right terminal loop)—was tested by inoculation onto cucumber and tomato seedlings. Chimeras CEHS and HSCE were not infectious, but CE/HS-TR and HS/CE-TR replicated stably and produced disease symptoms when inoculated onto tomato or cucumber, respectively. Progeny accumulation was reduced 10-fold or more compared to that of CEVd in tomato or HSVd in cucumber. The results suggested that the TR, like the TL and P regions, forms a relatively independent structural unit that contributes to the total function of a viroid. The effect of sequences in the right terminal loop on pathogenicity appears to be indirect, modulating the efficiency of viroid replication (or accumulation) efficiency rather than symptom expression per se.

Identification of a New Pathogen-induced Member of the Subtilisin-like Processing Protease Family from Plants

By using biochemical, immunological, and molecular strategies we have identified and cloned a cDNA encoding a protease from tomato (Lycopersicon esculentum) plants (P69B) that is part of a proteolytic system activated in the plant as a result of infection with citrus exocortis viroid. This new protease is closely related, in terms of amino acid sequence and structural organization, to the previously identified pathogenesis-related subtilisin-like protease (Tornero, P., Conejero, V., and Vera, P. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 6332-6337). The 745-residue amino acid sequence of P69B begins with a cleavable signal peptide, contains a prodomain and a 631-residue mature domain which is homologous to the catalytic modules of bacterial subtilisins and eukaryotic Kex2-like proteases. Within the catalytic domain, the essential Asp, His, and Ser residues that conform the catalytic triad of this family of proteases are conserved in P69B. Northern blot and reverse transcriptase-polymerase chain reaction analysis demonstrated widespread induced expression of the 2.5-kilobase hybridizing mRNA in plant tissues as a consequence of viroid infection. We propose that P69B is a member of a complex gene family of plant Kex2/subtilisin-like proteases presumably involved in a number of specific proteolytic events activated during pathogenesis in plants and that takes place in the extracellular matrix.

Direct Nucleotide Sequencing of Citrus Exocortis Viroid (CEV)

Citrus exocortis viroid (CEV), the causal agent of bark shelling disease in Poncirus trifoliata (L.) Raf, can be distinguished by its biological properties in Etrog citron and Gynura aurantiaca D. C. CEV isolate obtained from citrus trees of Çukurova region was reverse transcription-polymerase chain reaction assay (RT-PCR). The amplified products were gel purified, and subsequently sequenced without cloning, using the Promega fmol sequencing system. Both CEVc (citron) and CEVg (Gynura) were determined 371 nucleotides (nts) in length. Comparing the nucleotide sequence of CEV isolate from Çukurova region and California isolates of CEV, a number of exchanges were found in the pathogenic and variable domains.

Characterization of LRP, a leucine-rich repeat (LRR) protein from tomato plants that is processed during pathogenesis.

This paper describes the isolation and characterization of LRP, a new gene from tomato plants. The deduced amino acid sequence showed that the encoded protein is enriched in leucine, and contains interesting structural motifs. LRP contains four tandem repeats of a canonical 24 amino acid leucine-rich repeat (LRR) sequence present in different proteins that mediates molecular recognition and/or interaction processes. Genomic organization and intron-exon arrangement of LRP favor the hypothesis that the LRR domains present in LRP evolved by exon duplication and shuffling. LRP expression analysis and immunohistochemical localization studies of the encoded protein indicate that the gene is under developmental regulation exhibiting tissue-specificity, particularly in certain cell types of the stele, like phloem fibers, parenchyma cells of the protoxylem, and in the cell files that constitute the rays of the secondary xylem. It is shown that this gene is upregulated in diseased tomato plants infected with citrus exocortis viroid. However, in this pathogenic context, LRP is processed proteolytically to a lower molecular weight form by a host-induced extracellular protease. The structural characteristics of LRP, its spatio-temporal pattern of expression, and its post-translational processing during pathogenesis, suggest this protein as a candidate molecule that may mediate recognition and interaction events taking place in the plant extracellular matrix under normal and/or pathogenesis-related conditions.

Tissue and intra‐cellular distribution of coconut cadang cadang viroid and citrus exocortis viroid determined by in situ hybridization and confocal laser scanning and transmission electron microscopy

Confocal laser scanning microscopy and transmission electron microscopy (TEM) were used in conjunction with in situ hybridization techniques to compare and contrast the subnuclear (ultrastructural) and tissue (histological) localizations, respectively, of citrus exocortis viroid (CEV) and coconut cadang cadang viroid (CCCV). Both these viroids, which are members of the same taxonomic subgroup of viroids, were found in the vascular tissues as well as in the nuclei of mesophyll cells of infected host plants. At the subnuclear level, however, CEV was distributed across the entire nucleus, in contrast to CCCV which was mostly concentrated in the nucleolus with the remainder distributed throughout the nucleoplasm.

A rapid and sensitive dot-blot hybridization assay for the detection of citrus exocortis viroid in Citrus medica with digoxigenin-labelled RNA probes.

A rapid and sensitive dot-blot hybridization assay using in vitro-transcribed digoxigenin-labelled RNA probes (riboprobes) was developed aiming at detection of citrus exocortis viroid (CEVd) in crude sap of infected Citrus medica plants. The protocol includes a very quick and simple preparation of RNA extracts from samples using a denaturation step with formaldehyde. From our results, the employment of this step is highly recommended because the hybridization signals in formaldehyde-denatured samples were significantly stronger when compared with that of extracts without formaldehyde treatment. The assay was found to be sensitive enough to detect 0.1 ng of purified CEVd RNA and was able to detect viroid in 0.2 mg of symptomatic Citrus medica leaves. The use of riboprobes also allowed hybridization under high temperature conditions, avoiding non-specific background.

Naturally occurring variants of citrus exocortis viroid in vegetable crops

Four viroids were isolated in crops of tomato, eggplant, carrot and turnip. They were detected only after inoculation of ‘Rutgers’ tomato with nucleic acid extracts from field plants and were characterized as variants of citrus exocortis viroid (CEVd). The size of the variants ranged from 371 to 374 nucleotides and they contained nucleotide exchanges and insertions affecting all structural domains. Symptoms were induced in tomato and carrot whereas eggplant and turnip responded as symptomless carriers. As the samples came from an area where no citrus is grown, it appears that these viroids have a reproductive cycle independent of this crop, and perhaps involving wild plants or other vegetables.

Survey and Indexing for Citrus Exocortis and Other Viroids in Sichuan Province

Survey and Indexing for Citrus Exocortis and Other Viroids in Sichuan Province

Detection of Citrus Exocortis Viroid by In Vitro Inoculation of Callus Culture

Detection of Citrus Exocortis Viroid by In Vitro Inoculation of Callus Culture

A citrus exocortis viroid variant from broad bean (Vicia faba L.): infectivity and pathogenesis.

A viroid present in very low titres was isolated from symptomless field broad bean plants. It was identified as a variant of citrus exocortis viroid in the T2, V and C domains. Infection of several hosts resulted in a change in the composition of the viroid population. Serial passage through tomato and back to the host of origin, broad bean, resulted in major changes in replication efficiency, host range and pathogenicity. The unique nucleotide sequence differences identified in the original broad bean variant were not conserved after passage through alternative hosts. The effects of these sequence variations on viroid secondary structure result in nonpathogenic viroid variants which can remain unnoticed in certain plant species but may act as reservoirs of viroid disease.

The expression of antisense and ribozyme genes targeting citrus exocortis viroid in transgenic plants

Four ribozyme and antisense genes targeting citrus exocortis viroid (CEVd) positive- and negative-strand RNA molecules were constructed and used to transform the tomato Lycopersicon lycopersicum cv. UC82B. The tomato is a readily transformable plant and will support replication of CEVd following mechanical inoculation. The ribozyme genes contained three hammerhead catalytic motifs with long hybridizing arms and synthetic RNA transcripts were shown to cleave the target CEVd RNA molecule in vitro. Homozygous transgenic plants were produced from independent transformants expressing either ribozymes or antisense constructs. Inoculation of transgenic seedlings expressing antisense constructs targeting the negative-strand CEVd RNA molecule with CEVd resulted in a moderate reduction in the accumulation of CEVd RNA. In contrast, similarly inoculated transgenic plants expressing constructs targeting the positive-strand CEVd RNA molecule resulted in an increase in the rate of CEVd RNA accumulation. Addition of the ribozyme motifs to the antisense genes did not enhance their efficiency in the suppression of viroid replication and a moderation or elimination of the observed antisense effects was seen in plants expressing the corresponding catalytic RNA-encoding genes.

The movement and distribution of citrus tristeza virus and citrus exocortis viroid in citrus seedlings1

SUMMARYThe systemic movement of citrus tristeza virus (CTV) in sour orange (Citrus aurantium) seedlings and of citrus exocortis viroid (CEVd) in Etrog citron (C. medica) seedlings was studied. The movement of the two pathogens was analysed by detection in sections of roots and stems at different time intervals. Both pathogens were detected initially in the basal parts and the roots and subsequently spread to the shoot. CTV and CEVd moved in young citrus seedlings at similar rates. The findings are consistent with long distance phloem transport of the virus and the viroid. The practical implications of the pattern of systemic movement for diagnosis of infected trees are discussed.

Genomic Diversity Among Populations of Two Citrus Viroids from Different Graft-Transmissible Dwarfing Complexes in Israel

The nucleotide sequences of citrus bent leaf viroid (CBLVd) (formerly designated CV-Ib) and citrus exocortis viroid (CEVd), both found among the citrus viroid (CVd) populations from five graft-transmissible dwarfing complexes (GTDCs) originating from different source plants and geographical locations in Israel, were determined. The sequence homology varied only slightly among the CBLVd sequence variants, i.e., 0-7 nucleotides (nts); originating from a single GTDC and 2-8 nts between CBLVds that were obtained from different GTDCs. The lowest level of homology between CBLVd variants obtained from citrus was 97.5%. Considerably larger variation (8-15 nucleotide changes) was observed between CBLVd variants derived from citrus and the type strain that was passed through avocado seedlings. The type strain differed from all variants in six positions, located at 38, 62, 138, 179, 264, and 268 nts. The CEVd sequence variants showed considerable heterogeneity. Five variants derived from four GTDCs differed only in 2-9 nts. Five other variants derived from two GTDCs showed 27-50 nucleotide changes compared with the first group of CEVd variants. The largest variation within a single GTDC of up to 41 nucleotide changes was observed between CEVd variants derived from GTDC-G. Using CEVd # 225 as a reference strain, most of the nucleotide changes occurred in the V, LT, and RT domains. Additional changes in the P domain were found only among CEVd isolates derived from GTDC-M and GTDC-G. The sequence homologies to CEVd # 225 ranged from 89-2 (CEVd-G 2 ) to 99-0% (CEVd-NG 1 )

Induced Tolerance to Mal Secco Disease in Etrog Citron and Rangpur Lime by Infection with the Citrus Exocortis Viroid

Etrog citron and Rangpur lime plants were each inoculated with one of four isolates of the citrus exocortis viroid (CEVd) and incubated for 1 yr, during which time the viroid spread systemically. CEVd caused severe leaf curling and short internodes in Etrog citron but not in Rangpur lime. After leaf inoculation with Phoma tracheiphila, typical mal secco disease symptoms developed on leaves of both cultivars, regardless of prior infection by CEVd, but growth of the mycelium from the infected leaves into the branches was greatly affected by CEVd infection. In Etrog citron, P. tracheiphila was isolated from only 9.1% of the branches sampled from CEVd-infected plants, compared with 100% from CEVd-free plants. In Rangpur lime, the incidence of branches containing P. tracheiphila varied in CEVd-infected plants from 16.7 to 50% among CEVd isolates, compared with 70% in the viroid-free controls. This is believed to be the first report of systemic resistance induced in a woody plant by prior inoculation with a viroid

Growth of healthy and viroid-infected tomato cells in vitro

Cell cultures of a tomato hybrid infected with the citrus exocortis viroid (CEVd) display no adverse effects when compared with viroid-free cultures. The growth patterns of the viroid-containing cultures are not only similar to controls but more efficient as judged by longevity and biomass accumulation. Differences in growth characteristics can be accentuated by modification of the incubation temperature and the carbon sources present in the culture media. Supplementing the culture media with arabinose and xylose induced marked differences in growth and revealed the enhanced tolerance of viroid-containing cells to these sugars. Differences in carbohydrate metabolism and turnover are suggested to be responsible for the previously reported differences in the composition and properties of the cell wall.

Gene Diagnosis of Viroids: Comparisons of Return-PAGE and Hybridization Using DIG-labeled DNA and RNA Probes for Practical Diagnosis of Hop Stunt, Citrus Exocortis and Apple Scar Skin Viroids in Their Natural Host Plants.

Return-PAGE was reliable for diagnosis of HSVd in hop, CEVd in citron and ASSVd in apple, but was not reliable for HSVd in grapevine and citron, and ASSVd in pear, because of their low concentration. A disadvantage of return-PAGE was possible appearance of double or triple bands in case of mix-infecting samples such as hop or citron. Although DIG-labeled DNA probe was 2.5 to 25 times more sensitive than return-PAGE, the practical reliability of this method was not so superior to return-PAGE. DIG-labeled RNA probe was the most sensitive of the three methods examined in this experiment, which was 25-125 times and 100-625 times more sensitive than DIG-DNA probe and return-PAGE, respectively. Furthermore, DIG-RNA probe was sensitive enough to detect HSVd in grapevine, a symptomless host of the pathogen, although negative sample was emphasized to be re-examined by the other sensitive method such as cucumber bioassay or RT-PCR. DIG-RNA probe, however, is not sensitive enough to detect HSVd in citrus and ASSVd in pear. Based on the results obtained in this experiment, we proposed appropriate amounts of plant tissues for the successful diagnosis of the three viroids.

Infectivity of Linear Monomeric Transcripts of Citrus Exocortis Viroid: Terminal Sequence Requirements for Processing

Infectivity of linear monomeric viroid RNA transcripts is known to be dependent on the site of cloning of the viroid and the viroid and vector sequences at the termini of the RNA transcripts. We report here the further characterization of cloning site and sequence requirements using 11 different monomeric cDNA constructs of citrus exocortis viroid; these differed from one another by the site of cloning and by nonviroid residues adjacent to the 5′ and 3′ termini of the viroid sequence in the RNA transcript. The infectivity of these transcripts was assayed by inoculation onto tomato seedlings; all infectious transcripts were wild type, indicating accurate processing. The basic requirement for infectivity appeared to be the ability of the RNA transcripts to form a short double-stranded region of viroid and vector sequences at the junction of the two termini. The site of cloning of the viroid RNA used for cloning was not important as cDNA clones prepared at seven sites provided infectious transcripts.

A stable 463 nucleotide variant of citrus exocortis viroid produced by terminal repeats.

An unusual variant of citrus exocortis viroid (CEV) was detected when an inoculum source from Gynura aurantiaca D.C. was used to infect a hybrid tomato (Lycopersicon esculentum Mill. x L. peruvianum). The 92 nucleotide larger variant, CEV D-92, which displayed the characteristic circular and linear viroid structural forms, contained two repeated sequences spanning the V and T2 domains. A dramatic moderation of symptom expression in Gynura accompanied the incorporation of these repeated sequences. A comparison of the sequence and structure of CEV D-92 with coconut cadang-cadang viroid revealed similarities in the regions generating the naturally occurring terminal repeats suggesting a possible preferred site for RNA recombination between viroids.

Genes encoding acidic and basic class III ?-1,3-glucanases are expressed in tomato plants upon viroid infection

beta-1,3-glucanases are hydrolytic enzymes considered to constitute part of the general array of defense genes induced by pathogen infection in higher plants. We have isolated and characterized two complementary DNA clones, corresponding to new beta-1,3-glucanases from tomato plants (Lycopersicon esculentum) which are expressed upon challenge with citrus exocortis viroid. Amino acid sequence comparison revealed that they are most similar to beta-1,3-glucanases from tobacco, particularly to PR-Q', the unique component of the class III beta-1,3-glucanase. The deduced amino acid sequences of the two tomato beta-1,3-glucanases indicate that, although being highly similar in amino acid sequence, they have different isoelectric points: pI 10.5 for the basic isoform (Tom PR-Q'b) and pI 5.2 for the acidic one (Tom PR-Q'a). The expression of these two beta-1,3-glucanase messenger RNAs (mRNAs) in response to viroid infection and ethephon treatments was examined. mRNAs for these two isoforms are coordinately expressed and induced similarly to mRNAs for other PR proteins, indicating that they are part of a general and coordinate mechanism of response of tomato plants susceptible to viroid infection.

Isolates of citrus exocortis viroid recovered by host and tissue selection

Isolates of citrus exocortis viroid (CEV) from a single sweet orange citrus source have been selected by sequential passage through the alternative hosts citron, Gynura aurantiaca, a hybrid tomato Lycopersicon esculentum x L. peruvianum, and from disorganized callus culture of the hybrid tomato. The distinctions in symptom expression, titre and electrophoretic mobility among the CEV isolates, operationally termed CEVc (citron), CEVg (Gynura), CEVt (tomato) and CEVcls (callus) are supported by characteristically different nucleotide sequences. The nucleotide sequence of full-length cDNA clones of CEVc purified from citron shows exchanges not reported for any previously described CEV variant. An unusual number of exchanges have been localized in the terminal domains of all the isolates analysed here. A common pattern of nucleotide exchanges, described as a 'tomato signature', can be detected in all of the isolates derived from hybrid tomato tissues.