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Digoxigenin-labeled RNA probes were used to detect cherry leaf roll virus in infected plants. A dot-blot hybridization immunoenzymatic assay in both crude sap extracts and partially purified tissue with a colorigenic and chemiluminescent detection was developed. The use of the new AMPPD substrate was found to be effective in clarified sap extracts in conditions were the colorigenic detection method failed. Both detection assays were effective when using unfractionated nucleic acid preparations, the chemiluminescent being five times more sensitive than the colorigenic. The chemiluminescent hybridization assay makes it possible to detect the virus at the picogram level. The non-radioactive dot-blot hybridization techniques described here turned out to be very suitable for plant virus diagnosis. The sensitivity of this method and those obtained by ELISA or radioactive dot-blot described previously is compared.

Plant viruses move through plasmodesmata (PD) either as nucleoprotein complexes (NPCs) or as tubule-guided encapsidated particles with the help of movement proteins (MPs). To explore how and why MPs specialize in one mechanism or the other, we tested the exchangeability of MPs encoded by DNA and RNA virus genomes by means of an engineered alfalfa mosaic virus (AMV) system. We show that Caulimoviridae (DNA genome virus) MPs are competent for RNA virus particle transport but are unable to mediate NPC movement, and we discuss this restriction in terms of the evolution of DNA virus MPs as a means of mediating DNA viral genome entry into the RNA-trafficking PD pathway.

The cell-to-cell transport of plant viruses depends on one or more virus-encoded movement proteins (MPs). Some MPs are integral membrane proteins that interact with the membrane of the endoplasmic reticulum, but a detailed understanding of the interaction between MPs and biological membranes has been lacking. The cell-to-cell movement of the Prunus necrotic ringspot virus (PNRSV) is facilitated by a single MP of the 30K superfamily. Here, using a myriad of biochemical and biophysical approaches, we show that the PNRSV MP contains only one hydrophobic region (HR) that interacts with the membrane interface, as opposed to being a transmembrane protein. We also show that a proline residue located in the middle of the HR constrains the structural conformation of this region at the membrane interface, and its replacement precludes virus movement.

Multiplex Polymerase Chain Reaction (PCR) can be used for the simultaneous detection of plant viruses. Multiple primer pairs or polyvalent primer pairs can be used to detect and identify several viruses in a single PCR.

Evident stunting was observed for the first time on Prunus serrulata ‘Kwanzan’ indicator trees in Southern Italy during the indexing of two sour cherry accessions from cultivars ‘Marasca di Verona’ and ‘Spanska’. Bud break and shooting were delayed and the developing leaves remained small. During the third year many Kwanzan plants died, regardless of the indexed cultivar. Electrophoretic analysis showed the presence of dsRNA pattern in extracts of stunted Kwanzan with a similar size to that of viruses of the family Closteroviridae. An identical pattern of more abundant dsRNA bands was obtained from GF305 seedlings grafted with the same sour cherry accessions. Observations by electron microscopy revealed the presence of long flexuous virus particles in both indicators (Kwanzan and GF305), characteristic of closteroviruses. Subsequent cloning work, starting from the dsRNA extracts of cultivar Marasca di Verona grafted on GF305 indicator, yielded 7 different clones, all showing high identity to the Little cherry virus 1 genome. Full sequencing of this virus isolate (ITMAR) was then done resulting in a complete genome composed of 16,936 nt. Primers designed on the obtained sequences for RT-PCR detection confirmed the presence of Little cherry virus 1 in Kwanzan and GF305 trees, inoculated with both sour cherry cultivars. Phylogenetic analysis of the minor coat protein grouped virus isolates into two clusters: one including Italian isolates of sweet cherry, Japanese plum, peach and almond, together with German sweet cherry UW1 isolate, and a second one containing the Italian isolates of sour cherry (ITMAR and ITSPA), that were found associated with strong symptoms of ‘Kwanzan Stunting’.

The route of infection and the pattern of distribution of Prunus necrotic ringspot virus (PNRSV) in apricot pollen were studied. PNRSV was detected both within and on the surface of infected pollen grains. The virus invaded pollen during its early developmental stages, being detected in pollen mother cells. It was distributed uniformly within the cytoplasm of uni- and bicellular pollen grains and infected the generative cell. In mature pollen grains, characterized by their triangular shape, the virus was located mainly at the apertures, suggesting that PNRSV distribution follows the same pattern as the cellular components required for pollen tube germination and cell wall tube synthesis. PNRSV also was localized inside pollen tubes, especially in the growth zone. In vitro experiments demonstrated that infection with PNRSV decreases the germination percentage of pollen grains by more than half and delays the growth of pollen tubes by approximately 24 h. However, although PNRSV infection affected apricot pollen grain performance during germination, the presence of the virus did not completely prevent fertilization, because the infected apricot pollen tubes, once germinated, were able to reach the apricot embryo sacs, which, in the climatic conditions of southeastern Spain, mature later than in other climates. Thus, infected pollen still could play an important role in the vertical transmission of PNRSV in apricot.

Nonisotopic molecular dot blot hybridization technique and multiplex reverse transcription-polymerase chain reaction assay for the specific detection of Lettuce big-vein virus (LBVV) and Mirafiori lettuce virus (MiLV) in lettuce tissue were developed. Both procedures were suitable for the specific detection of both viruses in a range of naturally infected lettuce plants from various Spanish production areas and seven different cultivars. The study of the distribution of both viruses in the plant revealed that the highest concentration of LBVV and MiLV occurred in roots and old leaves, respectively. LBVV infection progress in a lettuce production area was faster than that observed for MiLV. In spite of different rates of virus infection progress, most lettuce plants became infected with both viruses about 100 days posttransplant. The appearance of both viruses in lettuce crops was preceded by a peak in the concentration of resting spores and zoosporangia of the fungus vector Olpidium brassicae in lettuce roots.

The translocation of Melon necrotic spot virus (MNSV) within tissues of inoculated and systemically infected Cucumis melo L. 'Galia' was studied by tissue-printing and in situ hybridization techniques. The results were compatible with the phloem vascular components being used to spread MNSV systemically by the same assimilate transport route that runs from source to sink organs. Virus RNAs were shown to move from the inoculated cotyledon toward the hypocotyl and root system via the external phloem, whereas the upward spread through the stem to the young tissues took place via the internal phloem. Virus infection was absent from non-inoculated source tissues as well as from both shoot and root apical meristems, but active sink tissues such as the young leaves and root system were highly infected. Finally, our results suggest that the MNSV invasion of roots is due to virus replication although a destination-selective process is probably necessary to explain the high levels of virus accumulation in roots. This efficient invasion of the root system is discussed in terms of natural transmission of MNSV by the soil-borne fungal vector.

This chapter covers the economic importance, geographical distribution, symptoms, causal agents, diagnosis, epidemiology and management (such as through the production of virus-free planting materials and quarantine) of the following diseases/pathogens of peach: Sharka or plum pox (caused by Plum pox virus), Ilarviruses, Tomato ringspot virus, Strawberry latent ringspot virus, Peach rosette mosaic virus, peach latent mosaic (induced by Peach latent mosaic viroid), peach dapple (caused by Hop stunt viroid), peach mosaic (caused by Peach mosaic virus or Peach latent mosaic viroid), and diseases of unknown aetiology and experimental infections in peach (Apricot latent virus, Peach sooty ringspot virus, Peach asteroid spot virus and Apple chlorotic leaf spot virus).

Viroids are small, single-stranded, circular, non-coding pathogenic RNAs. Hop stunt viroid (HSVd) is characterized by possesses rod-like structure and replicate in the host nuclei. Green fluorescent protein (GFP) fusions with transit sequences or entire proteins can be used for deliberate labelling of particular cell compartments. Different GFP-fusions have been obtained to selectively illuminate different organelles and membranes in many cell types. However, as far as we know, examples for established efficient markers for nucleoli are scarce. In this work, a viroid-RNA was made translatable by inserting an ATG at position 1 and fused to the GFP. The results showed that the resultant fusion can be used as an efficient "in vivo" nucleolar marker in "real time" cellular observations. Thus, this construct can be a very useful tool to study processes related with nucleolus functions.

Interactions between viral proteins are critical for virus viability. Bimolecular fluorescent complementation (BiFC) technique determines protein interactions in real-time under almost normal physiological conditions. The coat protein (CP) of Prunus necrotic ringspot virus is required for multiple functions in its replication cycle. In this study, the region involved in CP dimerization has been mapped by BiFC in both bacteria and plant tissue. Full-length and C-terminal deleted forms of the CP gene were fused in-frame to the N- and C-terminal fragments of the yellow fluorescent protein. The BiFC analysis showed that a domain located between residues 9 and 27 from the C-end plays a critical role in dimerization. The importance of this C-terminal region in dimer formation and the applicability of the BiFC technique to analyse viral protein interactions are discussed.

In the present work we have studied the accumulation of gentisic acid (2,5-dihydroxybenzoic acid, a metabolic derivative of salicylic acid, SA) in the plant-pathogen systems, Cucumis sativus and Gynura aurantiaca, infected with either prunus necrotic ringspot virus (PNRSV) or the exocortis viroid (CEVd), respectively. Both pathogens produced systemic infections and accumulated large amounts of the intermediary signal molecule gentisic acid as ascertained by electrospray ionization mass spectrometry (ESI-MS) coupled on line with high performance liquid chromatography (HPLC). The compound was found mostly in a conjugated (beta-glucoside) form. Gentisic acid has also been found to accumulate (although at lower levels) in cucumber inoculated with low doses of Pseudomonas syringae pv. tomato, producing a nonnecrotic reaction. In contrast, when cucumber was inoculated with high doses of this pathogen, a hypersensitive reaction occurred, but no gentisic-acid signal was induced. This is consistent with our results supporting the idea that gentisic-acid signaling may be restricted to nonnecrotizing reactions of the host plant (Belles et al. in Mol Plant-Microbe Interact 12:227-235, 1999). In cucumber and Gynura plants, the activity of gentisic acid as inducing signal was different to that of SA, thus confirming the data found for tomato. Exogenously supplied gentisic acid was able to induce peroxidase activity in both Gynura and cucumber plants in a similar way as SA or pathogens. However, gentisic-acid treatments strongly induced polyphenol oxidase activity in cucumber, whereas pathogen infection or SA treatment resulted in a lower induction of this enzyme. Nevertheless, gentisic acid did not induce other defensive proteins which are induced by SA in these plants. This indicates that gentisic acid could act as an additional signal to SA for the activation of plant defenses in cucumber and Gynura plants.

The movement protein (MP) of Prunus necrotic ringspot virus (PNRSV) is required for cell-to-cell movement. MP subcellular localization studies using a GFP fusion protein revealed highly punctate structures between neighboring cells, believed to represent plasmodesmata. Deletion of the RNA-binding domain (RBD) of PNRSV MP abolishes the cell-to-cell movement. A mutational analysis on this RBD was performed in order to identify in vivo the features that govern viral transport. Loss of positive charges prevented the cell-to-cell movement even though all mutants showed a similar accumulation level in protoplasts to those observed with the wild-type (wt) MP. Synthetic peptides representing the mutants and wild-type RBDs were used to study RNA-binding affinities by EMSA assays being approximately 20-fold lower in the mutants. Circular dichroism analyses revealed that the secondary structure of the peptides was not significantly affected by mutations. The involvement of the affinity changes between the viral RNA and the MP in the viral cell-to-cell movement is discussed.

A functional genomics project has been initiated to approach the molecular characterization of the main biological and agronomical traits of citrus. As a key part of this project, a citrus EST collection has been generated from 25 cDNA libraries covering different tissues, developmental stages and stress conditions. The collection includes a total of 22,635 high-quality ESTs, grouped in 11,836 putative unigenes, which represent at least one third of the estimated number of genes in the citrus genome. Functional annotation of unigenes which have Arabidopsis orthologues (68% of all unigenes) revealed gene representation in every major functional category, suggesting that a genome-wide EST collection was obtained. A Citrus clementina Hort. ex Tan. cv. Clemenules genomic library, that will contribute to further characterization of relevant genes, has also been constructed. To initiate the analysis of citrus transcriptome, we have developed a cDNA microarray containing 12,672 probes corresponding to 6875 putative unigenes of the collection. Technical characterization of the microarray showed high intra- and inter-array reproducibility, as well as a good range of sensitivity. We have also validated gene expression data achieved with this microarray through an independent technique such as RNA gel blot analysis.

Viroids are highly structured plant pathogenic RNAs that do not code for any protein, and thus, their long-distance movement within the plant must be mediated by direct interaction with cellular factors, the nature of which is presently unknown. In addition to this type of RNAs, recent evidence indicates that endogenous RNAs move through the phloem acting as macromolecular signals involved in plant defense and development. The form in which these RNA molecules are transported to distal parts of the plant is unclear. Viroids can be a good model system to try to identify translocatable proteins that could assist the vascular movement of RNA molecules. Here, we demonstrate by use of immunoprecipitation experiments, that the phloem protein 2 from cucumber (CsPP2) is able to interact in vivo with a viroid RNA. Intergeneric graft assays revealed that both the CsPP2 and the Hop stunt viroid RNA were translocated to the scion. The translocated viroid is symptomatic in the nonhost scion, indicating that the translocated RNA is functional. The CsPP2 gene was cloned and sequenced. The analysis of its primary structure revealed the existence of a potential double-spaced-RNA-binding motif, previously identified in a set of proteins that bind to highly structured RNAs, which could explain its RNA-binding properties. The possible involvement of this phloem protein in assisting the long-distance movement of the viroid RNA within the plant is discussed.

Available data suggests that lettuce big-vein disease is caused by the ophiovirus Mirafiori lettuce big-vein virus (MLBVV) but not by the varicosavirus Lettuce big-vein-associated virus (LBVaV), although the latter is frequently associated with the disease. Since the disease occurs worldwide, the putative coat protein (CP) open reading frames of geographically distinct isolates of MLBVV and LBVaV were sequenced. Comparison of both nucleotide and amino acid sequences showed a high level of sequence similarity among LBVaV isolates. Phylogenetic analysis of LBVaV CP nucleotide sequences showed that most of the Spanish isolates clustered in a phylogenetic group whereas English isolates were more similar to the USA isolate. An Australian isolate was closely related to the Dutch isolate. Genetic diversity among MLBVV CP nucleotide sequences was higher ranging from 0.2% to 12%. Phylogenetic analysis of MLBVV CP nucleotide sequences revealed two distinct subgroups. However, this grouping was not correlated with symptom development on lettuce or the geographic origin of the MLBVV isolates. Finally, a quick method based on RFLP analysis of RT-PCR amplicons was developed for assigning MLBVV isolates to the two subgroups.

A new strategy for the simultaneous detection of plant viruses by molecular hybridization has been developed. Two, four or six viral sequences were fused in tandem and transcribed to render unique riboprobes and designated as 'polyprobes'. The 'polyprobe four' (poly 4) covered the four ilarviruses affecting stone fruit trees including apple mosaic virus (ApMV), prunus necrotic ringspot virus (PNRSV), prune dwarf virus (PDV), and American plum line pattern virus (APLPV) whereas the 'polyprobe two' (poly 2) was designed to detect simultaneously, plum pox virus (PPV) and apple chlorotic leaf spot virus (ACLSV), the two more important viruses affecting these trees. Finally, a 'polyprobe six' (poly 6) was generated to detect any of the six viruses. The three polyprobes were comparable to the individual riboprobes in terms of end-point dilution limit and specificity. The validation of the new simultaneous detection strategy was confirmed by the analysis of 46 field samples from up to seven different hosts collected from 10 different geographical areas.

Melon necrotic spot virus (MNSV) is a water and soil-borne pathogen affecting species of the Cucurbitaceae family both in hydroponic and soil crops. Molecular methods for detecting MNSV in water samples, nutrient solutions and melon plants were developed. For this purpose, water samples from a water source pool of a hydroponic culture or from the recirculating nutrient solution were concentrated by ultracentrifugation or PEG precipitation followed by RT-PCR analysis. Both concentration methods were suitable to allow the detection of MNSV and represent, as far as we know, the first time that this virus has been detected in water samples. A non-isotopic riboprobe specific for MNSV was obtained and used to detect the virus in plant tissue. Different parts of mechanically infected plants were examined including the roots, stems, inoculated cotyledons and young leaves. Excluding the inoculated cotyledons, the tissues showing the highest accumulation levels of the virus were the roots. The potential inclusion of such tools in management programs is discussed.

Summary Measurements of the root hydraulic conductance (L0) of roots of Arabidopsis thaliana were carried out and the results were compared with the expression of aquaporins present in the plasma membrane of A. thaliana. L0 of plants treated with different NaCl concentrations was progressively reduced as NaCl concentration was increased compared to control plants. Also, L0 of plants treated with 60 mmol/L NaCl for different lengths of time was measured. Variations during the light period were seen, but only for the controls. A good correlation between mRNA expression and L0 was observed in both experiments. Control plants and plants treated with 60 mmol/L NaCl were incubated with Hg and then with DTT. For these plants, L0 and cell-to-cell pathway contributions to root water transport were determined. These results revealed that in control plants most water movement occurs via the cell-to-cell pathway, thus implying aquaporin involvement. But, in NaCl-stressed plants, the Hg-sensitive cell-to-cell pathway could be inhibited already by the effect of NaCl on water channels. Therefore, short periods of NaCl application to Arabidopsis plants are characterised by decreases in the L0 of roots, and are related to down-regulation of the expression of the PIP aquaporins. This finding indicates that the well known effect of salinity on L0 could involve regulation of aquaporin expression.

Time-course and in situ hybridization analyses were used to study the spatio-temporal distribution of Carnation mottle virus (CarMV) in Chenopodium quinoa plants. Genomic and subgenomic RNAs of plus polarity accumulated linearly with time, whereas the corresponding minus strands reached a peak during infection in inoculated leaves. Analyses of serial tissue sections showed that plus polarity strands were localized throughout the infection area, whereas minus strands were localized at the borders of the chlorotic lesions. The accumulation kinetics of the coat protein (CP) and the p7 movement protein (MP) as well as their subcellular localization were also studied. Unlike most MPs, CarMV p7 showed a non-transient expression and a mainly cytosolic location. However, as infection progressed the presence of p7 in the cell wall fraction increased significantly. These results are discussed on the basis of a recent model proposed for the mechanism of cell-to-cell movement operating in the genus Carmovirus.

The nucleotide sequences of the RNA 3 of fifteen isolates of Prunus necrotic ringspot virus (PNRSV) varying in the symptomatology they cause in six different Prunus spp. were determined. Analysis of the molecular variability has allowed, in addition to study the phylogenetic relationships among them, to evaluate the minimal requirements for the synthesis of the subgenomic RNA in Ilarvirus genus and their comparison to other members of the Bromoviridae family. Computer assisted comparisons led recently to Jaspars (Virus Genes 17, 233-242, 1998) to propose that a hairpin structure in viral minus strand RNA is required for subgenomic promoter activity of viruses from at least two, and possibly all five, genera in the family of Bromoviridae. For PNRSV and Apple mosaic virus two stable hairpins were proposed whereas for the rest of Ilarviruses and the other four genera of the Bromoviridae family only one stable hairpin was predicted. Comparative analysis of this region among the fifteen PNRSV isolates characterized in this study revealed that two of them showed a 12-nt deletion that led to the disappearance of the most proximal hairpin to the initiation site. Interestingly, the only hairpin found in these two isolates is very similar in primary and secondary structure to the one previously shown in Brome mosaic virus to be required for the synthesis of the subgenomic RNA. In this hairpin, the molecular diversity was concentrated mostly at the loop whereas compensatory mutations were observed at the base of the stem strongly suggesting its functional relevance. The evolutionary implications of these observations are discussed.

The complete nucleotide sequence of a Spanish isolate of Carnation mottle carmovirus (CarMV) has been determined. Phylogenetic analyses were carried out with the replicase, coat protein (CP) and the putative movement proteins (p7 and p9) of CarMV with the homologous proteins of representative members of the different genera included within the family Tombusviridae. These analyses revealed that phylogenetic trees obtained depended on the protein analyzed, and that the best correlation with taxonomy grouping was observed with the replicase and, to a lesser extent, with CP phylogenies. This result indicates that speciation has evolved as a consequence of different selection pressures to different genomic regions. In addition, the CP, p7 and p9 coding sequences of twenty-one CarMV isolates from nine different countries have been determined. Comparative analyses revealed that CarMV isolates separated in time and space show a very high genetic stability. A division in three protein motifs is proposed for the p7 movement protein, based on the homology data presented here and on our previous identification of RNA binding sequences and structural characterization of the protein. Interestingly, a remarkable covariation in the amino acid sequence was found for the CP between Pro164 (located at the S domain) and Lys331 (within the P domain), by which a change Pro164 → Ala correlated with a change Lys331 → Asn, strongly suggesting the existence of tertiary interactions between these two regions of the protein. In addition, this perfect covariation allows to segregate the 23 CarMV isolates characterised so far into two main groups that we propose to name as group PK and group AN for further studies.

Pelargonium flower break virus (PFBV) is one of the common viruses in the glasshouses of Western Europe and has been assigned to the genus Carmovirus. A Spanish isolate obtained from nursery-grown Pelargonium zonale plants (PFBV-m) has been characterized. The molecular weight of genomic RNA and coat protein of PFBV-m were determined to be 1.36 x 10(6) (corresponding to approximately 4 kb) and 36,000, respectively. Only genomic-size RNA was encapsidated in PFBV virions; making necessary to purify double-stranded RNA from infected tissue in order to detect putative PFBV subgenomic RNAs. PFBV RNA directed the synthesis of a major polypeptide of 34 kDa and three other relevant polypeptides of estimated sizes 88-90 kDa, 42 kDa and 35-36 kDa. Antisera specific to PFBV immunoprecipitated the in vitro translated 35-36 kDa polypeptide indicating that this polypeptide is the PFBV coat protein. The PFBV in vitro translation pattern was very similar to that of CarMV although the relative levels of translated coat protein differed dramatically between the two viruses, most probably due to the lack of encapsidation of subgenomic PFBV. In vitro translation studies with a different biological clone obtained from the same PFBV-m isolate revealed a prominent additional polypeptide which is postulated to be a truncation of the 5' proximal ORF.

The RNA binding properties of the prunus necrotic ringspot virus (PNRSV) coat protein (CP) were demonstrated by northwestern and dot-blot analyses. The capability to bind PNRSV RNA 4 was compared with viruses representing three different interactions prevailing in the assembly and architecture of virions. The results showed that cucumber mosaic virus (CMV) and PNRSV CPs, which stabilise their virions mainly through RNA-protein interactions bound PNRSV RNA 4 even at very high salt concentrations. The CP of cherry leaf roll nepovirus, whose virions are predominantly stabilised by protein-protein interactions did not bind even at the lowest salt concentration tested. Finally the CP of carnation mottle carmovirus, that has an intermediate position in which both RNA-protein and protein-protein interactions are equally important showed a salt-dependent RNA binding.

Biochemical and structural characterization studies on the p7 putative movement protein from a Spanish isolate of carnation mottle carmovirus (CarMV) have been conducted. The CarMV p7 gene was fused to a sequence coding for a six-histidine tag and expressed in bacteria, allowing the purification of CarMV p7 and the production of a specific antiserum. This antiserum led to the immunological identification of CarMV p7 in infected leaf tissue from the experimental host Chenopodium quinoa. Putative nucleic acid-binding properties of the CarMV p7 have been explored and demonstrated with both electrophoretic mobility shift and RNA-protein blot in vitro assays using digoxigenin-labeled riboprobes. CarMV p7 did not show preferential binding to any of the different regions of the CarMV genomic RNA tested, suggesting that RNA binding was sequence nonspecific. Quantitative analyses of the data allowed calculation of the apparent dissociation constant of the p7-RNA complex (Kd approximately 0.7 microM) and supported a cooperative type of binding. A small 19-amino-acid synthetic peptide whose sequence corresponds to the putative RNA-binding domain of CarMV p7, at the basic central part of the protein, was synthesized, and it was demonstrated that it binds viral RNA probes. Peptide RNA binding was as stable as p7 binding, although data indicated it was not cooperative, thus suggesting that this cooperative binding requires another motif or motifs within the p7 amino acid sequence. The peptide could be induced to fold into an alpha-helix structure in which amino acids that are conserved among carmovirus p7-like proteins are distributed on one side. This alpha-helix motif could define a new and previously uncharacterized RNA-binding domain for plant virus movement proteins.

One of the key issues regarding the biosafety of transgenic plants expressing plant virus genetic information concerns the potential for recombination between transgene-derived sequences and an incoming, more or less related virus. With no precise information currently available on the rate of appearance and potential fitness of such recombinants, the hypothesis that all viable possible recombinants have already been produced and selected in nature in the course of natural multiple infections has been the subject of a debate. One way to further our understanding of these mechanisms is to perform systematic searches, in order to identify natural viral isolates deriving from a recombination event and, therefore, make a first evaluation of the frequency of recombinant isolates in natural viral populations.

The long-distance movement of cherry leaf roll virus (CLRV) in tobacco plants was studied using a tissue printing technique with non-isotopic RNA probes. Time-course analysis revealed that CLRV RNA accumulated in the inoculated leaf at an early stage, such as 20 h post-inoculation. The virus accumulation reached a peak at 8–10 days post-inoculation (d.p.i.) and then progressively decreased. The virus RNA signal was detected before the appearance of symptoms. The virus invaded stem vascular tissues at 3 d.p.i., moving towards the roots before moving to the upper leaves. In systemically infected leaves, the virus appeared first in the basal regions and then moved to the distal parts through the vascular system. The distribution pattern of the virus coat protein in systemically infected leaves was parallel to that observed for the virus RNA, suggesting that CLRV requires the coat protein for long-distance movement. The movement of the virus was influenced by the phyllotactic position of the leaves. The viral symptoms and the virus RNA signal in older systemically infected leaves were asymmetrically distributed, being localized in the side of the lamina closest to the inoculated leaf. Virus distribution in infected plants as well as the susceptibility of the plant to systemic infection were also influenced by the developmental stage of the inoculated leaves. Inoculation of leaves at 95% of their final size resulted in virus replication but no systemic infection. In fully mature leaves the virus did not replicate.

A non-isotopic tissue-print hybridization technique was developed to study long-distance plant virus movement. By using digoxigenin-labeled RNA probes the distribution pattern of the viral RNA was observed in leaf, stem and petiole tissues. In leaf tissue viral RNA was confined preferentially to symptoms and veins, and in stem and petiole sections, the hybridization signal was observed in vascular tissues. Both chemiluminescent and colorigenic detection methods were used. The colorigenic method, though less sensitive, is advantageous in that it gives some anatomical information on the signal distribution. This non-isotopic tissue-print hybridization technique can provide considerable information about the spatial and temporal virus expression with regard to its symptoms.

Virus life cycle heavily depends on their ability to command the host machinery in order to translate their genomes. Animal viruses have been shown to interfere with host translation machinery by expressing viral proteins that either maintain or inhibit eIF2a function by phosphorylation. However, this interference mechanism has not been described for any plant virus yet. Prunnus necrotic ringspot virus (PNRSV) is a serious pathogen of cultivated stone fruit trees. The movement protein (MP) of PNRSV is necessary for the cell-to-cell movement of the virus. By using a yeast-based approach we have found that overexpression of the PNRSV MP caused a severe growth defect in yeast cells. cDNA microarrays analysis carried out to characterise at the molecular level the growth interference phenotype reported the induction of genes related to amino acid deprivation suggesting that expression of MP activates the GCN pathway in yeast cells. Accordingly, PNRSV MP triggered activation of the Gcn2p kinase, as judged by increased eIF2a phosphorylation. Activation of Gcn2p by MP expression required a functional Tor1p kinase, since rapamycin treatment alleviated the yeast cell growth defect and blocked eIF2a phosphorylation triggered by MP expression. Overall, these findings uncover a previously uncharacterised function for PNRSV MP viral protein, and point out at Tor1p and Gcn2p kinases as candidate susceptibility factors for plant viral infections., This work was supported by grants GV/2007/064 (Proyecto para grupos de investigacio´n emergentes) and (GV/2011/003) (Prometeo Project) from Conselleria d’Empresa, Universitat i Ciencia (Generalitat Valenciana). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Representing 2% of world production, 20,000 ha of apricot (Prunus armeniaca L.), are cultivated in Spain, primarily in the southeast. A survey was conducted during the spring of 2008 in orchards in the region of Murcia to assess the incidence of several stone fruit viruses. Leaf and fruit samples from 160 trees from 40 orchards were collected randomly for reverse transcription (RT)-PCR analysis. Total RNA extracted (3) from leaves and fruits was tested by a multiplex one-step RT-PCR protocol with a mix of primers that detect eight distinct viruses (4). Amplicons of 250 bp expected for Plum bark necrosis stem pitting-associated virus (PBNSPaV), corresponding to part of the heat shock 70 protein gene, were obtained from four trees and amplicons of 700 bp expected for Apricot latent virus (ApLV), corresponding to part of the coat protein (CP) gene, were obtained from two trees. In all cases, amplicons were obtained using RNA extracted from leaf and fruit tissues. RT-PCR results were confirmed by uniplex RT-PCR with primers specific for each virus and dot-blot hybridization with virus-specific digoxygenin-labeled RNA probes (2). To further characterize the new viruses, we designed primers to amplify specifically the CP gene of ApLV (5′-CCCGACCATGGCTACAAGC-3′ and 5′-TTGCCGTCCCGATTAGGTTG-3′) and the minor CP gene of PBNSPaV (5′-GAACAAACTACAGCAGCACC-3′ and 5′-CAAGGGTAGGACGGGTAACGC-3′). Amplicons of 1,500 and 950 bp corresponding to the ApLV and PBNSPaV CP genes, respectively, were purified from agarose gels and cloned in the pTZ57R plasmid (Fermentas, Burlington, Ontario, Canada). Blastp analysis of the full-length ApLV CP sequence from one infected tree (GenBank Accession No. GQ919051) revealed 86% amino acid (aa) similarity to the single full-length ApLV CP sequence available (No. AAC16234) and 79 and 66.9% similarity to Peach sooty ringspot virus (No. AAG48314) and Apple stem pitting virus (No. NP604468), respectively. Identity/similarity analysis of the full-length PBNSPaV minor CP genes using the Matrix Global Alignment Tool software, version 2.02 (1), revealed 98.8 to 99.6% aa similarity between the Spanish PBNSPaV isolates (Nos. GQ919047, GQ919048, GQ919049, and GQ919050) and 97.1 to 97.4% with the PBNSPaV isolate from the United States (No. EF546442). None of the six infected trees were associated with any particular field symptoms. Five infected trees were cv. Búlida and one was native cv. Murciana, which was infected with ApLV. All infected trees were located in geographically separated orchards. The incidence of ApLV and PBNSPaV was 1.25 and 2.5%, respectively. The low incidence of both viruses together with the scattered geographic distribution could be due to the recent introduction of virus-contaminated plants, although we cannot exclude that it is a consequence of a low dissemination rate. Even though no symptoms were observed, we cannot discard that the infection could affect fruit production or flowering or even cause a synergistic effect in mixed infection with other stone fruit viruses, a risk especially relevant considering the total area of cultivated apricot. To our knowledge, this is the first report of ApLV and PBNSPaV in Spain. References: (1) J. J. Campanella et al. BMC Bioinformatics 4:29, 2003. (2) M. C. Herranz et al. J. Virol. Methods 124:49, 2005. (3) D. J. Mackenzie et al. Plant Dis. 81:222, 1997. (4) J. A. Sánchez-Navarro et al. Eur. J. Plant Pathol. 111:77, 2005.

The State of Michoacán, México cultivates approximately 100,000 ha of avocados (Persea americana M.) (4). During a survey from 2006 to 2007 in cv. Hass avocado groves in Tingambato County, in the State of Michoacán, deep yellow spots and streaks, which sometimes became necrotic or reddish, were observed on the skin of fruits and the pulp of the fruit also showed big yellow spots. Some young shoots developed fine, yellow streaks, and leaves of symptomatic trees sometimes showed irregular, white-to-yellow spots. These symptoms were similar to those recorded for Avocado sunblotch viroid (ASBVd) (3). To determine if ABSVd was associated with these symptoms, total RNA extracted (1) from the skin and pulp of symptomatic and asymptomatic fruits and also from leaves and bark of shoots from five trees collected in a commercial plot in Tingambato County was tested by a one-step reverse transcription (RT)-PCR protocol using one primer pair to amplify specifically the complete ASBVd genome sequence (3). All 30 samples of skin and pulp of fruits, leaves, and cortex of shoots from symptomatic trees yielded two PCR fragments with estimated sizes of 250 and 500 base pairs (bp) corresponding to the putative monomeric and dimeric forms of ASBVd, respectively. The 500-bp RT-PCR fragments obtained from the different samples were purified from an agarose gel and cloned. The 249-bp nucleotide sequence of the ASBVd genomic monomer was determined using the clones from the fruit skin from sample Arb No. 3 (GenBank Accession No. EU888588), pulp from sample Arb No. 5 (GenBank Accession No. EU888590), leaves from samples Arb No. 15 (GenBank Accession No. EU888589) and Arb No. 8 (GenBank Accession Nos. EU888591 and EU888592), and cortex of shoots from sample Arb No. 16 (GenBank Accession Nos. EU888593, EU888594, EU888595, EU888596, and EU888597). BLAST analysis of the ASBVd sequences showed a range of 98 to 100% nucleotide identity to ASBVd sequences (GenBank Accession Nos. AF404064, AF404051, or AF229821). A clone of the Michoacán ASBVd (GenBank Accession No. EU888593) was used to synthesize a Dig-High Prime-UTP-T7 (Roche, Mannheim, Germany) fluorescent riboprobe complementary to the ASBVd plus strand to perform a dot-blot analysis as described previously (2). All ASBVd samples positive by RT-PCR gave a strong signal in the dot-blot analysis. This riboprobe will be used to index the ASBVd in other commercial avocado groves in Michoacán. To our knowledge, this is the first report of ASBVd in Michoacán, México. References: (1) D. J. Mackenzie et al. Plant Dis. 81:222, 1997. (2) J. A. Sánchez-Navarro et al. Plant Pathol. 47:780, 1998. (3) R. J. Schnell et al. Plant Dis. 81:1023, 1997. (4) D. Téliz and A. Mora. El aguacate y su Manejo Integrado. Mundiprensa, Mexico City, 2007.

“Tissue-printing” hybridization (3) for Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd) was used to assess the sanitary status of stone fruit accessions in the Canadian Clonal Genebank (CCG) located in Harrow (Ontario). The Prunus spp. accessions in the CCG are primarily of Canadian origin; other countries of origin include the United States, the United Kingdom, Hungary, the Czech Republic, the Former Soviet Union, Spain, New Zealand, and Italy. All Prunus spp. accessions were donated to the Genebank from Canadian or American sources. Leaves were harvested in November 2003 from 336 trees (116 peach and nectarine, 84 sweet and sour cherries, 54 plum, 44 apricot, and 38 of other cherries) representing 267 accessions. No visible symptoms were observed during the collection of the accessions to be evaluated. The petioles were excised at the base and imprinted on a nylon membrane in triplicate for each sample. The membranes were air dried and submitted by mail to the laboratory. The digoxigenin-labeled riboprobes used for hybridization were obtained by T7 RNA polymerase transcription of the linearized plasmids pHSVd (1) and pPLMVd (2). Thirty stone fruit samples were infected by viroids. PLMVd occurred in 28 peach and nectarine samples, representing the following cultivars and selections: Harblaze Hardired, Harko, Earlyvee, Harbelle, Harken, Harland, Harrow Beauty, Harrow Rubirose, HW264, Redhaven, Silver Gold, Suncling, V68101, Vanity, Veeglo, Velvet, Vesper, Villa Doria, and Vulcan. PLMVd-infected samples represented 24.1% of the tested peaches and nectarines. PLMVd finding confirms previous reports of the viroid in Canada from British Columbia and Ontario. Two CCG apricot accessions, ‘Bulida’ and ‘Velkopavlovicka’, were found to be infected with only HSVd, representing 4.5% of tested apricot samples. These samples, determined to be positive by tissue-printing hybridization, were also positive by reverse transcription-polymerase chain reaction (RT-PCR) (1). In addition, nucleotide sequences of the PCR products were obtained. The ‘Bulida’ isolate showed 100% homology to a Spanish isolate, apr9, while the ‘Velkopavlovicka’ isolate showed 99% homology to an Italian isolate. Since HSVd has not been previously reported in Canada (4), to our knowledge, this report documents its first detection in the country. This report may prompt the inclusion of regular testing for HSVd in existing Prunus spp. virus testing programs in Canada. References: (1) N. Astruc et al. Eur. J. Plant Pathol. 102:837, 1996. (2) M. Badenes et al. Acta Hortic. 472:565, 2001. (3) V. Pallás et al. Page 135 in: Virus and Virus-Like Diseases of Stone Fruits, with Particular Reference to the Mediterranean Region. A. Myrta et al., eds. CIHEAM-IAMB, 2003. (4) R. Singh et al. Page 255 in: Viroids. A. Hadidi et al., eds. CSIRO Publishing, Australia, 2003.

Peach latent mosaic viroid (PLMVd) (2) is widely distributed and causes yellow, chlorotic mosaics and delayed foliation, flowering, and ripening. Infected fruits display a cracked suture and are often dented, misshapen, frequently flattened, and discolored. In the greenhouse, PLMVd natural isolates are divided into severe or latent strains depending on whether they induce leaf symptoms on seedlings of the peach indicator GF-305. PLMVd was detected in 2001 during a survey in three locations in the Canelones Department, the main peach producing area in Uruguay. Fifty samples were tested for the presence of five viruses: Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), American plum line pattern virus (APLPV), Plum pox virus (PPV) and Apple chlorotic leaf spot virus (ACLSV); samples were also tested for the viroids affecting stone fruits, Hop stunt viroid (HSVd) and PLMVd. The analyses were completed with molecular hybridization using specific nonisotopic riboprobes for each virus (4). PLMVd, undescribed in Uruguay, was detected in 9 of 50 samples in three peach cultivars, Scarlet Pearl, EarliGrande, and Barcelo. The PLMVd-positive sample for ‘Scarlet Pearl’ showed mild mosaic symptoms on leaves whereas the two PLMVd-positives of ‘EarliGrande’ showed clear calico type symptoms. The remaining PLMVd-positive samples belonged to ‘Barcelo’ and showed no symptoms or mild chlorosis. The first two cultivars were imported from the United States, a source with a high percentage of PLMVd infections in peach germ plasm (1). In five of nine PLMVd-positive samples, the viroid occurred with PNRSV and in one with PDV and PNRSV. PLMVd has previously been reported in Brazil (3), but to our knowledge, this is the first report of PLMVd in Uruguay. These results reveal the importance of following strict sanitary practices with plant material used for propagation. Molecular tools are available to prescreen scion and rootstock sources for PLMVd. References: (1) M. L. Badenes and G. Llácer. Acta Hortic. 309:565, 1998. (2) R. Flores et al. Res. Virol. 141:109, 1990. (3) A. Hadidi et al. Plant Dis. 81:154, 1997. (4) V. Pallás et al. Detection of plant RNA viruses by non-isotopic dot-blot hybridization. Pages 461–468 in: Plant Virus Protocols: From Virus Isolation to Transgenic Resistance. G. Foster and S. Taylor, eds. Humana Press, Totowa, NJ. 1998.

SUMMARY A viroid-like RNA was detected in nucleic acid preparations from two of the three commercial hop varieties grown in Spain. It had a size very close to that of avocado sunblotch viroid (ASBV), although dot-blot analysis revealed that it was very different in base sequence from ASBV, coconut cadang-cadang viroid, hop stunt viroid (HSV) and citrus exocortis viroid. In its physical and biological properties, the viroid-like RNA differed from the previously characterized HSV.

SUMMARY Analysis by polyacrylamide gel electrophoresis of nucleic acid preparations, obtained from several varieties of grapevine by a procedure designed to isolate and purify viroids, revealed the presence of RNA species with some of the characteristic physical properties of viroids. Under non-denaturing conditions, a band with a mobility faster than that of citrus exocortis viroid (CEV) was detected, and under fully denaturing conditions two bands were observed, one co-migrating with the circular forms of CEV and a second migrating faster than the linear forms of this viroid. This RNA species did not hybridize with a cDNA probe to CEV. Some of the grapevine preparations were infective for Gynura aurantiaca, inducing symptoms similar to those caused by CEV, and the appearance of an RNA which had the same mobility as CEV in denaturing and non-denaturing electrophoretic systems and hybridized with cDNA to CEV. These results suggest that viroid-like and viroid RNAs can be recovered from grapevine, the former (with no detectable sequence homology to CEV) at a concentration sufficient to be observed as a physical entity in gels, and the latter (with close sequence homology to CEV) whose presence could only be revealed by bioassay. The possible involvement of these RNAs in some grapevine diseases of unknown aetiology is discussed.

Plants of Gynura aurantiaca and tomato (Lycopersicon esculentum) were inoculated with severe strains of citrus exocortis viroid (CEV) and potato spindle tuber viroid (PSTV). The progeny of both viroids was analyzed by two systems of PAGE which allowed discrimination between CEV and PSTV on the basis of their different sizes. When inoculated separately to G. aurantiaca CEV and PSTV induced severe and very mild symptoms, respectively, with CEV accumulating to a higher level than PSTV, whereas when tomato was the host, each viroid induced severe symptoms (although the reaction caused by PSTV was more intense) with PSTV reaching a higher steady-state concentration than CEV. The simultaneous inoculation of both viroids induced in G. aurantiaca the characteristic symptomatology of CEV, which was the only viroid that could be detected, whereas the response observed in tomato was that typical of PSTV, and only this viroid could be recovered. On the other hand, when G. aurantiaca plants were first inoculated with PSTV and later challenged with CEV, part of the plants displayed the characteristic severe reaction of CEV and both viroids were present in the corresponding extracts, but the other part showed the mild symptomatology induced by PSTV, and only this viroid could be isolated. However, when the protected plants were topped the new shoots exhibited severe symptoms and only CEV could be detected. These results suggest that CEV and PSTV compete for a limiting host factor needed for their replication, transport or accumulation, with CEV and PSTV having the highest affinity for the G. aurantiaca and tomato factors, respectively.

Binding of coat protein (CP) to the 3′ nontranslated region (3′-NTR) of viral RNAs is a crucial requirement to establish the infection of Alfamo- and Ilarviruses. In vitro binding properties of the Prunus necrotic ringspot ilarvirus (PNRSV) CP to the 3′-NTR of its genomic RNA using purified E. coli- expressed CP and different synthetic peptides corresponding to a 26-residue sequence near the N-terminus were investigated by electrophoretic mobility shift assays. PNRSV CP bound to, at least, three different sites existing on the 3′-NTR. Moreover, the N-terminal region between amino acid residues 25 to 50 of the protein could function as an independent RNA-binding domain. Single exchange of some arginine residues by alanine eliminated the RNA-interaction capacity of the synthetic peptides, consistent with a crucial role for Arg residues common to many RNA-binding proteins possessing Arg-rich domains. Circular dichroism spectroscopy revealed that the RNA conformation is altered when amino-terminal CP peptides bind to the viral RNA. Finally, mutational analysis of the 3′-NTR suggested the presence of a pseudoknotted structure at this region on the PNRSV RNA that, when stabilized by the presence of Mg2+, lost its capability to bind the coat protein. The existence of two mutually exclusive conformations for the 3′-NTR of PNRSV strongly suggests a similar regulatory mechanism at the 3′-NTR level in Alfamo- and Ilarvirus genera.

The p7A of Melon necrotic spot virus has been described to be a RNA-binding movement protein essential for cell-to-cell movement but its role in this process is still unknown. Here, we found that primary and secondary structure elements on p7A appear to form a composite RNA-binding site required for both RNA interaction and cell-to-cell movement in plants indicating a direct correlation between these activities. Furthermore, we found that fluorescent-tagged p7A was distributed in punctuate structures at the cell periphery but also in motile cytoplasmic inclusion bodies which were in close association with the actin MFs and most likely generated by self-interacting p7A molecules as shown by BiFC assays. Consistently, the p7A subcellular distribution was revealed to be sensitive to the actin inhibitor, latrunculin B. The involvement of the RNA-binding capabilities and the subcellular location of the p7A in the intracellular and intercellular virus movement is discussed.

Advances in structural and biochemical properties of carmovirus movement proteins (MPs) have only been obtained in p7 and p9 from Carnation mottle virus (CarMV). Alignment of carmovirus MPs revealed a low conservation of amino acid identity but interestingly, similarity was elevated in regions associated with the functional secondary structure elements reported for CarMV which were conserved in all studied proteins. Nevertheless, some differential features in relation with CarMV MPs were identified in those from Melon necrotic virus (MNSV) (p7A and p7B). p7A was a soluble non-sequence specific RNA-binding protein, but unlike CarMV p7, its central region alone could not account for the RNA-binding properties of the entire protein. In fact, a 22-amino acid synthetic peptide whose sequence corresponds to this central region rendered an apparent dissociation constant (Kd) significantly higher than that of the corresponding entire protein (9 mM vs. 0.83–25.7 μM). This p7A-derived peptide could be induced to fold into an alpha-helical structure as demonstrated for other carmovirus p7-like proteins. Additionally, in vitro fractionation of p7B transcription/translation mixtures in the presence of ER-derived microsomal membranes strongly suggested that p7B is an integral membrane protein. Both characteristics of these two small MPs forming the double gene block (DGB) of MNSV are discussed in the context of the intra- and intercellular movement of carmovirus.