Citrus tristeza virus (CTV) (genus Closterovirus, family Closteroviridae) is the causal agent of devastating epidemics that changed the course of the citrus industry. Adapted to replicate in phloem cells of a few species within the family Rutaceae and to transmission by a few aphid species, CTV and citrus probably coevolved for centuries at the site of origin of citrus plants. CTV dispersal to other regions and its interaction with new scion varieties and rootstock combinations resulted in three distinct syndromes named tristeza, stem pitting and seedling yellows. The first, inciting decline of varieties propagated on sour orange, has forced the rebuilding of many citrus industries using tristeza-tolerant rootstocks. The second, inducing stunting, stem pitting and low bearing of some varieties, causes economic losses in an increasing number of countries. The third is usually observed by biological indexing, but rarely in the field. CTV polar virions are composed of two capsid proteins and a single-stranded, positive-sense genomic RNA (gRNA) of approximately 20 kb, containing 12 open reading frames (ORFs) and two untranslated regions (UTRs). ORFs 1a and 1b, encoding proteins of the replicase complex, are directly translated from the gRNA, and together with the 5' and 3'UTRs are the only regions required for RNA replication. The remaining ORFs, expressed via 3'-coterminal subgenomic RNAs, encode proteins required for virion assembly and movement (p6, p65, p61, p27 and p25), asymmetrical accumulation of positive and negative strands during RNA replication (p23), or suppression of post-transcriptional gene silencing (p25, p20 and p23), with the role of proteins p33, p18 and p13 as yet unknown. Analysis of genetic variation in CTV isolates revealed (1) conservation of genomes in distant geographical regions, with a limited repertoire of genotypes, (2) uneven distribution of variation along the gRNA, (3) frequent recombination events and (4) different selection pressures shaping CTV populations. Measures to control CTV damage include quarantine and budwood certification programmes, elimination of infected trees, use of tristeza-tolerant rootstocks, or cross protection with mild isolates, depending on CTV incidence and on the virus strains and host varieties predominant in each region. Incorporating resistance genes into commercial varieties by conventional breeding is presently unfeasible, whereas incorporation of pathogen-derived resistance by plant transformation has yielded variable results, indicating that the CTV-citrus interaction may be more specific and complex than initially thought. A deep understanding of the interactions between viral proteins and host and vector factors will be necessary to develop reliable and sound control measures.

The peach latent mosaic viroid (PLMVd) is used to study the interactions between a viroid containing hammerhead ribozymes and its natural host, peach. To gain insight into the molecular basis of the phenotypic effects observed upon viroid infection, sequence variants from three PLMVd isolates that differ in symptom expression on the peach indicator GF-305 have been characterized. Analysis of the primary structures of a total of 29 different sequence variants derived from a severe and two latent isolates has revealed a large number of polymorphic positions in the viroid molecule. The variability pattern indicates that preservation of the stability of both hammerhead structures and conservation of a branched secondary structure of the viroid molecule may be factors limiting sequence heterogeneity in PLMVd. Moreover, compensatory mutations in two hairpin loops of the proposed secondary structure, suggesting that a pseudoknot-like interaction may exist between them, have also been observed. Phylogenetic analysis has allowed the allocation of PLMVd molecules into three major groups. This clustering does not strictly correlate with the source isolate from which the variants were obtained, providing insights into the complex mixture of molecules which make up each isolate. Bioassays of individual PLMVd sequence variants on GF-305 peach seedlings have shown that the biological properties of the PLMVd isolates may be correlated with both the complexity of their viroid populations and the presence of specific sequence variants.

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

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