Emergence and phylodynamics of Citrus tristeza virus in Sicily, Italy.

Citrus tristeza virus (CTV) outbreaks were detected in Sicily island, Italy for the first time in 2002. To gain insight into the evolutionary forces driving the emergence and phylogeography of these CTV populations, we determined and analyzed the nucleotide sequences of the p20 gene from 108 CTV isolates collected from 2002 to 2009. Bayesian phylogenetic analysis revealed that mild and severe CTV isolates belonging to five different clades (lineages) were introduced in Sicily in 2002. Phylogeographic analysis showed that four lineages co-circulated in the main citrus growing area located in Eastern Sicily. However, only one lineage (composed of mild isolates) spread to distant areas of Sicily and was detected after 2007. No correlation was found between genetic variation and citrus host, indicating that citrus cultivars did not exert differential selective pressures on the virus. The genetic variation of CTV was not structured according to geographical location or sampling time, likely due to the multiple introduction events and a complex migration pattern with intense co- and re-circulation of different lineages in the same area. The phylogenetic structure, statistical tests of neutrality and comparison of synonymous and nonsynonymous substitution rates suggest that weak negative selection and genetic drift following a rapid expansion may be the main causes of the CTV variability observed today in Sicily. Nonetheless, three adjacent amino acids at the p20 N-terminal region were found to be under positive selection, likely resulting from adaptation events.

Ectopic expression of the p23 silencing suppressor of Citrus tristeza virus differentially modifies viral accumulation and tropism in two transgenic woody hosts.

Citrus tristeza virus (CTV), a phloem-restricted closterovirus infecting citrus, encodes three different silencing suppressors (p25, p20 and p23), one of which (p23) is a pathogenicity determinant that induces aberrations resembling CTV symptoms when expressed ectopically in transgenic citrus hosts. In this article, the effect of p23 ectopic expression on virus infection was examined in sweet orange (SwO), a highly susceptible host, and sour orange (SO), which severely restricts CTV cell-to-cell movement. Transgenic plants of both species ectopically expressing p23, or transformed with an empty vector, were graft inoculated with the mild CTV isolate T385 or with CTV-BC1/GFP, a clonal strain derived from the severe isolate T36 carrying the gene for the green fluorescent protein (GFP). CTV distribution in infected tissues was assessed by direct tissue blot immunoassay and fluorescence emission, and virus accumulation was estimated by quantitative real-time reverse transcriptase-polymerase chain reaction. CTV accumulation in p23-expressing and control SwO plants was similar, whereas the viral load in transgenic SO expressing p23 was 10-10(5) times higher than in the cognate control plants. Although few infection foci composed of a single cell were observed in the phloem of CTV-infected control SO, the number of foci in p23-expressing plants was higher and usually comprised two to six cells, indicating viral cell-to-cell movement. CTV was detected in mesophyll protoplasts and cells from infected SO and SwO expressing p23, but not in similar protoplasts and cells from infected control plants. Our results show that the ectopic expression of p23 enables CTV to escape from the phloem and, in addition, facilitates systemic infection of the resistant SO host. This is the first report of a viral-encoded protein that enhances virus accumulation and distribution in woody hosts.

Isolates of Citrus tristeza virus that overcome Poncirus trifoliata resistance comprise a novel strain.

The economically important rootstock species Poncirus trifoliata is resistant to most isolates of Citrus tristeza virus (CTV), but not to members of the CTV resistance-breaking (RB) strain presently found in New Zealand. In this study, five known and suspected RB isolates were separated from field mixtures, and their genomes were sequenced in full. It was found that the RB isolates are members of a single phylogenetically distinct clade with an average of 90.3% genomic nucleotide sequence identity to the closest extant isolate, T36. These isolates also show evidence of multiple recombination events throughout their evolutionary history, with T36, T30 and VT-like isolates, and with each other. Finally, the genomic sequences of these isolates show that several genes contain unique polymorphisms that may or may not be involved in overcoming resistance. These data will aid in the understanding of host-virus interactions, and the mechanism of resistance in P. trifoliata.

Variations in two gene sequences of Citrus tristeza virus after host passage.

We estimated genetic variation in two groups of Citrus tristeza virus (CTV) isolates: one of them (isolates T385, T317, T318 and T305) derived from a Spanish source by successive host passages, and the other (isolates T388 and T390), obtained after aphid transmission of a Japanese source. The population structure of these isolates had been characterized by single-strand conformation polymorphism analysis of genes p18 and p20. The nucleotide sequences of representative haplotypes of each isolate and gene were used to estimate genetic diversity within and between isolates and to evaluate genetic differentiation between populations. Phylogenetic analysis of p18 and p20 sequence variants showed two main groups: one them included variants predominant in the severe isolates (T318, T305 and T388), and the other comprised variants present in both mild (T385, T317) and severe isolates. Most sequence variants of isolate T390 were not associated to these groups. In some isolates, within-isolate diversity was higher than diversity with other isolates because their population contained distantly related sequence variants, some of which were genetically close to variants predominant in the second isolate. Isolates T388 and T390 were genetically different for the two genes, as estimated by the F statistic. Furthermore, genetic differentiation between T385 and T317, T318 and T305 increased after each host passage. Our results suggest that aphid transmission and host passage may significantly alter the composition of CTV populations and thus be an important factor in their evolution.

Evolutionary analysis of genetic variation observed in citrus tristeza virus (CTV) after host passage.

We have studied the genetic variability in two genes (p18 and p20) from two groups of Citrus tristeza virus (CTV) isolates. One group (isolates T385, T317, T318, and T305) was derived from a Spanish source by successive host passages while the other (isolates T388 and T390) was obtained after aphid transmission from a Japanese source. A total of 274 sequences were obtained for gene p18 and 451 for p20. In the corresponding phylogenetic trees, sequences derived from the severe isolates (T318, T305, and T388) clustered together and separately from those derived from mild or moderate isolates (T385, T317, and T390), regardless of their geographic origin. Hierarchical analyses of molecular variance showed that up to 53% of the total genetic variability in p18 and up to 87% of the variation in p20 could be explained by differences in the pathogenicity features of the isolates. Neutrality tests revealed that different selection forces had been acting between isolates and between genes, with purifying selection being suggested for p18 from isolates T385 and T390 and for p20 from isolates T385, T317, and T388, and balancing selection for p18 from isolates T318, T305, and T388 and for p20 from isolates T318 and T390. Furthermore, several models of codon selection were observed, with purifying selection being the most notable one, compatible with low effective population size of the virus populations resulting from transmission bottlenecks. We found no evidence of recombination playing a significant role during p18 and p20 evolution in these isolates. These results suggest that hosts can be an important evolutionary factor for CTV isolates.

HSP70 homolog functions in cell-to-cell movement of a plant virus.

Plant closteroviruses encode a homolog of the HSP70 (heat shock protein, 70 kDa) family of cellular proteins. To facilitate studies of the function of HSP70 homolog (HSP70h) in viral infection, the beet yellows closterovirus (BYV) was modified to express green fluorescent protein. This tagged virus was competent in cell-to-cell movement, producing multicellular infection foci similar to those formed by the wild-type BYV. Inactivation of the HSP70h gene by replacement of the start codon or by deletion of 493 codons resulted in complete arrest of BYV translocation from cell to cell. Identical movement-deficient phenotypes were observed in BYV variants possessing HSP70h that lacked the computer-predicted ATPase domain or the C-terminal domain, or that harbored point mutations in the putative catalytic site of the ATPase. These results demonstrate that the virus-specific member of the HSP70 family of molecular chaperones functions in intercellular translocation and represents an additional type of a plant viral-movement protein.

A high-resolution linkage map of the citrus tristeza virus resistance gene region in Poncirus trifoliata (L.) Raf.

Resistance to citrus tristeza virus (CTV) was evaluated in 554 progeny of 10 populations derived from Poncirus trifoliata. A dominant gene (Ctv) controlled CTV resistance in P. trifoliata. Twenty-one dominant PCR-based DNA markers were identified as linked to Ctv by bulked segregant analysis. Of the 11 closest markers to Ctv, only 2 segregated in all populations. Ten of these markers were cloned and sequenced, and codominant RFLP markers were developed. Seven RFLP markers were then evaluated in 10 populations. Marker orders were consistent in all linkage maps based on data of single populations or on combined data of populations with similar segregation patterns. In a consensus map, the six closest marker loci spanned 5.3 cM of the Ctv region. Z16 cosegregated with Ctv. C19 and AD08 flanked Ctv at distances of 0.5 and 0.8 cM, respectively. These 3 markers were present as single copies in the Poncirus genome, and could be used directly for bacterial artificial chromosome library screening to initiate a walk toward Ctv. BLAST searches of the GenBank database revealed high sequence similarities between 2 markers and known plant disease resistance genes, indicating that a resistance gene cluster exists in the Ctv region in P. trifoliata.

Kinetics of accumulation of citrus tristeza virus RNAs.

Citrus tristeza virus (CTV), a member of the closterovirus group, is one of the more complex single-stranded RNA viruses. The 5' portion of its 19,296-nt, single-stranded RNA genome is expressed as an approximately 400-kDa polyprotein that is proteolytically processed, while the 10 3' open reading frames are expressed from 3'-coterminal subgenomic RNAs (sg RNAs). As an initial examination of the gene expression of this virus, we found that the kinetics of accumulation of genomic and sg RNAs and coat protein of the T36 isolate of CTV were similar in protoplasts of the natural host, citrus, and the experimental nonhost Nicotiana benthamiana. Newly synthesized genomic RNA was detected 2 days postinoculation and increased to a maximum at 3-5 days. The RNA complementary to the full-length virion RNA increased with similar kinetics, but at approximately one-tenth the concentration of genomic plus strands. Most of the abundant sg RNAs also accumulated in parallel to that of the genomic RNA. However, the smallest sg RNA, which corresponds to the p23 gene, increased earlier. The different sg RNAs accumulated in greatly differing amounts, in general with 3'-most sg RNAs accumulating to higher levels than 5' sg RNAs. However, some 3' sg RNAs (p13 and p18) accumulated to low levels. The two 3'-most sg RNAs (p23 and p20) accumulated to high levels approximately equal to that of the genomic RNA. The accumulation curve for coat protein paralleled that of its mRNA, suggesting that its regulation was transcriptional. Progeny virions from protoplasts were used to sequentially infect new protoplasts, serving as a potential source of virus that could evolve free from the genetic selection in intact plants for aphid transmission and movement.