Citrus Tristeza Virus Variants Research Articles

A Comprehensive Analysis of Citrus Tristeza Variants of Bhutan and Across the World.

Mandarin orange is economically one of the most important fruit crops in Bhutan. However, in recent years, orange productivity has dropped due to severe infection of citrus tristeza virus (CTV) associated with the gradual decline of citrus orchards. Although the disease incidence has been reported, very limited information is available on genetic variability among the Bhutanese CTV variants. This study used reverse transcription PCR (RT-PCR) to detect CTV in collected field samples and recorded disease incidence up to 71.11% in Bhutan’s prominent citrus-growing regions. To elucidate the extent of genetic variabilities among the Bhutanese CTV variants, we targeted four independent genomic regions (5′ORF1a, p25, p23, and p18) and analyzed a total of 64 collected isolates. These genomic regions were amplified and sequenced for further comparative bioinformatics analysis. Comprehensive phylogenetic reconstructions of the GenBank deposited sequences, including the corresponding genomic locations from 53 whole-genome sequences, revealed unexpected and rich diversity among Bhutanese CTV variants. A resistant-breaking (RB) variant was also identified for the first time from the Asian subcontinent. Our analyses unambiguously identified five (T36, T3, T68, VT, and HA16-5) major, well-recognized CTV strains. Bhutanese CTV variants form two additional newly identified distinct clades with higher confidence, B1 and B2, named after Bhutan. The origin of each of these nine clades can be traced back to their root in the north-eastern region of India and Bhutan. Together, our study established a definitive framework for categorizing global CTV variants into their distinctive clades and provided novel insights into multiple genomic region-based genetic diversity assessments, including their pathogenicity status.

The Intriguing Conundrum of a Nonconserved Multifunctional Protein of Citrus Tristeza Virus That Interacts with a Viral Long Non-Coding RNA.

Citrus tristeza virus (CTV), the largest non-segmented plant RNA virus, has several peculiar features, among which is the production of a 5′-terminal long non-coding RNA (lncRNA) termed low-molecular-weight tristeza 1 (LMT1). In this study, we found that p33, a unique viral protein that performs multiple functions in the virus infection cycle, specifically binds LMT1, both in vivo and in vitro. These results were obtained through the expression of p33 under the context of the wild type virus infection or along with a mutant CTV variant that does not produce LMT1 as well as via ectopic co-expression of p33 with LMT1 in Nicotiana benthamiana leaves followed by RNA immunoprecipitation and rapid amplification of cDNA ends assays. Further experiments in which a recombinant p33 protein and an in vitro transcribed full-length LMT1 RNA or its truncated fragments were subjected to an electrophoretic mobility shift assay demonstrated that p33 binds to at least two distinct regions within LMT1. To the best of our knowledge, this is the first report of a plant virus protein binding to a lncRNA produced by the same virus. The biological significance of the interaction between these two viral factors is discussed.

Walking Together: Cross-Protection, Genome Conservation, and the Replication Machinery of Citrus tristeza virus.

“Cross-protection”, a nearly 100 years-old virological term, is suggested to be changed to “close protection”. Evidence for the need of such change has accumulated over the past six decades from the laboratory experiments and field tests conducted by plant pathologists and plant virologists working with different plant viruses, and, in particular, from research on Citrus tristeza virus (CTV). A direct confirmation of such close protection came with the finding that “pre-immunization” of citrus plants with the variants of the T36 strain of CTV but not with variants of other virus strains was providing protection against a fluorescent protein-tagged T36-based recombinant virus variant. Under natural conditions close protection is functional and is closely associated both with the conservation of the CTV genome sequence and prevention of superinfection by closely similar isolates. It is suggested that the mechanism is primarily directed to prevent the danger of virus population collapse that could be expected to result through quasispecies divergence of large RNA genomes of the CTV variants continuously replicating within long-living and highly voluminous fruit trees. This review article provides an overview of the CTV cross-protection research, along with a discussion of the phenomenon in the context of the CTV biology and genetics.

A Long Non-Coding RNA of Citrus tristeza virus: Role in the Virus Interplay with the Host Immunity.

During infection, Citrus tristeza virus (CTV) produces a non-coding subgenomic RNA referred to as low-molecular-weight tristeza 1 (LMT1), which for a long time has been considered as a by-product of the complex CTV replication machinery. In this study, we investigated the role of LMT1 in the virus infection cycle using a CTV variant that does not produce LMT1 (CTV-LMT1d). We showed that lack of LMT1 did not halt virus ability to replicate or form proper virions. However, the mutant virus demonstrated significantly reduced invasiveness and systemic spread in Nicotiana benthamiana as well as an inability to establish infection in citrus. Introduction of CTV-LMT1d into the herbaceous host resulted in elevation of the levels of salicylic acid (SA) and SA-responsive pathogenesis-related genes beyond those upon inoculation with wild-type (WT) virus (CTV-WT). Further analysis showed that the LMT1 RNA produced by CTV-WT or via ectopic expression in the N. benthamiana leaves suppressed SA accumulation and up-regulated an alternative oxidase gene, which appeared to mitigate the accumulation of reactive oxygen species. To the best of our knowledge, this is the first report of a plant viral long non-coding RNA being involved in counter-acting host response by subverting the SA-mediated plant defense.

The p33 protein of Citrus tristeza virus affects viral pathogenicity by modulating a host immune response.

Accumulation of reactive oxygen species (ROS) is a general plant basal defense strategy against viruses. In this study, we show that infection by Citrus tristeza virus (CTV) triggered ROS burst in Nicotiana benthamiana and in the natural citrus host, the extent of which was virus-dose dependent. Using Agrobacterium-mediated expression of CTV-encoded proteins in N.benthamiana, we found that p33, a unique viral protein, contributed to the induction of ROS accumulation and programmed cell death. The role of p33 in CTV pathogenicity was assessed based on gene knockout and complementation in N.benthamiana. In the citrus-CTV pathosystem, deletion of the p33 open reading frame in a CTV variant resulted in a significant decrease in ROS production, compared to that of the wild type CTV, which correlated with invasion of the mutant virus into the immature xylem tracheid cells and abnormal differentiation of the vascular system. By contrast, the wild type CTV exhibited phloem-limited distribution with a minor effect on the vasculature. We conclude that the p33 protein is a CTV effector that negatively affects virus pathogenicity and suggest that N.benthamiana recognizes p33 to activate the host immune response to restrict CTV into the phloem tissue and minimize the disease syndrome.

Understanding superinfection exclusion by complex populations of Citrus tristeza virus

Superinfection exclusion (SIE) is a phenomenon in which a primary viral infection restricts a secondary infection with the same or closely related virus. Previously we showed that SIE by Citrus tristeza virus (CTV) occurs only between isolates of the same virus genotype. This work, however, was done using single genotype-containing isolates, while most field citrus trees harbor complex populations composed of different virus genotypes. Here we examined SIE in plants simultaneously infected with several CTV genotypes. The experiments showed that exclusion of a secondary infection by a CTV variant was triggered by the presence of another variant of the same genotype in the primary population, even under the conditions of its low-level accumulation, and was not affected by co-occurrence of additional heterologous genotypes. The same rule appeared to be in effect when SIE by mixed populations was tested in a series of different citrus varieties.

Membrane association of a nonconserved viral protein confers virus ability to extend its host range

Citrus tristeza virus (CTV), the largest and most complex member of the family Closteroviridae, encodes a unique protein, p33, which shows no homology with other known proteins, however, plays an important role in virus pathogenesis. In this study, we examined some of the characteristics of p33. We show that p33 is a membrane-associated protein that is inserted into the membrane via a transmembrane helix formed by hydrophobic amino acid residues at the C-terminal end of the protein. Removal of this transmembrane domain (TMD) dramatically altered the intracellular localization of p33. Moreover, the TMD alone was sufficient to confer membrane localization of an unrelated protein. Finally, a CTV variant that produced a truncated p33 lacking the TMD was unable to infect sour orange, one of the selected virus hosts, which infection requires p33, suggesting that membrane association of p33 is important for the ability of CTV to extend its host range.

CAPILLARY ELECTROPHORESIS-SINGLE STRAND CONFORMATION POLYMORPHISM ANALYSIS AND MULTIPLE MOLECULAR MARKER GENOTYPING ALLOW A RAPID DIFFERENTIATION OF CITRUS TRISTEZA VIRUS ISOLATES

According to recent studies, characterization of Citrus tristeza virus (CTV) isolates by molecular technologies is a prerequisite for developing a rational control strategy based on eradication or preimmunization (cross-protection). In this work we used the recently developed capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) method and the multiple molecular markers (MMM) analysis to evaluate a number of CTV isolates collected in different locations of Sicily. The screening, performed after a preliminary testing on indicator plants, led to the differentiation of three main CTV sub-groups or clusters: (i) severe isolates widespread in the area, (ii) mild isolates, and (iii) mixed isolates including both severe and mild CTV variants.

Superinfection exclusion by Citrus tristeza virus does not correlate with the production of viral small RNAs

Superinfection exclusion (SIE), a phenomenon in which a preexisting viral infection prevents a secondary infection with the same or closely related virus, has been described for different viruses, including important pathogens of humans, animals, and plants. Several mechanisms acting at various stages of the viral life cycle have been proposed to explain SIE. Most cases of SIE in plant virus systems were attributed to induction of RNA silencing, a host defense mechanism that is mediated by small RNAs. Here we show that SIE by Citrus tristeza virus (CTV) does not correlate with the production of viral small interfering RNAs (siRNAs). CTV variants, which differed in the SIE ability, had similar siRNAs profiles. Along with our previous observations that the exclusion phenomenon requires a specific viral protein, p33, the new data suggest that SIE by CTV is highly complex and appears to use different mechanisms than those proposed for other viruses.

A viral protein mediates superinfection exclusion at the whole-organism level but is not required for exclusion at the cellular level.

Superinfection exclusion (SIE), the ability of an established virus infection to interfere with a secondary infection by the same or a closely related virus, has been described for different viruses, including important pathogens of humans, animals, and plants. Citrus tristeza virus (CTV), a positive-sense RNA virus, represents a valuable model system for studying SIE due to the existence of several phylogenetically distinct strains. Furthermore, CTV allows SIE to be examined at the whole-organism level. Previously, we demonstrated that SIE by CTV is a virus-controlled function that requires the viral protein p33. In this study, we show that p33 mediates SIE at the whole-organism level, while it is not required for exclusion at the cellular level. Primary infection of a host with a fluorescent protein-tagged CTV variant lacking p33 did not interfere with the establishment of a secondary infection by the same virus labeled with a different fluorescent protein. However, cellular coinfection by both viruses was rare. The obtained observations, along with estimates of the cellular multiplicity of infection (MOI) and MOI model selection, suggested that low levels of cellular coinfection appear to be best explained by exclusion at the cellular level. Based on these results, we propose that SIE by CTV is operated at two levels--the cellular and the whole-organism levels--by two distinct mechanisms that could function independently. This novel aspect of viral SIE highlights the intriguing complexity of this phenomenon, further understanding of which may open up new avenues to manage virus diseases. Many viruses exhibit superinfection exclusion (SIE), the ability of an established virus infection to interfere with a secondary infection by related viruses. SIE plays an important role in the pathogenesis and evolution of virus populations. The observations described here suggest that SIE could be controlled independently at different levels of the host: the whole-organism level or the level of individual cells. The p33 protein of citrus tristeza virus (CTV), an RNA virus, was shown to mediate SIE at the whole-organism level, while it appeared not to be required for exclusion at the cellular level. SIE by CTV is, therefore, highly complex and appears to use mechanisms different from those proposed for other viruses. A better understanding of this phenomenon may lead to the development of new strategies for controlling viral diseases in human populations and agroecosystems.

Distribution, genetic diversity and recombination analysis of Citrus tristeza virus of India

Citrus tristeza virus (CTV) isolates representing all the citrus-growing geographical zones of India were analyzed for nucleotide sequence of the 5'ORF1a fragments of the partial LProI domain and for the coat protein (CP) gene. The nucleotide sequences were compared with previously reported Indian and CTV genotypes from GenBank. The Indian isolates had 80-99 % sequence identity for the 5'ORF1a and 89-99 % identity for the CP genes. In phylogenetic tree analysis, all the Indian and previously reported isolates segregated into eight clades or groups for the 5'ORF1a region. Indian CTV isolates were clustered in all the clades, four of which, D13, K5, BAN-1, and B165, consisted of only Indian isolates. Phylogenetic tree analysis of the CP genes resulted in seven clades. Indian CTV isolates clustered in six of them, and clades I and VI consisted of only Indian isolates. In the phylogenetic tree the Indian CTV isolates clustered in different groups regardless their geographical origin. Diversities in CTV isolates within individual citrus farms were highlighted. Because incongruent phylogenetic relationships were observed for both of the genomic regions, 5'ORF1a and CP gene, recombination analysis was performed using program RDP3. This analysis detected potential recombination events among the CTV isolates which involved exchange of sequences between divergent CTV variants. The SplitsTree analysis showed evidence of phylogenetic conflicts in evolutionary relationships among CTV isolates.

Discrimination Between Mild and Severe Citrus tristeza virus Isolates with a Rapid and Highly Specific Real-Time Reverse Transcription-Polymerase Chain Reaction Method Using TaqMan LNA Probes

Severe isolates of Citrus tristeza virus (CTV) inducing seedling yellows (SY) and/or stem pitting (SP) in grapefruit or sweet orange are a major threat for the citrus industry worldwide. Identification of these CTV variants was achieved by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) using a general primer set and three TaqMan locked nucleic acids (LNA) probes targeting sequences characteristic of severe, mild (non-SY, non-SP), and T36-like isolates. Successful amplification was achieved from fresh or silica-desiccated CTV-infected samples and all isolates but one reacted with one or more probes. Standard curves using RNA transcripts homologous to the three probes allowed a reproducible quantitative assay, with a wide dynamic range of detection starting with 10(2) copies. RT-PCR assays with homologous and heterologous transcript RNA mixes demonstrated that each probe reacted only with its cognate sequence which was detected even at ratios below 2.5%. Analysis of 56 pathogenically distinct CTV isolates from 20 countries showed that mild isolates reacted only with the mild probe, whereas severe SP and SY isolates reacted with the severe-SP or the T36-like probes, respectively, and often with a second probe. This procedure can be useful to identify and control potentially dangerous CTV isolates in areas affected only by mild isolates.

East Adriatic—a reservoir region of severe Citrus tristeza virus strains

Citrus tristeza virus (CTV) represents one of the major threats to citrus production worldwide. In the East Adriatic region, CTV symptoms are mostly absent due to traditional citrus grafting on trifoliate orange (Poncirus trifoliata), a CTV-tolerant rootstock. Therefore, the virus has been continuously spreading by the propagation of infected material. The genetic variability of CTV was studied on nineteen citrus samples, collected from orchards in the coastal region of Croatia, Montenegro and Albania, that previously tested positive by ELISA and immunocapture RT-PCR. Single-strand conformation polymorphism of the amplified coat protein gene demonstrated the presence of different CTV variants in each amplicon, while sequence analysis of cloned CP gene variants confirmed their clustering into six out of the seven phylogenetic groups so far delineated. Four of these groups include sequences of severe quick decline, seedling yellows and stem-pitting (SP) isolates, thought to be found only rarely in the Mediterranean region. Regardless of the lack of symptoms in the field, CTV isolates from the East Adriatic displayed high genetic variability and pathogenic potential, additionally confirmed by biological characterisation. The high percentage of mixed infections suggest the potential for further diversification and a greater risk of severe variants spreading into new areas.

Three genes of Citrus tristeza virus are dispensable for infection and movement throughout some varieties of citrus trees

Citrus tristeza virus (CTV), a member of the Closteroviridae, possesses a 19.3-kb positive-stranded RNA genome that is organized into twelve open reading frames (ORFs). The CTV genome contains two sets of conserved genes, which are characteristic of this virus group, the replication gene block (ORF 1a and 1b) and the quintuple gene block (p6, HSP70 h, p61, CPm, and CP). With the exception of the p6 gene, they are required for replication and virion assembly. CTV contains five additional genes, p33, p18, p13, p20 and p23, in the 3′ half of the genome, some of which (p33, p18 and p13) are not conserved among other members of this virus group, and have been proposed to have evolved for specific interactions with the citrus host. In the present study, the requirements for systemic infection of citrus trees of p33, p6, p18, p13 and p20 were examined. Viral mutants with a deletion in the p6 or the p20 ORF failed to infect citrus plants systemically, suggesting their possible roles in virus translocation/systemic infection. However, we found that deletions within the p33, p18 or p13 ORF individually resulted in no significant loss of ability of the virus to infect, multiply, and spread throughout citrus trees. Furthermore, deletions in the p33, p18 and p13 genes in all possible combinations including deletions in all three genes allowed the virus to systemically invade citrus trees. Green fluorescent protein-tagged CTV variants with deletions in the p33 ORF or the p33, p18 and p13 ORFs demonstrated that the movement and distribution of these deletion mutants were similar to that of the wild-type virus.

Genetic variation and recombination of RdRp and HSP 70h genes of Citrus tristeza virus isolates from orange trees showing symptoms of citrus sudden death disease.

BackgroundCitrus sudden death (CSD), a disease that rapidly kills orange trees, is an emerging threat to the Brazilian citrus industry. Although the causal agent of CSD has not been definitively determined, based on the disease's distribution and symptomatology it is suspected that the agent may be a new strain of Citrus tristeza virus (CTV). CTV genetic variation was therefore assessed in two Brazilian orange trees displaying CSD symptoms and a third with more conventional CTV symptoms.ResultsA total of 286 RNA-dependent-RNA polymerase (RdRp) and 284 heat shock protein 70 homolog (HSP70h) gene fragments were determined for CTV variants infecting the three trees. It was discovered that, despite differences in symptomatology, the trees were all apparently coinfected with similar populations of divergent CTV variants. While mixed CTV infections are common, the genetic distance between the most divergent population members observed (24.1% for RdRp and 11.0% for HSP70h) was far greater than that in previously described mixed infections. Recombinants of five distinct RdRp lineages and three distinct HSP70h lineages were easily detectable but respectively accounted for only 5.9 and 11.9% of the RdRp and HSP70h gene fragments analysed and there was no evidence of an association between particular recombinant mosaics and CSD. Also, comparisons of CTV population structures indicated that the two most similar CTV populations were those of one of the trees with CSD and the tree without CSD.ConclusionWe suggest that if CTV is the causal agent of CSD, it is most likely a subtle feature of population structures within mixed infections and not merely the presence (or absence) of a single CTV variant within these populations that triggers the disease.

Preferential accumulation of severe variants of Citrus tristeza virus in plants co-inoculated with mild and severe variants

The viral population in sweet orange plants, either healthy or pre-inoculated with the asymptomatic isolate of Citrus tristeza virus (CTV) T32, and then graft- or aphid-inoculated with the stem-pitting isolate T318, was characterized with respect to symptom expression, reaction with monoclonal antibody MCA13, single-strand conformation polymorphism (SSCP) of genes p18 and p20, bi-directional RT-PCR, and dot-blot hybridisation. All plants inoculated with T318, with or without pre-inoculation, showed stem pitting, reacted with MCA13, had the SSCP profile characteristic of this isolate, and in bi-directional RT-PCR yielded a 450-bp DNA product associated with severe isolates, indicating that T32 afforded no protection against T318. The latter isolate had two main sequence variants, the minor one of which was indistinguishable from the main T32 sequence, and both were detected in most plants that were graft-inoculated with T318. However, the T32 variant was not detected in plants that were aphid-inoculated only with T318 and also showed stem pitting. This suggested an association of symptoms with the major T318 sequence and preferential transmission of this variant by aphids. The T318-specific variant accumulated more than the T32 variant in plants in which both were replicating, suggesting a higher fitness of the former. Our results clearly emphasize the potential threat of severe CTV variants in areas where mild isolates are presently predominant.