Comparative analysis identifies amino acids critical for citrus tristeza virus (T36CA) encoded proteins involved in suppression of RNA silencing and differential systemic infection in two plant species.

Complementary (c)DNA clones corresponding to the full-length genome of T36CA (a Californian isolate of Citrus tristeza virus with the T36 genotype), which shares 99.1% identity with that of T36FL (a T36 isolate from Florida), were made into a vector system to express the green fluorescent protein (GFP). Agroinfiltration of two prototype T36CA-based vectors (pT36CA) to Nicotiana benthamiana plants resulted in local but not systemic GFP expression/viral infection. This contrasted with agroinfiltration of the T36FL-based vector (pT36FL), which resulted in both local and systemic GFP expression/viral infection. A prototype T36CA systemically infected RNA silencing-defective N. benthamiana lines, demonstrating that a genetic basis for its defective systemic infection was RNA silencing. We evaluated the in planta bioactivity of chimeric pT36CA-pT36FL constructs and the results suggested that nucleotide variants in several open reading frames of the prototype T36CA could be responsible for its defective systemic infection. A single amino acid substitution in each of two silencing suppressors, p20 (S107G) and p25 (G36D), of prototype T36CA facilitated its systemic infectivity in N. benthamiana (albeit with reduced titre relative to that of T36FL) but not in Citrus macrophylla plants. Enhanced virus accumulation and, remarkably, robust systemic infection of T36CA in N. benthamiana and C. macrophylla plants, respectively, required two additional amino acid substitutions engineered in p65 (N118S and S158L), a putative closterovirus movement protein. The availability of pT36CA provides a unique opportunity for comparative analysis to identify viral coding and noncoding nucleotides or sequences involved in functions that are vital for in planta infection.

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.

Citrus tristeza virus P33 Protein is Required for Efficient Transmission by the Aphid Aphis (Toxoptera) citricidus (Kirkaldy).

Plant viruses are threatening many valuable crops, and Citrus tristeza virus (CTV) is considered one of the most economically important plant viruses. CTV has destroyed millions of citrus trees in many regions of the world. Consequently, understanding of the transmission mechanism of CTV by its main vector, the brown citrus aphid, Aphis (Toxoptera) citricidus (Kirkaldy), may lead to better control strategies for CTV. The objective of this study was to understand the CTV-vector relationship by exploring the influence of viral genetic diversity on virus transmission. We built several infectious clones with different 5'-proximal ends from different CTV strains and assessed their transmission by the brown citrus aphid. Replacement of the 5'- end of the T36 isolate with that of the T30 strain (poorly transmitted) did not increase the transmission rate of T36, whereas replacement with that of the T68-1 isolate (highly transmitted) increased the transmission rate of T36 from 1.5 to 23%. Finally, substitution of p33 gene of the T36 strain with that of T68 increased the transmission rate from 1.5% to 17.8%. Although the underlying mechanisms that regulate the CTV transmission process by aphids have been explored in many ways, the roles of specific viral proteins are still not explicit. Our findings will improve our understanding of the transmission mechanisms of CTV by its aphid vector and may lead to the development of control strategies that interfere with its transmission by vector.

Fast-track breeding system to introduce CTV resistance of trifoliate orange into citrus germplasm, by integrating early flowering transgenic plants with marker-assisted selection.

BACKGROUND: Global warming will expand the range of new and invasive pathogens in orchards, and subsequently increase the risk of disease epidemics and economic losses. The development of new resistant plant varieties can help to reduce the impact of pathogens, however, the breeding speed can be extremely slow, due to the growth rates of the plants, and the availability of resistance genes. Citrus trees are suffering immense damage from serious diseases such as citrus canker (XCC), huanglongbing (HLB), and citrus tristeza virus (CTV). A fast-track breeding system, that aimed at shortening the duration for disease resistance breeding by incorporating the resistance genes from related species to commercial varieties, has been developed using the integration of precocious transgenic trifoliate orange with the overexpression of CiFT and MAS. It was applied here to incorporate CTV resistance of trifoliate orange into citrus germplasm. RESULTS: One generation of backcrossed breeding, that would normally take at least 5 years, was achieved in a single year by fast-track breeding system. Linkage analysis using the corresponding DNA markers revealed that CTV resistance and T-DNA integrated regions were found in different linkage groups, and they were independently segregated in the BC progenies. The CTV resistant null segregants, in which the T-DNA integrated region was removed from their genome, were feasibly obtained by MAS in each generation of the BC progenies, and their CTV resistance was confirmed by immunological analysis. Several BC3 null segregants, whose genetic backgrounds had been substituted into citrus germplasm, except for the haplotype block of CTV resistance, were successfully obtained. CGH and NGS analyses revealed that the T-DNA integrated region was safely segregated out in null segregants. CONCLUSION: Fast-track breeding systems are expected to shorten the required breeding time by more than one-fifth in comparison with conventional cross breeding techniques. Using this system, we obtained BC3-8, whose genetic background was successfully substituted except for the CTV resistance locus, and could be a novel mandarin breeding material. The fast-track breeding system will be useful to introduce important traits from related species to citrus germplasm while also drastically reducing the time required for breeding.

Biochemical changes in cultivars of sweet oranges infected with citrus tristeza virus / Mudanças bioquímicas em vários cultivares de laranjas doces infectadas com o vírus citrus tristeza

Abstract Citrus fruit production occupies a place of considerable importance in the economy of the world including Pakistan. Tristeza disease caused by Citrus Tristeza Virus (CTV) exists in various forms that may or may not cause symptoms in the plants. The bioactive compounds and antioxidants are naturally present in plants and provide a defense mechanism that is generally accelerated in response to a stress. The objective of the present study was to target and analyze the citrus plants that were CTV positive to observe the changes in the enzymatic and non-enzymatic antioxidants of citrus (Sweet Oranges only). It was observed that in response to CTV infection, both the non-enzymatic antioxidants (total flavonoid, ascorbic acid, phenolic acid) and enzymatic antioxidants (catalase, superoxide dismutase and peroxidase) activities showed an increasing trend overall. The profiling of antioxidants in response to a viral infection may help in the discovery of new biomarkers that can be used as a monitoring tool in disease management.

Resumo As frutas cítricas ocupam um lugar de considerável importância na economia do Paquistão, assim como o resto do mundo. A doença da tristeza causada pelo Vírus da Tristeza dos Citros (CTV) existe em várias formas que podem ou não apresentar sintomas nas plantas. Os compostos bioativos e antioxidantes estão naturalmente presentes nas plantas e fornecem um mecanismo de defesa que é geralmente acelerado em resposta a um estresse. O objetivo do presente estudo foi analisar as alterações causadas pelo CTV nos antioxidantes enzimáticos e não enzimáticos de laranjas doces. Foi observado que, em resposta ao ataque de CTV, os antioxidantes não enzimáticos como flavonoides totais, ácido ascórbico, ácido fenólico e antioxidantes enzimáticos, como as atividades de catalase, superóxido dismutase e peroxidase, geralmente mostram uma tendência crescente. O perfil de antioxidantes em resposta a um ataque viral pode ajudar na descoberta de novos biomarcadores que podem ser usados ​​como uma ferramenta de monitoramento no gerenciamento de doenças.

A Short Note on Reflections and Publications on Citrus tristeza virus (CTV) Methodologies.

My PhD thesis work of Citrus tristeza virus (CTV) purification was aimed to develop a rapid serological assay to replace biological indexing. The task turned difficult and was achieved after a lengthy struggle, rewarded by allowing (1) the rapid diagnosis of the first incidences of natural spread of a severe CTV-VT strain in our region and (2) finding that the CTV particle isolation protocol, with some modifications, was also useful for Beet yellows virus (BYV) particles, leading to their assignment in the Closterovirus group, the first group of elongated plant viruses with different modal lengths. Later, following the introduction of ELISA for large-scale diagnosis of tristeza-infected citrus trees, the CTV infection rates through the coastal citrus production areas were continually increasing, with many ELISA-positive samples appearing symptomless, prompting the need to develop strain-specific assays. Using CTV-VT cDNA fragments, as hybridization probes, the genetic diversity among local CTV isolates was demonstrated. With the emergence of the PCR technology, we developed a CTV-dsRNA cloning method based on the ligation of known oligonucleotide molecules to dsRNA ends and the use of complementary oligonucleotides for cDNA synthesis and PCR amplification. The method allowed the cloning of a cDNA molecule complementary to a defective dsRNA of 2.4 kb with intact 5 and 3 ends of the CTV-VT genome. A list of publications, resulting from continuous collaborative work with local and foreign associates and students on the development and adaptation of novel CTV methodologies, is present.

Shrink the giant: scale down the citrus tree to a model system to investigate the RNA interference efficiency.

Virus-induced silencing gene technology has been increasingly used; however, a controversy exists among researchers about whether using the sense or antisense orientation of the gene target is more efficient. Herein, instead of using the entire citrus tree, a reduced system consisting of a single leaf, 5 cm of the stem and a few roots was established to fairly compare between the sense or antisense orientation of phytoene desaturase gene (pds) in the Citrus tristeza virus vector, for improved RNAi efficiency. Although the virus titers were similar in the two cases, the gene expression of pds was significantly lower when using the antisense orientation than using the sense orientation. I hypothesize that the extra effect from use of antisense orientation is due to the role of subgenomic RNA as a supplemental source for complementary sequences, thus resulting in more RNAi.

Biochemical changes in cultivars of sweet oranges infected with citrus tristeza virus.

Citrus fruit production occupies a place of considerable importance in the economy of the world including Pakistan. Tristeza disease caused by Citrus Tristeza Virus (CTV) exists in various forms that may or may not cause symptoms in the plants. The bioactive compounds and antioxidants are naturally present in plants and provide a defense mechanism that is generally accelerated in response to a stress. The objective of the present study was to target and analyze the citrus plants that were CTV positive to observe the changes in the enzymatic and non-enzymatic antioxidants of citrus (Sweet Oranges only). It was observed that in response to CTV infection, both the non-enzymatic antioxidants (total flavonoid, ascorbic acid, phenolic acid) and enzymatic antioxidants (catalase, superoxide dismutase and peroxidase) activities showed an increasing trend overall. The profiling of antioxidants in response to a viral infection may help in the discovery of new biomarkers that can be used as a monitoring tool in disease management.

Titer Variation of Citrus Tristeza Virus in Aphids at Different Acquisition Access Periods and Its Association with Transmission Efficiency.

Tristeza, caused by citrus tristeza virus (CTV; Closterovirus, Closteroviridae), is of significant economic importance. Tristeza epidemics have caused severe declines in productivity, and even death, of millions of citrus trees on sour orange rootstock in many regions all over the world. In the field, CTV is most efficiently vectored by the brown citrus aphid (Toxoptera citricida (Kirkaldy)) in a semipersistent manner. The transmission efficiency of the vector is influenced by its acquisition access period (AAP) for CTV. A real-time RT-PCR assay using SYBR Green fluorescent dye was used to estimate the CTV titers in groups of 15 aphids under AAPs after 0.5 to 48 h for three CTV isolates (CT11A, CT16-2, and CTLJ). Similar trends for CTV titer in viruliferous aphids were displayed for the three isolates. The maximum CTV titer was at AAP 6 h for isolates CT11A and CT16-2, and at 4 h for isolate CTLJ. During the AAPs from 0.5 to 6 h, the mean CTV titer of CT16-2 increased from 7.8 × 104 to 1.71 × 107 copies per 15 aphids, and was correlated with an increase in transmission rate from 20 to 90.9%. This suggests that the transmission efficiency is positively correlated with viral titer in the insect from 0.5 h until 6 h AAPs. While a downward trend in CTV titer was observed after a 6-h AAP, the transmission rate remained higher than 90% up to 48 h. These results indicate that factors other than the virus titer in the vector contribute to successful transmission under long acquisition conditions. This is the first detailed quantitative analysis of CTV in its main vector species following different AAPs and its association with transmission efficiency, and should enhance our understanding of T. citricida-CTV interactions.

Citrus tristeza virus co-opts glyceraldehyde 3-phosphate dehydrogenase for its infectious cycle by interacting with the viral-encoded protein p23.

KEY MESSAGE: Citrus tristeza virus encodes a unique protein, p23, with multiple functional roles that include co-option of the cytoplasmic glyceraldehyde 3-phosphate dehydrogenase to facilitate the viral infectious cycle. The genome of citrus tristeza virus (CTV), genus Closterovirus family Closteroviridae, is a single-stranded (+) RNA potentially encoding at least 17 proteins. One (p23), an RNA-binding protein of 209 amino acids with a putative Zn-finger and some basic motifs, displays singular features: (i) it has no homologues in other closteroviruses, (ii) it accumulates mainly in the nucleolus and Cajal bodies, and in plasmodesmata, and (iii) it mediates asymmetric accumulation of CTV RNA strands, intracellular suppression of RNA silencing, induction of some CTV syndromes and enhancement of systemic infection when expressed as a transgene ectopically or in phloem-associated cells in several Citrus spp. Here, a yeast two-hybrid screening of an expression library of Nicotiana benthamiana (a symptomatic experimental host for CTV), identified a transducin/WD40 domain protein and the cytosolic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as potential host interactors with p23. Bimolecular fluorescence complementation corroborated the p23-GAPDH interaction in planta and showed that p23 interacts with itself in the nucleolus, Cajal bodies and plasmodesmata, and with GAPDH in the cytoplasm (forming aggregates) and in plasmodesmata. The latter interaction was preserved in a p23 deletion mutant affecting the C-terminal domain, but not in two others affecting the Zn-finger and one internal basic motif. Virus-induced gene silencing of GAPDH mRNA resulted in a decrease of CTV titer as revealed by real-time RT-quantitative PCR and RNA gel-blot hybridization. Thus, like other viruses, CTV seems to co-opt GAPDH, via interaction with p23, to facilitate its infectious cycle.

Bottlenecks and complementation in the aphid transmission of citrus tristeza virus populations.

Aphid transmission is a major factor in the formation of citrus tristeza virus (CTV) populations. Here, we examined the effect of population interaction on aphid transmissibility of different CTV genotypes. We found that there was no correlation between the proportion of viral genotypes in the source population and what was transmitted. We next examined the transmission of a poorly transmitted infectious cDNA clone (T36) in mixture with other CTV genotypes. T36 transmission increased from 0.5% alone, to up to 35.7%, depending on the coinfecting genotype. These results suggest that interaction between CTV genotypes affects the transmission of this virus.

Molecular and biological characterization of a novel mild strain of citrus tristeza virus in California.

Strain differentiating marker profiles of citrus tristeza virus (CTV) isolates from California have shown the presence of multiple genotypes. To better define the genetic diversity involved, full-length genome sequences from four California CTV isolates were determined by small-interfering RNA sequencing. Phylogenetic analysis and nucleotide sequence comparisons differentiated these isolates into the genotypes VT (CA-VT-AT39), T30 (CA-T30-AT4), and a new strain called S1 (CA-S1-L and CA-S1-L65). S1 isolates had three common recombination events within portions of genes from VT, T36 and RB strains and were transmissible by Aphis gossypii. Virus indexing showed that CA-VT-AT39 could be classified as a severe strain, whereas CA-T30-AT4, CA-S1-L and CA-S1-L65 were mild. CA-VT-AT39, CA-S1-L, and CA-S1-L65 reacted with monoclonal antibody MCA13, whereas CA-T30-AT4 did not. RT-PCR and RT-qPCR detection assays for the S1 strain were developed and used to screen MCA13-reactive isolates in a CTV collection from central California collected from 1968 to 2011. Forty-two isolates were found to contain the S1 strain, alone or in combinations with other genotypes. BLAST and phylogenetic analysis of the S1 p25 gene region with other extant CTV sequences from the NCBI database suggested that putative S1-like isolates might occur elsewhere (e.g., China, South Korea, Turkey, Bosnia and Croatia). This information is important for CTV evolution, detection of specific strains, and cross-protection.

Functional diversification upon leader protease domain duplication in the Citrus tristeza virus genome: Role of RNA sequences and the encoded proteins.

Viruses from the family Closteroviridae show an example of intra-genome duplications of more than one gene. In addition to the hallmark coat protein gene duplication, several members possess a tandem duplication of papain-like leader proteases. In this study, we demonstrate that domains encoding the L1 and L2 proteases in the Citrus tristeza virus genome underwent a significant functional divergence at the RNA and protein levels. We show that the L1 protease is crucial for viral accumulation and establishment of initial infection, whereas its coding region is vital for virus transport. On the other hand, the second protease is indispensable for virus infection of its natural citrus host, suggesting that L2 has evolved an important adaptive function that mediates virus interaction with the woody host.

Isolate fitness and tissue-tropism determine superinfection success.

The mechanism of cross-protection, the deliberate infection of plants with a "mild" virus isolate to protect against "severe" isolates, has long been a topic of debate. In our model system, Citrus tristeza virus (CTV), this appears to be genotype-specific superinfection-exclusion, suggesting a simple recipe for cross-protection. However, this concept failed in field trials, which led us to examine the process of superinfection-exclusion more closely. We found that exclusion relies on the relative fitness of the primary versus the challenge isolates, and the host infected, and that significant differences in superinfection success could occur between isolates that differ by as few as 3 nucleotides. Furthermore, we found that exclusion was not uniform throughout the plant, but was tissue-specific. These data suggest that cross-protection is not a simple like-for-like process but a complex interaction between the primary and challenge isolates and the host.

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.

Some like it hot: citrus tristeza virus strains react differently to elevated temperature.

Viruses often infect plants as a mixed population. The dynamics of viral populations dictate the success of the infection, yet there is little understanding of the factors that influence them. It is known that temperature can affect individual viruses; could it also affect a virus population? In order to study this, we observed citrus tristeza virus (CTV) populations in different hosts under winter and summer conditions (25 versus 36 °C). We found that only some CTV strains were affected by a higher summer temperature, which lead to a change in CTV population structure, and that this effect was host dependent.

A 5'-proximal region of the Citrus tristeza virus genome encoding two leader proteases is involved in virus superinfection exclusion.

Superinfection exclusion (SIE), a phenomenon in which a primary virus infection prevents a secondary infection with the same or closely related virus, has been observed with various viruses. Earlier we demonstrated that SIE by Citrus tristeza virus (CTV) requires viral p33 protein. In this work we show that p33 alone is not sufficient for virus exclusion. To define the additional viral components that are involved in this phenomenon, we engineered a hybrid virus in which a 5'-proximal region in the genome of the T36 isolate containing coding sequences for the two leader proteases L1 and L2 has been substituted with a corresponding region from the genome of a heterologous T68-1 isolate. Sequential inoculation of plants pre-infected with the CTV L1L2T68 hybrid with T36 CTV resulted in superinfection with the challenge virus, which indicated that the substitution of the L1-L2 coding region affected SIE ability of the virus.

Rootstock-scion interaction affecting citrus response to CTV infection: a proteomic view.

Citrus tristeza virus (CTV) is the causal agent of various diseases with dramatic effects on citrus crops worldwide. Most Citrus species, grown on their own roots, are symptomless hosts for many CTV isolates. However, depending on different scion-rootstock combination, CTV infection should result in distinct syndromes, being 'tristeza' the more severe one, leading to a complete decline of the susceptible plants in a few weeks. Transcriptomic analyses revealed several genes involved either in defense response, or systemic acquired resistance, as well as transcription factors and components of the phosphorylation cascades, to be differentially regulated during CTV infection in Citrus aurantifolia species. To date little is known about the molecular mechanism of this host-pathogen interaction, and about the rootstock effect on citrus response to CTV infection. In this work, the response to CTV infection has been investigated in tolerant and susceptible scion-rootstock combinations by two-dimensional gel electrophoresis (2DE). A total of 125 protein spots have been found to be differently accumulated and/or phosphorylated between the two rootstock combinations. Downregulation in tolerant plants upon CTV infection was detected for proteins involved in reactive oxygen species (ROS) scavenging and defense response, suggesting a probable acclimation response able to minimize the systemic effects of virus infection. Some of these proteins resulted to be modulated also in absence of virus infection, revealing a rootstock effect on scion proteome modulation. Moreover, the phospho-modulation of proteins involved in ROS scavenging and defense response, further supports their involvement either in scion-rootstock crosstalk or in the establishment of tolerance/susceptibility to CTV infection.

Finding balance: Virus populations reach equilibrium during the infection process.

Virus populations, mixtures of viral strains or species, are a common feature of viral infection, and influence many viral processes including infection, transmission, and the induction of disease. Yet, little is known of the rules that define the composition and structure of these populations. In this study, we used three distinct strains of Citrus tristeza virus (CTV) to examine the effect of inoculum composition, titer, and order, on the virus population. We found that CTV populations stabilized at the same equilibrium irrespective of how that population was introduced into a host. In addition, both field and experimental observations showed that these equilibria were relatively uniform between individual hosts of the same species and under the same conditions. We observed that the structure of the equilibria reached is determined primarily by the host, with the same inoculum reaching different equilibria in different species, and by the fitness of individual virus variants.