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William O. Dawson - One of the best experts on this subject based on the ideXlab platform.
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Citrus Tristeza Virus p33 protein is required for efficient transmission by the aphid aphis toxoptera citricidus kirkaldy
Viruses, 2020Co-Authors: Turksen Shilts, William O. Dawson, Choaa Elmohtar, Nabil KillinyAbstract: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.
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bottlenecks and complementation in the aphid transmission of Citrus Tristeza Virus populations
Archives of Virology, 2018Co-Authors: S. J. Harper, S J Cowell, William O. DawsonAbstract: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.
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minor coat and heat shock proteins are involved in the binding of Citrus Tristeza Virus to the foregut of its aphid vector toxoptera citricida
Applied and Environmental Microbiology, 2016Co-Authors: Nabil Killiny, S. J. Harper, Serine Alfaress, El C Mohtar, William O. DawsonAbstract:ABSTRACT Vector transmission is a critical stage in the viral life cycle, yet for most plant Viruses how they interact with their vector is unknown or is explained by analogy with previously described relatives. Here we examined the mechanism underlying the transmission of Citrus Tristeza Virus (CTV) by its aphid vector, Toxoptera citricida, with the objective of identifying what Virus-encoded proteins it uses to interact with the vector. Using fluorescently labeled virions, we demonstrated that CTV binds specifically to the lining of the cibarium of the aphid. Through in vitro competitive binding assays between fluorescent virions and free viral proteins, we determined that the minor coat protein is involved in vector interaction. We also found that the presence of two heat shock-like proteins, p61 and p65, reduces virion binding in vitro . Additionally, treating the dissected mouthparts with proteases did not affect the binding of CTV virions. In contrast, chitinase treatment reduced CTV binding to the foregut. Finally, competition with glucose, N -acetyl-β-d-glucosamine, chitobiose, and chitotriose reduced the binding. These findings together suggest that CTV binds to the sugar moieties of the cuticular surface of the aphid cibarium, and the binding involves the concerted activity of three Virus-encoded proteins. IMPORTANCE Limited information is known about the specific interactions between Citrus Tristeza Virus and its aphid vectors. These interactions are important for the process of successful transmission. In this study, we localized the CTV retention site as the cibarium of the aphid foregut. Moreover, we demonstrated that the nature of these interactions is protein-carbohydrate binding. The viral proteins, including the minor coat protein and two heat shock proteins, bind to sugar moieties on the surface of the foregut. These findings will help in understanding the transmission mechanism of CTV by the aphid vector and may help in developing control strategies which interfere with the CTV binding to its insect vector to block the transmission.
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sequence variation in two genes determines the efficacy of transmission of Citrus Tristeza Virus by the brown Citrus aphid
Archives of Virology, 2016Co-Authors: S. J. Harper, Satyanarayana Tatineni, Siddarame Gowda, Turksen Shilts, S J Cowell, Nabil Killiny, William O. DawsonAbstract:Vector transmission is an important part of the viral infection cycle, yet for many Viruses little is known about this process, or how viral sequence variation affects transmission efficacy. Here we examined the effect of substituting genes from the highly transmissible FS577 isolate of Citrus Tristeza Virus (CTV) in to the poorly transmissible T36-based infectious clone. We found that introducing p65 or p61 sequences from FS577 significantly increased transmission efficacy. Interestingly, replacement of both genes produced a greater increase than either gene alone, suggesting that CTV transmission requires the concerted action of co-evolved p65 and p61 proteins.
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Citrus Tristeza Virus making an ally from an enemy
Annual Review of Phytopathology, 2015Co-Authors: William O. Dawson, S M Garnsey, Moshe Barjoseph, P MorenoAbstract:Virus diseases of perennial trees and vines have characteristics not amenable to study using small model annual plants. Unique disease symptoms such as graft incompatibilities and stem pitting cause considerable crop losses. Also, Viruses in these long-living plants tend to accumulate complex populations of Viruses and strains. Considerable progress has been made in understanding the biology and genetics of Citrus Tristeza Virus (CTV) and in developing it into a tool for crop protection and improvement. The diseases in tree and vine crops have commonalities for which CTV can be used to develop a baseline. The purpose of this review is to provide a necessary background of systems and reagents developed for CTV that can be used for continued progress in this area and to point out the value of the CTV-Citrus system in answering important questions on plant-Virus interactions and developing new methods for controlling plant diseases.
Svetlana Y. Folimonova - One of the best experts on this subject based on the ideXlab platform.
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walking together cross protection genome conservation and the replication machinery of Citrus Tristeza Virus
Viruses, 2020Co-Authors: Svetlana Y. Folimonova, Diann Achor, Moshe BarjosephAbstract:“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.
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a non conserved p33 protein of Citrus Tristeza Virus interacts with multiple viral partners
Molecular Plant-microbe Interactions, 2020Co-Authors: Thi Nguyet Minh Dao, Sung-hwan Kang, Aurélie Bak, Svetlana Y. FolimonovaAbstract:The RNA genome of Citrus Tristeza Virus (CTV), one of the most damaging viral pathogens of Citrus, contains 12 open reading frames resulting in production of at least 19 proteins. Previous studies on the intraviral interactome of CTV revealed self-interaction of the viral RNA-dependent RNA polymerase, the major coat protein (CP), p20, p23, and p33 proteins, while heterologous interactions between the CTV proteins have not been characterized. In this work, we examined interactions between the p33 protein, a nonconserved protein of CTV, which performs multiple functions in the Virus infection cycle and is needed for Virus ability to infect the extended host range, with other CTV proteins shown to mediate Virus interactions with its plant hosts. Using yeast two-hybrid, bimolecular fluorescence complementation, and coimmunoprecipitation assays, we demonstrated that p33 interacts with three viral proteins, i.e., CP, p20, and p23, in vivo and in planta. Coexpression of p33, which is an integral membrane protein, resulted in a shift in the localization of the p20 and p23 proteins toward the subcellular crude-membrane fraction. Upon CTV infection, the four proteins colocalized in the CTV replication factories. In addition, three of them, CP, p20, and p23, were found in the p33-formed membranous structures. Using bioinformatic analyses and mutagenesis, we found that the N-terminus of p33 is involved in the interactions with all three protein partners. A potential role of these interactions in Virus ability to infect the extended host range is discussed.
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a long non coding rna of Citrus Tristeza Virus role in the Virus interplay with the host immunity
Viruses, 2019Co-Authors: Sung-hwan Kang, Yongduo Sun, Osama O. Atallah, Jose C Huguettapia, Jerald D Noble, Svetlana Y. FolimonovaAbstract: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.
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the p33 protein of Citrus Tristeza Virus affects viral pathogenicity by modulating a host immune response
New Phytologist, 2019Co-Authors: Yongduo Sun, Svetlana Y. FolimonovaAbstract: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.
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Functional diversification upon leader protease domain duplication in the Citrus Tristeza Virus genome: Role of RNA sequences and the encoded proteins.
Virology, 2017Co-Authors: Sung-hwan Kang, Yongduo Sun, Osama O. Atallah, Svetlana Y. FolimonovaAbstract: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.
Pedro Moreno - One of the best experts on this subject based on the ideXlab platform.
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Citrus Tristeza Virus p23 determinants for nucleolar localization and their influence on suppression of rna silencing and pathogenesis
Molecular Plant-microbe Interactions, 2013Co-Authors: Susana Ruizruiz, Pedro Moreno, Carmelo Lopez, Leandro Peña, Carmen Fagoaga, Luis Navarro, Nuria Soler, J A Sancheznavarro, Ricardo FloresAbstract:Citrus Tristeza Virus (CTV) encodes a singular protein (p23, 209 amino acids) with multiple functions, including RNA silencing suppression (RSS). Confocal laser-scanning microscopy of green fluorescent protein (GFP)-p23 agroexpressed in Nicotiana benthamiana revealed its accumulation in the nucleolus, Cajal bodies, and plasmodesmata. To dissect the nucleolar localization signal (NoLS) typically associated with basic motifs, seven truncated and 10 pointmutated versions of p23 were assayed. Deletion mutants showed that regions 50 to 86 and 100 to 157 (excluding fragment 106 to 114), both with basic motifs and the first with a zinc-finger, contain the (bipartite) NoLS. Alanine substitutions delimited this signal to three cysteines of the Zn-finger and some basic amino acids. RSS activity of p23 in N. benthamiana was abolished by essentially all mutants, indicating that it involves most p23 regions. The necroticinducing ability of p23 when launched in N. benthamiana from Potato Virus X was only retained by deletion mutant 158-209 and one substitution mutant, showing that the Znfinger and flanking basic motifs form part of the pathogenic determinant. Ectopic expression of p23 and some deletion mutants in transgenic Mexican lime demarcated a similar determinant, suggesting that p23 affects related pathways in Citrus and N. benthamiana. Both RSS activity and pathogenicity of p23 appear related to its nucleolar localization.
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Agroinoculation of Citrus Tristeza Virus Causes Systemic Infection and Symptoms in the Presumed Nonhost Nicotiana benthamiana
Molecular plant-microbe interactions : MPMI, 2011Co-Authors: Silvia Ambros, William O. Dawson, Jose Guerri, Choaa El-mohtar, Susana Ruiz-ruiz, Leandro Peña, Pedro MorenoAbstract: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 effic...
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accumulation of transgene derived sirnas is not sufficient for rnai mediated protection against Citrus Tristeza Virus in transgenic mexican lime
Molecular Plant Pathology, 2010Co-Authors: Carmelo Lopez, Pedro Moreno, Ricardo Flores, Carmen Fagoaga, Luis Navarro, Magdalena Cervera, Leandro PeñaAbstract:Mexican lime plants transformed with the 3'-terminal 549 nucleotides of the Citrus Tristeza Virus (CTV) genome in sense, antisense and intron-hairpin formats were analysed for transgene-derived transcript and short interfering RNA (siRNA) accumulation, and for CTV resistance. Propagations from all sense, antisense and empty-vector transgenic lines were susceptible to CTV, except for a single sense-line plant with a complex transgene integration pattern that showed transgene-derived siRNAs in association with low levels of the transgene-derived transcript. In contrast, nine of 30 intron-hairpin lines showed CTV resistance, with 9%-56% of bud-propagated plants, depending on the line, remaining uninfected on graft inoculation, and the others being susceptible. Although resistance was always associated with the presence of transgene-derived siRNAs, their level in different sense and intron-hairpin transformants was variable irrespective of the response to CTV infection. In intron-hairpin lines with single transgene integration, CTV resistance was correlated with low accumulation of the transgene-derived transcript rather than with high accumulation of transgene-derived siRNAs.
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contribution of recombination and selection to molecular evolution of Citrus Tristeza Virus
Journal of General Virology, 2009Co-Authors: S Martin, Patricia Moya, Jose Guerri, M C Vives, Luis Rubio, Santiago F. Elena, Adrian Sambade, Pedro MorenoAbstract:The genetic variation of Citrus Tristeza Virus (CTV) was analysed by comparing the predominant sequence variants in seven genomic regions (p33, p65, p61, p18, p13, p20 and p23) of 18 pathogenically distinct isolates from seven different countries. Analyses of the selective constraints acting on each codon suggest that most regions were under purifying selection. Phylogenetic analysis shows diverse patterns of molecular evolution for different genomic regions. A first clade composed of isolates that are genetically close to the reference mild isolates T385 or T30 was inferred from all genomic regions. A second clade, mostly comprising virulent isolates, was defined from regions p33, p65, p13 and p23. For regions p65, p61, p18, p13 and p23, a third clade that mostly included South American isolates could not be related to any reference genotype. Phylogenetic relationships among isolates did not reflect their geographical origin, suggesting significant gene flow between geographically distant areas. Incongruent phylogenetic trees for different genomic regions suggested recombination events, an extreme that was supported by several recombination-detecting methods. A phylogenetic network incorporating the effect of recombination showed an explosive radiation pattern for the evolution of some isolates and also grouped isolates by virulence. Taken together, the above results suggest that negative selection, gene flow, sequence recombination and virulence may be important factors driving CTV evolution.
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a real time rt pcr assay for detection and absolute quantitation of Citrus Tristeza Virus in different plant tissues
Journal of Virological Methods, 2007Co-Authors: Susana Ruizruiz, Pedro Moreno, Jose Guerri, Silvia AmbrosAbstract:A real-time RT-PCR assay using SYBR Green was developed for specific and reliable quantitative detection of Citrus Tristeza Virus (CTV) in infected plants. A general primer set designed from conserved sequences in ORFs 1b and 2 enabled amplification of the genomic RNA (gRNA) while excluding most subgenomic and defective RNAs. Single RT-PCR products of 204 bp (isolate T36) or 186 bp (other isolates) were obtained with no primer-dimer or non-specific amplifications detected. Melting curve analysis revealed distinct melting temperature peaks (T(m)) for severe and mild isolates. External standard curves using RNA transcripts of the selected target allowed a reproducible quantitative assay, with a wide dynamic range of detection starting with 10(2) gRNA copies and with very low variation coefficient values. This protocol enabled reliable assessments of CTV accumulation in different tissues and from different Citrus species, grown in the greenhouse or under field conditions, and infected with CTV isolates differing in their pathogenicity. CTV accumulation was higher in bark and fruits than in roots or leaves and showed minimal differences among several susceptible Citrus species, but it was significantly lower in sour orange. This quantitative detection assay will be a valuable tool for diagnosis and molecular studies on CTV biology.
Jose Guerri - One of the best experts on this subject based on the ideXlab platform.
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Agroinoculation of Citrus Tristeza Virus Causes Systemic Infection and Symptoms in the Presumed Nonhost Nicotiana benthamiana
Molecular plant-microbe interactions : MPMI, 2011Co-Authors: Silvia Ambros, William O. Dawson, Jose Guerri, Choaa El-mohtar, Susana Ruiz-ruiz, Leandro Peña, Pedro MorenoAbstract: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 effic...
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contribution of recombination and selection to molecular evolution of Citrus Tristeza Virus
Journal of General Virology, 2009Co-Authors: S Martin, Patricia Moya, Jose Guerri, M C Vives, Luis Rubio, Santiago F. Elena, Adrian Sambade, Pedro MorenoAbstract:The genetic variation of Citrus Tristeza Virus (CTV) was analysed by comparing the predominant sequence variants in seven genomic regions (p33, p65, p61, p18, p13, p20 and p23) of 18 pathogenically distinct isolates from seven different countries. Analyses of the selective constraints acting on each codon suggest that most regions were under purifying selection. Phylogenetic analysis shows diverse patterns of molecular evolution for different genomic regions. A first clade composed of isolates that are genetically close to the reference mild isolates T385 or T30 was inferred from all genomic regions. A second clade, mostly comprising virulent isolates, was defined from regions p33, p65, p13 and p23. For regions p65, p61, p18, p13 and p23, a third clade that mostly included South American isolates could not be related to any reference genotype. Phylogenetic relationships among isolates did not reflect their geographical origin, suggesting significant gene flow between geographically distant areas. Incongruent phylogenetic trees for different genomic regions suggested recombination events, an extreme that was supported by several recombination-detecting methods. A phylogenetic network incorporating the effect of recombination showed an explosive radiation pattern for the evolution of some isolates and also grouped isolates by virulence. Taken together, the above results suggest that negative selection, gene flow, sequence recombination and virulence may be important factors driving CTV evolution.
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a real time rt pcr assay for detection and absolute quantitation of Citrus Tristeza Virus in different plant tissues
Journal of Virological Methods, 2007Co-Authors: Susana Ruizruiz, Pedro Moreno, Jose Guerri, Silvia AmbrosAbstract:A real-time RT-PCR assay using SYBR Green was developed for specific and reliable quantitative detection of Citrus Tristeza Virus (CTV) in infected plants. A general primer set designed from conserved sequences in ORFs 1b and 2 enabled amplification of the genomic RNA (gRNA) while excluding most subgenomic and defective RNAs. Single RT-PCR products of 204 bp (isolate T36) or 186 bp (other isolates) were obtained with no primer-dimer or non-specific amplifications detected. Melting curve analysis revealed distinct melting temperature peaks (T(m)) for severe and mild isolates. External standard curves using RNA transcripts of the selected target allowed a reproducible quantitative assay, with a wide dynamic range of detection starting with 10(2) gRNA copies and with very low variation coefficient values. This protocol enabled reliable assessments of CTV accumulation in different tissues and from different Citrus species, grown in the greenhouse or under field conditions, and infected with CTV isolates differing in their pathogenicity. CTV accumulation was higher in bark and fruits than in roots or leaves and showed minimal differences among several susceptible Citrus species, but it was significantly lower in sour orange. This quantitative detection assay will be a valuable tool for diagnosis and molecular studies on CTV biology.
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transcriptional response of Citrus aurantifolia to infection by Citrus Tristeza Virus
Virology, 2007Co-Authors: Monica Gandia, Pedro Moreno, Ricardo Flores, Ana Conesa, Gema Ancillo, Jose Gadea, Javier Forment, Vicente Pallas, Nuria Duranvila, Jose GuerriAbstract:Changes in gene expression of Mexican lime plants in response to infection with a severe (T305) or a mild (T385) isolate of Citrus Tristeza Virus (CTV) were analyzed using a cDNA microarray containing 12,672 probes to 6875 different Citrus genes. Statistically significant (P<0.01) expression changes of 334 genes were detected in response to infection with isolate T305, whereas infection with T385 induced no significant change. Induced genes included 145 without significant similarity with known sequences and 189 that were classified in seven functional categories. Genes related with response to stress and defense were the main category and included 28% of the genes induced. Selected transcription changes detected by microarray analysis were confirmed by quantitative real-time RT-PCR. Changes detected in the transcriptome upon infecting lime with T305 may be associated either with symptom expression, with a strain-specific defense mechanism, or with a general response to stress.
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Variations in Two Gene Sequences of Citrus Tristeza Virus after Host Passage
Virus Genes, 2006Co-Authors: Maria A Ayllon, Jose Guerri, Luis Rubio, Vicente Sentandreu, Andrés Moya, Pedro MorenoAbstract: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.
Leandro Peña - One of the best experts on this subject based on the ideXlab platform.
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Citrus Tristeza Virus host rna silencing and Virus counteraction
Methods of Molecular Biology, 2019Co-Authors: Susana Ruizruiz, Leandro Peña, Pedro M D Moreno, Luis Navarro, Beatriz Navarro, Francesco Di Serio, Ricardo FloresAbstract:To dissect the host RNA silencing response incited by Citrus Tristeza Virus (CTV, genus ClosteroVirus), a (+) ssRNA of ~19300 nt, and the counter reaction deployed by the Virus via its three RNA silencing suppressors (RSS), the small RNAs (sRNAs) of three Virus-host combinations were deep sequenced. The subsequent analysis indicated that CTV sRNAs (1) constitute more than half of the total sRNAs in the susceptible Mexican lime and sweet orange, while only 3.5% in the restrictive sour orange; (2) are mostly of 21-22 nt, with those of (+) sense predominating slightly; and (3) derive from all the CTV genome, as evidenced by its entire recomposition from viral sRNA contigs but adopt an asymmetric pattern with a hotspot mapping at the 3'-terminal ~2500 nt. The Citrus homologues of Arabidopsis Dicer-like (DCL) 4 and 2 most likely generate the 21 and 22 nt CTV sRNAs, respectively, by dicing the gRNA and the 3' co-terminal sgRNAs and, particularly, their double-stranded forms accumulating in infected cells. The plant sRNA profile, very similar and dominated by the 24 nt sRNAs in the three mock-inoculated controls, displayed a major reduction of the 24 nt sRNAs in Mexican lime and sweet orange, but not in sour orange. CTV infection also influences the levels of certain microRNAs.The high accumulation of CTV sRNAs in two of the Citrus hosts examined suggests that it is not their synthesis, but their function, the target of the RSS encoded by CTV: p25 (intercellular), p23 (intracellular) and p20 (both). The two latter might block the loading of CTV sRNAs into the RNA silencing complex or interfere with it through alternative mechanisms. Of the three CTV RSS, p23 is the one that has been more thoroughly studied. It is a multifunctional RNA-binding protein with a putative Zn finger domain and basic motifs that (1) has no homologues in other closteroViruses, (2) accumulates in the nucleolus and plasmodesmata, (3) regulates the asymmetric balance of CTV (+) and (-) RNA strands, and (4) induces CTV syndromes and stimulates systemic infection in certain Citrus species when expressed as a transgene ectopically or in phloem-associated cells.
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Citrus Tristeza Virus p23 determinants for nucleolar localization and their influence on suppression of rna silencing and pathogenesis
Molecular Plant-microbe Interactions, 2013Co-Authors: Susana Ruizruiz, Pedro Moreno, Carmelo Lopez, Leandro Peña, Carmen Fagoaga, Luis Navarro, Nuria Soler, J A Sancheznavarro, Ricardo FloresAbstract:Citrus Tristeza Virus (CTV) encodes a singular protein (p23, 209 amino acids) with multiple functions, including RNA silencing suppression (RSS). Confocal laser-scanning microscopy of green fluorescent protein (GFP)-p23 agroexpressed in Nicotiana benthamiana revealed its accumulation in the nucleolus, Cajal bodies, and plasmodesmata. To dissect the nucleolar localization signal (NoLS) typically associated with basic motifs, seven truncated and 10 pointmutated versions of p23 were assayed. Deletion mutants showed that regions 50 to 86 and 100 to 157 (excluding fragment 106 to 114), both with basic motifs and the first with a zinc-finger, contain the (bipartite) NoLS. Alanine substitutions delimited this signal to three cysteines of the Zn-finger and some basic amino acids. RSS activity of p23 in N. benthamiana was abolished by essentially all mutants, indicating that it involves most p23 regions. The necroticinducing ability of p23 when launched in N. benthamiana from Potato Virus X was only retained by deletion mutant 158-209 and one substitution mutant, showing that the Znfinger and flanking basic motifs form part of the pathogenic determinant. Ectopic expression of p23 and some deletion mutants in transgenic Mexican lime demarcated a similar determinant, suggesting that p23 affects related pathways in Citrus and N. benthamiana. Both RSS activity and pathogenicity of p23 appear related to its nucleolar localization.
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Agroinoculation of Citrus Tristeza Virus Causes Systemic Infection and Symptoms in the Presumed Nonhost Nicotiana benthamiana
Molecular plant-microbe interactions : MPMI, 2011Co-Authors: Silvia Ambros, William O. Dawson, Jose Guerri, Choaa El-mohtar, Susana Ruiz-ruiz, Leandro Peña, Pedro MorenoAbstract: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 effic...
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accumulation of transgene derived sirnas is not sufficient for rnai mediated protection against Citrus Tristeza Virus in transgenic mexican lime
Molecular Plant Pathology, 2010Co-Authors: Carmelo Lopez, Pedro Moreno, Ricardo Flores, Carmen Fagoaga, Luis Navarro, Magdalena Cervera, Leandro PeñaAbstract:Mexican lime plants transformed with the 3'-terminal 549 nucleotides of the Citrus Tristeza Virus (CTV) genome in sense, antisense and intron-hairpin formats were analysed for transgene-derived transcript and short interfering RNA (siRNA) accumulation, and for CTV resistance. Propagations from all sense, antisense and empty-vector transgenic lines were susceptible to CTV, except for a single sense-line plant with a complex transgene integration pattern that showed transgene-derived siRNAs in association with low levels of the transgene-derived transcript. In contrast, nine of 30 intron-hairpin lines showed CTV resistance, with 9%-56% of bud-propagated plants, depending on the line, remaining uninfected on graft inoculation, and the others being susceptible. Although resistance was always associated with the presence of transgene-derived siRNAs, their level in different sense and intron-hairpin transformants was variable irrespective of the response to CTV infection. In intron-hairpin lines with single transgene integration, CTV resistance was correlated with low accumulation of the transgene-derived transcript rather than with high accumulation of transgene-derived siRNAs.
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Post-Transcriptional Gene Silencing of the p23 Silencing Suppressor of Citrus Tristeza Virus Confers Resistance to the Virus in Transgenic Mexican Lime
Plant Molecular Biology, 2006Co-Authors: Carmen Fagoaga, Pedro Moreno, Carmelo Lopez, Ricardo Flores, Alfonso Hermoso De Mendoza, Luis Navarro, Leandro PeñaAbstract:Previously, we have shown that most Mexican limes ( Citrus aurantifolia (Christ.) Swing.) expressing the p23 gene of Citrus Tristeza Virus (CTV) exhibit aberrations resembling viral leaf symptoms. Here we report that five independent transgenic lines having normal phenotype displayed characteristics typical of post-transcriptional gene silencing (PTGS): multiple copies of the transgene, low levels of the corresponding mRNA, methylation of the silenced transgene, and accumulation of p23 -specific small interfering RNAs (siRNAs). When graft- or aphid-inoculated with CTV, some propagations of these silenced lines were immune: they neither expressed symptoms nor accumulated virions and viral RNA as estimated by DAS-ELISA and Northern blot hybridization, respectively. Other propagations were moderately resistant because they became infected later and showed attenuated symptoms compared to controls. The susceptible propagations, in addition to symptom expression and elevated Virus titer, accumulated p23 -specific siRNAs at levels significantly higher than immune or non-inoculated propagations, and showed transgene demethylation. This variable response among clonal transformants indicates that factors other than the genetic background of the transgenic plants play a key role in PTGS-mediated resistance.