development of a dot-immunobinding assay for detection of citrus tristeza virus.the dot-immunobinding assay (diba) was adapted for detection of citrus tristeza virus (ctv) and compared with das-elisa and das-indirect elisa. diba was easy to perform and as sensitive as either elisa procedure for ctv diagnosis. the entire test could be performed in 2-3 h using polyclonal antibodies, with minimal laboratory equipment. three different polyclonal antibodies gave a strong positive reaction with 12 selected ctv isolates; however, each serum had to be cross-absorbed with sap from h ...19911744220
serological techniques for detection of citrus tristeza virus.citrus tristeza virus (ctv) is the most economically important virus disease of citrus. in the last ten years, remarkable progress has been achieved in the development and improvement of new serological methods for ctv detection so that serology has become a dependable tool for many research, extension and regulatory purposes worldwide. ctv-specific polyclonal antisera and monoclonal antibodies have been developed in different research laboratories and used extensively in a wide range of differe ...19911744221
detection of double-stranded rna by elisa and dot immunobinding assay using an antiserum to synthetic antiserum against polyinosinic-polycytidylic acid (in-cn) was used to detect double-stranded rna (dsrna) by several serological techniques. dsrna was readily detected by indirect elisa (elisa-i) and dot immunobinding assay (dia). addition of the antigen to poly-l-lysine-precoated plates and blocking with uncreamed milk powder allowed detection levels of 100 in-cn by elisa-i. concentrations as low as 1 were detected by dia using polyvinyliden difluoride (pvdf) membranes. detect ...19911939501
molecular cloning and nucleotide sequencing of the coat protein gene of citrus tristeza virus.citrus tristeza virus (ctv) contains approximately 20,000 bases of positive-sense ssrna, encapsidated by a coat protein of approximately 25,000 mr that has previously been reported to consist of at least two size variants, cp1 and cp2. in the present study, a cdna library of the t36 isolate of ctv was prepared in a protein expression vector and screened with a polyclonal antibody against the coat protein. five immunopositive clones produced proteins in escherichia coli that reacted with monoclon ...19912033386
flexuous rods in phloem cells of lime plants infected with citrus tristeza virus. 19665943539
a single temperature amplification technique applied to the detection of citrus tristeza viral rna in plant nucleic acid extracts.a procedure for the successful detection of citrus tristeza virus (ctv) rna in total crude nucleic acid extracts of infected citrus whole leaves and bark is described. the method requires the isolation and precipitation of total nucleic acids from either infected whole leaf or bark tissue. the ctv viral rna is then specifically amplified using a single temperature rna self-sustained sequence replication technique (3sr) performed at 42 degrees c for 60 minutes. the amplified negative-sense viral ...19948051221
a method combining immunocapture and pcr amplification in a microtiter plate for the detection of plant viruses and subviral pathogens.a method for the detection of rna viral and subviral plant pathogens was developed that combines pathogen partial purification by solid-phase adsorbed antibodies, reverse transcriptional-polymerase chain reaction (rt-pcr) and quantitation of the amplified products by fluorescence. the reverse transcription of the rna is performed directly on the retained material without any previous thermal or chemical disruption of the virus particles. the whole procedure can be carried out in a microtiter pla ...19938113346
nucleotide sequence and organization of eight 3' open reading frames of the citrus tristeza closterovirus genome.the citrus tristeza closterovirus (ctv) rna genome was cloned as cdna generated from both ctv-specific double-stranded rna and genomic rna, and the sequence of the 3' 7292 nucleotides was determined. the sequenced portion contained eight open reading frames potentially encoding, in the 5' to 3' direction, proteins with the apparent molecular weights of 65, 61, 27, 25 (capsid protein, cp), 18, 13, 20, and 23 kda, and a potential noncoding region of 277 nucleotides. the 65-kda protein is a viral h ...19948116253
the diverged copy of the citrus tristeza virus coat protein is expressed in vivo.nucleotide sequence analysis of a portion of the citrus tristeza closterovirus (ctv) genome revealed an open reading frame immediately upstream of the coat protein gene that can encode a protein with a calculated m(r) of 27,360 (p27). the deduced amino acid sequence indicated that this putative nonstructural gene product is highly homologous to the coat protein. to investigate whether p27 was expressed in ctv-infected plants, a fusion protein of p27 produced in escherichia coli was used to raise ...19948178485
screening of the closterovirus genome by degenerate primer-mediated polymerase chain reaction.the genome of beet yellows virus (byv), the type representative of the closterovirus group, encodes a homologue of the cellular heat-shock protein (hsp) 70 family. a pair of degenerate primers targeted to motifs a and e, which are highly conserved in hsp70s, was synthesized. genomes of several definite and possible members of the closterovirus group were screened for the presence of the hsp70 gene with pcr using these degenerate primers. byv, citrus tristeza virus (ctv), beet yellow stunt virus ...19948207405
comparative sequence analysis of the coat proteins of biologically distinct citrus tristeza closterovirus isolates.the genome of citrus tristeza closterovirus (ctv) consists of a 20 kb single-stranded rna encapsidated in a 2000 nm long, flexuous particle. double-stranded (replicative form) rnas purified from ctv-infected tissue were used to prepare complementary dna libraries that involved initial first-strand cdna synthesis followed by selective amplification of the coat protein gene. ctv-specific antisera were used to select clones expressing the coat protein. the coat protein genes of seven florida and fo ...19938279124
beet pseudo-yellows virus is an authentic closterovirus.we have previously used rt-pcr to amplify a 251 base pair fragment diagnostic for beet pseudo-yellows virus (bpyv) infection from several bpyv infected samples from europe and the usa. this has now been shown by sequencing to represent a fragment of a heat shock protein (hsp)70 gene. sequence analysis of the fragment revealed that it is closely similar to the c-terminal sequence of the hsp70 homologue gene of lettuce infectious yellows virus, a definitive closterovirus, and also has obvious sequ ...19968972572
kinetics of accumulation of citrus tristeza virus rnas.citrus tristeza virus (ctv), a member of the closterovirus group, is one of the more complex single-stranded rna viruses. the 5' portion of its 19,296-nt, single-stranded rna genome is expressed as an approximately 400-kda polyprotein that is proteolytically processed, while the 10 3' open reading frames are expressed from 3'-coterminal subgenomic rnas (sg rnas). as an initial examination of the gene expression of this virus, we found that the kinetics of accumulation of genomic and sg rnas and ...19979024813
characterization of the 3' proximal gene of the citrus tristeza closterovirus genome.the 3' proximal open reading frame (orf 11) in the citrus tristeza virus (ctv) genome potentially encodes a protein of 209 amino acids with an estimated molecular weight of 23 kda (p23). the p23 orf from the severe florida strain t36 of ctv was expressed in escherichia coli, and the expressed protein was used to raise polyclonal antibodies in a rabbit. using these antisera on a western blot, a protein of expected size (23 kda) was detected in tissue extracts from ctv-infected citrus but not from ...19979037736
transcriptional strategy of closteroviruses: mapping the 5' termini of the citrus tristeza virus subgenomic rnas.citrus tristeza virus (ctv) induces formation of a nested set of at least nine 3' coterminal subgenomic rnas (sgrnas) in infected tissue. the organization and expression of the 19,296-nucleotide (nt) ctv genome resembles that of coronaviruses, with polyprotein processing, translational frameshifting, and multiple sgrna formation, but phylogenetically the ctv polymerase, like polymerases of other closteroviruses, belongs to the sindbis virus-like lineage of rna virus polymerases. both positive-st ...19979223524
a cdna clone from a defective rna of citrus tristeza virus is infective in the presence of the helper virus.a naturally occurring defective rna of 2379 nt (d2.3) from the vt strain of citrus tristeza closterovirus (ctv) was cloned and sequenced. the d2.3 rna is a fusion of two regions of 1521 and 858 nt from the 5' and 3' ends of the ctv genome, respectively. a cdna clone of d2.3 rna was tagged by the insertion of a 0.47 kb chimeric dna fragment and the recombinant cdna was inserted downstream of the cauliflower mosaic virus 35s promoter. the resulting construct was bombarded into ctv-infected tissue, ...19979225053
booster immunization with a partially purified citrus tristeza virus (ctv) preparation after priming with recombinant ctv coat protein enhances the binding capacity of capture antibodies by elisa.groups of rabbits and young lambs were immunized subcutaneously and intramuscularly with a recombinant citrus tristeza virus (ctv) coat protein (rctv-cp) antigen. three weeks after primary immunization the animals were divided into two groups that were boosted either with rctv-cp or with a partially purified preparation of ctv particles (ppctv). twelve and 15 days after the last injection, the animals were bled and the binding capacity of the antisera for ctv detection was examined for capture a ...19979274814
involvement of a subgenomic mrna in the generation of a variable population of defective citrus tristeza virus molecules.the fusion sites between the termini of naturally occurring defective rnas (d-rnas) from three citrus tristeza virus (ctv) isolates were sequenced. seven of eight clones showed a common 3' terminus of 940 nucleotides (nt) fused to 5' termini with different sizes. an extra cytosine nucleotide was found at the junction site of the majority of the common 3' d-rnas. molecular analysis of the plus and minus strands of the 0.9-kbp double-stranded rna, corresponding to the ctv open reading frame 11 sub ...19979371649
nucleotide sequence and genome organization of grapevine leafroll-associated virus-2 are similar to beet yellows virus, the closterovirus type member.the entire genome of grapevine leafroll-associated closterovirus-2 (glrav-2), except the exact 5' terminus, was cloned and sequenced. the sequence encompasses nine open reading frames (orfs) which include, in the 5' to 3' direction, an incomplete orf1a encoding a putative viral polyprotein and eight orfs that encode proteins of 52 kda (orf1b), 6 kda (orf2), 65 kda (orf3), 63 kda (orf4), 25 kda (orf5), 22 kda (orf6), 19 kda (orf7) and 24 kda (orf8) respectively, and 216 nucleotides of the 3' untr ...19989603345
the haplotype distribution of two genes of citrus tristeza virus is altered after host change or aphid transmission.genetic variability of citrus tristeza virus (ctv) was studied using the haplotypes detected by single-strand conformation polymorphism (sscp) analysis of genes p18 and p20 in six virus populations of two origins. the spanish group included a ctv isolate and subisolates obtained by graft-transmission to different host species. the other included two subisolates aphid-transmitted from a single japanese isolate. the homozygosity observed for gene p20 was always significantly higher than that expec ...199910049819
d-rna molecules associated with subisolates of the vt strain of citrus tristeza virus which induce different seedling-yellows reactions.citrus tristeza virus (ctv) strains were previously catalogued as seedling-yellows (sy) and non-sy (nsy) types, according to their yellowing and stunting effects on indicator seedlings. among subisolates of the vt strain, which were selected from chronically infected alemow plants, there was a correlation between the presence of 2.4-, 2.7- and 4.5-kb d-rnas, and sy and nsy reactions, respectively. similarly, plants infected with mor-t subisolates, which cause sy, contained d-rnas of 2.6 to 2.8 k ...199910499445
molecular characterization of an isolate of citrus tristeza virus that causes severe symptoms in sweet orange.the complete sequence (19,249 nucleotides) of the genome of citrus tristeza virus (ctv) isolate sy568 was determined. the genome organization is identical to that of the previously determined ctv-t36 and ctv-vt isolates. sequence comparisons revealed that ctv-sy568, a severe stem-pitting isolate from california, has more than 87% overall sequence identity with ctv-vt, a seedling yellows isolate from israel. although sy568 has an overall sequence identity of 81% with ctv-t36, a quick decline isol ...199910541017
replication of heterologous combinations of helper and defective rna of citrus tristeza virus.citrus tristeza virus (ctv) populations are among the more complex of plant rna viruses with unusual mixtures of strains and defective rnas (drnas). citrus plants infected with different ctv isolates contain multiple drna molecules that differ in size and relative abundance within and between isolates. additionally, we found mixtures of heterologous drnas in populations. to examine the replication of ctv drnas, the protoplast system had to be extended to support helper-assisted amplification of ...200010662631
the p20 gene product of citrus tristeza virus accumulates in the amorphous inclusion bodies.citrus tristeza virus (ctv) has 10 3' open reading frames (orfs) of unknown function except for the two coat proteins. the highest produced subgenomic rnas are those of the major coat protein gene (p25) and the 3' most genes, p20 and p23. the proteins from three orfs, p25, p27, and p20, were examined in the yeast two-hybrid assay for the interactions between themselves and to one another. the p20 protein exhibited a high affinity for itself, suggesting that it might aggregate in infected cells. ...200010964768
the fitness of citrus tristeza virus defective rnas is affected by the lengths of their 5'- and 3'-termini and by the coding capacity.populations of the closterovirus citrus tristeza virus (ctv) generally contain defective rnas (drnas) that vary in size, abundance, and sequence. the variation in abundance of the different drnas in a population suggests selection for those of higher fitness. to examine factors affecting fitness of drnas, we investigated a series of in vitro constructed drnas for their ability to be amplified in protoplasts by an efficiently replicated ctv deletion mutant. the minimal sequences required for accu ...200011017786
closterovirus encoded hsp70 homolog and p61 in addition to both coat proteins function in efficient virion assembly.assembly of the viral genome into virions is a critical process of the virus life cycle often defining the ability of the virus to move within the plant and to be transmitted horizontally to other plants. closteroviridae virions are polar helical rods assembled primarily by a major coat protein, but with a related minor coat protein at one end. the closteroviridae is the only virus family that encodes a protein with similarity to cellular chaperones, a 70-kda heat-shock protein homolog (hsp70h). ...200011112500
population structure and genetic diversity within california citrus tristeza virus (ctv) isolates.the closterovirus, citrus tristeza virus (ctv) is an aphid-borne rna virus that is the causal agent of important worldwide economic losses in citrus. biological and molecular variation has been observed for many ctv isolates. in this work we detected and analyzed sequence variants (haplotypes) within individual ctv isolates. we studied the population structure of five california ctv isolates by single strand conformation polymorphism (sscp) analysis of four ctv genomic regions. also, we estimate ...200011129629
molecular characterization of a structural epitope that is largely conserved among severe isolates of a plant molecular evidence was obtained for the critical role of a single amino acid residue in a structural epitope distinguished by the monoclonal antibody mca-13, which reacts selectively with severe isolates of citrus tristeza virus (ctv). different ctv isolates cause a wide range of symptoms in the diverse citrus species they affect. severe symptoms include decline, stem pitting, and seedling yellows. plants infected by mild isolates are essentially symptomless. the monoclonal antibody mca-1 ...19937682715
hen egg yolk as a source of antiviral antibodies in the enzyme-linked immunosorbent assay (elisa): a comparison of two plant viruses.a method of raising antibodies against plant viruses in hen egg yolk is described. laying hens were immunized with citrus tristeza virus (ctv) or tobacco mosaic virus-avocado isolate (tmv-a). anti-viral antibodies in the yolks of sequentially laid eggs as well as in the serum were titrated by the (heterologous) antiglobulin double antibody sandwich form of the enzyme-linked immunosorbent assay (hadas-elisa). antibodies first appeared in yolk 7 days after injection and peak levels were attained o ...19807024292
a simple procedure for the extraction of double-stranded rna from virus-infected plants.a simple procedure for the isolation of double-stranded (ds) rna from virus-infected plants is described. the method is based on grinding plant tissue in 4% p-aminosalicylic acid and recovery of ds rna by phenol extraction and precipitation with 30% ethanol. the presence of both negative and positive virus rna strands in rna fractionated in agarose gels was verified by northern blot hybridization with polynucleotide kinase labelled genomic rna or complementary dna (cdna) probes. the procedure en ...19836833446
polymorphism of the 5' terminal region of citrus tristeza virus (ctv) rna: incidence of three sequence types in isolates of different origin and pathogenicity.sequences of the 5' terminal region of the genomic rna from eight isolates of citrus tristeza virus (ctv) were previously classified into three types (i, ii and iii), with intragroup sequence identity higher than 88% and intergroup sequence identity as low as 44%. sequencing of an additional 58 cdna clones from 15 virus isolates showed that all sequences could be unequivocally assigned to one of the three types previously established. the relative frequency of each sequence type was assessed in ...200111266215
5'-coterminal subgenomic rnas in citrus tristeza virus-infected cells.three unusual 5' coterminal positive-stranded subgenomic (sg) rnas, two of about 0.8 kb and one of 10 kb (designated lmt1, lmt2, and lamt, respectively), from citrus spp. plants and nicotiana benthamiana protoplasts infected with citrus tristeza virus (ctv) were characterized. the 5' termini of the lmt rnas were mapped by runoff reverse transcription and found to correspond with the 5' terminus of the genomic rna. the lmt 5'-coterminal sgrnas consisted of two modal lengths of 744--746 and 842--8 ...200111336562
construction of a 1.2-mb contig including the citrus tristeza virus resistance gene locus using a bacterial artificial chromosome library of poncirus trifoliata (l.) raf.the citrus tristeza virus resistance gene (ctv) is a single dominant gene in poncirus trifoliata, a sexually compatible relative of citrus. to clone this gene, a bacterial artificial chromosome (bac) library has been constructed from an individual plant that was homozygous for ctv. this library contains 45,696 clones with an average insert size of 80 kb, corresponding to 9.6 genome equivalents. screening of the bac library with five chloroplast dna probes indicated that 0.58% of the bac clones c ...200111444697
partial characterization of the lettuce infectious yellows virus genomic rnas, identification of the coat protein gene and comparison of its amino acid sequence with those of other filamentous rna plant viruses.purified virions of lettuce infectious yellows virus (liyv), a tentative member of the closterovirus group, contained two rnas of approximately 8500 and 7300 nucleotides (rnas 1 and 2 respectively) and a single coat protein species with m(r) of approximately 28,000. liyv-infected plants contained multiple dsrnas. the two largest were the correct size for the replicative forms of liyv virion rnas 1 and 2. to assess the relationships between liyv rnas 1 and 2, cdnas corresponding to the virion rna ...19948021583
diversity of citrus tristeza virus isolates indicated by dsrna major dsrna of molecular weight (mw) 13.3 x 10(6) and two others (mw 1.9 x 10(6) and 0.8 x 10(6] were routinely detected by polyacrylamide gel electrophoresis in extracts from sweet orange (citrus sinensis) or citron (citrus medica) infected with each of 66 isolates of citrus tristeza virus (ctv). several additional dsrna were also commonly detected, usually as weakly stained bands in reproducible positions in gels, but some were very prominent, e.g., a dsrna of mw 1.7 x 10(6) associated wit ...19873692724
incidence and epidemiology of citrus tristeza virus in the valencian community of spain.the first outbreak of citrus tristeza disease in spain caused by citrus tristeza virus (ctv) was recorded in 1957 in the valencian community (vc). in total c. 40 million trees, mainly of sweet orange and mandarin grafted on sour orange rootstocks, declined due to ctv. large-scale surveys in different municipalities of the vc indicated that the disease spread very fast. incidence increased from 11% in 1989 to 53% in 1998. toxoptera aurantii and aphis spiraecola (inefficient aphid vectors of ctv) ...200011137164
functional specialization and evolution of leader proteinases in the family closteroviridae.members of the closteroviridae and potyviridae families of the plant positive-strand rna viruses encode one or two papain-like leader proteinases. in addition to a c-terminal proteolytic domain, each of these proteinases possesses a nonproteolytic n-terminal domain. we compared functions of the several leader proteinases using a gene swapping approach. the leader proteinase (l-pro) of beet yellows virus (byv; a closterovirus) was replaced with l1 or l2 proteinases of citrus tristeza virus (ctv; ...200111711606
the p23 protein of citrus tristeza virus controls asymmetrical rna accumulation.citrus tristeza virus (ctv), a member of the closteroviridae, has a 19.3-kb positive-stranded rna genome that is organized into 12 open reading frames (orfs) with the 10 3' genes expressed via a nested set of nine or ten 3'-coterminal subgenomic mrnas (sgrnas). relatively large amounts of negative-stranded rnas complementary to both genomic and sgrnas accumulate in infected cells. as is characteristic of rna viruses, wild-type ctv produced more positive than negative strands, with the plus-to-mi ...200211752137
defective rna molecules associated with citrus tristeza virus.preparations of single-stranded (ss) rna extracted from particles of the israeli vt strain of citrus tristeza virus (ctv-vt), and ss- and double-stranded (ds) rna preparations extracted from infected alemow (citrus macrophylla) plants, contained a population of molecules with features that suggest that they are defective rnas. the prototype of 2424 nt was cloned and sequenced and was found to be composed of two genomic regions corresponding to the 5' (1151 nt) and the 3' (1259 nt) termini of the ...199511831725
a novel class of large and infectious defective rnas of citrus tristeza virus.citrus tristeza virus (ctv)-infected plants contain one or more populations of defective rnas (drnas), mostly with a size range of ca. 2.0 to 5.0 kb. several ctv drnas have been characterized and found to consist mainly of the two termini of the genomic rna, with extensive internal deletions. the present paper describes a new class of large ( approximately 12.0 kb) drnas from three different ctv isolates with two unusual features. first is their composition with intact replicase genes. these drn ...200212093181
differentiation of citrus tristeza virus isolates by serological analysis of p25 coat protein peptide maps.a procedure was developed to purify rapidly and easily a sufficient quantity of native p25 coat protein (cp) to allow comparison of five isolates of citrus tristeza virus (ctv) by serological analysis of peptide maps, using monoclonal and polyclonal antibodies. ctv particles were concentrated by centrifugation and purified by agarose gel electrophoresis. the cp was extracted from gel slices riched in virions and protein yields were about three times greater than those obtained previously and of ...200010921839
sequences of citrus tristeza virus separated in time and space are essentially identical.the first citrus tristeza virus (ctv) genomes completely sequenced (19.3-kb positive-sense rna), from four biologically distinct isolates, are unexpectedly divergent in nucleotide sequence (up to 60% divergence). understanding of whether these large sequence differences resulted from recent evolution is important for the design of disease management strategies, particularly the use of genetically engineered mild (essentially symptomless)-strain cross protection and rna-mediated transgenic resist ...200010888625
mutational analysis of the replication signals in the 3'-nontranslated region of citrus tristeza virus.citrus tristeza virus (ctv), a member of the closteroviridae, has a 19.3-kb messenger-sense rna genome consisting of 12 open reading frames with nontranslated regions (ntr) at the 5' and 3' termini. the 273 nucleotide (nt) 3'-ntr is highly conserved ( approximately 95%) among the sequenced ctv isolates in contrast to the highly diverse 5'-ntr sequences. the 3' replication signals were mapped to the 3' 234 nts within the ntr. this region of ctv does not contain a poly-a tract nor does it appear t ...200212202214
simultaneous and co-operational amplification (co-pcr): a new concept for detection of plant viruses.a new and highly sensitive method for the amplification of viral rna targets from plant material has been developed and patented. this technique called co-operational amplification (co-pcr) can be carried out easily in a simple tetraprimer reaction based on the simultaneous action of four primers. the reaction process consists of the simultaneous reverse transcription of two different fragments from the same target, one containing the other; the production of four amplicons by the combination of ...200212367729
a new procedure to differentiate citrus tristeza virus isolates by hybridisation with digoxigenin-labelled cdna probes.a non-isotopic hybridisation procedure was developed to differentiate isolates of citrus tristeza virus (ctv) using digoxigenin (dig)-labelled cdna probes and different kinds of target rna. hybridisation of dig-probes with purified double-stranded rna (dsrna) or concentrated total rna extracts spotted on nylon membranes allowed detection of ctv nucleic acid equivalent to as little as 0.1-1 mg infected tissue, when the reaction was developed with a chemiluminiscent substrate. this sensitivity was ...200010716341
an engineered closterovirus rna replicon and analysis of heterologous terminal sequences for replication.citrus tristeza virus (ctv) populations in citrus trees are unusually complex mixtures of viral genotypes and defective rnas developed during the long-term vegetative propagation of the virus and by additional mixing by aphid transmission. the viral replication process allows the maintenance of minor amounts of disparate genotypes and defective rnas in these populations. ctv is a member of the closteroviridae possessing a positive-stranded rna genome of approximately 20 kilobases that expresses ...199910377432
sequence analysis of a 282-kilobase region surrounding the citrus tristeza virus resistance gene (ctv) locus in poncirus trifoliata l. raf.citrus tristeza virus (ctv) is the major virus pathogen causing significant economic damage to citrus worldwide, and a single dominant gene, ctv, provides broad spectrum resistance to ctv in poncirus trifoliata l. raf. ctv was physically mapped to a 282-kb region using a p. trifoliata bacterial artificial chromosome library. this region was completely sequenced to about 8x coverage using a shotgun sequencing strategy and primer walking for gap closure. sequence analysis predicts 22 putative gene ...200312586873
new defective rnas from citrus tristeza virus: evidence for a replicase-driven template switching mechanism in their generation.defective rnas (d-rnas) ranging in size from 1968 to 2759 nt were detected in four citrus tristeza virus (ctv) isolates by hybridization of electroblotted dsrnas with two probes specific for the 5'- and 3'-terminal genomic regions. the rnas that hybridized with both probes were eluted, cloned and sequenced. comparison with the sequences of the corresponding genomic regions of the helper virus showed, in all cases, over 99% nucleotide identity and direct repeats of 4-5 nt flanking or in the vicin ...199910092024
grouping and comparison of indian citrus tristeza virus isolates based on coat protein gene sequences and restriction analysis patterns.citrus tristeza virus (ctv) is an aphid-transmitted closterovirus, which causes one of the most important citrus diseases worldwide. isolates of ctv differ widely in their biological properties. ctv-infected samples were collected from four locations in india: bangalore (ctv-b), delhi (ctv-d), nagpur (ctv-n), and pune (ctv-p), and were maintained by grafting into kagzi lime ( citrus aurantifolia (christm. swing.). all isolates produced typical vein clearing and flecking symptoms 6-8 weeks after ...200312664295
defective rnas of citrus tristeza virus analogous to crinivirus genomic rnas.the family closteroviridae includes the genera closterovirus and ampelovirus with monopartite genomes and the genus crinivirus with bipartite genomes. plants infected with the closterovirus, citrus tristeza virus (ctv), often contain one or more populations of defective rnas (drnas). although most drnas are comparatively small (2-5 kb) consisting of the genomic rna termini with large internal deletions, we recently characterized large drnas of approximately 12 kb that retained the open reading f ...200312781717
characterization of grapefruit plants (citrus paradisi macf.) transformed with citrus tristeza closterovirus genes.grapefruit (citrus paradisi macf. cv duncan) plants were transformed with several sequences from citrus tristeza closterovirus (ctv) that varied in terms of position in the ctv genome and virus strain origin in an attempt to obtain resistant plants. the sequences included the capsid protein gene from three different strains, a nontranslatable version of the capsid protein gene, the replicase (rdrp), the minor capsid protein (p27), a highly transcribed gene of unknown function (p20) and the more ...200312789444
the complete genome sequence of the major component of a mild citrus tristeza virus isolate.the genome of the spanish mild isolate t385 of citrus tristeza virus (ctv) was completely sequenced and compared with the genomes of the severe isolates t36 (florida), vt (israel) and sy568 (california). the genome of t385 was 19,259 nt in length, 37 nt shorter than the genome of t36, and 33 and 10 nt longer than those of vt and sy568, respectively, but their organization was identical. t385 had mean nucleotide identities of 81.3, 89.3 and 94% with t36, vt and sy568, respectively. the 3' utr had ...199910092023
new device and method for capture, reverse transcription and nested pcr in a single closed-tube.a device and improved method based on the use of a compartmentalized eppendorf tube that allows capture, reverse transcription and nested-pcr in a single closed-tube has been developed and patented. the main advantages of the system are the high sensitivity obtained, the simplicity, the low risk of contamination and the easy establishment of adequate conditions for nested-pcr. the method has been successfully applied to the detection and characterization of citrus tristeza closterovirus and plum ...199910037824
qtl analysis of citrus tristeza virus-citradia interaction.citrus tristeza virus (ctv) has caused the death of millions of trees grafted on sour orange ( citrus aurantium). however, this rootstock is very well adapted to the mediterranean, semi-arid conditions. the aim of the present research is to genetically analyze the accumulation of ctv in a progeny derived from the cross between c. aurantium and poncirus trifoliata, both resistant to ctv isolate t-346. graft propagation of 104 hybrids was done on healthy sweet orange as a rootstock. three months l ...200414614564
a high-resolution linkage map of the citrus tristeza virus resistance gene region in poncirus trifoliata (l.) raf.resistance to citrus tristeza virus (ctv) was evaluated in 554 progeny of 10 populations derived from poncirus trifoliata. a dominant gene (ctv) controlled ctv resistance in p. trifoliata. twenty-one dominant pcr-based dna markers were identified as linked to ctv by bulked segregant analysis. of the 11 closest markers to ctv, only 2 segregated in all populations. ten of these markers were cloned and sequenced, and codominant rflp markers were developed. seven rflp markers were then evaluated in ...19989755216
modulation of the antigenic reactivity of the citrus tristeza virus coat protein.trapping properties of a panel of monoclonal antibodies (mabs) raised against citrus tristeza virus (ctv) were analyzed in an indirect double-antibody sandwich elisa (i-das-elisa). these antibodies had been previously assigned by serological specificity into five groups (i to v). mabs from group v, which are directed to conformational epitopes, trapped significant amounts of virus antigen from ctv-infected plant tissue at igg concentration above 10 ng/ml. mabs from groups i to iv, which are dire ...19979328572
unusual sequence relationships between two isolates of citrus tristeza virus.the complete, 19,226 nt sequence of the rna genome from vt, a seedling yellows strain of citrus tristeza virus (ctv), was determined and found to have a genome organization identical with that of the previously determined ctv-t36 isolate, except that orf 1 of ctv-vt was 70 nt shorter due to two widely separated 18 nt deletions. sequence comparison of ctv-vt and ctv-t36 revealed approximately 89% identity throughout the ten 3' orfs, but only 60-70% identity throughout orf 1. the 5' nontranslated ...19968811037
multiple species of defective rnas in plants infected with citrus tristeza virus.alemow (citrus macrophylla) and sweet orange (c. sinensis) plants infected, respectively, with several israeli and florida isolates of the citrus tristeza virus (ctv) were found to contain multiple species of rna molecules with features similar to defective-interfering rnas. northern blot hybridizations of dsrnas extracted from serial passages of the israeli vt isolate (ctv-vt) and from different plants infected with a single source of inoculum showed considerable variation both in the presence ...19958525626
estimation of the number of aphids carrying citrus tristeza virus that visit adult citrus trees.aphid species were counted on citrus trees in orchards in valencia, spain, in the spring and autumn of 1997, 1998 and 1999. moericke yellow water traps, the 'sticky shoot' method and counts of established colonies were used in extensive surveys in which 29,502 aphids were recorded and identified. aphis spiraecola and aphis gossypii were the most abundant aphid species. the numbers of aphid species landing on mature trees of grapefruit, sweet orange, lemon and clementine and satsuma mandarins, we ...200415036840
nucleotide sequence of the coat protein gene of citrus tristeza virus: comparison of biologically diverse isolates collected in israel.the sequences of the coat protein genes of four seedling yellows (sy) and four non-sy (nsy) of citrus tristeza virus (ctv) isolates, which were collected in israel over a period of 30 years, were analyzed. pairwise comparisons showed extensive similarities in the nucleotide and amino acid sequences of six isolates designated the vt group. this group consists of three nsy isolates that cause a very mild ctv reaction on the sensitive combination of sweet orange (swo) grafted on sour orange (so), a ...19938279125
three distinct suppressors of rna silencing encoded by a 20-kb viral rna genome.viral infection in both plant and invertebrate hosts requires a virus-encoded function to block the rna silencing antiviral defense. here, we report the identification and characterization of three distinct suppressors of rna silencing encoded by the approximately 20-kb plus-strand rna genome of citrus tristeza virus (ctv). when introduced by genetic crosses into plants carrying a silencing transgene, both p20 and p23, but not coat protein (cp), restored expression of the transgene. although non ...200415505219
fine genetic mapping and bac contig development for the citrus tristeza virus resistance gene locus in poncirus trifoliata (raf.).a map-based cloning strategy has been employed to isolate ctv, a single dominant gene from poncirus trifoliata that confers resistance to citrus tristeza virus (ctv), the most important viral pathogen of citrus. cloning of this gene will allow development of commercially acceptable, virus-resistant cultivars. a high-resolution genetic linkage map of the ctv locus region was developed using a backcross population of 678 individuals. three dna markers that were closely linked or co-segregated with ...200111459195
characterisation of isolates and strains of citrus tristeza closterovirus using restriction analysis of the coat protein gene amplified by the polymerase chain reaction.citrus tristeza virus (ctv) exists as a large number of distinct strains differing in biological properties and with different distributions in citrus producing countries. strategies such as eradication or cross protection, aimed at controlling severe variants of the pathogen, require procedures to identify virus strains accurately and reliably. to fill the need for a rapid, reproducible assay, we have investigated the use of restriction analysis of the ctv coat protein gene amplified using the ...19937903310
aphid biology: expressed genes from alate toxoptera citricida, the brown citrus aphid.the brown citrus aphid, toxoptera citricida (kirkaldy), is considered the primary vector of citrus tristeza virus, a severe pathogen which causes losses to citrus industries worldwide. the alate (winged) form of this aphid can readily fly long distances with the wind, thus spreading citrus tristeza virus in citrus growing regions. to better understand the biology of the brown citrus aphid and the emergence of genes expressed during wing development, we undertook a large-scale 5' end sequencing p ...200315841239
populations of citrus tristeza virus contain smaller-than-full-length particles which encapsidate sub-genomic rna molecules.plants infected by citrus tristeza virus (ctv) contain, in addition to the 2000 nm full-length thread-like virions, a heterogeneous population of smaller particles. the ctv particles and rna extracts from purified ctv preparations were fractionated by sucrose gradient centrifugation and the rna molecules from different fractions translated in a reticulocyte translation system. fractions containing predominantly an rna band of approximately 3.2 kb directed the synthesis of ctv coat protein (cp), ...19957897354
a multiplex polymerase chain reaction method for reliable, sensitive and simultaneous detection of multiple viruses in citrus trees.a multiplex polymerase chain reaction (mpcr) assay was developed to detect six rna and one dna citrus virus: citrus leaf rugose virus (clrv), citrus psorosis virus (cpsv), citrus tatter leaf virus (ctlv), citrus tristeza virus (ctv), citrus variegation virus (cvv), citrus yellow mosaic virus (cymv), and indian citrus ringspot virus (icrsv) from citrus plants. these seven viruses are classified in six different virus genera. degenerate primers were designed based on the respective virus isolate s ...200515951030
assessment of sequence diversity in the 5'-terminal region of citrus tristeza virus from india.twenty-one citrus tristeza virus (ctv) isolates from india were characterized, using genotype-specific multiple molecular markers (mmm) from the 54'-terminal region and two other overlapping primer pairs (cn487/489 and cn488/491) from orf1a (697-1484 nucleotides (nt)). the 5'-terminal genotype-specific primer pairs amplified about 500 bases from the 5'-end of the ctv genomic rna (grna). with the three different mmm, the vt genotype-specific primers amplified 19 indian ctv isolates. the t30-speci ...200515963592
characterization of citrus tristeza virus subgenomic rnas in infected tissue.citrus tristeza virus (ctv) specific rnas extracted from infected citrus tissue were analyzed by northern blot hybridization. rnas were characterized by size and identified using cdna probes specific to nine open reading frames (orfs) identified by the analysis of sequence obtained from cdna clones of the t36 isolate of ctv. sequence specific cdna probes identified the genomic rna as well as subgenomic rnas representing the p33, p65, p61, p27, p25, p18, p13, p20, and p23 orfs in extracts of tota ...19957747429
engineering the genome of grapevine virus a into a vector for expression of proteins in herbaceous plants.grapevine virus a (gva), a species of the genus vitivirus, consists of a approximately 7.4 kb single-stranded rna genome of positive polarity, organized into five open reading frames (orfs). in addition to grape varieties, gva infects nicotiana benthamiana plants and protoplasts. we engineered the genome of gva as a vector that includes duplication of homologous sequences that contain the promoter of the movement protein (mp) sgrna, supplemented by enzymatic restriction sites to be used as a con ...200616298435
evolutionary analysis of genetic variation observed in citrus tristeza virus (ctv) after host passage.we have studied the genetic variability in two genes (p18 and p20) from two groups of citrus tristeza virus (ctv) isolates. one group (isolates t385, t317, t318, and t305) was derived from a spanish source by successive host passages while the other (isolates t388 and t390) was obtained after aphid transmission from a japanese source. a total of 274 sequences were obtained for gene p18 and 451 for p20. in the corresponding phylogenetic trees, sequences derived from the severe isolates (t318, t30 ...200616329002
post-transcriptional gene silencing of the p23 silencing suppressor of citrus tristeza virus confers resistance to the virus in transgenic mexican lime.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 s ...200616429257
citrus tristeza virus transmission by the toxoptera citricida vector: in vitro acquisition and transmission and infectivity immunoneutralization experiments.citrus tristeza virus (ctv) is transmitted by several aphid species in a semi-persistent manner with toxoptera citricida, the brown citrus aphid (brca), being the most efficient. as yet, the molecular interactions between the virus and its aphid vectors have not been determined. this is the first report of aphids acquiring ctv from preparations through an artificial membrane and then transmitting it to receptor plants. the brca fed across artificial membranes on crude tissue preparations made fr ...200616490262
complete sequence of the citrus tristeza virus rna genome.the sequence of the entire genome of citrus tristeza virus (ctv), florida isolate t36, was completed. the 19,296-nt ctv genome encodes 12 open reading frames (orfs) potentially coding for at least 17 protein products. the 5'-proximal orf 1a starts at nucleotide 108 and encodes a large polyprotein with calculated mw of 349 kda containing domains characteristic of (from 5' to 3') two papain-like proteases (p-pro), a methyltransferase (mt), and a helicase (hel). alignment of the putative p-pro sequ ...19957747424
progress on strain differentiation of citrus tristeza virus and its application to the epidemiology of citrus tristeza disease.citrus tristeza virus (ctv) occurs in most citrus producing regions of the world, and it is the most serious viral pathogen of citrus. with the recent establishment of the brown citrus aphid, toxoptera citricida, its most efficient vector, on madeira island (portugal) and in florida (usa) and the countries of the caribbean basin, the impact of ctv is likely to increase in these regions. since there are many strains of ctv and ctv infections frequently occur as mixtures of several strains, it is ...200011137165
[production of polyclonal and monoclonal antibodies against citrus tristeza virus and their efficiency for the detection of the virus].citrus tristeza virus (ctv) was purified from a citrus sample by a modified protocol, and the yield was about 1 mg from 100 g citrus tissues. polyclonal antibody was prepared by immunizing rabbits with the purified ctv preparation with a titer 1:25600 in indirect elisa test. eighteen hybridoma-cell lines secreting monoclonal antibodies (mabs) against ctv were screened after the fusion of mouse myeloma cells (sp2/0) with spleen cells from balb/c immunized with the virus preparation. four hybridom ...200616894900
transgenes consisting of a dsrna of an rnai suppressor plus the 3' utr provide resistance to citrus tristeza virus sequences in nicotiana benthamiana but not in an attempt to utilize post-transcriptional gene silencing (ptgs) as a means to impart resistance against citrus tristeza virus (ctv) into citrus plants, the p23 + 3'utr sequence (p23u) of the vt strain of ctv was engineered to fold into a double-stranded (ds) rna structure. the resulting construct (p23ui) was introduced into nicotiana benthamiana and alemow (citrus macrophylla) plants by agrobacterium-mediated transformation. transgenic p23ui- n. benthamiana were resistant to infection with a ...200616991003
amplification of citrus tristeza virus from a cdna clone and infection of citrus trees.isolates of the closterovirus, citrus tristeza virus (ctv), are populations of disparate genotypes and defective rnas developed during long periods of vegetative propagation of citrus trees. because it has not been possible to obtain pure cultures of the virus, it is not known what components of the population are primarily responsible for induction of diseases. we previously developed an infectious cdna clone from which in vitro-produced rna transcripts could infect protoplasts (satyanarayana e ...200111162822
characterization of the cis-acting elements controlling subgenomic mrnas of citrus tristeza virus: production of positive- and negative-stranded 3'-terminal and positive-stranded 5'-terminal rnas.citrus tristeza virus (ctv), a member of the closteroviridae, has an approximately 20-kb positive-sense rna genome with two 5' orfs translated from the genomic rna and 10 3' genes expressed via nine or ten 3'-terminal subgenomic (sg) rnas. the expression of the 3' genes appears to have properties intermediate between the smaller viruses of the "alphavirus supergroup" and the larger viruses of the coronaviridae. the sgrnas are contiguous with the genome, without a common 5' leader, and are associ ...200111448167
identification and characterization of raspberry mottle virus, a novel member of the closteroviridae.raspberry mosaic is one of the most important viral diseases of raspberry. four virus and virus-like agents, two of which are poorly characterized, have been implicated in the disease complex based on symptom development in rubus indicators. three novel viruses were identified in a red raspberry plant that caused typical raspberry mosaic symptoms when grafted onto indicators. this communication focuses on one of these viruses, raspberry mottle virus (rmov), a new member of the family closterovir ...200717448559
etiology of three recent diseases of citrus in são paulo state: sudden death, variegated chlorosis and huanglongbing.the state of são paulo (ssp) is the first sweet orange growing region in the world. yet, the ssp citrus industry has been, and still is, under constant attack from various diseases. in the 1940s, tristeza-quick decline (t-qd) was responsible for the death of 9 million trees in ssp. the causal agent was a new virus, citrus tristeza virus (ctv). the virus was efficiently spread by aphid vectors, and killed most of the trees grafted on sour orange rootstock. control of the disease resided in replac ...200717505974
genetic variation of citrus tristeza virus isolates from california and spain: evidence for mixed infections and recombination.we examined the population structure and genetic variation of four genomic regions within and between 30 citrus tristeza virus (ctv) isolates from spain and california. our analyses showed that most isolates contained a population of sequence variants, with one being predominant. four isolates showed two major sequence variants in some genomic regions. the two major variants of three of these isolates showed very low nucleotide identity to each other but were very similar to those of other isola ...200111483750
rapid isolation of double stranded rna segments from disulphide crosslinked polyacrylamide gels.a simple method was developed to isolate dsrna segments from disulphide crosslinked polyacrylamide gels. the dsrna preparations from the p4 killer virus strain 77 of ustilago maydis containing 7 genomic segments with molecular sizes ranging from 0.36 to 6.7 kbp, the 20 kbp dsrna associated with the '447' cytoplasmic male sterility in vicia faba and the 23 kbp genomic dsrna of citrus tristeza virus (ctv) were separated on disulphide crosslinked polyacrylamide gels. after uv visualisation, the dsr ...19892760164
transcriptional response of citrus aurantifolia to infection by citrus tristeza virus.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 ...200717617431
coat protein gene duplication in a filamentous rna virus of analysis revealed a striking sequence similarity between the putative 24-kda protein (p24) encoded by open reading frame (orf) 5 of beet yellows closterovirus and the coat protein of this virus encoded by the adjacent orf6. both of these proteins are closely related to the homologous proteins of another closterovirus, citrus tristeza virus. it is hypothesized that the genes for coat protein and its diverged tandem copy have evolved by duplication. phylogenetic analysis using va ...19921409617
regeneration of transgenic citrus plants under non selective conditions results in high-frequency recovery of plants with silenced transgenes.insertion of foreign dna into plant genomes frequently results in the recovery of transgenic plants with silenced transgenes. to investigate to what extent regeneration under selective conditions limits the recovery of transgenic plants showing gene silencing in woody species, mexican lime [ citrus aurantifolia (christm.) swing.] plants were transformed with the p25 coat protein gene of citrus tristeza virus (ctv) with or without selection for nptii and uida. strikingly, more than 30% of the tra ...200212111562
complete genome sequence and analyses of the subgenomic rnas of sweet potato chlorotic stunt virus reveal several new features for the genus crinivirus.the complete nucleotide sequences of genomic rna1 (9,407 nucleotides [nt]) and rna2 (8,223 nt) of sweet potato chlorotic stunt virus (spcsv; genus crinivirus, family closteroviridae) were determined, revealing that spcsv possesses the second largest identified positive-strand single-stranded rna genome among plant viruses after citrus tristeza virus. rna1 contains two overlapping open reading frames (orfs) that encode the replication module, consisting of the putative papain-like cysteine protei ...200212186910
transcription strategy in a closterovirus: a novel 5'-proximal controller element of citrus tristeza virus produces 5'- and 3'-terminal subgenomic rnas and differs from 3' open reading frame controller elements.citrus tristeza virus (ctv) produces more than thirty 3'- or 5'-terminal subgenomic rnas (sgrnas) that accumulate to various extents during replication in protoplasts and plants. among the most unusual species are two abundant populations of small 5'-terminal sgrnas of approximately 800 nucleotides (nt) termed low-molecular-weight tristeza (lmt1 and lmt2) rnas. remarkably, ctv replicons with all 10 3' genes deleted produce only the larger lmt1 rnas. these 5'-terminal positive-sense sgrnas do not ...200312477839
the 23-kda protein coded by the 3'-terminal gene of citrus tristeza virus is an rna-binding protein.the 23-kda protein (p23), encoded by the 3'-proximal gene of the rna of citrus tristeza virus (ctv), was overexpressed in escherichia coli fused to the maltose-binding protein and purified by affinity chromatography. gel retardation and uv crosslinking assays demonstrated that p23 has the ability to cooperatively bind single-stranded rna in a non-sequence-specific manner. formation of the p23-rna complex was dependent on the conformational state of p23 and on the presence of a basic region, but ...200010753724
variation of haplotype distributions of two genomic regions of citrus tristeza virus populations from eastern spain.genetic variation in natural populations of citrus tristeza virus (ctv) was studied using haplotypes detected by single-strand conformation polymorphism (sscp) analysis of two genomic regions (p20 gene and segment a, located in orf1a). analysis of 254 samples from 125 trees, collected at 12 different sites, yielded 8 different haplotypes for p20 and 5 for segment a. the most frequent haplotype of p20 was predominant at all sites, but several sites differed in the predominance of segment a haplot ...200312535101
high efficiency genetic transformation of sour orange (citrus aurantium) and production of transgenic trees containing the coat protein gene of citrus tristeza preliminary experiments on agrobacterium-mediated transformation of citrus, we found transformation events occurring in callus formed from the cambium. factors affecting agrobacterium-sour orange (citrus aurantium l.) interactions, such as culture medium, explant source and culture conditions, were studied to assess competence for transformation in such callus and to improve transformation frequency. cell divisions and redifferentiation from the transgenic cells leading to transformation even ...200012651494
in vivo and in vitro expression analysis of the rna-dependent rna polymerase of citrus tristeza virus.expression of the rna-dependent rna polymerase (rdrp) of citrus tristeza virus (ctv) was studied in vivo and in vitro using a polyclonal antiserum raised against the recombinant ctv-rdrp protein. although a 57-kda ctv-rdrp was expected to be expressed by a +1 translational frameshift at the carboxyl terminus of a 400-kda polyprotein, a 50-kda protein was detected in ctv-infected but not in healthy citrus tissue by western blot. this suggests that the rdrp was cleaved from the ctv polyprotein. th ...200818193157
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.citrus 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.200818199320
effects of modification of the transcription initiation site context on citrus tristeza virus subgenomic rna synthesis.citrus tristeza virus (ctv), a member of the closteroviridae, has a positive-sense rna genome of about 20 kb organized into 12 open reading frames (orfs). the last 10 orfs are expressed through 3'-coterminal subgenomic rnas (sgrnas) regulated in both amounts and timing. additionally, relatively large amounts of complementary sgrnas are produced. we have been unable to determine whether these sgrnas are produced by internal promotion from the full-length template minus strand or by transcription ...200312915539
frameshift mutations in infectious cdna clones of citrus tristeza virus: a strategy to minimize the toxicity of viral sequences to escherichia coli.the advent of reverse genetics revolutionized the study of positive-stranded rna viruses that were amenable for cloning as cdnas into high-copy-number plasmids of escherichia coli. however, some viruses are inherently refractory to cloning in high-copy-number plasmids due to toxicity of viral sequences to e. coli. we report a strategy that is a compromise between infectivity of the rna transcripts and toxicity to e. coli effected by introducing frameshift mutations into "slippery sequences" near ...200312954215
development and characterization of scar markers linked to the citrus tristeza virus resistance gene from poncirus trifoliata.twelve new dominant randomly amplified polymorphic dna (rapd) fragments associated with a single dominant gene for resistance to citrus tristeza virus (ctv) were identified using bulked segregant analysis of an intergeneric backcross family. these and eight previously reported rapds were mapped in the resistance gene (ctv) region; the resulting localized linkage map spans about 32 cm, with nine close flanking markers within 2.5 cm of ctv. seven of 20 rapd fragments linked with the resistance gen ...199718464859
the conserved structures of the 5' nontranslated region of citrus tristeza virus are involved in replication and virion assembly.the genomic rna of different isolates of citrus tristeza virus (ctv) reveals an unusual pattern of sequence diversity: the 3' halves are highly conserved (homology >90%), while the 5' halves show much more dissimilarity, with the 5' nontranslated region (ntr) containing the highest diversity (homology as low as 42%). yet, positive-sense sequences of the 5' ntr were predicted to fold into nearly identical structures consisting of two stem-loops (sl1 and sl2) separated by a short spacer region. th ...200314675624
expressed sequence enrichment for candidate gene analysis of citrus tristeza virus resistance.several studies have reported markers linked to a putative resistance gene from poncirus trifoliata ( ctv-r) located at linkage group 4 that confers resistance against one of the most important citrus pathogens, citrus tristeza virus (ctv). to be successful in both marker-assisted selection and transformation experiments, its accurate mapping is needed. several factors may affect its localization, among them two are considered here: the definition of resistance and the genetic background of prog ...200414624336
polymorphism of a specific region in gene p23 of citrus tristeza virus allows discrimination between mild and severe isolates.the pathogenicity determinants of citrus tristeza virus (ctv) are presently unknown, although transgenic mexican limes over-expressing ctv p23, an rna-binding protein involved in regulating the asymmetrical accumulation of viral rna strands, display typical ctv symptoms. here we compared the predominant sequence variants of gene p23 from 18 ctv isolates of different geographic origin and pathogenicity characteristics. phylogenetic analysis of these sequences revealed three groups of isolates: i) ...200314648289
transgenic resistance to citrus tristeza virus in grapefruit.grapefruit (citrus paradisi) transgenic plants transformed with a variety of constructs derived from the citrus tristeza virus (ctv) genome were tested for their resistance to the virus. most transgenic lines were susceptible (27 lines), a few were partially resistant (6 lines) and only one line, transformed with the 3' end of ctv was resistant. transgene expression levels and sirna accumulation were determined to identify whether the resistance observed was rna-mediated. the responses were vari ...200817882423
citrus tristeza virus: survival at the edge of the movement continuum.systemic invasion of plants by viruses is thought to involve two processes: cell-to-cell movement between adjacent cells and long-distance movement that allows the virus to rapidly move through sieve elements and unload at the growing parts of the plant. there is a continuum of proportions of these processes that determines the degrees of systemic infection of different plants by different viruses. we examined the systemic distribution of citrus tristeza virus (ctv) in citrus species with a rang ...200818434397
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