柑橘衰退病毒基因组的简单重复序列分布分析

柑橘衰退病毒（Citrus tristeza virus,CTV）属于长线性病毒科（Closteroviridae）,是目前已知植物病毒中基因组最大的病毒,其引起的柑橘衰退病对全世界的柑橘产业造成着严重影响。本文以在GenBank登录的32条全长CTV基因组序列为材料,分析简单重复序列（Simple Sequence Repeats,SSRs）在其基因组序列中的分布情况。研究结果显示,在所有的CTV基因组中均有SSRs的分布,SSRs重复次数较少,二型SSRs占主导地位,未在CTV基因组序列中发现五型和六型SSRs。在32条基因组全长序列中仅在5条序列中发现四型SSRs。这是首次以柑橘病毒为材料进行的SSRs分析研究。     

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 progeny.Two progenies derived from P. trifoliata, by self-pollination and by crossing with sour orange (Citrus aurantium), a citrus rootstock well-adapted to arid and semi-arid areas, were used for linkage group-4 marker enrichment. Two new methodologies were used to enrich this region with expressed sequences. The enrichment of group 4 resulted in the fusion of several C. aurantium linkage groups. The new one A(7+3+4) is now saturated with 48 markers including expressed sequences. Surprisingly, sour orange was as resistant to the CTV isolate tested as was P. trifoliata, and three hybrids that carry Ctv-R, as deduced from its flanking markers, are susceptible to CTV. The new linkage maps were used to map Ctv-R under the hypothesis of monogenic inheritance. Its position on linkage group 4 of P. trifoliata differs from the location previously reported in other progenies. The genetic analysis of virus-plant interaction in the family derived from C. aurantium after a CTV chronic infection showed the segregation of five types of interaction, which is not compatible with the hypothesis of a single gene controlling resistance. Two major issues are discussed: another type of genetic analysis of CTV resistance is needed to avoid the assumption of monogenic inheritance, and transferring Ctv-R from P. trifoliata to sour orange might not avoid the CTV decline of sweet orange trees.Communicated by C. Möllers     

The movement and distribution of citrus tristeza virus and citrus exocortis viroid in citrus seedlings1

The systemic movement of citrus tristeza virus (CTV) in sour orange (Citrus aurantium) seedlings and of citrus exocortis viroid (CEVd) in Etrog citron (C. medica) seedlings was studied. The movement of the two pathogens was analysed by detection in sections of roots and stems at different time intervals. Both pathogens were detected initially in the basal parts and the roots and subsequently spread to the shoot. CTV and CEVd moved in young citrus seedlings at similar rates. The findings are consistent with long distance phloem transport of the virus and the viroid. The practical implications of the pattern of systemic movement for diagnosis of infected trees are discussed.     

Serological characterisation of citrus tristeza virus isolates from Israel

Seven monoclonal antibodies (MAbs) showing homologous reactions with the VT strain of citrus tristeza virus (CTV) were tested against 21 CTV strains or isolates, representing the range of biological diversity and the geographical distribution of CTV in Israel. All the CTV strains gave positive reactions in ELISA with polyclonal antibodies and with one MAb (#25#2). Two MAbs; #13#21 and #3/7 reacted with 19 and 12 out of the 21 CTV strains, respectively. Seventeen CTV strains, including all those previously assigned by sequencing their coat protein gene (CPG) to the CPG-VT group (Mawassi, Gafny & Bar-Joseph, 1993), reacted with five or more MAbs. Four CTV strains of minor epidemiological importance, including two members of the CPG-MT group, did not react with five or more of the MAbs. These results indicated the existence of two serogroups of Israeli CTV strains that can be differentiated by MAbs and which closely correlate with and extend the previous CPG grouping. The extensive biological variation within each CPG group confirms recent analyses suggesting that the CTV pathogenic traits are not necessarily associated with a sequence or antigenic variation of the CTV-CPG.     

Efficient production of transgenic citrus plants expressing the coat protein gene of citrus tristeza virus

 The coat protein gene of citrus tristeza virus (CTV) has been introduced into Mexican lime (Citrus aurantifolia Swing.) plants by using an improved Agrobacterium-mediated genetic transformation system. Internodal stem segments from greenhouse-grown seedlings were co-cultivated with A. tumefaciens strain EHA 105 carrying the binary plasmid pBI 121/CTV-CP in a medium rich in auxins that provided the explant cells with the proper treatment to shift them to a competent state for transformation. The transformation frequency was enhanced, and this allowed us to recover 42 transgenic plants from 1200 explants. Regenerated shoots were identified as transformants by performing β-glucuronidase (GUS) assays and subsequently by PCR amplifications of the CTV-CP transgene. Southern analyses revealed that at least one copy of the CTV-CP gene was integrated in all PCR positive plants. Interestingly, 70% of them had linked T-DNAs arranged at one locus. Copy number of the CTV-CP gene varied from one to six among the transgenic lines. Half of them showed truncated T-DNAs in which the left border was lost. Expression of the CTV-CP transgene was demonstrated in 38 out of 42 plants by western analysis and DASI-ELISA. No correlation was found between coat protein expression and transgene copy number or integration pattern. Received: 7 April 1999 / Revision received: 17 June 1999 · Accepted: 24 June 1999     

Transgenic citrus plants expressing the citrus tristeza virus p23 protein exhibit viral-like symptoms

The 23 kDa protein (p23) coded by the 3'-terminal gene of Citrus tristeza virus (CTV), a member of the genus Closterovirus with the largest genome among plant RNA viruses, is an RNA-binding protein that contains a motif rich in cysteine and histidine residues in the core of a putative zinc-finger domain. On this basis, a regulatory role for CTV replication or gene expression has been suggested for p23. To explore whether over-expression of this protein in transgenic plants could affect the normal CTV infection process, transgenic Mexican lime plants were generated carrying the p23 transgene, or a truncated version thereof, under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Constitutive expression of p23 induced phenotypic aberrations that resembled symptoms incited by CTV in non-transgenic lime plants, whereas transgenic plants expressing the p23 truncated version were normal. The onset of CTV-like symptoms in p23 -transgenic plants was associated with the expression of p23, and its accumulation level paralleled the intensity of the symptoms. This demonstrates that p23 is involved in symptom development and that it most likely plays a key role in CTV pathogenesis. This is the first case in which a protein encoded by a woody plant-infecting RNA virus has been identified as being directly involved in pathogenesis in its natural host. This finding also delimits a small region of the large CTV genome for the future mapping of specific pathogenic determinants.     

A localized linkage map of the citrus tristeza virus resistance gene region

A localized genetic linkage map was developed of the region surrounding the citrus tristeza virus (CTV) resistance gene (designated Ctv) from Poncirus trifoliate L., a sexually compatible Citrus relative. Bulked segregant analysis (BSA) was used to identify potential resistance-associated RAPD fragment markers in four intergeneric backcross families that were segregating for CTV resistance. Eight RAPD fragments were found that were consistently linked to Ctv in the four families. Map distances and locus order were determined with MAPMAKER 3.0, using the results obtained from 59 individuals in the largest family. Also, a consensus map was constructed with JOINMAP 1.3, using pooled results from the four backcross families. Marker orders were identical, except for 1 marker, on these independently developed maps. Family-specific resistance-associated markers were also identified, as were numerous susceptibility-associated markers. The identification of markers tightly linked to Ctv will enable citrus breeders to identify plants likely to be CTV-resistant by indirect, marker-assisted selection, rather than by labor-intensive direct challenge with the pathogen. These markers also provide a basis for future efforts to isolate Ctv for subsequent genetic manipulation.Florida Agricultural Experimental Station Journal Series No. R-04491     

Monoclonal antibody-based serological methods for detecting Citrus tristeza virus in citrus groves

Citrus tristeza virus(CTV) is one of the most economically important citrus viruses and harms the citrus industry worldwide.To develop reliable and effective serological detection assays of CTV,the major capsid protein(CP) gene of CTV was expressed in Escherichia coli BL21(DE3) using the expression vector p ET-28 a and purified through Ni~+-NTA affinity chromatography.The recombinant protein was used to immunize BALB/c mice.Four hybridoma cell lines(14B10,14H11,20D5,and20G12) secreting monoclonal antibodies(MAbs) against CTV were obtained through conventional hybridoma technology.The titers of MAb-containing ascitic fluids secreted by the four hybridoma lines ranged from 10-6 to 10-7 in indirect enzyme-linked immunosorbent assay(ELISA).Western blots showed that all four MAbs could specifically react with CTV CP.Using the prepared MAbs,dot-ELISA,Tissue print-ELISA,and triple antibody sandwich(TAS)-ELISA were developed to detect CTV in tree nurseries and epidemiological studies.The developed dot-ELISA and TAS-ELISA methods could detect CTV in crude extracts of infected citrus leaves with dilutions of 1:2560 and1:10,240(w/v,g/m L),respectively.Tissue print-ELISA was particularly useful for large-scale field sample detection,mainly owing to its simplicity and lack of sample preparation requirements.The field survey revealed that CTV is prevalent on citrus trees in the Chongqing Municipality,Jiangxi Province,and Zhejiang Province of China.The coincidence rate of serological and RT-PCR test results reached more than 99.5%.The prepared MAbs against CTV and established sensitive and specific serological assays have a significant role in the detection and prevention and control of CTV in our country.     

Differentiation of citrus tristeza closterovirus (CTV) isolates by single-strand conformation polymorphism analysis of the coat protein gene

Citrus tristeza closterovirus (CTV) isolates of several geographical origins were compared for variations in their coat protein (CP) gene by analysis of single-strand conformation polymorphism (SSCP). The CP gene of 17 isolates was reverse transcribed, amplified by polymerase chain reaction (PCR), and 22 clones were inserted into a plasmid vector. These clones were sequenced and found to have between 91.7% and 99.8% sequence homology. Clones were amplified and the PCR products denatured and compared by SSCP analysis in 8% polyacrylamide gels. Using two different electrophoretic conditions, the patterns were different for 16 or 17 clones. Four pairs of clones (T36/T66, P1/Q2, 03/8Q, and E1/E2) differing by 10, 2, 1 and 1 nucleotides, respectively, could not be distinguished using either condition. When these clones were compared by SSCP after digestion with Eco91I (BstEII) three of the pairs (T36/T66, P1/Q2, and 03/8Q) could be differentiated, whereas the clones E1 and E2 (differing by 1 nucleotide) remained indistinguishable. Thus, SSCP analysis combining two electrophoretic conditions and restriction of eight clones with Eco91I allowed discrimination between 21 of the 22 CP gene clones selected. SSCP analysis may provide a procedure to identify and differentiate CTV isolates based on comparisons of several genes or gene regions. It is rapid and cheap and may drastically reduce the amount of sequencing necessary for accurate comparisons.     

Citrus tristeza virus replicates and forms infectious virions in protoplasts of resistant citrus relatives

Citrus tristeza virus (CTV) is the most economically important viral disease of citrus worldwide. Cultivars with improved CTV tolerance or resistance are needed to manage CTV-induced diseases. The citrus relatives Poncirus trifoliata (L.) Raf., Swinglea glutinosa (Blanco) Merr., and Severinia buxifolia (Poir) Ten. are potential sources of CTV resistance, but their resistance mechanisms are poorly characterized. As a first step to examine the mechanisms of resistance to CTV in these citrus relatives and selected Citrus × Poncirus hybrids, it was necessary to develop methods for protoplast isolation and viral inoculation to allow examination of CTV multiplication in this range of citrus varieties and relatives. Leaf and/or cultured cell protoplasts were isolated and inoculated with four biologically distinct CTV isolates. Northern-blot hybridization analyses for progeny RNAs and immuno-electron microscopy assays for newly produced virions showed that CTV replicated and produced infectious particles in protoplasts from all of the resistant plants tested. These results suggest that resistance to CTV observed at the plant level results from a lack of virus movement and/or some induced resistance response, rather than lack of viral multiplication at the cellular level.     

Citrus tristeza virus (CTV) resistance in transgenic citrus based on virus challenge of protoplasts

Summary A strategy for the sereening of candidate virus-derived sequences to provide RNA-mediated citrus tristeza virus (CTV) resistance and early selection of virus-resistant citrus is presented. The system is based on the polyethylene glycol-(PEG) mediated cotransformation of protoplasts using virus-derived sequences and green fluorescent protein as a single selectable marker, followed by an in vitro assay of virus inoculation into transgenic protoplasts to determine the level of citrus tristeza virus replication. A cotransformation rate higher than 20% allowed selection of several clones carrying the desired transgenes. Efficient in vitro inoculation of virus in transgenic protoplasts was performed. Tobacco mosaic virus virions were used as a control in order to check eitrus protoplast viability. Different CTV replication levels were detected in transgenic clones. Only one clone showed no replication of CTV. Considerations regarding selection of candidate virusderived sequences and virus challenge of transgenic cells are presented.     

Sscp analysis of variations in haplotypes of citrus tristeza virus isolated from yuzu (Citrus junos) in geographically separate regions of Korea

Yuzu (Cittus junos) trees were examined from six geographically separate provinces in the Republic of Korea, including four islands (Geoje, Namhae, Wan, and Jeju), 1 peninsula (Goheung), and 1 inland area (Boseong). The population of sequence variants of citrus tristeza virus (CTV) was isolated and analyzed by single-strand conformation polymorphism (SSCP) analysis of cDNA from thep20 gene. SSCP profiles of 65 PCR products showed different band patterns but with similar intensities. Sixteen haplotypes were subgrouped according to their SSCP profiles and severity of symptoms. Their genomes were sequenced and compared. DNA analysis of thep20 genes revealed nucleotide identities ranging from 88-99.8%. Based on SSCP analysis, the pathologically mild isolates of CTV yielded two to three DNA bands, whereas the most virulent isolates contained more than two bands. Comparisons of these physically separate haplotypes suggest that CTV isolates with multiple SSCP profiles could have arisen as a result of a mixed infection and genetic recombination of two divergent isolates. Plants with severe disease symptoms, such as stem pitting, closely corresponded to a CTV strain showing typical SSCP profiles in Florida (USA) and Japan.     

Efficient search for new resistant genotypes to the citrus tristeza closterovirus in the orange subfamily Aurantioideae

 Virulent isolates of the citrus tristeza virus (CTV) are continuously arising and their spread threatens the world citrus industry. Methods for effective utilization of material conserved in germplasm banks are needed in plant improvement. Two objectives are pursued in the present paper: a search for new CTV-resistant genotypes and tests of two strategies for this search. One of these tests is based on a study of genetic relationships among genera and species of the orange subfamily and the other on scores of molecular markers known to be linked to the CTV-resistant locus. Sampled plants were graft-inoculated with a mild CTV isolate (T-346) and two virulent ones (T-388 and T-305). Susceptible plants were those where CTV multiplication was detected beyond 4 months after inoculation. All cultivars of Poncirus trifoliata tested, as well as Severinia buxifolia and Atalantia ceylanica, were resistant to the three CTV isolates; Fortunella crassifolia (Meiwa kumquat) resists two of them. The finding of CTV resistance in this species, closely related to cultivated Citrus species, opens a new arena for CTV-resistance improvement of oranges and mandarines by sexual hybridization. The searching strategy based on phylogenetic data has been successful, whereas the other one may be worthwhile only when the search is restricted to the species where linkage analysis is available. A good documentation system that allows quick sampling of accessions to build up core collections and where the location of new and useful genes could be easily worked out, is suggested to enhance germplasm utilization. Received: 27 April 1997 / Accepted: 5 June 1997     

Molecular markers flanking citrus tristeza virus resistance gene from Poncirus trifoliata (L.) Raf.

 Two segregating populations for citrus tristeza virus (CTV) resistance derived from Poncirus trifoliata var ‘Flying Dragon’ by self-pollination and pollination to Citrus medica L. var ethrog ‘Arizona’ were inoculated with a common CTV isolate. The presence of virus was checked by the Double Antibody Sandwich Enzyme-Linked Assay and Direct Tissue Blot Inmunoassay at 3, 6, and 12 months after inoculation. Seven RAPDs were found linked to the CTV resistance gene by bulked segregant analysis. The closest linked RAPDs were cloned to obtain linked codominant RFLPs and to increase the precision of the genetic distance estimation. The CTV resistance gene seems to be located between cW18 and cK16. Differences in genetic distances among progenies are large and can be explained by genome-wide reduction in the recombination of progeny derived from male versus female gametes. Received: 5 June 1996 / Accepted: 26 July 1996     

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 gene were cloned and sequenced, and their sequences were used to design longer primers to develop sequence characterized amplified region (SCAR) markers that can be utilized reliably in marker-assisted selection, high-resolution mapping, and map-based cloning of the resistance gene. All seven cloned RAPDs were converted successfully into SCARs by redesigning primers, optimizing PCR parameters (especially the annealing temperature), or digesting amplification products with restriction enzymes. Four of the seven remained dominant markers, displaying presence-absence polymorphism patterns; the other three detected restriction site changes or length variations and thus were transformed into codominant markers. Two genomic regions rich in variability were also detected by two codominant SCAR markers.     

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 subgenomic RNA (sgRNA), showed that they were identical in length and sequence to the common 3' sequence of the D-RNAs. These results imply that viral sgRNA messengers also function as building components for genomic rearrangement and exchange of complete viral genes.     

Gibberellin-ethylene interaction controls radial expansion in citrus roots

The involvement of gibberellins (GAs) and ethylene in the process of root radial expansion was studied in young seedlings of Carrizo citrange [Citrus sinensis (L.) Osb. × Poncirus trifoliata (L.) Raf.]. The GA inhibitors cycocel, paclobutrazol, and tetcyclacis enhanced radial expansion of the root tip (up to 2.3-fold) as a result of increases in stele diameter and inner cortex width. The GA deficiency increased cell number and width, and changed the polarity of growth, generating wider and shorter cortical cells in the elongation zone. In the presence or absence of GA inhibitors, GA₃ decreased root tip width and reduced all parameters related to radial expansion. The ethylene inhibitors (aminooxyacetic acid; cobalt ions, CoCl₂; silver thiosulfate) suppressed swelling induced by GA deficiency, generating thinner cells just as GA₃ did. In contrast to GA₃, ethylene inhibitors produced longer cells strongly resembling those of the untreated seedlings. Ethylene released by ethephon did not modify root tip width in control plants, while root diameter behind the root tip was increased. In the presence of low and ineffective concentrations of cycocel, the ethylene precursor 1-aminocyclopropane-1-carboxylic acid increased radial expansion of root tips (1.3-fold) and changed the polarity of growth, producing wider and shorter inner cortical cells as GA inhibitors did. These observations imply, first, that ethylene is the hormonal effector of the process of root radial expansion and, second, that the endogenous GAs modulate the promotive response of ethylene. Received: 4 October 1996 / Accepted: 25 December 1996