RESUMEN
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.
Asunto(s)
Closterovirus/genética , Closterovirus/inmunología , Interacciones Huésped-Patógeno/inmunología , Inmunidad de la Planta/inmunología , ARN Largo no Codificante/inmunología , ARN Viral/inmunología , Citrus/virología , Virus ADN/genética , Genoma Viral , Interacciones Huésped-Patógeno/genética , Proteínas Mitocondriales , Oxidorreductasas , Enfermedades de las Plantas/virología , Hojas de la Planta/virología , Proteínas de Plantas , ARN Viral/genética , Ácido Salicílico , Nicotiana/virología , Carga Viral , Replicación ViralRESUMEN
Citrus tristeza virus (CTV) is the most economically important virus found on citrus and influences production worldwide. The 3' half of the RNA genome is generally conserved amongst sources, whereas the 5' portion is more divergent, allowing for the classification of the virus into a number of genotypes based on sequence diversity. The acknowledged genotypes of CTV are continually being expanded, and thus far include T36, T30, T3, VT, B165, HA16-5, T68 and RB. The genotype composition of the CTV populations of a potential cross protection source in Mexican lime was studied whilst comparing different techniques of viral population characterization. Cloning and sequencing of an ORF 1a fragment, genotype specific RT-PCRs and Illumina sequencing of the p33 gene as well as RNA template enrichment through immuno-capture was done. Primers used in the cloning and sequencing proved to be biased towards detection of the VT genotype. RT-PCR and Illumina sequencing using the two different templates provided relatively comparable results, even though the immuno-captured enriched template provided less than expected CTV specific data, while the RT-PCRs and p33 sequencing cannot be used to make inferences about the rest of the genome; which may vary due to recombination. The source was found to contain multiple genotypes, including RB and VT. When choosing a characterization method, the features of the virus under study should be considered. It was found that Illumina sequencing offers an opportunity to gain a large amount of information regarding the entire viral genome, but challenges encountered are discussed.
Asunto(s)
Citrus/virología , Closterovirus/genética , Genoma Viral , Análisis de Secuencia de ADN/métodos , Closterovirus/inmunología , Protección Cruzada , Cartilla de ADN , Variación Genética , Genotipo , Sistemas de Lectura Abierta , Filogenia , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/virología , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Análisis de Secuencia de ADN/instrumentaciónRESUMEN
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 pET-28a and purified through Ni+-NTA affinity chromatography. The recombinant protein was used to immunize BALB/c mice. Four hybridoma cell lines (14B10, 14H11, 20D5, and 20G12) 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 and 1:10, 240 (w/v, g/mL), 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.
Asunto(s)
Citrus/virología , Closterovirus/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática/métodos , Enfermedades de las Plantas/virología , Animales , Anticuerpos Monoclonales/análisis , Anticuerpos Antivirales/análisis , Proteínas de la Cápside/análisis , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Closterovirus/genética , Closterovirus/inmunología , Ensayo de Inmunoadsorción Enzimática/instrumentación , Inmunización , Ratones , Ratones Endogámicos BALB CRESUMEN
The complete genome sequence of a highly divergent strain of Grapevine leafroll-associated virus 4 (GLRaV-4) was determined using 454 pyrosequencing technology. This virus, designated GLRaV-4 Ob, was detected in Vitis vinifera 'Otcha bala' from our grapevine virus collection at Agroscope. The GLRaV-4 Ob genome length and organization share similarities with members of subgroup II in the genus Ampelovirus (family Closteroviridae). Otcha bala was graft-inoculated onto indicator plants of cultivar Gamay to evaluate the biological properties of this new strain, and typical leafroll symptoms were induced. A monoclonal antibody for the rapid detection of GLRaV-4 Ob by enzyme-linked immunosorbent assay is available, thus facilitating large-scale diagnostics of this virus. Based on the relatively small size of the coat protein, the reduced amino acid identity and the distinct serological properties, our study clearly shows that GLRaV-4 Ob is a divergent strain of GLRaV-4. Furthermore, molecular and serological data revealed that the AA42 accession from which GLRaV-7 was originally reported is in fact co-infected with GLRaV-4 Ob and GLRaV-7. This finding challenges the idea that GLRaV-7 is a leafroll-causing agent.
Asunto(s)
Closterovirus/aislamiento & purificación , Genoma Viral/genética , Enfermedades de las Plantas/virología , Vitis/virología , Secuencia de Bases , Closterovirus/genética , Closterovirus/inmunología , Closterovirus/ultraestructura , Ensayo de Inmunoadsorción Enzimática , Secuenciación de Nucleótidos de Alto Rendimiento , Datos de Secuencia Molecular , Filogenia , Hojas de la Planta/virología , ARN Viral/química , ARN Viral/genética , Análisis de Secuencia de ARNRESUMEN
It has been known that there exists serological differentiation among Citrus tristeza virus (CTV) isolates. The present study reports three linear epitopes (aa 48-63, 97-104, and 114-125) identified by using bacterially expressed truncated coat proteins and ten monoclonal antibodies against the native virions of CTV-S4. Site-directed mutagenesis analysis demonstrated that the mutation D98G within the newly identified epitope (97)DDDSTGIT(104) abolished its reaction to MAbs 1, 4, and 10, and the presence of G98 in HB1-CP also resulted in its failure to recognize the three MAbs. Our results suggest that the conformational differences in the epitope I (48)LGTQQNAALNRDLFLT(63) between the CPs of isolates S4 and HB1 might contribute to the different reactions of two isolates to MAbs 5 and 6. This study provides new information for the antigenic structures of CTV, and will extend the understanding of the processes required for antibody binding and aid the development of epitope-based diagnostic tools.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Proteínas de la Cápside/química , Proteínas de la Cápside/inmunología , Citrus/virología , Closterovirus/inmunología , Enfermedades de las Plantas/virología , Secuencia de Aminoácidos , Proteínas de la Cápside/genética , Closterovirus/química , Closterovirus/genética , Mapeo Epitopo , Datos de Secuencia MolecularRESUMEN
Viral cross protection in plants is known as an acquired immunity phenomenon, where a mild virus isolate/strain can protect plants against economic damage caused by a severe challenge strain/isolate of the same virus. Mild strain cross protection (MSCP) has been used extensively to control losses caused by a few major virus diseases in some parts of the world. So far, none of the many proposed mechanisms can fully explain the intact process of MSCP. In fact, it may be that different mechanisms are involved in MSCP against different viruses, even when different research approaches are used for the same virus, different mechanisms could be proposed. The molecular detail of MSCP still remains unclear, although several lines of evidence imply that the resistance is protein and/or RNA mediated. Some data to date have shown that a minimum time (a few days to less than a month) is required for the mild virus strain to establish MSCP. To investigate interference among virus strains and the plant host at an early stage of MSCP at a subcellular level, we developed a rapid micro-extraction method for the preparation of total nucleic acid (TNA), combined with other molecular methods, to monitor the interaction of virus strains at short time intervals in young plants. This method was initially developed to further study the mechanism of MSCP against Citrus tristeza virus, but has potentially widespread application to other viruses after having been efficiently used to extract over 50,000 TNA samples of citrus viruses, viroids, and bacteria.
Asunto(s)
Citrus/virología , Closterovirus/genética , Protección Cruzada , Enfermedades de las Plantas/prevención & control , Citrus/genética , Closterovirus/inmunología , Closterovirus/patogenicidad , Protección Cruzada/genética , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/virologíaRESUMEN
Citrus tristeza virus (CTV) causes one of the most destructive viral diseases of citrus worldwide. Generation of resistant citrus genotypes through genetic engineering could be a good alternative to control CTV. To study whether production of single-chain variable fragment (scFv) antibodies in citrus could interfere and immunomodulate CTV infection, transgenic Mexican lime plants expressing two different scFv constructs, separately and simultaneously, were generated. These constructs derived from the well-referenced monoclonal antibodies 3DF1 and 3CA5, specific against CTV p25 major coat protein, whose mixture is able to detect all CTV isolates characterized so far. ScFv accumulation levels were low and could be readily detected just in four transgenic lines. Twelve homogeneous and vigorous lines were propagated and CTV-challenged by graft inoculation with an aggressive CTV strain. A clear protective effect was observed in most transgenic lines, which showed resistance in up to 40-60% of propagations. Besides, both a delay in symptom appearance and attenuation of symptom intensity were observed in infected transgenic plants compared with control plants. This effect was more evident in lines carrying the 3DF1scFv transgene, being probably related to the biological functions of the epitope recognized by this antibody. This is the first report describing successful protection against a pathogen in woody transgenic plants by ectopic expression of scFv recombinant antibodies.
Asunto(s)
Citrus aurantiifolia/genética , Citrus aurantiifolia/virología , Closterovirus/inmunología , Enfermedades de las Plantas/prevención & control , Anticuerpos de Cadena Única/genética , Anticuerpos Antivirales/genética , Secuencia de Bases , Citrus aurantiifolia/inmunología , Closterovirus/patogenicidad , Cartilla de ADN/genética , Expresión Génica , Ingeniería Genética , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/virología , Planticuerpos/genética , Plantas Modificadas GenéticamenteRESUMEN
The viral population in sweet orange plants, either healthy or pre-inoculated with the asymptomatic isolate of Citrus tristeza virus (CTV) T32, and then graft- or aphid-inoculated with the stem-pitting isolate T318, was characterized with respect to symptom expression, reaction with monoclonal antibody MCA13, single-strand conformation polymorphism (SSCP) of genes p18 and p20, bi-directional RT-PCR, and dot-blot hybridisation. All plants inoculated with T318, with or without pre-inoculation, showed stem pitting, reacted with MCA13, had the SSCP profile characteristic of this isolate, and in bi-directional RT-PCR yielded a 450-bp DNA product associated with severe isolates, indicating that T32 afforded no protection against T318. The latter isolate had two main sequence variants, the minor one of which was indistinguishable from the main T32 sequence, and both were detected in most plants that were graft-inoculated with T318. However, the T32 variant was not detected in plants that were aphid-inoculated only with T318 and also showed stem pitting. This suggested an association of symptoms with the major T318 sequence and preferential transmission of this variant by aphids. The T318-specific variant accumulated more than the T32 variant in plants in which both were replicating, suggesting a higher fitness of the former. Our results clearly emphasize the potential threat of severe CTV variants in areas where mild isolates are presently predominant.
Asunto(s)
Citrus sinensis/virología , Closterovirus/genética , Closterovirus/patogenicidad , Animales , Áfidos/virología , Secuencia de Bases , Closterovirus/inmunología , Closterovirus/aislamiento & purificación , Cartilla de ADN/genética , ADN Viral/genética , Variación Genética , Insectos Vectores/virología , Enfermedades de las Plantas/virología , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Virulencia/genéticaRESUMEN
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 hybridoma-cell lines were selected randomly for later analysis. The results indicated that the titers of ascetic fluids against these hybridoma cell lines ranged from 1:51200 to 1:204800 in indirect ELISA, and their isotypes and subclasses were IgG2a for 2G and 3H and IgG2b for IE and 4H. These four Mabs were used to detect CTV in citrus samples in different sources. Results showed that TAS-ELISA with polyclonal antibody as trapping antibody and monoclonal antibody as testing antibody had a higher specificity and sensitivity than PAS-ELISA. Four Mabs showed different intensities of serological reaction with different CTV isolates. However, much work remains for realizing the characteristics and the serological relationships among these isolates.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Anticuerpos Antivirales/biosíntesis , Citrus/virología , Closterovirus/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Western Blotting , Closterovirus/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática , Ratones , Ratones Endogámicos BALB C , ConejosRESUMEN
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 from CTV-infected bark tissue were able to transmit CTV to virus-free receptor plants at low rates. CTV p20, p27 and p25 proteins, detected by Western blots, were present in all crude tissue preparations from CTV-infected plants. Partially purified CTV preparations were not transmitted by the BrCA in this manner. Infectivity immunoneutralization experiments were conducted where aphids were forced to feed in vitro on three CTV-specific antibodies (p25, p27 and p20) before being placed on receptor plants following a 48h acquisition feed on CTV-infected source plants. There were no differences in transmission rates among the majority of treatments and the control treatments. However, in one infectivity immunoneutralization experiment, the CTV p20 antibodies significantly enhanced CTV transmission compared to buffer only, pre-immune antiserum or no antibody control treatments. This suggests the inactivity of CTV p20 aids BrCA transmission of virions.
Asunto(s)
Áfidos/virología , Closterovirus , Insectos Vectores/virología , Enfermedades de las Plantas/virología , Animales , Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos , Western Blotting , Citrus/metabolismo , Citrus/virología , Closterovirus/química , Closterovirus/inmunología , Ecosistema , Pruebas de Neutralización , Proteínas Virales/análisis , Proteínas Virales/inmunología , Proteínas Virales/metabolismoRESUMEN
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 comparable purity. The purified CP was partially digested with either V8 or papain endo-protease, and the peptides generated were separated and electroblotted to a membrane. Protein blots were tested with four monoclonal antibodies and one source of polyclonal antibodies. The serological maps generated by papain allowed differentiation of all the isolates examined, and those generated by V8 endoprotease allowed discrimination of four of the five isolates tested. Some of these isolates had been indistinguishable based on their reactivity in DASI-ELISA, dsRNA pattern and biological characterization. Serological analysis of peptide maps, as described below, allowed accurate comparison of CTV isolates with minimum amounts of p25 CP and proved superior to other techniques for discriminating CTV isolates.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Cápside/inmunología , Citrus/virología , Closterovirus/clasificación , Mapeo Peptídico , Cápside/aislamiento & purificación , Closterovirus/inmunología , Closterovirus/aislamiento & purificación , Immunoblotting , Papaína/metabolismo , Virión/químicaRESUMEN
The 3'-proximal open reading frames (ORFs) of beet yellows closterovirus, California isolate (BYV-CA), were sequenced and the expression of the corresponding proteins analyzed. The nucleotide sequence of ORF 5 (coding for p24) was most conserved compared with ORF 7 (coding for p20) and ORF 8 (coding for p21) among the isolates analyzed. Polyclonal antisera were produced to GST fusion proteins of p24, p20, and p21. Accumulation of p24, CP, p20 and p21 was studied in infected Tetragonia expansa plants and Chenopodium quinoa protoplasts. All four proteins were expressed in all tissues (old leaves, young leaves and stems), and most abundantly in young leaves. The subcellular localization of each protein in different tissues showed that compared with p24, CP and p21, p20 accumulated less in transfected protoplasts. Immunogold labeling in sugarbeet with p24 and CP antisera demonstrated co-localization of p24 and CP in vascular petiole tissues. In infectivity neutralization tests, antisera against p24 and CP greatly reduced transmission of BYV by viruliferous aphids compared with viruliferous aphids fed on preimmune serum or antiserum to p21.
Asunto(s)
Closterovirus/genética , Closterovirus/inmunología , Proteínas Virales/genética , Proteínas Virales/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales , Áfidos/virología , Secuencia de Bases , Closterovirus/patogenicidad , Cartilla de ADN/genética , ADN Viral/genética , Escherichia coli/genética , Expresión Génica , Inmunohistoquímica , Microscopía Electrónica , Datos de Secuencia Molecular , Pruebas de Neutralización , Sistemas de Lectura Abierta , Plantas/ultraestructura , Plantas/virología , Reacción en Cadena de la Polimerasa , Protoplastos/virología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Proteínas Virales/metabolismoRESUMEN
A reverse-transcription polymerase chain reaction (RT-PCR) was developed to detect beet yellows closterovirus (BYV) in plants and single aphids using primers spanning the conserved regions of the published sequences of the coat protein gene and open reading frames 7 and 8. Three total RNA extraction procedures were examined and all were found to produce RNA of sufficient quality for RT-PCR, although the RNA extraction kit supplied by Flowgen was found to be the most versatile for the extraction of BYV from individual aphids. When 60 aphids, which had fed on virus infected sugar beet were tested by RT-PCR, 55% of individuals were found to contain BYV. Two groups of 36 individual aphids were tested by TAS-ELISA using a specific BYV monoclonal antibody; 53% gave positive absorbance values when a substrate amplification system was used, but none was found to contain virus when the system was replaced with the substrate p-nitrophenyl phosphate. An immunocapture RT-PCR method was shown to detect BYV regardless of whether the RNA had been extracted from the trapped particles or the reverse transcription and PCR mixtures were added to the wells containing intact particles. The RT-PCR method is now used to determine the numbers of BYV carrying aphids migrating into sugar-beet crops.
Asunto(s)
Closterovirus/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática/métodos , Reacción en Cadena de la Polimerasa/métodos , Animales , Áfidos/virología , Closterovirus/genética , Closterovirus/inmunología , Transcripción Genética , Verduras/virología , AguaRESUMEN
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 antibodies by the indirect ELISA. Considerably higher ELISA titers were obtained from animals that were boosted with ppCTV than with rCP. Boosting with partially purified native antigens after priming with recombinant antigens is expected to extend the applicability of the antisera for detecting other structural and non-structural viral antigens by trapping ELISA.
Asunto(s)
Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Citrus/virología , Closterovirus/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Animales , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/sangre , Antígenos Virales/genética , Cápside/genética , Cápside/inmunología , Pollos , Closterovirus/genética , Closterovirus/aislamiento & purificación , Inmunización Secundaria , Conejos , Proteínas Recombinantes de Fusión , OvinosRESUMEN
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 directed to linear, continuous epitopes, performed poorly as coating antibodies, even at a 1 microgram/ml concentration of the IgG's, indicating that the respective linear epitopes were inaccessible. However, when Mabs from groups I to IV were combined with a small amount of Mabs from group V, a substantial increase in trapping of the CTV antigen was recorded. In this 'two antibody-binding assay' previously cryptic, linear epitopes of the CTV CP apparently became accessible to the Mabs from groups I to IV. Modulation of the antigenic reactivity of the CTV CP was also recorded upon binding of the Mabs directed to the conformational epitopes in solution. Induced exposure of the linear epitopes of the CTV CP was revealed in 'two antibody-binding assays' with pairwise combinations of different mouse Mabs and several rabbit and chicken polyclonal antisera with different serological specificities, including antisera to bacterially expressed CP fragments. This mixed coating in I-DAS-ELISA resulted in substantially increased efficiency of the virus antigen trapping by antisera produced against bacterially expressed protein fragments and an increased sensitivity of the CTV detection after optimization of the ratio between conformational and linear antibodies.
Asunto(s)
Proteínas de la Cápside , Cápside/inmunología , Citrus/virología , Closterovirus/inmunología , Epítopos/inmunología , Anticuerpos Antivirales/química , Anticuerpos Antivirales/metabolismo , Especificidad de Anticuerpos , Sitios de Unión de Anticuerpos , Cápside/química , Closterovirus/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática , Epítopos/química , Sueros Inmunes/química , Conformación Proteica , SolucionesRESUMEN
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 uninfected citrus. Most of the p23 protein was found in the soluble, cytoplasmic fraction. Comparison of the sequence of p23 genes from several biologically and geographically diverse CTV isolates indicated a high degree of conservation for this gene and for the RNA binding motif in particular. A cluster dendrogram of the deduced amino acid sequences correlated with the biological properties of the isolates, forming distinct groups of mild, quick decline on stem pitting-inducing isolates. Therefore it is possible that, in addition to the capsid protein gene, the p23 gene also may serve as an indicator for disease severity.