Application of cDNA Macroarray for Simultaneous Detection of 12 Potato Viruses.

A complementary DNA (cDNA) macroarray was developed for simultaneous detection of 12 different potato viruses. A suitable region in the viral genome for each was selected for Alfalfa mosaic virus, Cucumber mosaic virus, Potato aucuba mosaic virus, Potato leafroll virus, Potato mop-top virus, Potato virus A, Potato virus M, Potato virus S, Potato virus X, Potato virus Y, Tomato ringspot virus, and Tomato spotted wilt virus, and their respective cDNAs were cloned into plasmid vectors. Capture probes for each virus ranging from 290 to 577 bp were generated by polymerase chain reaction (PCR) and immobilized on a nylon membrane. Total RNAs were extracted from each of these virus infected-plants, and cDNAs were synthesized from the RNA extracts using a random 9-mer primer. Subsequently, PCR reactions were performed using one primer pair for each of the 12 viruses. During PCR, amplified cDNAs were labeled with biotin and used as a target for hybridization analyses on a macroarray membrane. Hybridization signals between capture probes for the 12 viruses and their respective target cDNAs were observed using chemiluminescent or colorimetric detection. In all viruses, hybridization signals with capture probes were detected only when homologous virus targets were examined, and no hybridization to healthy plant extract was observed, facilitating identification of each virus. The results by colorimetric detection agreed with those obtained using chemiluminescence. The macroarray method developed was 5 × 102 to 4 × 106 times more sensitive than enzyme-linked immunosorbent assay and 5 to 5 × 104 times more sensitive than reverse-transcription PCR, except for Alfalfa mosaic virus. Colorimetric detection and substantial reduction in cross-hybridization signals much improved the method compared with other array-based detection methods for practical use.

Molecular characterization of Hop mosaic virus: its serological and molecular relationships to Hop latent virus.

The 3'-terminal sequence of hop mosaic virus (HpMV) genomic RNA was determined. A cDNA of approximately 1.8 kbp was amplified from the HpMV genome by 3' RACE using a degenerate primer, which was designed to anneal to the overlapping region of open reading frames (ORFs) 2 and 3 of eight carlavirus genomes. The sequence contained three ORFs, encoding proteins of 7-, 34-, and 11-kDa, which corresponded to ORFs 4, 5, and 6 of the carlavirus genome, respectively. The amino acid sequence of ORF 5, encoding the coat protein (CP) of HpMV, shows the highest identity (67%) to that of Hop latent virus (HpLV). The HpMV CP N-terminal sequence differs from that of HpLV, but the central and C-terminal sequences of the CP of both viruses are similar. The sequence similarity possibly causes the cross-reaction of heterologous antibodies of HpMV and HpLV. Phylogenetic analyses based on the CP amino acid and 3' non-coding region sequences indicate close relationships among HpMV, HpLV, and Potato virus M. We report here the first molecular characterization of HpMV genomic RNA.

Molecular characterization of Hop latent virus and phylogenetic relationships among viruses closely related to carlaviruses.

The complete nucleotide sequence of the hop latent virus (HpLV) genome was determined. The viral RNA genome is 8,612 nucleotides long, excluding the poly(A) tail, and contains six open reading frames (ORFs), which encode putative proteins of 224-kDa (ORF 1), 25-kDa (ORF 2), 11-kDa (ORF 3), 7-kDa (ORF 4), 34-kDa (ORF 5), and 12-kDa (ORF 6). ORF 5 encodes the coat protein as demonstrated by N-terminal sequencing of three proteolytic peptides derived from the virus particle. The genome organization of HpLV is similar to that of other species in the genus Carlavirus, and the overall sequence of HpLV is more similar to that of Potato virus M than to sequences of other carlaviruses reported to date. The amino acid sequences of the putative methyltransferase, RNA helicase, and RNA-dependent RNA polymerase encoded in ORF 1 and an 'accessory' helicase encoded in ORF 2 of the HpLV genome were compared with those of viruses in the 'tymo' lineage: the genera Carlavirus, Potexvirus, Allexivirus, Foveavirus, Trichovirus, Capillovirus, Vitivirus, and Tymovirus. The phylogenetic relationships among the viruses in these genera are discussed. This is the first molecular characterization of a carlavirus infecting hop plants.

A simple, rapid method of nucleic acid extraction without tissue homogenization for detecting viroids by hybridization and RT-PCR.

A simple, rapid method of nucleic acid extraction on a microcentrifuge tube scale for detecting viroids is presented. Five distinct citrus viroids (CVds), chrysanthemum stunt viroid (CSVd), hop stunt viroid (HSVd), hop latent viroid (HLVd) and potato spindle tuber viroid (PSTVd) were detected in their natural host plants by hybridization using cRNA probes and reverse transcription-polymerase chain reaction (RT-PCR). Nucleic acids (NA) were liberated from tissues by incubation in a buffer containing potassium ethyl xanthogenate (PEX) without tissue homogenization, and then precipitated with ethanol (NA-PEX). All the viroids except CVd-IV could be detected clearly in NA-PEX by hybridization. HSVd, HLVd and PSTVd could also be detected in NA-PEX by RT-PCR. Although CVds and CSVd could not be detected in NA-PEX by RT-PCR, they were detected after further purification: differential precipitation with 2-butoxyethanol and HCl treatment followed by ethanol-precipitation. In addition, PCR in the presence of tetramethylammonium chloride specifically amplified the cDNA of all five distinct CVds under the same temperature and cycle conditions. Since all the viroids could be detected in NA liberated by PEX, the amount of NA extracted by the method described here is sufficient for detecting viroids, enabling the processing of a large number of samples.

A single amino Acid change in viral genome-associated protein of potato virus y correlates with resistance breaking in 'virgin a mutant' tobacco.

ABSTRACT Tobacco cultivar Virgin A Mutant (VAM) is reported to have the recessive potyvirus resistance gene va. Varied levels of resistance were observed in VAM plants inoculated with Japanese potato virus Y (PVY) isolates. VAM was highly resistant to most of the PVY isolates tested and tolerant to three necrotic strain isolates of PVY-T. Based on data obtained from tissue printing and press blotting, the resistance appeared to be mainly at the level of cell-to-cell movement. PVY replicated in VAM proto-plasts, but the replication was 30% lower than in susceptible tobacco, suggesting that impairment of replication also contributes to resistance. To identify the viral gene product or products involved in VAM resistance, we isolated spontaneous resistance-breaking mutants by passing vein-banding (O strain) isolates several times through VAM plants. By comparing the amino acid sequences of the mutants with their original isolates, we identified a single amino acid substitution in the viral genome-associated protein (VPg) domain that is correlated with VAM resistance breaking. Together, these results suggest that, in addition to its role in replication, VPg plays an important role in the cell-to-cell movement of PVY.

Citrus viroid Ia is a derivative of citrus bent leaf viroid (CVd-Ib) by partial sequence duplications in the right terminal region.

Nucleotide sequences of group I citrus viroids Ia (CVd-Ia) and citrus bent leaf viroid (CBLVd, formerly designated CVd-Ib) isolated from citrus plants in Japan, the Philippines and China have been determined. Citrus samples in Japan and the Philippines contained CVd-Ia, which consists of 328 nucleotides(nt). Although 10 nt longer than the type CBLVd-225A in Israel they share 94% identity in overall nucleotide sequence. The Philippines sample also contained a 329-nt long CVd-Ia sequence variant, in which one base insertion and three substitutions were observed. A citrus in China contained CBLVd, which consists of 318 nt and shares 98% identity to CBLVd-225A. CVd-Ia was clearly separated from CBLVd by two 5-nt insertions located in upper (5'-AGCUG-3') and the lower (5'-CUUCU-3') strand of the right terminal region (which is also designated T2 domain) in rod-like secondary structure. Since both of the additional 5-nt sequences are similar to the adjacent sequences (5'-AGUUG-3' and 5'-CUUCU-3'), we hypothesize that CVd-Ia is a derivative of CBLVd caused by partial sequence duplications and substitutions taking place in the right terminal region.

A mixture of synthetic oligonucleotide probes labeled with biotin for the sensitive detection of potato spindle tuber viroid.

Five kinds of synthetic oligonucleotide probes labeled with biotin (BIO) were designed for the detection of potato spindle tuber viroid (PSTVd), and their sensitivities were compared with that of a digoxigenin (DIG)- or BIO-labeled cDNA probe. Although each oligonucleotide probe alone was less sensitive than the DIG-cDNA probe, sensitivity was increased by using a mixture of two or all of the five oligonucleotide probes. The sensitivity of a PSmix1-5 probe, which was a mixture of five oligonucleotides, was the same as that of a DIG-labeled cDNA probe, which can detect 7.8 pg of purified PSTVd and PSTVd in nucleic acids, equivalent to extracts from 20 microg of infected potato leaf and 310 microg of infected potato tuber. Using the PSmix1-5 probe, PSTVd in all leaves and tubers of seven potato cultivars could be detected without any background. Moreover, with the PSmix1-5 probe, the hybridization time could be shortened to 2 h without any decrease in sensitivity, whereas the sensitivity of the cDNA probes clearly decreased when the hybridization time was shortened. Hybridization using a mixture of several oligonucleotide probes may be applicable to the gene diagnosis of other viroids and viruses.

Inosine 5'-triphosphate can dramatically increase the yield of NASBA products targeting GC-rich and intramolecular base-paired viroid RNA.

Nucleic acid sequence-based amplification (NASBA) according to the standard protocol failed to amplify cRNA of viroids, probably because of their GC-rich and intramolecular base-paired structure. However, NASBA in the presence of inosine 5'-triphosphate successfully amplified the cRNAs to viroids in total nucleic acid extracts from citrus plants. As sequence specificity of the cRNA to viroids was confirmed by northern analysis, the amplification and fidelity of cRNAs are sufficient for the sensitive and specific detection of viroids.

Rice ragged stunt Oryzavirus genome segment 9 encodes a 38 600 Mr structural protein.

The complete nucleotide sequence of rice ragged stunt virus genome segment 9 (S9) was determined. The S9 segment is 1132 nucleotides long and has a long open reading frame starting from the first AUG codon at nucleotide position 14-16 and terminating at a UAG codon located at 1028-1030, which could encode a polypeptide with an Mr of 38 600 (P9). The encoded polypeptide has no sequence homology to polypeptides of any other plant reoviruses published previously. An immunological study demonstrated that P9 was the smallest of the structural proteins. The P9 polypeptide was expressed as a fusion protein with maltose binding protein in Escherichia coli. Antisera to purified virions and to the fusion protein reacted with both the bacterially expressed polypeptide and the smallest polypeptide of the purified virus in immunoblotting analyses.

A PCR-microplate hybridization method for plant virus detection.

A sensitive nonradioactive method for detection of plant viruses was evaluated. A cDNA fragment from the coat protein coding region of the potato virus Y (PVY) RNA genome amplified by reverse transcription (RT) followed by polymerase chain reaction (PCR), was directly adsorbed onto polystyrene microplate wells after heat denaturation. The adsorbed cDNA was hybridized with a digoxigenin (DIG)-labelled cDNA probe without a prehybridization step. The probe was also prepared by PCR using the same pair of primers to amplify the target sequence from a cDNA clone of part of the viral genome. The hybrid of the adsorbed cDNA and DIG-labelled probe was reacted with alkaline phosphatase-conjugated anti-DIG antibody. The enzyme activity was then detected by hydrolysis of a substrate, and the absorbance values were measured using a microplate reader. Highest absorbance values were obtained when the amplified DNA fragments were diluted 100 or 125-fold in 10 x SSC (1.5 M NaCl, 0.15 M sodium citrate) for adsorption. Highly concentrated DNA gave lower absorbance values. The absorbance values differed depending on the microplates used, and the highest value was obtained using Nunc Immunoplate II-Maxisorp microplates. DNA fragments longer than 300 bp all gave similar absorbance values, which were twice as high as those obtained with shorter fragments. When the amplified DNA was diluted 100-fold, 10 fg of PVY genomic RNA could be detected by this method, which is called PCR-microplate hybridization. This is about 10,000 times more sensitive than enzyme-linked immunosorbent assay (ELISA). By this method, PVY was detected from five field potato samples showing to be free from PVY by ELISA. PCR-microplate hybridization is nucleic acid-based ELISA-like highly sensitive diagnostic method, and may be generally applicable for detection of plant viruses, viroids, and possibly other plant pathogens.

Characterization and strain identification of a potato virus Y isolate non-reactive with monoclonal antibodies specific to the ordinary and necrotic strains.

An isolate of potato virus Y, named PVY-36, reacted with polyclonal antibody against PVY-O (an ordinary strain), but not with any of eight monoclonal antibodies (MAbs) specific to PVY-O or with two MAbs specific to PVY-T (a necrotic strain). From its host range and symptomatology, PVY-36 belongs to the PVYO group. The nucleotide sequence of the coat protein (CP) coding region of the PVY-36 genome was determined and the amino acid sequence was predicted. Based on the CP amino acid sequence, PVY-36 is more closely related to PVY-O than to PVY-T. There were eight amino acid differences between the CPs of PVY-36 and PVY-O in the N-terminal 30 amino acids. It is suggested that amino acids 8-15 and/or 26-30 from the N-terminus may determine a PVY-O-specific epitope (or epitopes).

Production and characteristics of strain common antibodies against a synthetic polypeptide corresponding to the C-terminal region of potato virus Y coat protein.

Comparing the predicted amino acid sequence between two Japanese potato virus Y (PVY) strains, necrotic strain and ordinary strain, it was found that the C-terminal regions (H2N-HTTEDVSPSMHTLLGVKNM-COOH) of the coat proteins in the two strains were completely conserved. The conserved amino acid sequence was also found in the C-terminal region coat protein of PVY-36, a strain which did not react with monoclonal antibodies specific to the necrotic and the ordinary strain respectively. Antibodies were produced against a synthetic polypeptide PVY-C19 consisting of 19 amino acids, which correspond to the C-terminal region of the coat protein, using 4 coupling combinations of polypeptide PVY-C19 to protein carriers. Carrier-free polypeptides and those coupled to ovalbumin with ECDI (ethyl-dimethylaminopropyl carbodiimide) produced high titer of antibodies and detected PVY strains from PVY-infected plants by Western blot analysis and by ELISA.

Polymerase chain-reaction-mediated cloning and expression of the coat protein gene of potato virus Y in Escherichia coli.

Complementary DNAs (cDNAs) to the RNA genome of a necrotic strain of potato virus Y in Japan (Hokkaido Univ. isolate of PVY-T:PVY-TH) were synthesized and cloned into a plasmid pBR322. About 4.3 kbp of the cDNA sequence containing the 3'-poly(A) tract of PVY-TH was inserted into a recombinant plasmid pBRYT88. The coat protein coding region (CP gene) in pBRYT88 was amplified using the polymerase chain reaction (PCR) and subcloned into a plasmid pUC119. The nucleotide sequence of the CP gene was determined from both the PCR-mediated clones and pBRYT88. The CP gene of PVY-TH consisted of 801 nucleotides, corresponding to 267 amino acids of Mr 29,811. The predicted amino acid sequence of the PVY-TH CP gene was different from that of PVYN (1) in only five amino acids and displayed 98.1% sequence homology. This result indicates that PVY-TH is closely related to PVYN (1). The cDNAs of the PVY-TH CP gene containing an additional initiation codon (ATG) at the 5' end and a stop codon at the 3' end were constructed by PCR amplification and subcloned into an E. coli expression vector, pKK223-3. Five transformed E. coli colonies expressing the PVY-TH CP were identified by immunoscreening using both polyclonal rabbit antiserum against PVY-TH and mouse monoclonal antibody (MoAb) specific to PVY-T. The CP of PVY-TH produced in the E. coli colonies had an electrophoretic mobility identical to that of native PVY-TH CP and reacted strongly to a specific MoAb to PVY-T, but did not react to a specific MoAb to an ordinary strain of PVY (PVY-O). The maximum expression of the CP in E. coli was approximately 7% of the total soluble proteins. The result indicates that the CP gene cloned by PCR was functional and the PCR procedure was useful for producing biologically active cDNA clones from a single, long positive-sense RNA genome encoding a single, large polyprotein precursor, such as potyviruses.

Hop stunt viroid strains from dapple fruit disease of plum and peach in Japan.

Viroids have been isolated from plum trees (Prunus salicina Lindley) affected with plum dapple fruit disease and from peach trees (Prunus persica Batsch) showing dapple fruit symptoms. The viroids were inoculated mechanically to cucurbitaceous plants, in which symptoms typical of hop stunt viroid (HSV) infection appeared. The complete nucleotide sequences of an isolate from plum and an isolate from peach (AF isolate) were shown to be identical, consisting of 297 nucleotides with a 93.6% sequence homology to HSV-hop. Another isolate from peach (A9 isolate) also consists of 297 nucleotides, but the sequence homology to HSV-hop is 99.7%, showing only one nucleotide replacement. These results indicate that these three viroids are strains of HSV, which we designate HSV-plum, HSV-peach (AF) and HSV-peach (A9), respectively. Comparative analysis of the nucleotide sequences of HSV strains from hop, grapevine, citrus, cucumber, plum and peach revealed variable and conserved regions in the HSV molecule. In Japan, these viroids are closely related not only to dapple fruit disease in plum cv. Taiyo, but also to dapple fruit symptoms on peach cv. Asama-Hakutou.