Production of polyclonal antibodies to a recombinant coat protein of potato virus Y.

The gene encoding the coat protein (CP) of a potato virus Y (PVY) was cloned into expression vector pMPM-A4Omega. PVY CP was expressed in Escherichia coli and the purified recombinant protein was used for raising rabbit polyclonal antibodies. The sera and antibodies were tested for the detection of PVY in the laboratory host Nicotiana tabacum cv. Petit Havana SR1 and in various cultivars of the natural host Solanum tuberosum by ELISA as well as by Western blots. The antibodies can be used for the detection of the whole strain spectrum of PVY by indirect plate trapped antigen ELISA and Western blot, but not by double antigen sandwich ELISA.

Experimental transmission of pospiviroid populations to weed species characteristic of potato and hop fields.

Weed plants characteristic for potato and hop fields have not been considered in the past as potential hosts that could transmit and lead to spreading of potato spindle tuber (PSTVd) and hop stunt (HSVd) viroids, respectively. To gain insight into this problem, we biolistically inoculated these weed plants with viroid populations either as RNA or as cDNA. New potential viroid host species, collected in central Europe, were discovered. From 12 weed species characteristic for potato fields, high viroid levels, detectable by molecular hybridization, were maintained after both RNA and DNA transfers in Chamomilla reculita and Anthemis arvensis. Low viroid levels, detectable by reverse transcription-PCR (RT-PCR) only, were maintained after plant inoculations with cDNA in Veronica argensis and Amaranthus retroflexus. In these two species PSTVd concentrations were 10(5) and 10(3) times, respectively, lower than in tomato as estimated by real-time PCR. From 14 weeds characteristic for hop fields, high HSVd levels were detected in Galinsoga ciliata after both RNA and DNA transfers. HSVd was found, however, not to be transmissible by seeds of this weed species. Traces of HSVd were detectable by RT-PCR in HSVd-cDNA-inoculated Amaranthus retroflexus. Characteristic monomeric (+)-circular and linear viroid RNAs were present in extracts from weed species propagating viroids to high levels, indicating regular replication, processing, and circularization of viroid RNA in these weed species. Sequence analyses of PSTVd progenies propagated in C. reculita and A. arvensis showed a wide spectrum of variants related to various strains, from mild to lethal variants; the sequence variants isolated from A. retroflexus and V. argensis exhibited similarity or identity to the superlethal AS1 viroid variant. All HSVd clones from G. ciliata corresponded to a HSVdg variant, which is strongly pathogenic for European hops.

Sequence analysis of the Czech potato mop-top virus (PMTV) isolate Korneta-Nemilkov.

The nucleotide sequence was determined for Czech potato mop-top virus (PMTV) isolate Korneta-Nemilkov, found in the potato field situated in South Bohemia. The nucleotide and amino acid sequences were compared with other PMTV isolates available in databases. The sequence identity was always >99% when Czech isolate RNA 2 and RNA 3 sequences were compared with each of the 3 Danish isolates and with Sw isolate, and slightly lower when compared to Scottish isolates. Similarity of deduced proteins was 100% for 5 out of 6 proteins used in comparison of Czech isolate with Danish isolate 54-15. The only difference between 2 isolates was found in coat protein (CP) gene. Interestingly, the CP of the Czech isolate seems to be 100% identical to the one of Sw, while many changes were found in the region encoding TGBp2, TGBp3 and cysteine-rich protein (CRP) for these 2 isolates. The lowest similarity scores were found when comparing the Czech isolate CRP with CRP of Scottish isolates.

Complete nucleotide sequence and molecular probing of potato virus S genome.

Complete genomes of three isolates of Potato virus S (PVS) were cloned and sequenced. The PVS ORF-1 was characterized for the first time. It encodes a putative replication protein (RPT) that shares the highest homology (about 52%) with that of Blueberry scorch virus (BlScV). ORF-1 motifs, characteristic for carlaviruses were found for methyltransferase (MTR), helicase (HEL) and RNA-dependent RNA polymerase (RdRp). The complete sequence of PVS genome enabled to develop an immunocapture RT-PCR probing of the PVS genome. Using this system, the sequence variability of 11 genome zones was examined for 34 PVS isolates including 15 PVS-CS variants that caused a systemic infection in Chenopodium quinoa. A broad variability between PVS isolates and diverse sequence variants was found. cDNA fragments covering the coat protein (CP) leader and CP-coding region (approx. 420 bp) were pooled for PVS-O and Chenopodium-systemic PVS isolates (PVS-CS) and corresponding cDNA libraries were screened for sequence variants. Both cDNA pools differred mainly in the 5'-end of the CP gene. Methionine at the position 17 in combination with serine at the position 34 were frequently associated with the CS character of PVS. In general, hydrophobic and polar amino acids were characteristic for the positions 17 and 34, respectively in PVS-CS isolates. Genome probing and evolutionary distances suggested that the PVS-CS isolates analyzed were close to the ordinary European isolates of ordinary strain of PVS (PVS-O) but distant to the original Andean strain of PVS (PVS-A).

DNA microarray: parallel detection of potato viruses.

DNA microarray assay has become a useful tool for gene expression studies. Less frequent is its application to detection of viruses or diagnostics of virus diseases. Here we show design of a microscope slide-based microarray assay for simultaneous identification of several potato viruses. Different primer pairs were designed or adopted to obtain specific amplicons from six potato viruses: Potato virus A (PVA), Potato virus S (PVS), Potato virus X (PVX), Potato virus Y (PVY), Potato mop-top virus (PMTV) and Potato leaf-roll virus (PLRV). Purified viral DNA probes were spotted on a microscope slide coated with poly-L-lysine. The same primers were used for preparation of fluorochrome-labeled targets. The latter were denatured and hybridized on the microarray slide (chip). An example of simultaneous assay of two pathogens is given and possibilities of practical application of this type of assay are discussed.

Immunocapture RT-PCR probing of potato virus Y isolates.

Twenty potato virus Y (PVY) isolates were characterized. They represented two strains only, PVY(O) (three isolates) and PVY(N) (17 isolates). However, application of serological and molecular genetic methods led to a more complicated characterization. For example, five isolates induced necrotic symptoms on tobacco plants typical of PVY(N), despite reacting as PVY(O) serologically. Moreover, the PVY isolates were not identical according to molecular genetic properties. Typical PVY(NTN) PCR products were observed for 14 isolates, but five of them (Hr 220-5, Hr 387-7, Nord 242, Syn1Scot, and 41-97) did not produce potato tuber necrotic symptoms in infected cultivars. An immunocapture reverse transcription-polymerase chain reaction (RT-PCR) probing was developed using a set of 24 primer pairs derived from eight regions of the PVY genome. Using this method, five out of seven PVY(NTN) isolates including the Czech standard PVY(NTN) from the potato cv. Nicola were found to be identical. However, two PVY(NTN) isolates and all the other probed PVY samples showed unique patterns, suggesting specific differences at the nucleotide level. This method enabled specific identification of individual isolates variability even within different PVY strains.

Analysis of variability of P1 gene region of N strain of potato virus Y using temperature-gradient gel electrophoresis and DNA heteroduplex analysis.

Reaction conditions specific for reverse taranscription-polymerase chain reaction (RT-PCR) of potato virus Y strain NTN (PVYNTN) were used to amplify a 394 bp fragment of the P1 gene from selected PVY isolates with the aim to study the PVY variability within this genomic region. The P1 gene fragment from the Nicola isolate (Nicola P1/1 clone) was sequenced and characterized by temperature-gradient gel electrophoresis (TGGE). The Nicola P1/1 clone differed from that from the Hungarian isolate by double point mutation resulting in two changes at the deduced amino acid level. The clone showed simple transition from double-stranded to single-stranded form with two characteristic melting end points of about 41 degrees C and 48 degrees C. A more complicated TGGE pattern was, however, found for the whole P1 cDNA library of the Nicola isolate, suggesting accumulation of some minor sequence variants of PVY in this isolate. Based on the TGGE pattern, 46 degrees C was selected as the standard temperature for electrophoretic analysis of heteroduplex DNAs formed with the Nicola P1/1 DNA as reference. More than 40 other PVY isolates from PVYN group were analysed using this method. In most cases only minor fractions of electrophoretically distinguishable DNA heteroduplexes were found, however, in most isolates of PVYN-Wilga type, mixtures of the major sequence variants were observed. Two of these variants from the hybrid 220-5 (Czech Republic) were sequenced. Both of them differed from the Nicola P1/1 clone by 6 point mutations, which led to several changes at the amino acid level.

Inhibition of viroid infection by antisense RNA expression in transgenic plants.

Complex formation between different antisense RNAs directed against either plus-strand or minus-strand sequences of the potato spindle tuber viroid (PSTVd) was studied using temperature-gradient gel electrophoresis and immunochemical detection with an antibody specific for double-stranded RNA. Short minus-strand sequences were directed against the upper central conserved region (UCCR) of plus-strand viroid replication intermediates, a plus-strand corresponding to the left half of the rod-like secondary structure (VL+) against minus-strand replication intermediates. It was shown that antisense RNA forms complexes with the corresponding target RNA only with low yield during incubation at low (physiological) temperatures but with high yield during in vitro transcription of the target RNA when the antisense RNA is already present in the solution. The antisense RNA sequences were integrated into Solanum tuberosum L. by Agrobacterium tumefaciens transformation. Antisense RNA expression in vivo was analyzed by Northern analysis. Infection tests were performed using the transgenic potato lines in order to evaluate their degree of resistance against PSTVd infection. Although some lines showed a significant inhibition of viroid accumulation, a high variability of viroid infection in different transgenic potato lines was obtained. Since strongly infected plants were observed in all transgenic lines 6 to 8 weeks post inoculation, a threshold concentration of viroid, overcoming the antisense effect has to be assumed. When the rate of viroid accumulation was tested using agroinfection assays on leaf discs, a stronger antisense effect could be achieved.

Inhibition of potato spindle tuber viroid (PSTVd) infection with DNA oligonucleotides.

The effects of DNA oligonucleotides complementary to potato spindle tuber viroid (PSTVd) on viroid infection were investigated. The oligonucleotides were used to form hybrids with PSTVd in the infection mixture. A 75% reduction of viroid infection was found when an oligonucleotide complementary to nucleotides 79-110 of PSTVd was hybridized with PSTVd at a molar ratio of approximately 5000:1, respectively. A total inhibition of PSTVd infection was observed using an oligonucleotide complementary to nucleotides 42-78 at the same molar excess of DNA over PSTVd, although a 200-fold molar excess was found to be sufficient for the complete blocking of infection by PSTVd. This oligonucleotide caused a significant reduction (about 83%) of viroid infection even if the hybridization was done at a low (30 degrees C) temperature. Shorter oligonucleotides containing 22 and 15 bases corresponding to position 42-62 and 63-78, respectively, exhibited a significant effect only at a high (80 degrees C) initial temperature of molecular hybridization. Heteroduplexes formed between PSTVd RNA and antisense DNA were found to be less stable in a crude nuclease extract from tomato leaves as compared with PSTVd RNA alone. RNase H was demonstrated to cleave the molecular hybrids in vitro.