Effects of heat treatment on metabolism of tobacco plants infected with Potato virus Y.

Many factors affect successful virus propagation and plant defence responses. Heat shock protein (Hsp) expression after heat shock plays an ambiguous role in viral infection. On the one hand, Hsp70 participates in plant defence response; on the other hand, Hsp70 could interact with viral proteins and facilitate virus propagation. Here, we studied metabolic adaptations of Nicotiana tabacum L. subjected to heat shock (42 °C, 2 h) before or after inoculating the plants with Potato virus Y (potyvirus). RT-qPCR and ELISA were used for potyvirus quantification. Hsp70 and Hsp90 isoforms were analysed by Western blotting. Salicylic, quinic and chlorogenic acid content was determined by LC-MS. The activity of Hatch-Slack enzymes (as markers of potyviral infection in tobacco) and glycosidases was assayed. Application of heat shock before or after inoculation showed accelerated potyviral propagation in comparison with only inoculated plants. Plants exposed to heat shock and concurrently inoculated showed higher potyviral content, higher amount of Hsp70, together with late decline of quinic acid content and low chlorogenic acid content. Spread of potyviral infection correlated with enhanced salicylic acid content and activities of enzymes of the Hatch-Slack cycle, α- and ß-galactosidase, α-mannosidase, α-glucosidase and ß-N-acetylhexosaminidase. Heat shock proteins accelerate potyviral propagation. The lower weight cytosolic and mitochondrial Hsp70 (~50-75 kDa) persist throughout the viral infection. Also, the plant defense response results in increase of salicylic and chlorogenic acids but decrease of quinic acid content.

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.

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.

The activity and isoforms of NADP-malic enzyme in Nicotiana benthamiana plants under biotic stress.

The activity and presence of isoforms of NADP-dependent malic enzyme (NADP-ME, EC 1.1.1.40) were studied in non-transgenic and transgenic Nicotiana benthamiana plants containing potyviral gene for helper component protease (HC-pro) and in plants infected by Potato virus Y strain NTN (PVY(NTN)). No significant changes in enzyme activities and isoenzyme pattern were observed due to foreign gene introduction and PVY(NTN) infection. However, the activity and isoenzyme composition of NADP-ME measured in extracts from different parts of the plants showed significant differences. Non-denaturating electrophoresis followed by specific detection of NADP-ME activity in polyacrylamide gel detected the presence of only one isoform in roots and younger leaves. Two isoforms of NADP-ME were detected in older leaves and stem (relative molecular mass approximately 248,000 and approximately 280,000) and three isoforms corresponding to tetramer, dimer and monomer were found in flowers. The activity of NADP-ME and the isoenzyme pattern was discussed in relation to its role in plant metabolism within distinct plant parts.

Nucleotide sequences of coat protein coding regions of six potato mop-top virus isolates.

Coat protein (CP) coding regions of six Potato mop-top virus (PMTV) isolates from the Czech Republic and Denmark (54-10, 54-11, 54-15, 54-19, Korneta and Pacov) were sequenced. Comparison of the obtained nucleotide sequences as well as alignment of the deduced amino acid sequences were performed. The obtained results showed that the isolates from different parts of Europe seem to have highly conserved coding regions which is unexpected for a viral RNA genome known for its high mutation rate. Thus considerable differences in virulence and significant variation in biological properties of these isolates should not be attributed to CP but to some other part of the genome.

The detection of recombinant, tuber necrosing isolates of Potato virus Y (PVY(NTN)) using a three-primer PCR based in the coat protein gene.

A simple and reliable procedure for reverse transcription-polymerase chain reaction (RT-PCR) detection and strain differentiation of Potato virus Y (PVY) was developed. Three primers were designed within the coat protein (CP) and nuclear inclusion protein b (NIb) region, exploiting a single base polymorphism identified as being present in all the recombinant PVY(NTN) isolates published. Samples infected with PVY produce a single band of 569 bp, while isolates belonging to PVY(NTN) strain give an additional band of 334 bp. The technique was tested on a collection of well-characterised isolates of PVY from a range of strains and was found to detect all of the isolates reported as belonging to the PVY(NTN) strain. All of the isolates detected possess a recombination event within the coat protein. Further sequence analysis revealed that all the recombinant PVY(NTN) isolates reported thus far would be detected using this assay, whilst isolates thought to be PVY(NTN) that do not possess the coat protein recombination event would not be detected.

Polyclonal antibodies to a recombinant coat protein of Potato virus A.

Specific mouse antibodies against a recombinant coat protein (CP) of Potato virus A (PVA) were produced. The PVA CP gene was cloned in an expression vector pMPM4omega. After expression in Escherichia coli the presence of the expressed CP was proved by Western blot analysis using polyclonal and monoclonal antibodies (MAbs). The expressed CP was purified by centrifugation in CsCl density gradient or on a sucrose cushion. The production of virus-like particles (VLPs) was proved by electron microscopy. The purified CP was used for preparation of a mouse antiserum which had a titer of 1:1024 in ELISA and reacted specifically in Western blot analysis and indirect plate-trapped antigen ELISA (PTA-ELISA).

Nucleotide sequences of 5'-terminal parts of coat protein genes of various isolates of NTN strain of potato virus Y.

Sequences of the first 300 nucleotides of coat protein (CP) genes of 7 isolates of NTN strain of potato virus Y (PVY, PVY(NTN)) were determined and compared with analogous published sequences of various isolates and strains of PVY. The sequence identity among the sequenced isolates ranged from 96 to 100%. The differences were found at different positions. The nucleotide sequence of this part of CP gene seems to be very conservative among the isolates tested that means that PVY(NTN) is the evolutionary youngest among all PVY strains.

Partial antigenic characterization of different potato virus Y NTN strain isolates.

Eight isolates of potato virus Y NTN strain (PVY-NTN) of different origin were studied by means of monoclonal antibodies (MAbs) in non-competitive and competitive enzyme-linked immunosorbent assay (ELISA), and by immunoblot analysis of the viral coat protein (CP). As the MAbs reacted with the denatured viral CP, their epitopes must be continuous. The ELISA data demonstrate that the epitopes are topologically different. The epitopes may be located on the N-terminal part of CP as showed its partial amino acid sequencing and the immunoblot analysis.

Potato virus A detection by reverse transcription-polymerase chain reaction.

Simple and reliable procedure for sample preparation and reverse transcription-polymerase chain reaction (RT-PCR) detection of potato virus A (PVA) is described. PVA-specific primers used in the RT-PCR defined a target sequence of 321 bp and did not produce amplification product(s) with potato virus Y.

Electron microscopic observation of potato virus A using murine monoclonal antibodies.

Six monoclonal antibodies (MoAbs) against potato virus A (PVA) were prepared and used in enzyme-linked immunosorbent assay (ELISA), immunoblot analysis and electron microscopic study of the virus. Four MoAbs, 151, 290, 328 and 634, reacted with purified virus preparation in dot blot test and showed strong reaction also with virus coat protein (CP) denatured by sodium dodecyl sulphate (SDS), while two MoAbs, 534 and 187, gave significantly weaker reaction with denatured CP than with purified virus. On electron micrographs, MoAb 534 effected binding only on few separate locations of the virus surface after prolonged storage. We presume that this MoAb recognized a conformation-dependent epitope.

Optimization of purification procedure for potato virus Y strain NN.

Potato virus Y strain NN (PVYNN) was purified from mechanically infected plants Nicotiana tabaccum cv. Samsun by extraction of the plants with various buffers, clarification of the suspensions with chloroform or Triton X-100, high speed centrifugation of the virus through sucrose cushion and resuspension of the sedimented virus is different buffers. The two optimal combinations of different procedures tested were either (1) extraction of the plants with the buffer containing 0.3% ascorbic, 0.3% mercaptoethanol, 0.01 mol/l diethyl pyrocarbonate (DEPC) and 5 mmol/l phenylmethylsulphonyl fluoride (PMSF), pH 5.3, clarification with cold chloroform, PEG precipitation, high speed centrifugation through sucrose cushion and resuspension of the sedimented virus in 0.02 mol/l Na-borate buffer pH 7.8, or (2) extraction of the plants with the buffer containing 0.5 mol/l Na-citrate, 1% mercaptoethanol and 5 mmol/l PMSF pH 6.5, clarification with 2% Triton X-100, PEG precipitation, high speed centrifugation through sucrose cushion and resuspension of the sedimented virus in 0.02 mol/l K-phosphate, 0.5 mol/l urea and 0.1% mercaptoethanol, pH 7.5.

Monoclonal antibodies against potato virus A -- immunoblot analysis.

Monoclonal antibodies (MoAbs) against potato virus A (PVA) were examined in their reactivity with PVA and its denatured capsid protein (PVA-CP) bound to the nitrocellulose membrane. Five MoAbs reacted with native PVA, three of them also with PVA-CP. One MoAb gave no reaction in dot-blot test. In polyacrylamide electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE) PVA-CP migrated as two major bands. In immunoblot analysis, two MoAbs reacted only with the slower band, one only with the faster one. We presume that those bands do not correspond to the intact CP but they do to truncated N- and C-terminal CP molecules, respectively, and that the corresponding epitopes reacting with MoAbs are localized near to both termini of CP molecules. After mild trypsinolysis of PVA particles no MoAb reacted with resulting "core" CP.

Partial antigenic characterization of potato virus S (Andean strain) by monoclonal antibodies.

Four mouse monoclonal antibodies (MoAbs)) against potato virus S Andean strain (PVSA) were tested. While MoAbs 2 and 3 reacted only with complete virions and were apparently specific for epitopes dependent on quaternary structure, MoAbs 1 and 4 appared to be conformation independent and reacted with exposed regions on native virions as well as on the surface of dissociated coat protein subunits. This seems to be an evidence of metatope existence. The results of competitive binding tests together with reaction patterns of individual MoAbs suggest that the used MoAbs reacted with at least two different epitopes on PVSA particles or polypeptide subunits. Immunoblot analysis of proteolytically cleaved PVSA capsid protein (CP) confirmed close proximity of epitopes recognized by MoAbs 1 and 4. Anti-PVS polyclonal antibody recognized both intact CP and its natural or artificial digest, while the MoAbs bound to intact CP only. These results indicate that the surface virus-specific epitopes are located near the terminus of CP molecule as it is characteristic for potyviruses.

Metabolic activation of m-phenylenediamine to products mutagenic in Salmonella typhimurium by medium isolated from tobacco suspension cell cultures.

Both tobacco cells in suspension and the medium recovered from the suspension cultures (TX1MX) activated the aromatic amine m-phenylenediamine (m-PDA) into a product that was mutagenic in Salmonella typhimurium TA98 and YG1024. Medium recovered from stationary-phase tobacco cell cultures exhibited the highest level of m-PDA activation. No cytochrome P-450 was detected in the activating medium. A high molecular weight matrix having the highest m-PDA activating capacity and associated with a substantial fraction of the total peroxidase activity was isolated by Centricon-100 ultrafiltration of TX1MX. The data suggest that the peroxidases present in the recovered cell culture medium or in the high molecular weight matrix are responsible for the plant activation of m-PDA.

Optimum conditions for the storage of potato virus S.

The effect of storage conditions on the serological activity of two strains of potato virus S (PVS), Andean and the ordinary, was studied by ELISA. Virus purificates, infected leaves and their homogenates, stored in lyophilized, frozen and dissolved form at various temperatures were tested. Virus purificates were most stable in lyophilized form, their activity decreased after 9 months only by 20-30%. Also non-purified virus was most stable as a lyophilized leaf homogenate, its activity decreased after 12 months by 30%. When lyophilized leaves were stored, the virus activity dropped after 12 months by 45%. Both the Andean and the ordinary strain of PVS behaved similarly during storage under the conditions tested.

Monoclonal antibodies against potato virus A--competitive binding tests.

Six mouse monoclonal antibodies (MoAbs) against potato virus A (PVA) were tested. One of them (PVA 534) reacted only with complete virions and was apparently specific for epitopes dependent on quaternary structure. MoAb PVA 328 recognized the virus antigen only after its dissociation into subunits. MoAb PVA 328 must have reacted with a cryptotope of the antigen. MoAb PVA 151 and 290 appeared to be conformation independent and reacted with exposed regions on native virus particles as well as on the surface of dissociated coat protein subunits. Two other MoAbs (PVA 187 and 634) did not recognize subunits or the virions adsorbed directly to the microtiter plate. This seems to be an evidence of metatope existence. The results of competitive binding tests combined with the reaction patterns of individual MoAbs to different potyviruses indicate that the MoAbs are specific for 6 distinct epitopes.

Some factors influencing purification of potato virus A (PVA).

Potato virus A (PVA) was purified from mechanically infected plants Nicotiana tabacum cv Samsun by high speed centrifugation and subsequent isopycnic density gradient centrifugation in CsCl gradient. From different procedures tested 0.05 mol/l phosphate buffer (PBS) pH 8 seemed optimal for virus purification and 0.1 mol/l borate buffer pH 8.0 for virus storage; other pH of PBS, Tris and Hepes buffers were less suitable. Additives preventing virus aggregation were beneficial. The average yield ranged about 40 mg virus protein per kg starting material. The purified virus has remained serologically active after 30 days storage.

A comparative study of the effect of triazine herbicides on alcohol dehydrogenases isolated from various sources.

The studied herbicides (terbutylazine, simazine) inhibit the activity of plant, animal, and yeast alcohol dehydrogenases. The inhibition constant Ki for alcohol dehydrogenase (ADH) isolated from peas and bakers' yeast equals approximately 10(-4) M, and that for ADH isolated from horse liver is of the order of 10(-5) M. The character of inhibition for all the herbicides studied for the reaction catalyzed by pea, liver, and yeast ADH is always noncompetitive toward ethanol and competitive with respect to NAD. The inhibition constants for the enzyme isolated from peas are pH independent. The interaction constants found for terbutylazine and simazine and for o-phenanthroline, nicotinamide, and ATP indicate that the herbicides are bonded through the metal component of the enzyme, similar to the nicotinamide part of NAD. The interaction constant less than unity found for the herbicide-ATP system indicates that the bonding site in the active center of the enzyme is different for the herbicides and the adenine part of NAD.