Ervin Balázs

Comparative sequence analysis of four complete primary structures of plum pox virus strains

The complete nucleotide sequence of plum pox virus (PPV) strain SK 68 was determined from a series of overlapping cDNA clones. The exact 5′ terminus was determined by direct RNA sequencing. The RNA sequence was 9786 nucleotides in length, excluding a 3′ terminal poly(A) sequence. The large open reading frame starts at nucleotide position 147 and is terminated at position 9568. Comparison of cistrons from other plum pox virus strains with those predicted for the SK 68 strain indicated the same genomic organizations. Comparison of sequences leads to the following conclusions: (1) The genetic organization of all four PPV strains is identical, containing one large polyprotein gene and two noncoding regions at the 5′ and 3′ ends; (2) pairwise comparison of the genomic sequence of PPV SK 68 with other PPV strains shows 11% alteration. Sequence differences among strains are spread in a uniform manner upon the genome, except for the P1, HC-pro, and two noncoding regions, which are more conserved (with a 4% and 6.6% change). The stability of the noncoding regions is probably linked to their role in replication. The sequence variation has little effect on the amino acid sequence of the corresponding polypeptides, as changes occur preferentially in the third position of the reading frame triplets, except in the case of the 5′ end of the coat protein gene (2.7% average difference in amino acid level, while in the case of coat protein it is 7.7%). The sequence analysis of the coat protein region of the four complete and one partial sequence indicates that the Hungarian plum pox virus strain diverges at the larger extent, similar to the El Amar strain, from which only less than half of the sequence is available.

Virus-resistant transgenic plants : potential ecological impact

Evolution the past, a window on the future?-Systematic search for recombination events in plant viruses and viroids- Different mechanisms of homologous and nonhomologous recombination in brome mosaic virus-Studies on RNA recombination in vivo and in vitro-RNA recombination in viral protein mediated virus resistant transgenic plants- Transgenic plants expressing viral sequences create a favourable environment for recombination between viral sequences-Behaviour of cucumovirus pseudorecombinant and recombinant strains in solanaceous hosts-

The necrotic pathotype of the cucumber mosaic virus (CMV) ns strain is solely determined by amino acid 461 of the 1a protein.

The unique Ns isolate of Cucumber mosaic virus (CMV) induces necrotic lesions on several Nicotiana spp. in contrast to other strains that cause systemic mosaic on these plants. By using biologically active RNA transcripts from cDNAs of Ns-CMV and a reference subgroup I strain Rs-CMV, we confined the genetic determinant solely responsible for necrosis induction to amino acid 461 of the la protein translated from genomic RNA1. An Arg to Cys change at this position (R461C) rendered Rs-CMV necrotic, whereas the reciprocal C461R mutation reverted the necrotic phenotype of Ns-CMV. Necrotic (Ns-CMV, R461C) and non-necrotic (Rs-CMV and C461R) viruses accumulated to similar levels in Nicotiana clevelandii protoplasts. Deletion of the residue at position 461 abolished replicase activity of the Ns-CMV 1a protein. The R461C mutation also was introduced into the 1a protein of Trk7-CMV, a subgroup II isolate. Symptoms induced by the Trk7/R461C mutant were identical to those caused by wild-type Trk7-CMV, even when the mutant Trk7 RNA1 was co-inoculated with RNA2 and 3 of the necrotic Ns strain.

Nicotiana benthamiana plants transformed with the plum pox virus helicase gene are resistant to virus infection.

Nicotiana benthamiana Domin. plants were transformed with the cytoplasmic inclusion protein (CI) gene of plum pox potyvirus (PPV) to investigate, whether this non-structural protein would be able to confer resistance. The CI protein is an RNA helicase, which contains a conserved nucleotide binding motif (NTBM) and plays an important role in viral replication. Two gene constructions were developed for plant transformation. The first contains the original coding sequence of the CI gene under the control of 35S-promoter and nos terminator signal, the second is mutated in the NTBM region. Several transgenic plant lines were obtained following Agrobacterium tumefaciens-mediated transformation. The integration of the viral genes into the plant genome was confirmed using the polymerase chain reaction and the transgene derived mRNAs were detected by Northern blot hybridization. The CI protein in the transgenic plants could not be detected by Western blot analyses. One transgenic line containing the mutated CI gene remained completely symptomless after PPV infection, indicating that the putative defective helicase gene was capable of eliciting virus resistance.

Effect of light on the gene expression and hormonal status of winter and spring wheat plants during cold hardening

The effect of light on gene expression and hormonal status during the development of freezing tolerance was studied in winter wheat (Triticum aestivum var. Mv Emese) and in the spring wheat variety Nadro. Ten-day-old plants (3-leaf stage) were cold hardened at 5°C for 12 days under either normal (250 µmol m(-2) s(-1) ) or low (20 µmol m(-2) s(-1) ) light conditions. Comprehensive analysis was carried out to explore the background of frost tolerance and the differences between these wheat varieties. Global genome analysis was performed, enquiring about the details of the cold signaling pathways. The expression level of a large number of genes is affected by light, and this effect may differ in different wheat genotypes. Photosynthesis-related processes probably play a key role in the enhancement of freezing tolerance; however, there are several other genes whose induction is light-dependent, so either there is cross-talk between signaling of chloroplast originating and other protective mechanisms or there are other light sensors that transduce signals to the components responsible for stress tolerance. Changes in the level of both plant hormones (indole-3-acetic acid, cytokinins, nitric oxide and ethylene precursor 1-aminocyclopropane-1-carboxylic acid) and other stress-related protective substances (proline, phenolics) were investigated during the phases of the hardening period. Hormonal levels were also affected by light and their dynamics indicate that wheat plants try to keep growing during the cold-hardening period. The data from this experiment may provide a new insight into the cross talk between cold and light signaling in wheat.

Salicylic acid treatment of pea seeds induces its de novo synthesis

Salicylic acid (SA), which is known as a signal molecule in the induction of defense mechanisms in plants, could be a promising compound for the reduction of stress sensitivity. The aim of the present work was to investigate the distribution of SA in young pea (Pisum sativum L.) seedlings grown from seeds soaked in (3)H-labeled SA solution before sowing, and to study the physiological changes induced by this seed treatment. The most pronounced changes in SA levels occurred in the epicotyl and the seeds. Radioactivity was detected only in the bound form of SA, the majority of which was localized in the seeds, and only a very low level of radioactivity was detected in the epicotyl. SA pre-treatment increased the expression of the chorismate synthase and isochorismate synthase genes in the epicotyl. Pre-soaking the seeds in SA increased the activities of some antioxidant enzymes, namely ascorbate peroxidase (EC 1.11.1.11) and guaiacol peroxidase (EC 1.11.1.7) and the level of ortho-hydroxycinnamic acid, but decreased the level of polyamines. These results suggest that the increased level of free and bound SA detected in plants growing from seeds soaked in SA solution before sowing is the product of de novo synthesis, rather than having been taken up and mobilized by the plants.

Limited utility of blue fluorescent protein (BFP) in monitoring plant virus movement

While the green fluorescent protein (GFP) is a routinely used marker gene in higher plants, there are only a few data concerning the use of blue fluorescent protein (BFP). These proteins together are used for dual colour tagging experiments in various biological systems; however, the benefits of this technique in plant virology have not been exploited yet. In this work, our aim was to determine whether the BFP is a suitable second marker in conjunction with GFP for following the progress of virus infection. Nicotiana clevelandii, N. benthamiana and N. tabacum cv. Xanthi-nc plants were infected with potato virus X vector carrying the GFP or the Y66H type BFP gene. While GFP was brightly fluorescent in all species, the fluorescence intensity of BFP varied widely, from the bright fluorescence observed in N. clevelandii to the absence of fluorescence in N. tabacum cv. Xanthi-nc. Since at even mild acidic pH BFP rapidly fades, the more acidic cytosol of N. tabacum could be responsible for impaired in vivo fluorescence. After infiltration of the infected leaves of N. clevelandii with pH 5 phosphate buffer, the fluorescence faded thus confirming this situation.

Antiauxin enhanced microshoot initiation and plant regeneration from epicotyl-originated thin-layer explants of sugarbeet (Beta vulgaris L.)

An in vitro method was developed for microshoot initiation from thin-layer explants prepared from the elongated epicotyls of sugarbeet (Beta vulgaris L.). Intact epicotyls of 14-day-old seedlings were excised from the hypocotyls above the cotyledons and allowed to elongate on De Greef and Jacobs (1979) medium supplemented with 0.2 mg/l 6-benzyladenine, 0.2 mg/l gibberellic acid and 0.1 mg/l indole-3-acetic acid in darkness. After a 21-day-incubation, the elongated epicotyls were halved to obtain apical and basal segments prior to removing the leaves and lateral buds. Subsequently, 5–8 mm long, 2–3 mm wide and 0.8–1.0 mm thick tangential sections were prepared longitudinally from the exterior parts of the halved epicotyls. These thin-layer explants were incubated on microshoot initiating media containing various growth regulators. The combination of 1.0 mg/l 6-benzyladenine and the antiauxin 2,3,5-triiodobenzoic acid (1.0 mg/l) resulted in maximum microshoot development (6.3±0.2 microshoots/thin-layer explant). The final efficiency of our tissue culture system was significantly increased by the NaCl (100 mg/l) initiated in vitro rooting of microshoot originated plantlets.

A Cucumber Mosaic Virus Based Expression System for the Production of Porcine Circovirus Specific Vaccines

Potential porcine circovirus type 2 (PCV2) capsid protein epitopes, suitable for expression on the surface of cucumber mosaic virus (CMV) particles were determined by a thorough analysis of the predicted PCV capsid protein structure. The ab initio protein structure prediction was carried out with fold recognition and threading methods. The putative PCV epitopes were selected on the basis of PCV virion models and integrated into the plant virus coat protein, after amino acid position 131. The recombinants were tested for infectivity and stability on different Nicotiana species and stable recombinant virus particles were purified. The particles were tested for their ability to bind to PCV induced porcine antibodies and used for specific antibody induction in mice and pigs. The results showed that PCV epitopes expressed on the CMV surface were recognized by the porcine antibodies and they were also able to induce PCV specific antibody response. Challenge experiment with PCV2 carried out in immunized pigs showed partial protection against the infection. Based on these results it was concluded that specific antiviral vaccine production for the given pathogen was feasible, offering an inexpensive way for the mass production of such vaccines.

Engineering resistance to PVY in different potato cultivars in a marker-free transformation system using a ‘shooter mutant’ A. tumefaciens

In this work, Potato virus Y (PVY) resistant potatoes were generated using an environmentally safe construct. For this purpose, a ‘shooter’ mutant Agrobacterium-based transformation system was used. The isopentenyl transferase gene (ipt) present on the Ti plasmid of ‘shooter’ strains enhances shoot regeneration and can be used as a phenotypic selection marker. The introduced marker-free binary vector carried a hairpin construct derived from the coat protein gene of PVY-NTN strain in order to induce gene silencing. Transformation resulted in high regeneration rates (1.4–5.7 shoots per explant). With pre-selection for the ipt+ phenotype the transformation frequency was 24–53%, while without selection 12–28% of the shoots were PCR positive. The presence of the transgene was verified by Southern hybridization. In 16 of 31 challenged transformant lines PVY could be detected neither by RT-PCR nor by back inoculation. A 62.5% of these resistant lines proved to be also ipt-free. This transformation system was reproducible in four potato cultivars, suggesting that it could easily be adapted for other species.