E. A. Filipenko

PR-proteins with ribonuclease activity and plant resistance against pathogenic fungi

Pathogenesis-related (PR) proteins participate in complex plant defense responses to pathogens. It is known that members of two PR-protein families (PR4 and PR10) exhibit ribonuclease activity in some cases. These proteins were found to be able to inhibit the growth of pathogenic fungi, and ribonuclease activity is necessary for the manifestation of this effect. This paper presents current data on molecular mechanisms governing the antifungal activity of PR-ribonucleases, associated both with their direct cytotoxic impact on pathogen cells and with their possible participation in the induction of plant cell apoptosis and development of hypersensitive reactions (HR).

Comparative analysis of HBV M-antigen production in leaves of individual transgenic carrot plants

76 The advantages of plants as alternative systems for expression of heterologous proteins are illustrated by numerous examples of accumulation in plant tissues of complex functionally active immunoglobulins, secretory granules, collagen, hemoglobin, and cytokines [1]. Transgenic plants accumulating antigens against various pathogens can be used as edible vaccines, including those against hepatitis B virus [2].

Specific features of T-DNA insertion regions in transgenic plants

Experimental data from analysis of exogenous DNA (T-DNA) insertion sites in transgenic plants are summarized. Arguments are considered in favor and against the existence of genome DNA regions preferred for transgene integration that are determined by distinctive features characterizing the organization and nucleotide composition of the plant genome and the structure and conformational state of the chromatin. The main stages of T-DNA integration into a plant chromosome and possible molecular mechanisms of this process are discussed.

Analysis of integration sites of T-DNA insertions in transgenic tobacco plants

DNA fragments containing T-DNA/plant DNA junctions isolated from 17 transgenic tobacco plants were amplified using inverse PCR. Analysis of the nucleotide sequences of 34 cloned DNA fragments revealed 100% homology with vector sequences outside T-DNA in 10 cases. Nine nucleotide sequences had homology with the repeats in the tobacco genome. The percentage of homology varied from 70 to 90%, with the identified repeats belonging to different types. In most clones no homology was revealed with the GENEBANK sequences. Alignment of the sequences truncated during the integration of the left and the right borders of the T-DNA insertions demonstrated significant clusterization (10 bp region) of truncation sites for the left border. Five sequences had identical truncation sites (+23 T) that showed the perferable use of this nucleotide. The AT content varied from 51 to 72% which was close to the total percentage of AT pairs in the tobacco genome.

Transgenic expression of Serratia marcescens native and mutant nucleases modulates tobacco mosaic virus resistance in Nicotiana tabacum L.

Extracellular Serratia marcescens nuclease is an extremely active enzyme which non-specifically degrades RNA and DNA. Its antiviral activity was previously shown both in animals and in plants when applied exogenously. Transgenic tobacco plants (Nicotiana tabacum L. cv. SR1) expressing S. marcescens chimeric, mutant, and intracellular mutant nuclease gene variants were regenerated and challenged with tobacco mosaic virus. The transgenic plants exhibited a higher level of resistance to the virus infection than the control non-transgenic plants. The resistance was evidenced by the delay of the appearance of mosaic symptoms and the retarded accumulation of viral antigen. Thus, these results reveal that modulations of both extracellular nuclease activity and intracellular RNA/DNA binding can protect plants against viral diseases.

Evaluation of Salt Tolerance of Transgenic Tobacco Plants Bearing with P5CS1 Gene of Arabidopsis thaliana

Arabidopsis thaliana Δ1-pyrroline-5-carhoxylate synthetase 1 (P5CS1) cDNA was cloned under the control of the potent constitutive 35S RNA promoter of the cauliflower mosaic virus and transferred into genome of tobacco cv. Petit Havana SR-1 (Nicotiana tabacum L.) plants. It is shown that the constitutive level of proline in the transgenic plants T0 exceeds that of the SR1 reference line by 1.5 to 4 times. Under conditions of salt stress (200, 300 mM NaCl) T1-generation transgenic plants in early stages of development formed a large biomass, developed more quickly, and had a higher rate of root growth compared to the control, which confirms the involvement of the P5CS1 gene in molecular mechanisms of stress resistance in plants.

Transgenic tobacco plants producing human interleukin-18.

Studies conducted over the last decade have shown that genetically modified plants are potentially less expensive and safer sources of recombinant proteins than the commonly used expression systems based on bacteria, yeast, and cultured mammalian and insect cells. World’s leading biotech companies have already created transgenic plants producing hormones, cytokines, growth factors, and enzymes. The recombinant proteins from plants were found to be similar in biological activity to their counterparts derived from other expression systems [1]. Transgenic plants that produce epitopes of human and animal pathogens are promising for oral immunization [2]. Vaccines against hepatitis B, rabies, Norwalk virus, and diarrhea, obtained from plants, have now passed first clinical trials [3, 4]. However, when subunit vaccines are used, immunological tolerance may develop to the pathogen [5] (for example, no immune response is observed to antigens consumed every day in food). To improve the immunization efficacy of “edible vaccines” and to prevent the development of immunological tolerance, researchers actively seek proteins that can act as adjuvants [6]. Interleukin-18 (IL-18) was found to be a promising adjuvant in laboratory animals subjected to intranasal vaccination [7]. It was possible to avoid the induction of immunological tolerance to ovalbumin in newborn mice by combining this antigen with oral administration of IL-18; their combination elicited the systemic and mucosal immune responses to ovalbumin in the mice [8]. In this context, it is of interest to study the possibility of supplementing edible vaccines with IL18 as an adjuvant. We believe that transgenic plants may be convenient vehicles for delivery of IL-18 by the oral route to gastrointestinal mucosae. It is also of interest to study whether pure recombinant IL-18 can be obtained by affine chromatography.

Induced expression of Serratia marcescens ribonuclease III gene in transgenic Nicotiana tabacum L. cv. SR1 tobacco plants

Transgenic Nicotiana tabacum L. cv. SR1 plants, characterized by an increase in the level of dsRNA-specific hydrolytic activity after induction by wounding, were obtained. The Solanum lycopersicum anionic peroxidase gene promoter (new for plant genetic engineering) was for the first time used for the induced expression of the target Serratia marcescens RNase III gene. Upon infection with the tobacco mosaic virus (TMV), the transgenic plants of the obtained lines did not differ significantly from the control group in the level of TMV capsid protein accumulation. In general, no delay in the development of the infection symptoms was observed in transgenic plants as compared with the control group. The obtained transgenic plants represent a new model for the study of the biological role of endoribonucleases from the RNase III family, including in molecular mechanisms of resistance to pathogens.

Effect of Sexual Maturation onDD2RGene Expression in Fat Body ofDrosophila melanogasterFemales

The expression of the DD2R gene was studied by in situ hybridization in the fat body (place of the synthesis of enzymes that degrade juvenile hormone) and ovarian follicular cells (place of the synthesis of 20-hydroxyecdysone) in young and sexually mature D. melanogaster females. It was demonstrated that the DD2R gene is expressed in the fat body, and its expression is higher in young females than in sexually mature females. The DD2R gene expression was not detected in ovarian follicular cells.

Effect of Sexual Maturation on DD2R Gene Expression in Fat Body of Drosophila melanogaster Females

The expression of the DD2R gene was studied by in situ hybridization in the fat body (place of the synthesis of enzymes that degrade juvenile hormone) and ovarian follicular cells (place of the synthesis of 20-hydroxyecdysone) in young and sexually mature D. melanogaster females. It was demonstrated that the DD2R gene is expressed in the fat body, and its expression is higher in young females than in sexually mature females. The DD2R gene expression was not detected in ovarian follicular cells.