Expression of a Stilbene Synthase Gene from the Vitis labrusca x Vitis vinifera L. Hybrid Increases the Resistance of Transgenic Nicotiana tabacum L. Plants to Erwinia carotovora.

'Isabel' grape (Vitis labrusca x V. vinifera L. hybrid) is one of the main grape cultivars in Russia and some other countries for processing, due to its vigor, tolerance to the main fungal diseases, high yield and potential for sugar accumulation. The stilbene synthase gene VlvSTS was isolated from the hybrid grape cv. Isabel and cloned into a pSS plant transformation vector under the control of a constitutive 35S RNA double promoter of the cauliflower mosaic virus, CaMV 35SS. VlvSTS-gene containing transgenic tobacco lines were obtained and analyzed. For the first time plants expressing the VlvSTS gene were shown to have an enhanced resistance to the bacterial pathogen Erwinia carotovora subsp. carotovora B15. Transgenic plants were tested for resistance to a number of fungal pathogens. The plants were resistant to the grey mould fungus Botrytis cinerea, but not to the fungi Fusarium oxysporum, F. sporotrichioides, or F. culmorum. According to the results of a high performance liquid chromatography-mass spectrometry analysis, the amount of trans-resveratrol in leaves of transgenic plants with the highest expression of the VlvSTS gene was in a range from 150 to 170 µg/g of raw biomass. Change in the color and a decreased anthocyanin content in the flower corollas of transgenic plants were observed in transgenic lines with the highest expression of VlvSTS. A decrease in total flavonoid content was found in the flower petals but not the leaves of these tobacco lines. High expression of the VlvSTS gene influenced pollen development and seed productivity in transgenic plants. The size of pollen grains increased, while their total number per anther decreased. A decrease in the number of fertile pollen grains resulted in a decreased average weight of a seed boll in transgenic plants.

Amyloidogenic Propensities of Ribosomal S1 Proteins: Bioinformatics Screening and Experimental Checking.

Structural S1 domains belong to the superfamily of oligosaccharide/oligonucleotide-binding fold domains, which are highly conserved from prokaryotes to higher eukaryotes and able to function in RNA binding. An important feature of this family is the presence of several copies of the structural domain, the number of which is determined in a strictly limited range from one to six. Despite the strong tendency for the aggregation of several amyloidogenic regions in the family of the ribosomal S1 proteins, their fibril formation process is still poorly understood. Here, we combined computational and experimental approaches for studying some features of the amyloidogenic regions in this protein family. The FoldAmyloid, Waltz, PASTA 2.0 and Aggrescan programs were used to assess the amyloidogenic propensities in the ribosomal S1 proteins and to identify such regions in various structural domains. The thioflavin T fluorescence assay and electron microscopy were used to check the chosen amyloidogenic peptides' ability to form fibrils. The bioinformatics tools were used to study the amyloidogenic propensities in 1331 ribosomal S1 proteins. We found that amyloidogenicity decreases with increasing sizes of proteins. Inside one domain, the amyloidogenicity is higher in the terminal parts. We selected and synthesized 11 amyloidogenic peptides from the Escherichia coli and Thermus thermophilus ribosomal S1 proteins and checked their ability to form amyloids using the thioflavin T fluorescence assay and electron microscopy. All 11 amyloidogenic peptides form amyloid-like fibrils. The described specific amyloidogenic regions are actually responsible for the fibrillogenesis process and may be potential targets for modulating the amyloid properties of bacterial ribosomal S1 proteins.

Solvent effects on the secondary structure of the membrane-active zervamicin determined by PELDOR spectroscopy.

Zervamicin is a voltage-gated ion-channel-forming peptide. Channels are generally considered to be formed by first insertion of amphipathic molecules into the phospholipid bilayer, followed by self-assembly of a variable number of transmembrane helices. We have studied the length of the peptide structure to address the question whether this peptide is long enough to span the phospholipid bilayer. The pulsed electron-electron double resonance (PELDOR) spectroscopic technique was used to determine the length of the helical molecule in membrane-mimicking solvents. This was achieved from the distance-related dipole-dipole interaction between spin labels, which were located at both ends of the linear peptide chain. The data were obtained by using samples of frozen glassy solutions of MeOH, MeOH/toluene, and MeOH/CHCl(3). Contributions of inter- and intramolecular interactions of spin labels were separated to analyze the intramolecular interaction and the distance distribution function between the labels. It is shown that the main maximum of the distribution functions is located at a distance of ca. 3.3 nm, and this distance appears to be only slightly dependent on the solvent composition. The distribution function was observed to narrow after addition of either CHCl(3) or toluene to MeOH. This effect is rationalized in terms of a decreased mobility of the terminal amino acid residues. By molecular-dynamics simulations, it was shown that the conformation, corresponding with the predominant distance found by PELDOR, agrees well with the mixed alpha/3(10)-helical that was previously determined by NMR. However, in the case toluene was added to the MeOH solution to further increase the hydrophobicity of the environment of the membrane-active peptide, the distribution function gives rise to a minor fraction (7-8%) with a distance of 4.2 nm. This distance corresponds most likely to the more extended 2(7)-helix structure.