The aim of our work was to study the colonization of potato, tomato, rapeseed and camelina by associative microorganisms Methylobacterium mesophilicum, Pseudomonas aureofaciens BS1393 and Pseudomonas putida BS3701; examine the resistance of colonized plants to biotic (phytopathogens Erwinia carotovora and Sclerotinia sclerotiorum) and abiotic (naphthalene, oil) stressors. Colonized plants were characterized by an increased growth rate (1.5–2.0 times higher) compared to non-colonized ones; flower-bud formation, flowering and fructification of the colonized plants also started earlier. An increased resistance of colonized plants to phytopathogens, naphthalene (100 mµ/ml) and oil (0.7 %) was noted, too. The level of superoxide dismutase (SOD) in control plants on a medium with naphthalene or oil increased by 160–150%; in colonized plants – by 20–18 %. Colonized plants were more viable because of the presence of P. putida BS3701 on the roots.

The in vivo and in vitro interactions between tomato (Lycopersicon esculentum Mill.) and tobacco (Nicotiana tabacum L.) and the bacteria Pseudomonas fluorescens, Acinetobacter baumannii, Rhodococcus erythropolis,Pseudomonas aureofaciens, Pseudomonas putida, Methylovorus mays have been studied. These microorganisms were shown to be in stable associations with plants. The colonized plants were characterized by more rapid growth, a higher yield, and better adaptation to in vivo conditions. The colonized plants were more resistant to bacterial phytopathogens Erwinia carotovora and Pseudomonas syringae. Plants colonized by naphthalene-resistant bacteria can grow steadily on a medium containing this compound. The results demonstrate the prospects of the use of beneficial associative microorganisms in the development of technologies for plant protection against biotic and abiotic stressors.

Marker-free transgenic Camelina sativa (L.) plants carrying a synthetic gene for cecropin P1, an antimicrobial peptide, under the control of the cauliflower mosaic virus 35S RNA promoter have been obtained and analyzed. The plants were transformed with an agrobacterial binary vector free of selective genes of antibiotic and herbicide resistance. The marker-free transformants were screened via measurement of the antibacterial activity of cecropin P1 and enzyme immunoassay. The obtained plants exhibited an increased resistance to infection with the bacteria Erwinia carotovora, the fungi Fusarium graminearum, and oxidative stress during infection. Analysis of the fatty acid composition of seed oil showed an increased amount of α-linolenic acid in the transgenic Camelina lines as compared to unmodified plants. The results indicate that the cecropin P1 gene can be included in an integral antistress plant-protective system.

A study of the plants rapeseed (Brassica napus L.) with artificial gene antimicrobial peptide cecropin P1 has shown enhanced resistance of transgenic plants to phytopathogenic microorganisms Erwinia carotovora, Fusarium oxysporum, Sclerotinia sclerotiorum and oxidative stress, as compared to the nontransformed plants.

Transgenic tobacco (Nicotiana tabacum L.) plants with an artificial gene from the antimicrobial peptide bombinin (bom) have been obtained and studied. The presence of the bom gene in the genome of kanamycin-resistant plants was shown by PCR. Expression of the bom gene was confirmed by antimicrobial activity measurements in leaf extracts. The obtained plants were morphogenetically resistant to Erwinia carotovora bacteria and Rhizoctonia solani fungi phytopathogens. In addition, the protective oxidative reaction to the infection, i.e., the SOD activity and proline content, were lower in transgenic plants than in the infected nontransgenic plants. Plants with the expression of the antimicrobial bombinin peptide gene are promising for use in agricultural biotechnology as plant protectors.

Получены безмаркерные растения камелины (Camelina sativa (L.)), с геном антимикробного пептида цекропина Р1

Kalanchoe pinnata L. plants bearing an artificial CP1 gene encoding the cecropin P1 antimicrobial peptide have been obtained. The presence of the CP1 gene in the plant genome has been confirmed by PCR. Cecropin P1 synthesis in transgenic plants has been shown by MALDI mass spectrometry and Western blotting. The obtained plants have been highly resistant to bacterial and fungal phytopathogens, and their extracts have demonstrated antimicrobial activity towards human and animal pathogens. It has been shown that transgenic plants bearing the CP1 gene can be colonized by the beneficial associative microorganisms Methylovorus mays.

Transgenic kalanchoe plants (Kalanchoe pinnata L.) expressing the antimicrobial peptide cecropin P1 gene (cecP1) under the control of the 35S cauliflower mosaic virus 35S RNA promoter and the selective neomycin phosphotransferase II (nptII) gene under the control of the nopaline synthase gene promoter were studied. The 35S promoter methylation and the cecropin P1 biosynthesis levels were compared in plants growing on media with and without kanamycin. The low level of active 35S promoter methylation further decreases upon cultivation on kanamycin-containing medium, while cecropin P1 synthesis increases.

Ген антимикробного пептида цекропина Р1 (СP1) встраивали в векторную плазмиду pPCV91 под контролем промотора 35S РНК вируса мозаики цветной капусты (CaMV 35S), содержащего 4 энхансерных последовательности CaMV 35S и нетранслируемую лидерную последовательность РНК вируса табачной мозаики. С помощью полученного рекомбинантного вектора проведена агробактериальная трансформация растений табака (Nicotiana tabacum L.) сорта Самсун. Присутствие гена СР1 в геноме растений подтверждено методом полимеразной цепной реакции. Экспрессия гена СР1 в трансгенных растениях доказана вестерн-блот анализом и тестированием антибиотической активности растительных экстрактов. Уровень синтеза цекропина Р1 в различных линиях составлял 0.02–0.2% от общего растворимого белка листьев растений. Трансгенные растения проявляли по сравнению с контрольными растениями повышенную устойчивость к фитопатогенным микроорганизмам и к окислительному стрессу. Показано, что способность трансгенных растений экспрессировать цекропин Р1 передавалась потомству.

A gene of antimicrobial peptide cecropin P1 (CP1) was inserted into the vector plasmid pPCV91 under the control of promoter 35S RNA of cauliflower mosaic virus (CaMV 35S) containing four enhancer sequences CaMV 35S and nontranslated leader sequence Ω RNA of tobacco mosaic virus. The recombinant vector obtained was used for agrobacterial transformation of tobacco plants (Nicotiana tabacum L., variety Samsun) with the polymerase chain reaction (PCR)-based method. The presence of gene CP1 in the genome of plants was proven by western-blot analysis and testing the antibiotic activity of plant extracts. In different plant lines, the level of cecropin P1 synthesis amounted 0.02–0.2% of total soluble plant leaf protein. The transgenic plants, unlike the control ones, displayed enhanced tolerance to phytopathogenic microorganisms and oxidative stress. It was established that the ability of the transgenic plants to express cecropin P1 is transmitted to progeny.

Design of the transcriptionally-fused protein MoHhaI-EGFP, composed of bacterial DNA-methyltransferase MoHhaI and enhanced green fluorescent protein (GFP) is described. The mentioned MoHhaI-EGFP was expressed in Escherichia coli ER1821 and purified by affinity chromatography on Ni-NTA agarose. According to expectations MoHhaI-EGFP fused protein retained significant features of corresponding original proteins: the ability to transfer methyl group to the C5 carbon atom of internal cytosine in CGCG site and absorption-emission spectral characteristics. The created transcriptionally-fused protein MoHhaI-EGFP could be used in various experiments in molecular biology. (C) 2014 Elsevier Ltd. All rights reserved.

DNA methyltransferases might be used as powerful tools for studies in molecular and cell biology due to their ability to recognize and modify nitrogen bases in specific sequences of the genome. Methylation of the eukaryotic genome using exogenous DNA methyltransferases appears to be a promising approach for studies on chromatin structure. Currently, the development of new methods for targeted methylation of specific genetic loci using DNA methyltransferases fused with DNA-binding proteins is especially interesting. In the present review, expression of exogenous DNA methyltransferase for purposes of in vivo analysis of the functional chromatin structure along with investigation of the functional role of DNA methylation in cell processes are discussed, as well as future prospects for application of DNA methyltransferases in epigenetic therapy and in plant selection.

The karyotype of the common ice plant Mesembryanthemum crystallinum L. (Aizoaceae) was studied using Chromomycin A(3) (CMA)/4',6-diamidino-2-phenylindole (DAPI) staining, fluorescence in situ hybridization with 5S and 18S-5.8S-25S rDNA probes, DAPI/C-banding and immunodetection of 5-methylcytosine. A single bright CMA-band was revealed on the satellite chromosome, whose location was coincided with a position of a site of 18S-5.8S-25S rRNA genes. A site of 5S rRNA genes was observed on one of the other chromosomes. Relatively large DAPI/C-bands were mainly localized in the pericentromeric regions of the chromosomes. DAPI/C-banding patterns allowed us to identify all the chromosomes in the karyotype of M. crystallinum. The methylation of euchromatic chromosome regions was weaker as compared with heterochromatic DAPI/C-bands, which were hypermethylated. The obtained results may provide opportunities for investigating, at the chromosomal level, the genomic changes occurring in M. crystallinum either under salinization or under the action of other stress factors.

DNA methylation is an integral part of the mechanism of a remodeling and modification of the chromatin structure. The global complex net of chromatin modification and remodeling reactions is still to be determined, and studies of the mechanisms controlling the epigenetic processes of histone modification and DNA methylation are in their infancy. Cytosine methylation occurs predominantly in CpG sequences of the eukaryotic genome, and it also takes place at symmetric CpHpG and nonsymmetric CpHpH sites (where H is A, T, or C). The modification efficiency of the three types of DNA methylation sites depends on their genomic localization. Different regions of the eukaryotic genome are remarkable for their methylation features: CpG-islands, CpG-island shores, differentially methylated regions of imprinted genes, and regions of nonalternative site-specific modification. The three canonical sites (CpG, CpHpG, and CpHpH) differ in DNA methylation efficiency depending on their nucleotide context. An epigenetic code of DNA methylation can be assumed with context differences playing a specific functional role. The review summarizes the main up-to-date data on the structural and functional features of site-specific cytosine methylation in eukaryotic genomes. Pathogenesis-related alterations in the methylation pattern of the eukaryotic genome are considered.