N. S. Zakharchenko

Effect of Hypermethylation of CCWGG Sequences in DNA of Mesembryanthemum crystallinum Plants on Their Adaptation to Salt Stress

Under salt stress conditions, the level of CpNpG-methylation (N is any nucleoside) of the nuclear genome of the facultative halophyte Mesembryanthemum crystallinum in the CCWGG sequences (W = A or T) increases two-fold and is coupled with hypermethylation of satellite DNA on switching-over of C3-photosynthesis to the crassulacean acid metabolism (CAM) pathway of carbon dioxide assimilation. The methylation pattern of the CCWGG sequences is not changed in both the 5′-promoter region of the gene of phosphoenolpyruvate carboxylase, the key enzyme of C4-photosynthesis and CAM, and in the nuclear ribosomal DNA. Thus, a specific CpNpG-hypermethylation of satellite DNA has been found under conditions of expression of a new metabolic program. The functional role of the CpNpG-hypermethylation of satellite DNA is probably associated with formation of a specialized chromatin structure simultaneously regulating expression of a large number of genes in the cells of M. crystallinum plants on their adaptation to salt stress and switching-over to CAM metabolism.

Enhanced resistance to phytopathogenic bacteria in transgenic tobacco plants with synthetic gene of antimicrobial peptide cecropin P1

Plasmids with a synthetic gene of the mammalian antimicrobial peptide cecropin P1 (cecP1) controlled by the constitutive promoter 35S RNA of cauliflower mosaic virus were constructed. Agrobacterial transformation of tobacco plants was conducted using the obtained recombinant binary vector. The presence of gene cecP1 in the plant genome was confirmed by PCR. The expression of gene cecP1 in transgenic plants was shown by Northern blot analysis. The obtained transgenic plants exhibit enhanced resistance to phytopathogenic bacteria Pseudomonas syringae, P. marginata, and Erwinia carotovora. The ability of transgenic plants to express cecropin P1 was transmitted to the progeny. F0 and F1 plants had the normal phenotype (except for a changed coloration of flowers) and retained the ability to produce normal viable seeds upon self-pollination. Lines of F1 plants with Mendelian segregation of transgenic traits were selected.

The Effect of Aerobic Methylotrophic Bacteria on the in vitro Morphogenesis of Soft Wheat (Triticum aestivum)

The effects of four aerobic methylotrophic bacteria on the morphogenesis of soft wheat (Triticum aestivum) were studied in vitro using immature embryos as explants. The inoculation of the embryos with methylotrophic bacteria led to their stable colonization with the bacteria. The colonization of the explants with the strains of Methylobacterium sp. D10 and Methylophilus glucoseoxidans stimulated the formation of morphogenic calli and shoots and also promoted development of the regenerated plants. These regenerated plants manifested bright green leaves and a well-developed root system. The colonization of immature wheat embryos with methylotrophic bacteria can be employed as a tool for raising the efficiency of genetic transformation of various wheat cultivars.

Hypermethylation of 5′-Region of the Human Calcitonin Gene in Leukemias: Structural Features and Diagnostic Significance

Methylation of the 5′-region of the calcitonin gene was investigated in bone marrow and peripheral blood cells of 27 healthy volunteers and 25 leukemic patients. In all patients suffering from various forms of myeloid and lymphoid leukemia, hypermethylation of CpG sequences was observed in this region of the calcitonin gene. Cytosine hypermethylation in the CpG sequence did not involve cytosines of adjacent CpNpG sequences (where N is any nucleoside). The 5′-region of the calcitonin gene lacked CpNpG methylation both in healthy controls and in leukemic patients; this apparently represents specific “non-alternative” type of CpG methylation in the extended DNA sequence. Methylation of the calcitonin gene was monitored in 18 leukemic patients during malignant progression and medical treatment. Hypermethylation of the calcitonin gene was not observed on long-term clinical hematological remission. In ten patients characterized by unstable (or incomplete) remission hypermethylation of the calcitonin gene persisted through the whole period of observation. In relapses, hypermethylation of the calcitonin gene appeared again and in six patients, this “molecular relapse” being registered 1-8 months before onset of clinical and laboratory signs of disease progression. The leukemia-specific hypermethylation of CpG sequences of the 5′-region of the calcitonin gene is a promising prognostic and diagnostic marker of leukemias and might be useful for monitoring of this disease.

The ways to produce biologically safe marker-free transgenic plants

The review considers the basic strategies used to produce biologically safe marker-free transgenic plants and analyzes their advantages and disadvantages. The systems of positive and negative selection as safer approaches for transformant identification are briefly described. The application of co-transformation, transposition, and site-specific recombination for production of marker-free plants is described. Special attention is paid to novel approaches to create marker-free plants initially containing no selective genes in their genomes.

Expression of cecropin P1 gene increases resistance of Camelina sativa (L.) plants to microbial phytopathogenes

Transgenic plants of camelina (Camelina sativa (L.) Crantz) with the synthetic gene of antimicrobial peptide cecropin P1 (cecP1) were obtained. Agrobacterium-mediated transformation is performed using the binary vector pGA482::cecP1 by vacuum infiltration of flower buds. The presence of the cecP1 gene in the genome of plants was confirmed by PCR. CecP1 gene expression in transgenic plants was shown by Western blot analysis and by antimicrobial activity of plant extracts against the bacterial phytopathogene Erwinia carotovora. The plants of F0 and F1 generations had the normal phenotype and retained the ability to form viable seeds in self-pollination. cecP1 plants exhibit enhanced resistance to bacterial and fungal phytopathogens: Erwinia carotovora and Fusarium sporotrichioides. The increased sustainability of cecropin P1-expressing plants against salt stress is shown. The possibility of the integration of the cecP1 gene into the overall protective system of plants against biotic and abiotic stresses is discussed.

Use of the gene of antimicrobial peptide cecropin P1 for producing marker-free transgenic plants

The marker-free transgenic tobacco plants carrying a synthetic gene encoding the antimicrobial peptide cecropin P1 (cecP1) under the control of the cauliflower mosaic virus 35S RNA promoter were produced. The binary vector pBM, free of any selective genes of resistance to antibiotics or herbicides intended for selecting transgenic plants, was used for transformation. The transformants were screened on a nonselective medium by detecting cecropin P1 in plant cells according to the antibacterial activity of plant extracts and enzyme immunoassay. According to the two used methods, 2% of the analyzed regenerants were transformants. The resulting marker-free plants displayed a considerably increased resistance to microbial phytopathogens—the bacterium Erwinia carotovora and fungus Sclerotinia sclerotiorum. Thus, the gene cecP1 can be concurrently used as a target gene and a screening marker. The utility of cecP1 as a selective gene for direct selection of transformed plants is discussed.

Physiological features of rapeseed plants expressing the gene for an antimicrobial peptide cecropin P1

Transgenic rapeseed (Brassica napus L.) plants carrying an artificial gene for the antimicrobial peptide cecropin P1 (cecP1) were obtained and characterized. The agrobacterial transformation was done by vacuum infiltration of seeds with agrobacterium GV3101(pMP90RK) containing a binary vector pGA482::cecP1. The cec1 gene expression was analyzed by Western blotting and confirmed by antimicrobial activity measurements of plant extracts. The obtained plants showed the resistance to the bacterial and fungal pathogens Erwinia carotovora and Fusarium sporotrichioides. The photosynthetic activities of control and transgenic plants under biotic stress conditions of E. carotovora infection were comparatively studied. The higher tolerance of the cecP1 plants to the oxidative stress caused by paraquat was shown. The results obtained point to the possibility of incorporation of the cecropin P1 gene into the integral stress protection system of plants.

Effect of rhizosphere bacteria Pseudomonas aureofaciens on the resistance of micropropagated plants to phytopathogens

Effects of colonization of micropropagated potato (Solanum tuberosum L.) and strawberry (Fragaria L.) plants by the rhizosphere bacterium Pseudomonas aureofaciens strain BS1393 (VKM B-2188 D) on plant growth and resistance to bacterial and fungal phytopathogens were studied. Pseudomonad colonization improved the physiological characteristics of plants and enhanced their adaptation to in vivo conditions. The presence of P. aureofaciens cells in various plant tissues (leaves, stems, and roots) in vitro was demonstrated on the background of plant cocolonization by two associative strains—P. aureofaciens strain BS1393 (VKM-B-2188 D) and Methylovorus mays (VKM-B-2221). The colonized plants displayed an increased resistance to the phytopathogens Erwinia carotovora, Sclerotinia sclerotiorum, and Phytophthora infestans. These results demonstrate that pseudomonades are promising for practical application in the microbial protection of plants against phytopathogens.

Effects of associative pseudomonads and methylobacteria on plant growth and resistance to phytopathogens and xenobiotics

The in vivo and in vitro interactions between tobacco (Nicotiana tabacum L.), tomato (Lycopersicon esculentum Mill.), cabbage (Brassica oleracea var. capitata L.), rape (B. napus L.), and the common ice plant (Mesembryanthemum crystallinum L.) and bacteria Pseudomonas aureofaciens, P. putida, and Methylovorus mays were studied. Stable associations of these microorganisms with plants are demonstrated. Colonized plants were characterized by accelerated growth, more efficient rooting, better adaptation to in vivo conditions, and enhanced resistance to bacterial and fungal phytopathogens (Erwinia carotovora, Sclerotinia sclerotiorum and Phytophthora infestans). Plants colonized by bacteria resistant to kanamycin and naphthalene can grow steadily on the medium containing these compounds. The results obtained indicate a promising usage of beneficial associative microorganisms for the development of technologies for plant protection against biotic and abiotic stressors.