Department of Plant Molecular Biology and Biotechnology

All publications (show selected)

Sergey Zavriev

  • Russia, Moscow, Ul. Miklukho-Maklaya 16/10 — On the map
  • IBCh RAS, build. БОН, office. 405
  • Phone: +7(495)995-55-57#2044
  • E-mail: szavriev@ibch.ru

Production of marker-free tomato plants expressing the supersweet protein thaumatin II gene under the control of predominantly fruit-specific promoters

Artificial Climate Station “BIOTRON”

Despite the lack of evidence of the danger of genetically modified organisms the presence of marker and antibiotic-resistant genes in transgenic plants causes concern to consumers. Genetically modified plants with viral and bacterial genes are adopted by consumers, but with concerns; in addition, constitutive promoters have a number of disadvantages in industrial-scale cultivation of plants. In our study, we used the pMF vector system (Wageningen Plant Research, Wageningen, Netherlands), which combines inducible site-specific recombinase and a bifunctional selectable gene to obtain marker-free tomato plants. The gene of interest was the supersweet thaumatin II protein from the tropical plant Thaumatococcus daniellii under the control of tomato predominantly fruit-specific early-light inducible protein (ELIP) or E8 promoters and tomato Rubisco terminator. The use of this gene in our laboratory allowed enhancing sweetness, as well as improving the taste characteristics of fruit such as apple, strawberries, carrots, tomatoes, and pears. By using different strategies of early and delayed selection we developed a protocol for obtaining fully marker-free tomato plants, which was checked by polymerase chain reaction and Southern blotting. The thaumatin II gene expression was confirmed by reverse transcription-PCR and western blotting analyses. The fruit of transgenic and marker-free tomato plants displayed a sweet taste. A quantitative comparative assessment of the level of expression of the thaumatin protein under the control of two promoters was carried out using enzyme-linked immunosorbent assay. Multiple and/or incomplete T-DNA inserts that often occur during transformation of Solanaceae greatly reduced the efficiency of the system used.

Small open reading frames encode regulatory microproteins in plants

Laboratory of functional genomics and plant proteomics

Genomes contain millions of short (<100 codons) open reading frames (sORFs), which are usually dismissed during gene annotation. Nevertheless, peptides encoded by such sORFs can play important biological roles, and their impact on cellular processes has long been underestimated. Here, we analyzed approximately 70,000 transcribed sORFs in the model plant Physcomitrella patens (moss). Several distinct classes of sORFs that differ in terms of their position on transcripts and the level of evolutionary conservation are present in the moss genome. Over 5000 sORFs were conserved in at least one of 10 plant species examined. Mass spectrometry analysis of proteomic and peptidomic data sets suggested that tens of sORFs located on distinct parts of mRNAs and long noncoding RNAs (lncRNAs) are translated, including conserved sORFs. Translational analysis of the sORFs and main ORFs at a single locus suggested the existence of genes that code for multiple proteins and peptides with tissue-specific expression. Functional analysis of four lncRNA-encoded peptides showed that sORFs-encoded peptides are involved in regulation of growth and differentiation in moss. Knocking out lncRNA-encoded peptides resulted in a decrease of moss growth. In contrast, the overexpression of these peptides resulted in a diverse range of phenotypic effects. Our results thus open new avenues for discovering novel, biologically active peptides in the plant kingdom.

Publications

  1. Fesenko I, Kirov I, Kniazev A, Khazigaleeva R, Lazarev V, Kharlampieva D, Grafskaia E, Zgoda V, Butenko I, Arapidi G, Mamaeva A, Ivanov V, Govorun V (2019). Distinct types of short open reading frames are translated in plant cells. Genome Res 29 (9), 1464–1477

Obtaining and Analysis of Marker-free Oil Plants Camelina sativa (L.) Expressing Gene for Antimicrobial Peptide Cecropin P1

Laboratory of Plant Biotechnology

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 selection 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.

Diversity of rhodopsins in cultivated bacteria of the family Geodermatophilaceae associated with non-aquatic environments

Laboratory of Plant Biotechnology

A large number of different rhodopsins were found in cultivated bacteria isolated from hot and arid ecological niches. A total of 31 rhodopsin genes were identified in 51 analyzed genomes of strains belonging to the family Geodermatophilaceae. Overall, 88% of the strains harbouring rhodopsins are isolated from non-aquatic environments. It was found that 82% of strains belonging to the genus Geodermatophilus have at least one gene as compared to 38% of strains of other genera which contain rhodopsins. Analysis of key amino acids revealed two types of the studied proteins: DTE type (putative proton pump) and NDQ type (putative sodium pump). Proton pumps were divided into two subtypes (DTEW and DTEF) according to phylogenetic analysis and the presence of highly conserved tryptophan or phenylalanine at position 182. Among all studied rhodopsins DTEF subtype is the most unique one, identified only in this family.

The picture shows phylogenetic tree based on rhodopsin protein sequences found in bacteria of the family Geodermatophilaceae.

Publications

  1. Tarlachkov SV, Shevchuk TV, Montero-Calasanz MDC, Starodumova IP (2019). Diversity of rhodopsins in cultivated bacteria of the family Geodermatophilaceae associated with non-aquatic environments. Bioinformatics 36 (6), 1668–1672

Cluster of differentially expressed methionine cycle proteins in potato cultivars differing in responses to combined stress

Laboratory of functional genomics and plant proteomics,  Laboratory of Molecular Bases of Plant Stress Resistance

Comparative quantitative proteomic analysis of potato cultivars Gala and Chicago differing in responses to biotic stress (infection with potato virus Y) and abiotic stress (elevated temperature; 28°С) identifies a cluster of differentially expressed protein members of methionine cycle. It is well known that RNA/DNA methylation is an important factor affecting plant resistance to virus infections and possibly abiotic stress tolerance.

In plants of susceptible cv Chicago, accumulation of five proteins involved in methylation processes is reduced (shown in green), whereas in plants of resistant cv Gala, accumulation of cystathionine beta lyase and SAM-dependent methyltransferase (shown in red), involved in metabolism of  S-adenosyl methionine (methyl-group donor) is significantly increased. We have also analysed expression levels of some methionine cycle proteins using quantitative RT-PCR. Comparison of RT-PCR and proteomic data has demonstrated that decrease in accumulation of methionine cycle proteins in susceptible Chicago plants occurs at the translational rather than transcriptional level. It is suggested that susceptibility to stresses in potato cv Chicago plants may be related to reduced amount of proteins involved in methylation.

Identification of novel Fusarium taxa by multiphasic approach

Laboratory of Molecular Diagnostics

A comprehensive analysis of the taxonomic status and characteristics of a number of collection strains of fungi of the genus Fusarium was carried out. The use of multilocus phylogenetic analysis, morphological characteristics and the study of toxin formation allowed us to identify a number of cultures that are believed to be new, previously uncharacterized species. The most prominent example of such a strain is strain F-846, originally identified as F.poae. He formed a separate branch on phylogenetic trees constructed using sequences of 5 marker genes. A study using HPLC methods has demonstrated that strain F-846 is capable of synthesizing DON and its acetylated derivatives. Microscopic analysis confirmed that this strain appears to be a new species of the genus Fusarium.

Novel plant antiviral mechanism involves interaction of subnuclear structures and a viral protein.

Laboratory of functional genomics and plant proteomics,  Laboratory of Molecular Bases of Plant Stress Resistance

The nucleolus and Cajal bodies (CBs) are sub-nuclear domains with well-known roles in RNA metabolism and RNA-protein assembly. However, they also participate in other important aspects of cell functioning. In collaboration with the James Hutton Institute we have described a previously unrecognised mechanism by which these bodies and their components regulate host defence against pathogen attack. It has been shown that the major proteins of CBs (coilin) and the nucleolus (fibrillarin) interact with poly(ADP-ribose) polymerase (PARP) and modify its function, and that this interaction is modulated by the tobacco rattle virus 16K protein.

Our results reveal that PARP a nuclear protein, modifies the function and subcellular localisation of a variety of nuclear “target” proteins (acceptors) by attaching chains of ADP ribose (PAR) to them. To re-activate these target proteins PARP shuttles them from both the nucleolus (NO) and chromatin to CBs for PAR removal and recycling (Fig 1a). In response to TRV infection plants initiate defence responses as evidenced by the over-accumulation of reactive oxygen species (ROS) and some increase in levels of pathogenesis-related (PR) PR-1a transcripts and callose deposits (markers of antivirus defence response). Upon TRV infection, the 16K protein is produced in the cytoplasm and is targeted to the nucleus (Fig 1b). In the nucleus (CBs and nucleoplasm) the 16K protein interacts with coilin and re-localizes it to the nucleolus, which in turn traps PARP within this sub-nuclear domain, preventing its trafficking from the nucleolus for PAR cleavage and recycling. This leads to over-accumulation of PAR/PARylated proteins and significant enhancement of the defence responses (such as PR-1a gene expression and callose deposition). Callose deposition presumably directly restricts TRV spread to newly emerging leaves (Fig. 1c).

Study of molecular biological mechanisms and the development of molecular breeding methods for obtaining plants of agricultural crops that are resistant to environmental stress factors and with improved crop quality

Artificial Climate Station “BIOTRON”

An analysis of the inheritance of transgenic traits in the offspring of 54 primary transgenic lines of T0 common wheat, containing the genes for resistance to the herbicide bar and salt stress hvnxh2 or agnhx, was carried out. 165 T2-T3 populations inheriting stress resistance genes as homozygous alleles were identified. Molecular and biological analysis confirmed the expression of salt tolerance genes in the homozygous progeny of 16 T0 lines. To study the post-transcriptional regulation of the ripening time of fruits, 27 transgenic apple lines adapted to ex vitro conditions, which contain expression cassettes to suppress ethylene synthesis, were successfully grafted onto rootstocks in order to accelerate their flowering and fruiting.

Study of molecular biological mechanisms and the development of molecular breeding methods for obtaining plants of agricultural crops that are resistant to environmental stress factors and with improved crop quality. № 0101-2014-0047, № 01201352435

Early Detection and Identification of the Main Fungal Pathogens for Resistance Evaluation of New Genotypes of Forest Trees

Group of Forest Biotechnology

Growing importance of forest plantations increases the demand for phytopathogen resistant forest trees. This study describes an effective method for the early detection and identification of the main fungal phytopathogens in planting material of silver birch (Betula pendula) and downy birch (B. pubescens) based on the estimation of size of the internal transcribed spacers (ITS1 and ITS2) in the 18S-5.8S-28S rDNA gene cluster, which are species-specific for most micromycetes. Electrophoretic assay of the ITS1 and ITS2 loci allowed us to identify the predominant phytopathogenic fungal species in downy and silver birch in planta. This new molecular genetic method can be used to screen birch and other forest trees for different fungal pathogens in evaluation of disease resistance. This information can be useful in breeding of new genotypes of forest trees including transgenic clones with modified wood composition. Example of two computer-generated electrophoregrams with multiple peaks representing species-specific fungal and host plant (B. pendula) amplicon DNA fragments from the two PCR-amplified samples of DNA isolated from infected silver birch leaf (A) and shoot (B)tissues, amplified using the ITS1-ITS2 primer pair combination and separated in the capillary gel electrophoresis using an ABI Prism 310 Genetic Analyzer. Brown peaks represent DNA fragments of the GeneScan™ 500 LIZ™ dye Size Standard.

The test on laboratory animals of the recombinant vaccine protein M2e-RTB, obtained in the duckweed plants.

Artificial Climate Station “BIOTRON”

In accordance with the research plan in 2018, a molecular-biological analysis of duckweed plants was carried out; the duckweed lines, producers of recombinant vaccine protein M2e-RTB, were obtained for the first time. The recombinant protein M2e-RTB obtained in plants of the duckweed was orally immunized to mice, and induction of antibodies to the peptide M2e of the avian influenza virus was shown.

"Development of systems for the synthesis of recombinant proteins based on plant expression platforms (biofarming)". № 01201352434

Resistance of bombinin-expressing plants to phytopathogens

Laboratory of Plant Biotechnology

Transgenic plants expressing artificial gene of an antimicrobial peptide bombinin have been produced and investigated. This plants were resistant to the Erwinia carotovora bacteria and Rhizoctonia solani fungi phytopathogens. Such plants expressing the small antimicrobial peptide gene are promising for new approach in plant protection agriculture technology.

The first recombinant viper three finger toxins - antagonists of the nicotinic acetylcholine receptors of muscle and neuronal types

Laboratory of ligand-receptor interactions,  Laboratory of Molecular Diagnostics,  Science-Educational center,  Laboratory of molecular toxinology

One of the main components of the venom of the snakes from the Elapidae family are three finger toxins, which possess various types of biological activity, including inhibition of synaptic transmission by blocking the nicotinic acetylcholine receptors. So far, three finger toxins have not been found in the venom of the Viperidae snakes, although the mRNA encoding these toxins has been found in the venom glands of snakes from this family. Genes encoding two three-finger toxins TFT-AF and TFT-VN, nucleotide sequences of which were earlier determined by cloning cDNA from venom glands of vipers Azemiops feae and Vipera nikolskii, respectively, were expressed for the first time in E. coli cells. The biological activity of these toxins was studied by electrophysiological techniques, calcium imaging, and radioligand analysis. We have shown for the first time that viper three-finger toxins are antagonists of nicotinic acetylcholine receptors both of neuronal and muscle type.

Subcellular localization and detection of Tobacco mosaic virus ORF6 protein by immunoelectron microscopy

Laboratory of Molecular Diagnostics

Previous studies have shown that genomes of some tobamoviruses contain not only genes coding for coat protein, movement protein, and the cistron coding for different domains of RNA-polymerase, but also a gene, named ORF6, coding for a poorly conserved small protein. In this study, using biochemical and immunological methods, we have shown that ORF6 peptide is accumulated after infection in case of two isolates of Tobacco mosaic virus strain U1 (TMV-U1 common and TMV-U1 isolate A15). Unlike virus particles accumulating in the cytoplasm, the product of the ORF6 gene is found mainly in nuclei, which correlates with previously published data about transient expression of ORF6 isolated from TMV-U1. Moreover, we present new data showing the presence of ORF6 genes in genomes of several tobamoviruses.

Publications

  1. Erokhina TN, Lazareva EA, Richert-Pöggeler KR, Sheval EV, Solovyev AG, Morozov SY (2017). Subcellular localization and detection of Tobacco mosaic virus ORF6 protein by immunoelectron microscopy. Biochemistry (Mosc) 82 (1), 60–66

Variety of downy birch (Betula pubescens Ehrh.) Belros-3

Group of Forest Biotechnology

Birch is the main forest-forming deciduous tree species of the Russian Federation, occupying 15% of the forest area. Until now, not one birch variety has been registered in the State Register of Breeding Achievements. For the first time in Russia, a new variety of downy birch was created that differs from non-selective genotypes by high stability of various growth characteristics, increased productivity and increased sizes of wood fiber. The genotype can be used in short-rotation forest plantations.

A covalent DNA antibody conjugate was prepared for use in immuno-PCR

Laboratory of Molecular Diagnostics

The conjugate of antibody and single-stranded oligonucleotide  was prepared using a bioorthogonal strain promoted azide-alkyne cycloaddition reaction. The antibody was modified by sulphotetrafluorophenyl ester of azidocaproic acid; N-hydroxysuccinimide ester of dibenzocyclooctin was used to modify the oligonucleotide with the amino group introduced during the synthesis. Conjugates are obtained with a degree of  labeling 1-3  of the oligonucleotide to the antibody.

Mega-grant from the Government of the Russian Federation

Artificial Climate Station “BIOTRON”,  Laboratory of Molecular Bases of Plant Stress Resistance

The Laboratory of Molecular Bases of Plant Stress Resistance was established in 2017 when Professor M. Taliansky was awarded a mega-grant from the Russian government on the topic "New functions of the cell nucleus and complex resistance of potato plants to diseases and physiological stresses". The activity of the laboratory is aimed at obtaining comprehensive knowledge of the complex stresses in plants mechanisms and identifying key target genes necessary to protect plants from the impact of various stresses on the growth, development and productivity of plants. 

Study of gene expression of trichothecene cluster genes in Fusarium graminearum and F. ussurianum under different culture conditions and their toxigenicity

Laboratory of Molecular Diagnostics

Expression dynamics of genes belonging to the trichothecene cluster which includes 12 genes, responsible for regulation and different steps of mycotoxin biosynthesis has been investigated. Strains of F. graminearum (St-6 and St-9) and F. ussurianum (g. 120 and g. 267) were selected for this study. 3 groups of genes with different levels and periods of expression were identified. A special attention was paid to genes TRI9 and TRI14, which functions are not known at the moment. The expression of these genes kept on the same level during all the cultivation process. Nucleotide sequences of these genes and predicted protein sequences for TRI14 gene of F. poae, F. sambucinum, F. langsethiae, F. venenatum, F. cerealis were determined for the first time.     

Biological activity of water extract of cecropin P1-expressing Kalanchoe plants

Laboratory of Plant Biotechnology

Water extract of constructed Kalanchoe pinnata plants expressing antimicrobial peptide cecropin P1 (cecP1) has been analyzed in the experiments on plants and animals. The extract has effectively induced rhizogenesis on callus culture of Mesembryanthemum crystallinum plants, difficult object for differentiation in vitro. High wound healing (Fig. 1) and and microbiocide activity of the cecP1-plant extract, better than efficiency of Cefazolin antibiotic against Staphylococcus aureus and Pseudomonas aeruginosa, causative agents of purulent infections (Fig. 2), and efficiency of Clotrimazolum antifungal drug against highly virulenr clinical isolate Candida albicans were found. These data demonstrate good prospects for use cecP1-kalanchoe plants in experimental biology and pharmaceutics.

Elucidation of structure of elicitor protein CS20EP from Fusarium oxysporum strain CS-20

Laboratory of Molecular Diagnostics

The structure of cDNA of a protein expressed by F. oxysporum strain CS-20 has been determined based on preliminary data of N-terminus sequence. The use of bioinformatics approach allowed to deduce the full amino-acid sequence of the protein. The calculated molecular mass corresponded well to the data obtained earlier by MALDI-TOF (10 kDa). It was demonstrated that the CS20EP possesses elicitor activity and stimulates tomato plants’ response to infection by virulent strain of F. oxysporum, causing vascular wilt and necrosis of plant tissue. Several structural differences between CS20EP and similar proteins from other Fusarium species have been shown. The nucleotide sequence of the gene encoding CS20EP protein has been deposited to the GenBank   (accession number KR028481).

The molecular genetics study of toxigenic Fusarium fungi diversity on the territory of Russia. Search of new high polymorphic markers in the fungi genome for their particular diagnostic and identification.

Laboratory of Molecular Diagnostics

Partial sequences of frataxin and phosphate permease genes of major Fusarium pathogens common in Russia were first identified and presented in the international databases. As well, the phylogenetic potential of these loci was found.

Developing of the bank of hybridomas producing monoclonal antibodies to the HLA

Laboratory of Molecular Diagnostics

Hybridoma clones producing monoclonal antibodies to three different HLA-I variants were obtained. The 5 clones produced monoclonal antibodies to HLA-A03, 9 clones – to HLA-A11, and 6 clones – to HLA-B51. All the clones produced monoclonal antibodies of G isotype. The obtained monoclonal antibodies have no domestic analogues.

Obtaining of plants with increased expression of HBsAg as edible vaccine against hepatitis B

Laboratory of Plant Biotechnology

The new expression system created for increased synthesis of proteins in plants 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 immunogenicity of HBsAg synthesized by potato tubers was shown in experiments on mice. Increased synthesis of target proteins allows the use of transgenic plants as edible vaccine against the hepatitis B without additional injections of recombinant vaccine.