Laboratory of Molecular Diagnostics

Department of Molecular Biology and Biotechnology of Plants

Head: Sergey Zavriev, corresponding member of the academy of sciences
szavriev@ibch.ru+7(495)336-45-11

The laboratory of Molecular Diagnostics was founded in 2015, on basis of Molecular Diagnostics group existing from 2005 in IBCh (before 2005 - group of phytoimmunodiagnostics). Principal work directions of the laboratory are development of the immunochemical test systems for plant pathogens (viruses, bacteria, fungi) detection, and fundamental research in the field of plant virology and molecular biology.

  1. Development of PCR diagnostics systems for identification variable plant and animal pathogens (viruses, bacteria, fungi, nematodes etc.)
  2. Optimization of DNA/RNA separation methods for analysis of plant and animal specimens and microorganisms identification
  3. Genetic engineering of plants
  4. Utilizing of PCR technologies for rapid evaluation of the efficiency of plant growth regulators
  5. Studies of structure and functions a number of phytoviral and plant proteins — interactions with viruses in infected plants
  6. Investigation of inter- and intracellular protein and viruses transport machine in plants, protein functioning and subcellular localization, virus-host interactions
  7. Viral suppresoors of post-transcriptional genes silencing (for example, gamma-b protein of Hordeivirus)
  8. Producing of poly- and monoclonal antibodies
  9. Developing of immunochemical assays

Basic research methods are genetic engineering and immunochemical methods and DNA technologies. Our studies are carried out in cooperation with some other IBCh labs, Belozersky Institute of Physico-Chemical Biology MSU, ZAO “DNA Technologies”, Institute of Phytopathology of the Russian Academy of Agricultural Sciences and a number of foreign institutes and companies.

Awards

The Laboratory was awarded with a lot of Russian grants as “Researching of ways and mechanisms of protein translocation through plasmodesma with the model of fitoviruses’ transport proteins” or EU grants “Researching of new and rentable methods for a noninvasive diagnostic of human pathogens microorganisms”.

Talks and lectures

  • “Modern approaches for use of monoclonal antibodies in plant protection and studies of virus-induced pathogenic processes”; Erokhina T., 200;
  • “Studies on plant cell protein At 4/1 capable of interacting with viral movement proteins”; Erokhina T., Minina E., Schepetilnikov M., Solovyev A., Kellmann J., Morozov S.Y., 2005.

...and many Russian talks.

International contacts

  • Dr. Matthias Leiser, Nexttec Gmbh Biotechnologie Hemmerlrather Weg 201 51377 Leverkusen, Germany;
  • Dr. Joachim Schiemann, Julius Kuhn Institute, Federal Research Centre for Cultivated Plants, Messeweg 11/12, Braunshweig, Germany;
  • Dr. Chet Sutula, Agdia Incorporated, 30380 County Road 6 Elkhart, Indiana 46514 USA.
  • Prof. G. Brem, Agrobiogen GmbH (ABG), established in Thalmannsdorf 25, Hilgertshausen 86567,Germany;
  • Dr. C.-D. Bauermeister, Labor Dr. Bauermeister & Co. (LB), established in Franz-Haniel-Str. 20 47403 Moers, Germany;
  • Dr. Radovan Haluza, Generi Biotech s.r.o (GB), established in Machkova 587, 50011Hradec Králové 11 Czech Republic;
  • Dr. Elisa Gargiullo, DIATHEVA S. r. I (DT), established in Viale Piceno 137/F, 61032 FANO (PU), Italy;
  • Dr. Ronald Bosch, HLB Research and Consultancy in Agriculture (HLB), established in Kampsweg 27 Wijster, The Netherlands;
  • Dr. René Pellaux, preenTec AG (PRT), established in Esc. Du Court-Chemin 19, Fribourg CH-1704, Switzerland;
  • Dr. H. Balayan, Institute of Fine Organic Chemistry, Armenian National Academy of Sciences (IFOC), established in Azatulyan ave. 26, Yerevan 375005, Republic of Armenia;
  • Dr. Christoph Krukenkamp, Charite — Universitaetsmedizin Berlin (CHA), established Charitéplatz 1, 10117 Berlin, Germany.
NamePositionContacts
Sergey Zavriev, corresponding member of the academy of sciencesdepart. dir.szavriev@ibch.ru+7(495)336-45-11
Dmitry Ryazantsev, Ph.D.s. r. f.d.yu.ryazantsev@gmail.com+7(495)336-45-11
Tatiana Erokhina, Ph.D.s. r. f.tne@mx.ibch.ru+7(495)336-45-11
Maria Simonova, Ph.D.r. f.simonova@ibch.ru+7(495)335-33-22
Ravilja Komalevar. f.+7(495)000-00-00
Ol'ga Lahtinar. f.+7(495)000-00-00
Elena Petrova, Ph.D.r. f.petrova@ibch.ru+7(495)000-00-00
Alexander Stakheev, Ph.D.r. f.ast@mx.ibch.ru
Larisa Samohvalovaj. r. f.+7(495)000-00-00
Yuliya Zvezdinat. q. - lab. as.
Viktorina Budkovskaja, Ph.D.k. eng.vnb@ibch.ru+7(495)330-63-38
Polina Drobyazinak. eng.

Former members:

Tat'jana Valjakina, Ph.D.s. r. f.valyakina@ibch.ru
Tat'jana Chernichkos. r. f.tcher@mx.ibch.ru

Selected publications

  1. Simonova M.A., Pivovarov V.D., Ryazantsev D.Y., Dolgova A.S., Berzhets V.M., Zavriev S.K., Svirshchevskaya E.V. (2018). Comparative diagnostics of allergy using quantitative immuno-PCR and ELISA. Bioanalysis 10 (10), 757–767 [+]

    Estimation of specific IgE is essential for the prevention of allergy progression. Quantitative immuno-PCR (qiPCR) can increase the sensitivity of IgE detection. We aimed to develop qiPCR and compare it to the conventional ELISA in identification of IgE to Alt a 1 and Fel d 1 allergens.

    ID:2179
  2. Пивоваров В.Д., Рязанцев Д.Ю., Симонова М.А., Димитриева Т.В., Хлгатян С.В., Завриев С.К., Свирщевская Е.В. (2018). Разработка тест-систем для анализа антител к вирусу Эпштейна-Барр методом иммуно-ПЦР. Мол. биол. 52, 727–734 ID:2178
  3. Симонова М.А., Пивоваров В.Д., Рязанцев Д.Ю., Костромина М.А., Муравьева Т.И., Мокроносова М.А., Хлгатьян С.В., Есипов Р.С., Завриев С.К. (2018). Определение специфических иммуноглобулинов класса Е к аллергену березы Bet v 1 методом иммуно-ПЦР. Биоорг. хим. 44, 203–211 ID:2180
  4. Смирнов И., Завриев С. (2018). Химическое оружие: современное состояние и контроль за выполнением международных соглашений. Мировая экономика и международные отношения 62 (1), 76–84 ID:2191
  5. Стахеев А.А., Звездина Ю.К., Микитюк О.Д., Завриев С.К. (2018). Изучение токсинообразования и полиморфизма трихотеценовых генов у грибов рода Fusarium российских коллекций. Успехи медицинской микологии XIX, 337–343 ID:2192
  6. Стахеев А.А., Рязанцев Д.Ю., Звездина Ю.К., Баранов М.С., Завриев С.К. (2018). Новая метка для количественной ПЦР на основе синтетического аналога хромофора зелёного флуоресцентного белка. Биохимия 87 (7), 1089–1095 ID:2193
  7. Башкирова И.Г., Матяшова Г.Н., Завриев С.К., Рязанцев Д.Ю., Шнейдер Ю.А. (2018). Апробация тест-систем для детекции фитоплазм яблони и груши. Защита и карантин растений 7, 40–41 ID:2195
  8. Rogozhin E., Ryazantsev D., Smirnov A., Zavriev S. (2018). Primary Structure Analysis of Antifungal Peptides from Cultivated and Wild Cereals. Plants 7 (3), 74 ID:2196
  9. Можаев А.А., Ерохина Т.Н., Серова О.В., Деев И.Е., Петренко А.Г. (2017). Получение и иммунохимическая характеристика моноклонального антитела к эктодомену рецептора, подобного рецептору инсулина (IRR). Биоорг. хим. 43 (6), 631–636 [+]
    Insulin receptor-related receptor (IRR) is the only known metabotropic sensor of extracellular alkaline
    medium involved in the regulation of the acid-base balance in the body. IRR is expressed in separate cell
    populations of the kidney, stomach and pancreas, that can contact with the extracellular fluids with alkaline
    pH. To study IRR structure and function we obtained a stable hybridoma cell line producing antibody to the
    extracellular portion of the receptor. The monoclonal antibody isolated from ascitic fluids showed positive
    reaction with the antigen in the ELISA test. The minimum working concentration of antibodies was 12.5 ng/ml.
    The ability of the antibodies to specifically recognize purified ectodomain IRR and the full-length receptor
    was confirmed by Western-blot, immunoprecipitation and immunocytochemistry.
    ID:1871
  10. Gushchin V.A., Karlin D.G., Makhotenko A.V., Khromov A.V., Erokhina T.N., Solovyev A.G., Morozov S.Y., Agranovsky A.A. (2017). A conserved region in the Closterovirus 1a polyprotein drives extensive remodeling of endoplasmic reticulum membranes and induces motile globules in Nicotiana benthamiana cells. Virology 502, 106–113 [+]

    In infected plant cells, closterovirus replicative polyproteins 1a and 1ab drive membrane remodeling and formation of multivesicular replication platforms. Polyprotein 1a contains a variable Central Region (CR) between the methyltransferase and helicase domains. In a previous study, we have found that transient expression of the Beet yellows virus CR-2 segment (aa 1305-1494) in Nicotiana benthamiana induces the formation of ~1µm mobile globules originating from the ER membranes. In the present study, sequence analysis has shown that a part of the CR named the "Zemlya region" (overlapping the CR-2), is conserved in all members of the Closterovirus genus and contains a predicted amphipathic helix (aa 1368-1385). By deletion analysis, the CR-2 region responsible for the induction of 1-μm globules has been mapped to aa 1368-1432. We suggest that the conserved membrane-modifying region of the BYV 1a may be involved in the biogenesis of closterovirus replication platforms.

    ID:1695
  11. Erokhina N., Lazareva A., RichertPoeggeler R., Sheval V., Solovyev G., Morozov Y. (2017). Subcellular localization and detectiomn of Tobacco mosaic virus ORF6 protein by immunoelectron microscopy. Biochemistry-Moscow 82 (1), 60–66 [+]
    The Tobamovirus genus is one of the bestcharacterized groups of plant viruses. In previous studies, it was found that
    the genomes of some tobamoviruses encode, in addition to wellknown polypeptides (RNA polymerase, movement
    protein, and coat protein), a positively charged and poorly conserved small protein encoded by a small cistron (so
    called open reading frame 6 – «ORF6»). The amino acid sequences of the ORF6 proteins encoded by different
    tobamoviruses are often only marginally related. In this work, we used immunogold labeling and electron microscopy
    to confirm the in vivo expression of ORF6 and to study its subcellular localization. It was experimentally found that
    two isolates of tobacco mosaic virus strain U1 directed the synthesis of ORF6 peptide. The product of ORF6 is main
    ly found in nuclei, which is in accordance with previously published data on transient expression of the isolated ORF6.
    In addition, we present new data on the diversity of ORF6 proteins and other poorly conserved proteins encoded by
    tobamoviral RNA viromes.
    ID:1765
  12. Erokhina T.N., Lazareva E.A., RichertPöggeler K.R., Sheval E.V., Solovyev A.G., Morozov S.Y. (2017). Subcellular Localization and Detection of Tobacco mosaic virus ORF6 Protein by Immunoelectron Microscopy. Biochemistry Mosc. 82 (1), 60–66 [+]
    The Tobamovirus genus is one of the bestcharacterized groups of plant viruses. In previous studies, it was found that
    the genomes of some tobamoviruses encode, in addition to wellknown polypeptides (RNA polymerase, movement
    protein, and coat protein), a positively charged and poorly conserved small protein encoded by a small cistron (so
    called open reading frame 6 – «ORF6»). The amino acid sequences of the ORF6 proteins encoded by different
    tobamoviruses are often only marginally related. In this work, we used immunogold labeling and electron microscopy
    to confirm the in vivo expression of ORF6 and to study its subcellular localization. It was experimentally found that
    two isolates of tobacco mosaic virus strain U1 directed the synthesis of ORF6 peptide. The product of ORF6 is main
    ly found in nuclei, which is in accordance with previously published data on transient expression of the isolated ORF6.
    In addition, we present new data on the diversity of ORF6 proteins and other poorly conserved proteins encoded by
    tobamoviral RNA viromes.
    ID:1872
  13. Vasilchenko A.S., Smirnov A.N., Zavriev S.K., Grishin E.V., Vasilchenko A.V., Rogozhin E.A. (2017). Novel thionins from black seed (Nigella sativa L.) Demonstrate antimicrobial activity. International Journal of Peptide Research and Therapeutics 23, 171–180 ID:2184
  14. Стахеев А.А., Кондратьев М.О., Приходько Ю.Н., Завриев С.К. (2017). Диагностика карантинных вирусов рода Nepovirus методом количественной ПЦР. Защита и карантин растений 3, 35–38 ID:2185
  15. Свирщевская Е.В., Фаттахова Г.В., Хлгатян С.В., Бержец В.М., Завриев С.К. (2017). Сенсибилизация к грибным аллергенам. Успехи медицинской микологии XVII, 401–406 ID:2186
  16. Артыков Ф.Ф., Фурсова К.К., Рязанцев Д.Ю., Щанникова М.П., Лоскутова И.В., Шепеляковская А.О., Ламан А.Г., Завриев С.К., Бровко Ф.А. (2017). Детекция стафилоккокового энтеротоксина А методом фаговой иммуно-ПЦР. Биоорг. хим. 43, 518–522 ID:2187
  17. Beishova I., Chuzhebaeva G., Ulyanov V., Zharlygassov Z.h., Sultangazina G., Stakheev A., Zavriev S. (2017). Development of sensitive, highly specific express tests based on DNA markers to diagnose the casual fungus Puccinia and Pyrenophora that causes diseases of cereal crops. Current Science 112 (7), 1693–1699 ID:2188
  18. Завриев С.К. (2017). Биобезопасность в современном мире. Ежегодник СИПРИ 2016: Безопасность, разоружение и международная безопасность , 980–984 ID:2189
  19. Рогожин Е.А., Кисиль О.В., Чертаев И.В., Завриев С.К. (2017). Характеристика белково- пептидного экстракта семян мари белой ( Chenopodium album L.): изучение компонентного состава, антимикробных и анальгетических свойств. Антибиотики и химиотерапия 62, 3–8 ID:2190
  20. Ерохина Николаевн.а., Лазарева Алексеевн.а., РихертПоеггелер , Шеваль Валерьеви.ч., Соловьев Геннадьев.и., Морозов Юрьеви.ч. (2016). Субклеточная локализация и детекция белка ОРТ6 вируса табачной мозаики с помощью иммуноэлектронной микроскопии. Биохимия 82 (1), 149–156 [+]

    The genus Tobamovirus is one of the best-characterized groups of plant viruses. It previous studies it was found that the genomes of some tobamoviruses encode, in addition to well-known polypeptides (RNA-polymerase, movement protein and coat protein), a positively charged and poorly conserved small protein encoded by small cistron (so-called open reading frame 6 - “ORF6”). The amino acid sequences of the ORF6 proteins encoded by different tobamoviruses are often only marginally related. In this paper, we used the immunogold labelling and electron microscopy to confirm the in vivo expression of ORF6  and to study its subcellular localization. It was experimentally found that two isolates of tobacco mosaic virus strain U1 directed the synthesis of ORF6 peptide. The product of ORF6 is found in nuclei mainly that is in accordance with previously published data on transient expression of the isolated ORF6. In addition, we present new data on the diversity of ORF6 proteins and other poorly conserved proteins encoded by tobamoviral RNA viromes. 

    ID:1618
  21. Erokhina , Solovyev , Morozov  (2016). Diversity of Small Non-Conserved Predicted proteins in Tobamoviruses. Ann Virol Res 2 (1), 1009 [+]
    The genus Tobamovirus is one of the biologically and molecularly best-characterized
    groups of plant viruses varying widely in host range. We have previously found that the
    genomes of tobacco mosaic virus and tomato mosaic virus encode, in addition to wellknown
    polypeptides, a positively charged small protein encoded by small cistron (socalled
    “ORF6”), which overlaps the virus MP and CP genes. The amino acid sequences
    of the ORF6 proteins encoded by different tobamoviruses are often only marginally
    related. Although the Tobacco Mosaic Virus and Tomato Mosaic Virus ORF6 proteins
    have been shown to enhance the virulence of the heterologous viruses, their mutations
    had no significant effect on homologous virus pathogenicity. A combination of the
    features that are characteristic for ORF6 protein makes this protein a good candidate viral
    security protein. Here, we present comprehensive data on the diversity of ORF6 proteins
    and other poorly conserved proteins encoded by tobamoviral RNA viromes. Particularly,
    an additional extended ORF coding for hydrophibic protein is embedded into the
    movement protein gene of Cucumber fruit mottle mosaic virus, Cucumber mottle virus
    and Cucumber green mottle mosaic virus. Moreover, Passion fruit mosaic virus and
    Maracuja mosaic virus genomes contain additional ORF encoding the 195 residue-long
    cysteine-rich polypeptide and located downstream the replicase gene termination codon.
    One can propose that these polypeptides represent novel unique tobamoviral security
    ORFs in addition to ORF6.
    Keywords:
    Tobamovirus;
    ID:1619
  22. Stakheev A.A., Khairulina D.R., Zavriev S.K. (2016). Four-locus phylogeny of Fusarium avenaceum and related species and their species-specific identification based on partial phosphate permease gene sequences. International Journal of Food Microbiology 225, 27–37 [+]

    The fungus Fusarium avenaceum and its closest relatives are responsible for contamination of agricultural plants and their products by mycotoxins such as enniatins and moniliformin. Precise identification of mycotoxin producers is necessary for estimation of the accumulation risk of those compounds and for preventing the consumption of highly contaminated products. Nucleic acids amplification-based techniques proved to be the most rapid and reliable approach for pathogen diagnostics and identification. In this study partial phosphate permease gene (PHO) sequences were determined for Fusarium avenaceum (including one isolate identified as F. arthrosporioides), F. tricinctum, F. acuminatum and F. torulosum. Phylogenetic analysis of 40 isolates of those species from different climates and geographical regions of Russia and some neighboring countries based on sequences of PHO, translation elongation factor 1 alpha (TEF1α), beta-tubulin (β-TUB), enniatin synthetase (Esyn1) genes and combined data set demonstrated that the PHO gene possesses the highest rate of variability among them and can be considered as an informative marker for phylogenetic studies of these species. According to the combined data set phylogeny, the isolates of each species formed clusters with a high bootstrap support. Analysis of PHO sequences revealed a high intraspecific variability of F. avenaceum: there were 5 independent clusters on the dendrogram, including one cluster which was closer to F. torulosum than to other F. avenaceum isolates. Variable sites in PHO sequences have been used for the design of species-specific primers and a fluorescent hydrolysis probe. The specificity of the assay was shown for DNA samples extracted from 68 isolates of 23 Fusarium species. Quantitative PCR approach was applied to estimate the contamination rate of 17 naturally infected oat and barley samples, previously characterized by microbiological procedures.

    ID:1613
  23. Shcherbakova L.A., Odintsova T.I., Stakheev A.A., Fravel D.R., Zavriev S.K. (2016). Identification of a Novel Small Cysteine-Rich Protein in the Fraction from the Biocontrol Fusarium oxysporum Strain CS-20 that Mitigates Fusarium Wilt Symptoms and Triggers Defense Responses in Tomato. Front Plant Sci 6, 1207 [+]

    The biocontrol effect of the non-pathogenic Fusarium oxysporum strain CS-20 against the tomato wilt pathogen F. oxysporum f. sp. lycopersici (FOL) has been previously reported to be primarily plant-mediated. This study shows that CS-20 produces proteins, which elicit defense responses in tomato plants. Three protein-containing fractions were isolated from CS-20 biomass using size exclusion chromatography. Exposure of seedling roots to one of these fractions prior to inoculation with pathogenic FOL strains significantly reduced wilt severity. This fraction initiated an ion exchange response in cultured tomato cells resulting in a reversible alteration of extracellular pH; increased tomato chitinase activity, and induced systemic resistance by enhancing PR-1 expression in tomato leaves. Two other protein fractions were inactive in seedling protection. The main polypeptide (designated CS20EP), which was specifically present in the defense-inducing fraction and was not detected in inactive protein fractions, was identified. The nucleotide sequence encoding this protein was determined, and its complete amino acid sequence was deduced from direct Edman degradation (25 N-terminal amino acid residues) and DNA sequencing. The CS20EP was found to be a small basic cysteine-rich protein with a pI of 9.87 and 23.43% of hydrophobic amino acid residues. BLAST search in the NCBI database showed that the protein is new; however, it displays 48% sequence similarity with a hypothetical protein FGSG_10784 from F. graminearum strain PH-1. The contribution of CS20EP to elicitation of tomato defense responses resulting in wilt mitigating is discussed.

    ID:1427
  24. Svirshchevskaya E., Fattakhova G., Khlgatian S., Chudakov D., Kashirina E., Ryazantsev D., Kotsareva O., Zavriev S. (2016). Direct versus sequential Immunoglobulin switch in allergy and antiviral response. Clin. Immunol. 170, 31–38 [+]

    Allergy is characterized by IgE production to innocuous antigens. The question whether the switch to IgE synthesis occurs via direct or sequential pathways is still unresolved. The aim of this work was to analyze the distribution of immunoglobulins (Ig) to house dust mite D. farinae and A. alternata fungus in allergic children with primarily established diagnosis and compare it to Epstein-Barr antiviral (EBV) response in the same patients. In allergy patients the only significant difference was found in allergen specific IgE, likely mediated by a direct isotype switch, while antiviral response was dominated by EBV specific IgG and low level of concordant IgA and IgG4 production consistent with a minor sequential Ig switches. Taken collectively, we concluded that sequential isotype switch is likely to be a much rarer event than a direct one

    ID:1578
  25. Стахеев А.А., Самохвалова Л.В., Рязанцев Д.Ю., Завриев С.К. (2016). Молекулярно-генетические методы в исследовании таксономии и специфической идентификации токсинпродуцирующих грибов рода Fusarium: успехи и проблемы (обзор). Сельскохозяйственная биология 51 (3), 275– 284 ID:2177
  26. Рязанцев Д.Ю., Воронина Д.В., Завриев С.К. (2016). Иммуно-ПЦР: достижения и перспективы. Успехи биологической химии 56, 377–410 ID:2181
  27. Стахеев А.А., Самохвалова Л.В., Рязанцев Д.Ю., Завриев С.К. (2016). Генетический полиморфизм российской популяции продуцентов фузариотоксинов. Материалы мемориальной конференции по медицинской микологии (к 120-летию А.М. Ариевича). Успехи медицинской микологии XVI, 229–234 ID:2183
  28. Morozov S.Y., Miliytina I.A., Bobrova V.K., Ryazantsev D.Y., Erokhina T.N., Zavriev S.K., Agranovsky A.A., Solovyev A.G., Troitsky A.V. (2015). Structural evolution of the 4/1 genes and proteins in non-vascular and lower vascular plants. Biochimie , [+]

    The 4/1 protein of unknown function is encoded by a single-copy gene in most higher plants. The 4/1 protein of Nicotiana tabacum (Nt-4/1 protein) has been shown to be alpha-helical and predominantly expressed in conductive tissues. Here, we report the analysis of 4/1 genes and the encoded proteins of lower land plants. Sequences of a number of 4/1 genes from liverworts, lycophytes, ferns and gymnosperms were determined and analyzed together with sequences available in databases. Most of the vascular plants were found to encode Magnoliophyta-like 4/1 proteins exhibiting previously described gene structure and protein properties. Identification of the 4/1-like proteins in hornworts, liverworts and charophyte algae (sister lineage to all land plants) but not in mosses suggests that 4/1 proteins are likely important for plant development but not required for a primary metabolic function of plant cell.

    ID:1343
  29. Чудаков Д.Б., Рязанцев Д.Ю., Каширина Е.И., Бержец В.М., Свирщевская Е.В. (2014). Роль дозы аллергена в индукции у мышей IgE антител на белки клещей домашней пыли. Иммунология 35 (6), 321–328 [+]

    Aim: The aim of this work was to study of humoral immune response to different house dust mite protein doses in mice.

    Results: Specific IgE antibody production was observed only upon multiple administration of low (1ng/injection) recombinant protein doses. The production of IgG and IgA antibodies ware observed only upon administration of relatively high (1-10 microgrammes) allergen doses. IgG and IgA production was also observed during the immunisation with adjuvant upon administration of 0,1 microgramm of proteins. IgE antibodies in our allergic model have low affinity.

    ID:1268
  30. Ryazantsev D.Y., Kvach M.V., Tsybulsky D.A., Prokhorenko I.A., Stepanova I.A., Martynenko Y.V., Gontarev S.V., Shmanai V.V., Zavriev S.K., Korshun V.A. (2014). Design of molecular beacons: 3' couple quenchers improve fluorogenic properties of a probe in real-time PCR assay. Analyst 139 (11), 2867–72 [+]

    Convenient preparation of fluorogenic hairpin DNA probes (molecular beacons) carrying a pair of FAM fluorophores (located close to 5'-terminus of the probe) or a pair of BHQ1 quenchers on 3'-terminus (with (BHQ1)2 or BHQ1-BHQ1 composition) is reported. These probes were used for the first time in a real-time PCR assay and showed considerable improvements in fluorogenic properties (the total fluorescence increase or signal-to-background ratio) in assay conditions vs. conventional one-FAM-one-BHQ1 molecular beacon probes as well as vs. hydrolyzable one-FAM-one-BHQ1 TaqMan probes. At the same time, such multiple modifications of the probe do not influence its Cq (a fractional PCR cycle used for quantification). The probe MB14 containing a BHQ1-BHQ1 pair showed a PCR fluorescence/background value of 9.6 which is more than two times higher than that of a regular probe MB2 (4.6). This study demonstrates prospects for the design of highly fluorogenic molecular beacon probes suitable for quantitative real-time PCR and for other potential applications (e.g. intracellular RNA detection and SNP/mutation analysis).

    ID:1019
  31. Morozov S.Y., Makarova S.S., Erokhina T.N., Kopertekh L., Schiemann J., Owens R.A., Solovyev A.G. (2014). Plant 4/1 protein: potential player in intracellular, cell-to-cell and long-distance signaling. Front Plant Sci 5, 26 [+]

    Originally isolated as a result of its ability to interact with the movement protein of Tomato spotted wilt virus in a yeast two-hybrid system, the 4/1 protein is proving to be an excellent tool for studying intracellular protein trafficking and intercellular communication. Expression of 4/1 in vivo is tightly regulated, first appearing in the veins of the cotyledon and later in the vasculature of the leaf and stem in association with the xylem parenchyma and phloem parenchyma. Structural studies indicate that 4/1 proteins contain as many as five coiled-coil (CC) domains; indeed, the highest level of sequence identity among 4/1 proteins involves their C-terminal CC domains, suggesting that protein-protein interaction is important for biological function. Recent data predict that the tertiary structure of this C-terminal CC domain is strikingly similar to that of yeast protein She2p; furthermore, like She2p, 4/1 protein exhibits RNA-binding activity, and mutational analysis has shown that the C-terminal CC domain is responsible for RNA binding. The 4/1 protein contains a nuclear export signal. Additional microscopy studies involving leptomycin and computer prediction suggest the presence of a nuclear localization signal as well.

    ID:1008
  32. Gushchin V.A., Solovyev A.G., Erokhina T.N., Morozov S.Y.u., Agranovsky A.A. (2013). Beet yellows virus replicase 1a: parallels with induction of replication-associated membrane compartments by other RNA viruses,. Frontiers in Virology , [+]

    In eukaryotic virus systems, infection leads to induction of membranous compartments in which replication occurs. Virus-encoded subunits of the replication complex mediate its interaction with membranes. As replication platforms, RNA viruses use the cytoplasmic surfaces of different membrane compartments, e.g., endoplasmic reticulum (ER), Golgi, endo/lysosomes, mitochondria, chloroplasts and peroxisomes. Closterovirus infections are accompanied by formation of multivesicular complexes from cell membranes of ER or mitochondrial origin. So far the mechanisms for vesicles formation have been obscure. In the replication-associated 1a polyprotein of Beet yellows virus (BYV) and other closteroviruses, the region between the methyltransferase (MTR) and helicase (HEL) domains (1a central region, 1a CR) exhibits little or no significant similarity. Computer-assisted analysis predicts several putative membrane-binding domains in the BYV 1a CR. Transient expression of a hydrophobic segment (referred to here as CR-2) of the BYV 1a in Nicotiana benthamiana led to reorganization of the ER and formation of ~1-mm mobile globules. We propose that the CR-2 may be involved in the formation of multivesicular complexes in BYV-infected cells. This provides analogy with membrane-associated proteins mediating the build-up of “virus factories” in cells infected with diverse positive-strand RNA viruses (alpha-like viruses, picorna-like viruses, flaviviruses, and nidoviruses) and negative-strand RNA viruses (bunyaviruses).

    ID:806
  33. Rogozhin E.A., Ryazantsev D.Y., Grishin E.V., Egorov T.A., Zavriev S.K. (2012). Defense peptides from barnyard grass (Echinochloa crusgalli L.) seeds. Peptides 38 (1), 33–40 [+]

    A number of defense polypeptides from latent seeds of weed cereal barnyard grass (Echinochloa crusgalli L.) has been isolated and characterized using an acidic extraction and high performance liquid chromatography methods in combination with MALDI-TOF mass spectrometry and Edman sequencing. Members of three antimicrobial peptide families and two protease inhibitor families were found to be localized in barnyard grass seeds. Their biological activity concerning to Gram-Positive and Gram-Negative phytopathogenic bacteria, as well as oomycete Phytophthora infestans, has been investigated. Diversity of barnyard grass defense peptides is a significant factor that provides a resistance of E. crusgalli seeds to germination and latent phases.

    ID:805
  34. Ryazantsev D.Y., Tsybulsky D.A., Prokhorenko I.A., Kvach M.V., Martynenko Y.V., Philipchenko P.M., Shmanai V.V., Korshun V.A., Zavriev S.K. (2012). Two-dye and one- or two-quencher DNA probes for real-time PCR assay: synthesis and comparison with a TaqMan™ probe. Analytical and bioanalytical chemistry 404 (1), 59–68 [+]

    A typical TaqMan™ real-time PCR probe contains a 5'-fluorescent dye and a 3'-quencher. In the course of the amplification, the probe is degraded starting from the 5'-end, thus releasing fluorescent dye. Some fluorophores (including fluorescein) are known to be prone to self-quenching when located near each other. This work is aimed at studying dye-dye and dye-quencher interactions in multiply modified DNA probes. Twenty-one fluorogenic probes containing one and two fluoresceins (FAM), or a FAM-JOE pair, and one or two BHQ1 quenchers were synthesized using non-nucleoside reagents and "click chemistry" post-modification on solid phase and in solution. The probes were tested in real-time PCR using an ~300-bp-long natural DNA fragment as a template. The structural prerequisites for lowering the probe background fluorescence and increasing the end-plateau fluorescence intensity were evaluated and discussed.

    ID:753
  35. Ryazantsev D.Y.u., Petrova E., Kalinina N.A., Valyakina T.I., Grishin E.V., Zavriev S.K. (2012). Application of supramolecular DNA-streptavidin complexes for ultrasensitive detection of several toxins by immuno-PCR. 14. Global J. Anal. Chem. 3 (17), [+]
    ID:650
  36. Makarova S.S., Minina E.A., Makarov V.V., Semenyuk P.I., Kopertekh L., Schiemann J., Serebryakova M.V., Erokhina T.N., Solovyev A.G., Morozov S.Y. (2011). Orthologues of a plant-specific At-4/1 gene in the genus Nicotiana and the structural properties of bacterially expressed 4/1 protein. Biochimie 93 (10), 1770–8 [+]

    Arabidopsis thaliana At-4/1 is the protein of unknown function capable of polar localization in plant cells and intercellular trafficking. In this work, we cloned cDNAs and chromosomal genes of At-4/1 orthologues from several Nicotiana species. Similarly to the 4/1 genes of A. thaliana and Oryza sativa, Nicotiana 4/1 genes have eight exons and seven introns but are considerably longer due to their larger introns. The allotetraploid genome of Nicotiana tabacum, which is known to consist of the 'S genome' originated from Nicotiana sylvestris and the 'T genome' derived from Nicotiana tomentosiformis, encodes two 4/1 genes. The T genome-encoded 4/1 gene, but not that of the S genome, contains a SINE-like transposable element in its intron 2. The 4/1 genes of Nicotiana hesperis and Nicotiana benthamiana lack such an element in the intron 2, but possess a related SINE-like sequence in their intron 4. Collectively, the sequence analysis data provide an insight into the organization of 4/1 genes in flowering plants and the patterns of evolution in the genus Nicotiana. The Nicotiana 4/1 proteins and those of other flowering plants show a significant level of sequence similarity. Computer-assisted analysis was further used to compare their predicted secondary structures. Several algorithms confidently predicted the presence of several coiled-coil domains occupying similar positions in different 4/1 proteins. Analysis of circular dichroism spectra carried out for bacterially expressed N. tabacum 4/1 protein (Nt-4/1) and its N- and C-terminally truncated mutants confirmed that the secondary structure of Nt-4/1 is generally alpha-helical. The C-terminal region of Nt-4/1 was found to undergo a partial proteolysis in Escherichia coli cells. Differential scanning calorimetry of Nt-4/1 protein and its mutants revealed three calorimetric domains most probably corresponding to the N-terminal, central, and C-terminal structural domains of the protein.

    ID:521
  37. Shemyakina E.A., Erokhina T.N., Gorshkova E.N., Schiemann J., Solovyev A.G., Morozov S.Y. (2011). Formation of protein complexes containing plant virus movement protein TGBp3 is necessary for its intracellular trafficking. Biochimie 93 (4), 742–8 [+]

    Cell-to-cell movement of Poa semilatent virus (genus Hordeivirus) in infected plants is mediated by three viral 'triple gene block' (TGB) proteins. One of those termed TGBp3 is an integral membrane protein essential for intracellular transport of other TGB proteins and viral genomic RNA to plasmodesmata. TGBp3 targeting to plasmodesmata-associated sites is believed to involve an unconventional mechanism which does not employ endoplasmic reticulum-derived transport vesicles. Previously TGBp3 has been shown to contain a composite transport signal consisting of the central hydrophilic protein region which includes a conserved pentapeptide YQDLN and the C-terminal transmembrane segment. This study demonstrates that these TGBp3 structural elements have distinct functions in protein transport. The YQDLN-containing region is essential for TGBp3 incorporation into high-molecular-mass protein complexes. In transient expression assay formation of such complexes is necessary for entering the TGBp3-specific pathway of intracellular transport and protein delivery to plasmodesmata-associated sites. In virus-infected plants TGBp3 is also found predominantly in the form of high-molecular-mass complexes. When the complex-formation function of YQDLN-containing region is disabled by a mutation, targeting to plasmodesmata-associated sites can be complemented by a heterologous peptide capable of formation multimeric complexes. The C-terminal transmembrane segment is found to be an essential signal of TGBp3 intracellular transport to peripheral sites.

    ID:522
  38. Стахеев А.А., Рязанцев Д.Ю., Завриев С.К. (2011). Выявление новых генетических маркеров для таксономической характеристики и идентификации грибов рода Fusarium. Биоорг. хим. 37, 662–671 [+]
    ID:649
  39. Kapustin D.V., Prostyakova A.I., Ryazantsev D.Y., Zubov V.P. (2011). Novel composite matrices modified with nanolayers of polymers as perspective materials for separation of biomolecules and bioanalysis. Nanomedicine (Lond) 6 (2), 241–55 [+]

    A new approach for the preparation of adsorbents for one-step isolation/purification of DNA from different samples (e.g., bacterial lysates, smears and blood) has been developed.

    ID:458
  40. Makarova S., Erokhina N., Solovyev G., Schiemann , Owens , Morozov Y. (2011). Subcellular localization and structural properties of tobacco 4/1 protein. FEBS J 278 (SI), 465 [+]

    Here we report isolation of Nt 4/1 gene, the 4/1 gene from tobacco (Nicotiana tabacum), and the subcellular localization of the encoded protein as well as its structural properties. The Nt 4/1 protein fused to GFP, localizes in numerous small vesicular structures dispersed throughout the cytoplasm and, in addition, in the cell nucleus. Dynamic laser light scattering analysis revealed that the bacterially expressed Nt 4/1 protein can form dimers and trimers in vitro. Analysis of circular dichroism spectrademonstrated that it has a pronounced alfa-helical secondary structure.

    ID:697
  41. Stakheev A.A., Ryazantsev D.Y.u., Gagkaeva T.Y.u., Zavriev S.K. (2010). PCR detection of Fusarium fungi with similar profiles of the produced mycotoxins. Food Control 22 (3-4), 462–468 [+]
    ID:648
  42. Рязанцев Д.Ю., Абрамов Д.Д., Завриев С.К. (2009). Диагностика карантинных фитопатогенов методом ПЦР в формате FLASH. Сельскохозяйственная биология  (3), 114–117 ID:161
  43. Рязанцев Д.Ю., Завриев С.К. (2009). Эффективный метод диагностики и идентификации вирусных патогенов картофеля. Мол. биол. 43 (3), 558–567 ID:160
  44. Lukhovitskaia N.I., Solov'eva A.G., Koshkina T.E., Zavriev S.K., Morozov S.I.u. (2009). [Interaction of cysteine-rich protein of Carlavirus with plant defense system]. Mol. Biol. (Mosk.) 39 (5), 896–904 [+]

    Viruses of genus Carlavirus encode a small cysteine-rich protein (CRP) of unknown function. To investigate the role of CRP of carlavirus chrysanthemum virus B (CVB), a recombinant potato virus X (PVX) genome was constructed, which carried the CVB CRP gene. Expression of CVB CRP in the PVX genetic background drastically changed the PVX symptom phenotype in N. benthamiana. Instead of symptomless infection and mild mosaic, which are characteristic of PVX in this plant host, the recombinant virus expressing CVB CRP induced formation of necrotic local lesions on inoculated leaves and necrosis of the apical leaves. In N. tabacum, the infection pattern depended on the host genotype: the recombinant PVX was able to spread systemically only in N gene-carrying plants. In agroinfiltration-mediated transient expression assay, CVB CRP did not exhibit the properties of avirulence factor in N. benthamiana and was unable to suppress post-transcriptional gene silencing. Thus, CVB CRP is the viral pathogenicity determinant controlling the virus interaction with plant hosts in a manner which depends on plant defense mediated by resistance genes such as the N gene.

    ID:164
  45. Minina E.A., Erokhina T.N., Garushyants S.K., Solovyev A.G., Morozov S.Y. (2009). Subcellular localization of the new plant protein 4/1 and analysis of heterologous protein-protein interactions indicate its ability for nuclear-cytoplasmic transport. Dokl. Biochem. Biophys. 429, 296–300 ID:523
  46. Iaroslavov A.A., Kaplan I.V., Erokhina T.N., Morozov S.I.u., Solovev A.G., Leshchiner A.D., Riakhnianskaia A.A., Malinin A.C., Stepanova L.A., Kiselev O.I., Atabekov I.G. (2009). [A new method of producing biologically active nanocomplexes by non-covalent conjugation of proteins with viral particles]. Bioorg. Khim. 37 (4), 496–503 [+]

    Currently, a range of biologically active molecules have been attached to plant and bacterial viras nanoscaffolds, yielding stable nanoparticles that display multiple copies of the desired molecule. In this paper we propose a new method of non-covalent attachment of peptides to the surface of virios. We have demonstrated that this method is efficient in a model system that includes tobacco mosaic virus particles, synthetic polycation (quaternized poly(4-vinylpyridine) carrying ethyl ethyl pendant radicals) and polypeptide of interest. This principle of step-by-step binding to the surface of virions was used for electrostatic association with hydrophilic fragment of influenza virus haemagglutinin.

    ID:645
  47. Abramova S.L., Riazantsev D.Y.u., Voinova T.M., Zavriev S.K. (2009). [Diagnostics of phytopathogen fungi Septoria tritici and Stagonospora nodorum by fluorescent amplification-based specific hybridization (FLASH) PCR]. Bioorg. Khim. 34 (1), 107–13 [+]
    ID:646
  48. Riazantsev D.Y.u., Abramova S.L., Evstratova S.V., Gagkaeva T.Y.u., Zavriev S.K. (2009). [FLASH-PCR diagnostics of toxigenic fungi of the genus Fusarium]. Bioorg. Khim. 34 (6), 799–807 [+]
    ID:647
  49. Стахеев A.A., Самохвалова Л.В., Микитюк О.Д., Завриев С.К. (2009). Филогенетический анализ и молекулярное типирование трихотеценпродуцирующих грибов рода Fusarium из российских коллекций. Acta Naturae 10 (2), 85–99 ID:2194
  50. Абрамова С.Л., Рязанцев Д.Ю., Воинова Т.М., Завриев С.К. (2008). Диагностика фитопатогенных грибов Septoria tritice и Stragonaspora nodorum методом FLASH–ПЦР. Биоорг. хим. 34, 107–113 ID:158
  51. Рязанцев Д.Ю., Абрамова С.Л., Евстратова С.В., Гагкаева Т.Ю., Завриев С.К. (2008). Диагностика токсиногенных грибов рода Fusarium методом FLASH-PCR. Биоорг. хим. 35, 799–807 ID:29
  52. Кокарев Н.В., Кошкина Т.Е., Рязанцев Д.Ю., Завриев С.К. (2007). Влияние дефектной РНК вируса крапчатости ежи сборной на накопление вирусного капсидного белка в растениях пшеницы. Доклады РАСХН  (3б), 13–15 ID:157
  53. Минина Е.А., Ерохина Т.Н., Сошникова Н.В., Соловьев А.Г., Морозов С.Ю. (2006). Иммунологическая детекция белка растений At-4/1, способного взаимодействовать с вирусными белками межклеточного транспорта. ДАН 411 (5), 689–693 ID:156
  54. Mitioushkina T.Y.u., Dolgov S.V., Zavriev S.K., Kharchenko P.N. (2006). Molecular biology approach for improving chrysanthemum resistance to virus B. Acta Horticulturae. Acta Horticulturae 772, 327–332 ID:166
  55. Paape M., Solovyev A.G., Erokhina T.N., Minina E.A., Schepetilnikov M.V., Lesemann D.E., Schiemann J., Morozov S.Y., Kellmann J.W. (2006). At-4/1, an interactor of the Tomato spotted wilt virus movement protein, belongs to a new family of plant proteins capable of directed intra- and intercellular trafficking. Mol. Plant Microbe Interact. 19 (8), 874–83 [+]

    The Tomato spotted wilt virus (TSWV) encoded NSm movement protein facilitates cell-to-cell spread of the viral genome through structurally modified plasmodesmata. NSm has been utilized as bait in yeast two-hybrid interaction trap screenings. As a result, a protein of unknown function, called At-4/1, was isolated from an Arabidopsis thaliana GAL4 activation domain-tagged cDNA library. Using polyclonal antibodies against bacterially expressed At-4/1, Western blot analysis of protein extracts isolated from different plant species as well as genome database screenings showed that homologues of At-4/1 seemed to be encoded by many vascular plants. For subcellular localization studies, At-4/1 was fused to green fluorescent protein, and corresponding expression vectors were used in particle bombardment and agroinfiltration assays. Confocal laser scannings revealed that At-4/1 assembled in punctate spots at the cell periphery. The protein accumulated intracellularly in a polarized fashion, appearing in only one-half of a bombarded epidermal cell, and, moreover, moved from cell to cell, forming twin-structured bodies seemingly located at both orifices of the plasmodesmatal pore. In coexpression studies, At-4/1 colocalized with a plant virus movement protein TGBp3 known to reside in endoplasmic reticulum-derived membrane structures located in close vicinity to plasmodesmata. Thus, At-4/1 belongs to a new family of plant proteins capable of directed intra- and intercellular trafficking.

    ID:28
  56. Вишниченко В.К., Рязанцев Д.Ю., Завриев С.К. (2005). Экспрессия капсидного белка Х вируса шалота в различных органах растений Allium cepa var. Aggregatum. Сельскохозяйственная биология  (1), 104–109 ID:155
  57. Yelina N.E., Erokhina T.N., Lukhovitskaya N.I., Minina E.A., Schepetilnikov M.V., Lesemann D.E., Schiemann J., Solovyev A.G., Morozov S.Y. (2005). Localization of Poa semilatent virus cysteine-rich protein in peroxisomes is dispensable for its ability to suppress RNA silencing. J. Gen. Virol. 86 (Pt 2), 479–89 [+]

    Subcellular localization of the Poa semilatent virus cysteine-rich gammab protein was studied by using different approaches. In infected tissue, gammab was detected mainly in the P30 fraction as monomers, dimers and oligomers. Green fluorescent protein-fused gammab was found to localize in punctate bodies in the cytoplasm. Colocalization with marker proteins demonstrated that these bodies represent peroxisomes. Immunoelectron microscopy revealed that gammab was localized in the peroxisomal matrix and that localization of gammab in peroxisomes required the C-terminal signal tripeptide SKL. An SKL-deletion mutant exhibited a diffuse localization, but retained the protein's ability to suppress RNA silencing, determine infection phenotype and support virus systemic spread. These data indicate that gammab functions are not associated with the protein's localization to peroxisomes.

    ID:165
  58. Adams M.J., Antoniw J.F., Bar-Joseph M., Brunt A.A., Candresse T., Foster G.D., Martelli G.P., Milne R.G., Zavriev S.K., Fauquet C.M. (2004). The new plant virus family Flexiviridae and assessment of molecular criteria for species demarcation. Arch. Virol. 149 (5), 1045–60 [+]

    The new plant virus family Flexiviridae is described. The family is named because its members have flexuous virions and it includes the existing genera Allexivirus, Capillovirus, Carlavirus, Foveavirus, Potexvirus, Trichovirus and Vitivirus, plus the new genus Mandarivirus together with some related viruses not assigned to any genus. The family is justified from phylogenetic analyses of the polymerase and coat protein (CP) sequences. To help to define suitable molecular criteria for demarcation of species, a complete set of pairwise comparisons was made using the nucleotide (nt) and amino acid (aa) sequences of each fully-sequenced gene from every available accession in the family. Based on the distributions and on inspection of the data, it was concluded that, as a general rule, distinct species have less than ca. 72% identical nt or 80% identical aa between their entire CP or replication protein genes.

    ID:163
  59. Кошкина Т.Е., Баранова Е.Н., Завриев С.К. (2003). Точечная мутация в гене белка оболочки влияет на дальний транспорт вируса табачной мозаики. Мол. биол. 37, 742–748 ID:154
  60. Zinovkin R.A., Erokhina T.N., Lesemann D.E., Jelkmann W., Agranovsky A.A. (2003). Processing and subcellular localization of the leader papain-like proteinase of Beet yellows closterovirus. J. Gen. Virol. 84 (Pt 8), 2265–70 [+]

    ORF 1a of Beet yellows closterovirus (BYV) encodes the domains of the papain-like proteinase (PCP), methyltransferase (MT) and RNA helicase. BYV cDNA inserts encoding the PCP-MT region were cloned in pGEX vectors next to the glutathione S-transferase gene (GST). In a 'double tag' construct, the GST-PCP-MT cDNA was flanked by the 3'-terminal six histidine triplets. Following expression in E. coli, the fusion proteins were specifically self-cleaved into the GST-PCP and MT fragments. MT-His(6) was purified on Ni-NTA agarose and its N-terminal sequence determined by Edman degradation as GVEEEA, thus providing direct evidence for the Gly(588)/Gly(589) bond cleavage. The GST-PCP fragment purified on glutathione S-agarose was used as an immunogen to produce anti-PCP monoclonal antibodies (mAbs). On Western blots of proteins from virus-infected Tetragonia expansa, the mAbs recognized the 66 kDa protein. Immunogold labelling of BYV-infected tissue clearly indicated association of the PCP with the BYV-induced membranous vesicle aggregates, structures related to closterovirus replication.

    ID:162
  61. Gorshkova E.N., Erokhina T.N., Stroganova T.A., Yelina N.E., Zamyatnin A.A. Jr, Kalinina N.O., Schiemann J., Solovyev A.G., Morozov S.Y. (2003). Immunodetection and fluorescent microscopy of transgenically expressed hordeivirus TGBp3 movement protein reveals its association with endoplasmic reticulum elements in close proximity to plasmodesmata. J. Gen. Virol. 84 (Pt 4), 985–94 [+]

    The subcellular localization of the hydrophobic TGBp3 protein of Poa semilatent virus (PSLV, genus Hordeivirus) was studied in transgenic plants using fluorescent microscopy to detect green fluorescent protein (GFP)-tagged protein and immunodetection with monoclonal antibodies (mAbs) raised against the GFP-based fusion expressed in E. coli. In Western blot analysis, mAbs efficiently recognized the wild-type and GFP-fused PSLV TGBp3 proteins expressed in transgenic Nicotiana benthamiana, but failed to detect TGBp3 in hordeivirus-infected plants. It was found that PSLV TGBp3 and GFP-TGBp3 had a tendency to form large protein complexes of an unknown nature. Fractionation studies revealed that TGBp3 represented an integral membrane protein and probably co-localized with an endoplasmic reticulum-derived domain. Microscopy of epidermal cells in transgenic plants demonstrated that GFP-TGBp3 localized to cell wall-associated punctate bodies, which often formed pairs of opposing discrete structures that co-localized with callose, indicating their association with the plasmodesmata-enriched cell wall fields. After mannitol-induced plasmolysis of the leaf epidermal cells in the transgenic plants, TGBp3 appeared within the cytoplasm and not at cell walls. Although TGBp3-induced bodies were normally static, most of them became motile after plasmolysis and displayed stochastic motion in the cytoplasm.

    ID:27
  62. Завриев С.К., Вишниченко В.К., Келдыш М.А. (2002). Обнаружение аллексивирусов в составе вирусных комплексов, поражающих декоративные луковичные культуры. Доклады РАСХН  (1), 11–13 ID:152
  63. Maroon C.J.M., Zavriev S. (2002). PCR-BASED TESTS FOR THE DETECTION OF TOBAMOVIRUSES AND CARLAVIRUSES. Acta Horticulturae 598, 117–122 [+]

    Routine testing of ornamental plants involves ELISA screens that include a number of specific tests to detect the presence of a virus belonging to a particular group. This approach has been desirable for a number of reasons: cost-effectiveness, simplicity and speed. Despite these advantages, the screens can become quite complicated due to the number of specific tests that need to be conducted. Furthermore, with certain host species, non-specific tissue reaction is not at all uncommon thus prompting the use of more than one ELISA format for a particular test. As an alternative to our ELISA screens, we have developed two PCR group tests that respectively detect members of the tobamovirus and the carlavirus groups. These tests are coupled with reverse transcription using random hexamers or oligo d(T)16 primer, respectively. For the PCR part, primers are designed based on the conserved regions of the viral genome and are used under optimized conditions for the amplification of the target sequences. Using our tobamovirus group PCR test, we successfully detected CGMMV, KGMMV, ORSV, PMMoV, RMV, SHMV, TMV and ToMV from leaves, stems, fruits and seeds of infected plants. Our carlavirus group PCR test could detect all carlaviruses examined: BBScV, CLV, CVB, KLV, LSV, PotLV, PVM, PVS and SLV.

    ID:25
  64. Вишниченко В.К., Стельмащик В.Я., Завриев С.К. (2002). 42K белок Х вируса шалота участвует в формировании вирусных частиц. Мол. биол. 36, 1080–1084 ID:26
  65. Кошкина Т.Е., Новиков В.К., Завриев С.К. (2002). Исследование биологических свойств и структуры генома изолята К3 казахского штамма вируса табачной мозаики. Доклады РАСХН  (3), 14–15 ID:153
  66. Erokhina T.N., Vitushkina M.V., Zinovkin R.A., Lesemann D.E., Jelkmann W., Koonin E.V., Agranovsky A.A. (2001). Ultrastructural localization and epitope mapping of the methyltransferase-like and helicase-like proteins of Beet yellows virus. J. Gen. Virol. 82 (Pt 8), 1983–94 [+]

    Monoclonal antibodies (MAbs) specific to the methyltransferase (MT) and helicase (HEL) domains of the closterovirus Beet yellows virus (BYV) were used for immunogold labelling of ultrathin sections of virus-infected Tetragonia expansa plants. MAbs 4A2 and 4A5 from the MT panel, and 1C4 from the HEL panel, specifically labelled distinct closterovirus-induced membranous structures, the 'BYV-type vesicles', thus suggesting that the closterovirus MT-like and HEL-like proteins co-localize in these structures. Probing of the MT and HEL MAbs with synthetic octapeptides spanning the sequences of the recombinant MT and HEL fragments that had been used as immunogens showed that 4A5 and 4A2 recognized a single epitope, SRLLENET (aa 686-692 in the BYV 1a protein), and 1C4 reacted with the DDPF epitope (aa 2493-2496). These epitopes apparently reside on the exposed parts of the membrane-associated molecules of the closterovirus MT-like and HEL-like proteins. Two other epitopes determined for the MT MAbs that were nonreactive in the immunogold labelling, namely TMVTPGEL (aa 750-757; MAbs 3C5, 4B4 and 4C5) and SREQLVEA (aa 806-813; MAb 2A4), are possibly buried in the MT domain fold or shielded by membranes or other proteins involved in the viral replicative complex.

    ID:23
  67. Vishnichenko V.K., Zavriev S.K. (2001). Detection of infectious viral particles in plant protoplasts inoculated with transcripts of full-length shallot virus X cDNA. Arch. Virol. 146 (6), 1213–7 [+]

    Flexible filamentous shallot virus X (ShVX) particles were detected in extracts of Beta vulgaris protoplasts inoculated with transcripts from a full-length ShVX cDNA. Extracts from ShVX-infected protoplast were infectious for ShVX-healthy shallot seedlings. Western blot analysis of inoculated plants revealed the accumulation of the ShVX coat protein, while electron microscopy confirmed the presence of ShVX virions. The results suggest that the in vitro RNA transcripts from full-length ShVX cDNA give rise to infectious viral particles.

    ID:24
  68. Erokhina , Solovyev , Morozov  (1970). Diversity of small non-conserved predicted proteins in Tobamoviruses. Ann Virol Res 2 (1), 1009–1016 [+]
    The genus Tobamovirus is one of the biologically and molecularly best-characterized
    groups of plant viruses varying widely in host range. We have previously found that the
    genomes of tobacco mosaic virus and tomato mosaic virus encode, in addition to wellknown
    polypeptides, a positively charged small protein encoded by small cistron (socalled
    “ORF6”), which overlaps the virus MP and CP genes. The amino acid sequences
    of the ORF6 proteins encoded by different tobamoviruses are often only marginally
    related. Although the Tobacco Mosaic Virus and Tomato Mosaic Virus ORF6 proteins
    have been shown to enhance the virulence of the heterologous viruses, their mutations
    had no significant effect on homologous virus pathogenicity. A combination of the
    features that are characteristic for ORF6 protein makes this protein a good candidate viral
    security protein. Here, we present comprehensive data on the diversity of ORF6 proteins
    and other poorly conserved proteins encoded by tobamoviral RNA viromes. Particularly,
    an additional extended ORF coding for hydrophibic protein is embedded into the
    movement protein gene of Cucumber fruit mottle mosaic virus, Cucumber mottle virus
    and Cucumber green mottle mosaic virus. Moreover, Passion fruit mosaic virus and
    Maracuja mosaic virus genomes contain additional ORF encoding the 195 residue-long
    cysteine-rich polypeptide and located downstream the replicase gene termination codon.
    One can propose that these polypeptides represent novel unique tobamoviral security
    ORFs in addition to ORF6.
    ID:1608

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

Subcellular localization and detection of Tobacco mosaic virus ORF6 protein by immunoelectron microscopy (2017-11-27)

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 N., Lazareva A., RichertPoeggeler R., Sheval V., Solovyev G., Morozov Y. (2017). Subcellular localization and detectiomn of Tobacco mosaic virus ORF6 protein by immunoelectron microscopy. Biochemistry-Moscow 82 (1), 60–66 [+]
    The Tobamovirus genus is one of the bestcharacterized groups of plant viruses. In previous studies, it was found that
    the genomes of some tobamoviruses encode, in addition to wellknown polypeptides (RNA polymerase, movement
    protein, and coat protein), a positively charged and poorly conserved small protein encoded by a small cistron (so
    called open reading frame 6 – «ORF6»). The amino acid sequences of the ORF6 proteins encoded by different
    tobamoviruses are often only marginally related. In this work, we used immunogold labeling and electron microscopy
    to confirm the in vivo expression of ORF6 and to study its subcellular localization. It was experimentally found that
    two isolates of tobacco mosaic virus strain U1 directed the synthesis of ORF6 peptide. The product of ORF6 is main
    ly found in nuclei, which is in accordance with previously published data on transient expression of the isolated ORF6.
    In addition, we present new data on the diversity of ORF6 proteins and other poorly conserved proteins encoded by
    tobamoviral RNA viromes.
    ID:1765

A covalent DNA antibody conjugate was prepared for use in immuno-PCR (2017-11-27)

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.

Study of gene expression of trichothecene cluster genes in Fusarium graminearum and F. ussurianum under different culture conditions and their toxigenicity (2017-11-27)

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.     

Elucidation of structure of elicitor protein CS20EP from Fusarium oxysporum strain CS-20 (2016-11-18)

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).

Publications

  1. Shcherbakova L.A., Odintsova T.I., Stakheev A.A., Fravel D.R., Zavriev S.K. (2016). Identification of a Novel Small Cysteine-Rich Protein in the Fraction from the Biocontrol Fusarium oxysporum Strain CS-20 that Mitigates Fusarium Wilt Symptoms and Triggers Defense Responses in Tomato. Front Plant Sci 6, 1207 [+]

    The biocontrol effect of the non-pathogenic Fusarium oxysporum strain CS-20 against the tomato wilt pathogen F. oxysporum f. sp. lycopersici (FOL) has been previously reported to be primarily plant-mediated. This study shows that CS-20 produces proteins, which elicit defense responses in tomato plants. Three protein-containing fractions were isolated from CS-20 biomass using size exclusion chromatography. Exposure of seedling roots to one of these fractions prior to inoculation with pathogenic FOL strains significantly reduced wilt severity. This fraction initiated an ion exchange response in cultured tomato cells resulting in a reversible alteration of extracellular pH; increased tomato chitinase activity, and induced systemic resistance by enhancing PR-1 expression in tomato leaves. Two other protein fractions were inactive in seedling protection. The main polypeptide (designated CS20EP), which was specifically present in the defense-inducing fraction and was not detected in inactive protein fractions, was identified. The nucleotide sequence encoding this protein was determined, and its complete amino acid sequence was deduced from direct Edman degradation (25 N-terminal amino acid residues) and DNA sequencing. The CS20EP was found to be a small basic cysteine-rich protein with a pI of 9.87 and 23.43% of hydrophobic amino acid residues. BLAST search in the NCBI database showed that the protein is new; however, it displays 48% sequence similarity with a hypothetical protein FGSG_10784 from F. graminearum strain PH-1. The contribution of CS20EP to elicitation of tomato defense responses resulting in wilt mitigating is discussed.

    ID:1427

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. (2016-03-16)

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.

Publications

  1. Shcherbakova L.A., Odintsova T.I., Stakheev A.A., Fravel D.R., Zavriev S.K. (2016). Identification of a Novel Small Cysteine-Rich Protein in the Fraction from the Biocontrol Fusarium oxysporum Strain CS-20 that Mitigates Fusarium Wilt Symptoms and Triggers Defense Responses in Tomato. Front Plant Sci 6, 1207 [+]

    The biocontrol effect of the non-pathogenic Fusarium oxysporum strain CS-20 against the tomato wilt pathogen F. oxysporum f. sp. lycopersici (FOL) has been previously reported to be primarily plant-mediated. This study shows that CS-20 produces proteins, which elicit defense responses in tomato plants. Three protein-containing fractions were isolated from CS-20 biomass using size exclusion chromatography. Exposure of seedling roots to one of these fractions prior to inoculation with pathogenic FOL strains significantly reduced wilt severity. This fraction initiated an ion exchange response in cultured tomato cells resulting in a reversible alteration of extracellular pH; increased tomato chitinase activity, and induced systemic resistance by enhancing PR-1 expression in tomato leaves. Two other protein fractions were inactive in seedling protection. The main polypeptide (designated CS20EP), which was specifically present in the defense-inducing fraction and was not detected in inactive protein fractions, was identified. The nucleotide sequence encoding this protein was determined, and its complete amino acid sequence was deduced from direct Edman degradation (25 N-terminal amino acid residues) and DNA sequencing. The CS20EP was found to be a small basic cysteine-rich protein with a pI of 9.87 and 23.43% of hydrophobic amino acid residues. BLAST search in the NCBI database showed that the protein is new; however, it displays 48% sequence similarity with a hypothetical protein FGSG_10784 from F. graminearum strain PH-1. The contribution of CS20EP to elicitation of tomato defense responses resulting in wilt mitigating is discussed.

    ID:1427

Developing of the bank of hybridomas producing monoclonal antibodies to the HLA (2016-03-16)

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.