Лаборатория молекулярной диагностики

Группа молекулярной диагностики была образована в 2005 году на основе существовавшей в ИБХ РАН с 1987 года группы фитоиммунодиагностики. Основными направлениями работы этой группы стали разработка иммуноферментных диагностических тест-систем для диагностики различных патогенов растений (вирусов, бактерий, грибов), а также работа в области фундаментальных исследований вирусов растений и молекулярной биологии растений. В 2015 году группа была преобразована в лабораторию молекулярной диагностики в составе отдела молекулярной нейробиологии

  1. Разработка систем ПЦР-диагностики и идентификации различных патогенов растений и животных (вирусы, бактерии, грибы, нематоды и др.)
  2. Оптимизация методов выделения ДНК и РНК для анализа материала растительного и животного происхождения и идентификации микроорганизмов
  3. Генная инженерия растений
  4. Использование ПЦР-технологий для быстрой оценки эффективности регуляторов роста растений
  5. Изучение структуры и функций ряда белков фитовирусов и растительных белков, с которыми взаимодействуют вирусные белки в инфицированных растениях
  6. Изучение процессов внутри- и межклеточного транспорта белков и вирусов в растениях, механизмы действия этих белков, их внутриклеточная локализация и взаимодействие с белками растения-хозяина
  7. Вирусные белки-супрессоры посттранскрипционного умолкания генов (например, гамма-b белок гордеивирусов)
  8. Получение поли- и моноклональных антител
  9. Разработка иммунохимических тест-систем

Основными методами исследований являются генно-инженерные и иммунохимические методы, а также ДНК-технологии. Часть исследований проводится в сотрудничестве с другими подразделениями ИБХ РАН, НИИ им. Белозерского МГУ, ЗАО «ДНК-технологии», Институтом фитопатологии РАСХН и рядом зарубежных Институтов и фирм.

Гранты и проекты

1.Грант РФФИ № 06-04-48211а,2006–2008 гг. «РНК-репликаза клостеровируса желтухи свеклы: протеолитический процессинг белка-предшественника, зрелые компоненты репликативных комплексов и их цитопатогенные эффекты»

2.Грант РФФИ № 06-04-49129а,2006–2008 гг. «Изучение путей и механизмов транслокации белков через плазмодесмы на модели транспортных белков фитовирусов»

3.Проект МНТЦ № 3721p 2007–2010 гг. Идентификация молекулярными методами фитопаразитических нематод из Национальной Российской Коллекции Нематод, создание электронной базы данных молекулярных, морфологических и других характеристик фитонематод, создание исследовательской группы молекулярной диагностики нематод (совместно с ВНИИФ)

4.Грант Европейского Сообщества FP6 № 037212 (LSHB-CT-2006-037212) 2006–2009 гг. «Разработка новых и рентабельных методов неинвазивной диагностики патогенных микроорганизмов человека».

5.Госконтракт «Разработка биочипа и тест-систем для определения белковых токсинов, включая стафилококковые энтеротоксины, и антидота против ботулинического нейротоксина А, а так же ПЦР-тест систем для выявления ряда значимых патогенов растений» (Шифр «Биопатоген-ИБХРАН») по Договору № 34–405/07 от 5.10.2007. в рамках Государственного контракта №ГП/07/538/НТБ/К от 11.09.2007 г. ФЦП «Национальная технологическая база» на 2007–2011 гг.

6.Грант РФФИ 11-04-01493-а. «Съедобные вакцины против аллергии: изучение механизмов индукции толерантности мукозальными вакцинами на основе рекомбинантных белков клещей домашней пыли Dermatofagoides farina Der f 1 и Der f 2, полученных в листьях табака». 2011-2013 гг.

7. Грант РФФИ 11-04-00767-а. «Изучение динамики экспрессии генов, кодирующих антимикробные пептиды ежовника обыкновенного (Echinochloa crusgalli L.) под воздействием абиотических и биотических стрессов». 2011-2013 гг.

8. Госконтракт «Разработка опытно-промышленной технологии производства тест-систем на основе иммуно-ПЦР и мультиплексного флуоресцентного иммуноанализа, проведение медицинских испытаний и подготовка досье для государственной регистрации», 2010-2013 гг.

9. СЧ НИР "Исследования по созданию банка гибридом-продуцентов моноклональных антител к антигенам главного комплекса гистосовместимости человека", 2015-2017 гг.

10. Грант РФФИ 14-04-00997а. «Белок ОРТ6 вируса табачной мозаики –потенциальный белок вирусной безопасности: тканеспецифичность экспрессии и динамическая субклеточная локализация». 

11. Грант РФФИ 13-04-00094а, (2015 г.). «Вирусные фабрики: взаимодействие репликативных белков клостеровируса желтухи свеклы (ВЖС) с органеллами клетки, трансформация мембран , формирование и передвижение глобулярных сайтов репликации».

12. Грант РФФИ 15-29-02527 (офи-м).  «Изучение разнообразия токсигенных грибов рода Fusarium на территории России молекулярно-генетическими методами и поиск новых высокополиморфных маркеров для их специфической диагностики и идентификации».

Доклады и лекции.

«Modern approaches for use of monoclonal antibodies in plant protection and studies of virus-induced pathogenic processes»; Erokhina T., 2005
  • «Изучение роли нового класса белков растений в межклеточном транспорте макромолекул через плазмодесмы»; Ерохина Т. Н., 2005
  • «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
  • «At 4/1-подобные белки растений: свойства и возможная роль во внутри- и межклеточном транспорте макромолекул»; Ерохина Т. Н., 2007
  • «Клонирование гена, мутагенез и субклеточная локализация нового белка табака Nt-4/1»; Ерохина Т. Н., Минина Е. А., Гарушянц С. К., Соловьев А. Г., Морозов С. Ю., 2008
  • «Эффективный и экономичный метод чувствительной диагностики и идентификации патогенов картофеля»; Завриев С. К., Рязанцев Д. Ю., Кошкина Т. Е., Абрамов Д. Д., 2007
  • «ДНК-технологии для диагностики и идентификации токсигенных патогенов зерна и продуктов его переработки»; Завриев С. К., Рязанцев Д. Ю., Абрамова С. Л., Евстратова С. В., Гагкаева Т. Ю., 2008
  • «Новые белки из Arabidopsis thaliana: структура и свойства»; Рязанцев Д. Ю., Минина Е. А., 2008
  • «Детекция ряда карантинных фитопатогенов методом ПЦР в формате FLASH»; Рязанцев Д. Ю., Абрамов Д. Д, Завриев С. К., 2008
  •  
Сотрудники лаборатории (слева направо): Ерохина Татьяна Николаевна, кхн, снс; Стахеев Александр Александрович, аспирант; Завриев Сергей Кириакович (руководитель), дбн, проф., чл-корр. РАСХН; Рязанцев Дмитрий Юрьевич, кбн, мнс.

 

Избранные публикации

  1. 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
  2. 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
  3. 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
  4. 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 [+]

    Цель: изучение гуморального иммунного ответа на различные дозы белков клещей домашней пыли у мышей.

    Результаты: показана продукция антител класса IgE (но не IgG, IgA) при многократном введении низких доз (1 нг/инъекция) рекомбинантных белков. Продукция антител класса IgG и IgA возникала при введении относительно высоких (1-10 мкг) доз антигенов, при использовании адьювантов - начиная с 0,1 мкг. Антитела класса IgE были низкоаффинны.

    ID:1268
  5. 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
  6. 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
  7. 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 , ID:806
  8. 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
  9. 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
  10. 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
  11. 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
  12. 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
  13. 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
  14. 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
  15. 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 ID:697
  16. 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
  17. 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
  18. Рязанцев Д.Ю., Завриев С.К. (2009). Эффективный метод диагностики и идентификации вирусных патогенов картофеля. Мол. биол. 43 (3), 558–567 ID:160
  19. 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
  20. 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 [+]

    В статье исследуется субклеточная локализация гомологов обнаруженного ранее растительного белка At-4/1 Arabidopsisthalianaс неизвестной функцией. Белок At-4/1 локализуется в мембранных везикулярных структурах вблизи плазмодесм и способен к межклеточному транспорту. Гены гомологов этого белка были обнаружены у многих наземных растений, включая древние примитивные семенные растения. Нами был клонирован ген гомолога этого белка Nt4/1 из растения табака и сшит с геном  зеленого флуоресцирующего белка. Целью работы являлось изучение экспрессии и субклеточной локализации белка

    Nt4/1-GFPв эпидермальных клетках листьев табака. Было показано, что Nt4/1-GFPна второй день после агроинокуляции обнаруживается не только в цитоплазме, но и в ядре клеток табака, однако позднее исчезает из ядра. Методами конфокальной и электронной микроскопии показано, что белки 4/1 действительно локализуются в мембранных везикулах неизвестного происхождения. Доказано, что мембранные везикулы, в которых содержится белок, не являются производными мембран аппарата Гольджи. 

    ID:523
  21. 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
  22. 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
  23. 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
  24. 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
  25. 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
  26. 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
  27. 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
  28. 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
  29. 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
  30. 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
  31. 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
  32. 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
  33. 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
  34. 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
  35. 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
  36. 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
  37. 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
  38. 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
  39. 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
  40. 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
  41. 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

Завриев Сергей Кириакович

  • Москва, ул. Миклухо-Маклая, 16/10 — На карте
  • ИБХ РАН, корп. БОН, комн. 405
  • Тел.: +7(495)335-15-11
  • Эл. почта: szavriev@ibch.ru

Определение структуры элиситорного белка CS20EP из штамма CS-20 гриба Fusarium oxysporum (2016-11-18)

На основе предварительных данных об N-концевой аминокислотной последовательности белка, экспрессируемого штаммом  CS-20 гриба F. oxysporum, была определена структура его кДНК. С помощью методов биоинформатического анализа предсказана полная аминокислотная последовательность белка CS20EP, расчётная  масса которого соответствовала полученным ранее данным MALDI-TOF (~10 кDa). Продемонстрировано, что белок CS20EP обладает элиситорной активностью, стимулируя защитную реакцию растений томата в ответ на их заражение вирулентным штаммов F. oxysporum, вызывающим сосудистый вилт и некроз проводящих тканей. Показано, что изучаемый белок имеет ряд структурных отличий от сходных по последовательности белков вирулентных штаммов F. oxysporum и других видов рода Fusarium.  Последовательность нуклеотидов гена, кодирующего белок CS20EP, депонирована в базу данных GenBank NCBI (accession number KR028481).

Публикации

  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

Изучение разнообразия токсигенных грибов рода Fusarium на территории России молекулярно-генетическими методами и поиск новых высокополиморфных маркеров для их специфической диагностики и идентификации (2016-03-16)

Впервые определены и представлены в международных базах данных частичные последовательности генов фратаксина и фосфатпермеазы основных возбудителей фузариоза, распространённых на территории России и установлен филогенетический потенциал этих локусов

Публикации

  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

Исследования по созданию банка гибридом-продуцентов моноклональных антител к антигенам главного комплекса гистосовместимости человека (2016-03-16)

Получены клоны гибридом, продуцирующие моноклональные антитела к трем различным вариантам антигенов главного комплекса гистосовместимости человека 1 класса. 5 клонов гибридом продуцируют моноклональные антитела к HLA-A03, 9 клонов – к HLA-A11 и 6 клонов – к HLA-B51. Все полученные клоны гибридом продуцируют моноклональные антитела класса IgG. Отечественных аналогов выше указанных моноклональных антител не обнаружено.