Thesis (MSc (Plant Biotechnology))--University of Stellenboscg, 2010. / Includes bibliography. / Title page: Dept. of Genetics, Faculty of Natural Sciences. / ENGLISH ABSTRACT: Novel plant growth regulating substances (PGRs) are emerging as a useful tool to investigate important growth traits in plants. This study reports on growth promotion pathways leading to enhanced biomass accumulation in two PGRs sharing a common α, β-unsaturated furanone moiety. Growth promotion by GR24, a synthetic strigolactone, and an aqueous smoke solution (including the active compound, KAR1) in physiologically normal seedlings was characterized by enhanced biomass accumulation and higher seedling vigour. Root architecture (lateral root number and root length) and shoot size (fresh and dry shoot weight and leaf area) were also dramatically improved following GR24 and smoke/KAR1 treatment. Despite these apparent similarities, parallel transcript and phytohormone profiling identified only a limited number of overlapping entities. Four common up-regulated and nineteen down-regulated mRNA transcripts were identified; whilst amongst the phytohormones that were analyzed, only ABA and JA levels were commonly increased between the treatments. This suggests that, whilst the phenotypic end response(s) was similar, it was attained via distinct pathways. The limited number of co-expressed transcripts between these treatments, as well as repressed biomass accumulation when combining GR24 and aqueous smoke in a single treatment suggests, however, that a certain degree of cross-talk in either signal perception/transduction and/or biomass regulation could not be ruled out. In light of the structural similarity between the strigolactone and KAR1 molecules and the degree of redundancy between these treatments, it is possible that these two molecules might share a common receptor/perception pathway. Two silencing vectors were constructed, specifically aimed at silencing Nicotiana benthamiana genes MAX4 and MAX2 which are known to function in the strigolactone biosynthesis pathway and signal transduction pathway, respectively. Transgenes designed to express single- or double-stranded-self- complementary hairpin RNA have a post translational gene silencing effect. The pHELLSGATE2 plasmid a binary vector that incorporates GATEWAY cloning technology which makes use of λ-phage-based site specific recombination, rather than restriction endonucleases and ligation, was used to construct these gene silencing vectors. These constructs can in future be used to produce Nicotiana plants with impaired strigolactone production and perception abilities and may provide evidence as to whether the signaling cascade of KAR1 and strigolactone share a degree of crosstalk. / AFRIKAANSE OPSOMMING: Aanvraag na plantmateriaal is besig om toe te neem, hetsy vir gebruik as mens- en diervoeding of vir die produksie van biobrandstof. Om aan hierdie behoefte te voldoen, word verskeie pogings geloods wat fokus op die optimisering van plantproduksiestelsels. Om plantgroei te stimuleer/verbeter, is ’n ingewikkelde proses en is oor die algemeen moeilik om te begryp. Die produksie van plantbiomassa is nou gekoppel aan primêre metabolisme en enige verandering in hierdie biochemiese padweë kan lei tot ongewenste newe-effekte. Gevolglik word primêre metabolisme streng beheer deur reguleringsmeganismes. ’n Nuttige alternatief tot metaboliese wysiging is deur bio-aktiewe agente te karakteriseer op grond van die veranderinge aan plantgroei wat waargeneem word. Nuwe stowwe met biologiese aktiwiteite in plantontwikkeling word elke dag ontdek en speel ’n belangrike rol in die studie van plantgroei en -ontwikkeling. Hier word verslag gelewer van twee plantgroei-stimulerende stowwe wat albei lei tot die aktivering van verbeterde plantbiomassa-akkumulasie-padweë. Swaarder plantjies met ’n verhoogde oorlewingsvermoё is waargeneem in fisiologies normale saailinge wat met ’n sintetiese strigolaktoon (GR24) of met rookwater (met aktiewe bestanddeel, KAR1) behandel is. Behandeling met hierdie twee stowwe het gelei tot soortgelyke plantbiomassa-akkummulasie- vermoё. Hierdie twee stowwe (GR24 en KAR1) deel ’n ooreenstemmende molekulêre struktuur in die vorm van ’n α, β-onversadigde furanone-moieteit. Ten spyte van die groeiverbeteringsooreenkomste, gesien in saalinge behandel met GR24 en rook/KAR1, dui verskille in transkripsie- en hormoonprofiel op twee verskillende groeistimuleringspadweë. Saailinge wat gelyktydig behandel is met ’n kombinasie van die twee stowwe het egter ’n stremming in groei getoon in vergelyking met die kontroleplantjies. Dit is egter waargeneem dat daar wel ’n mate van oorvleueling in die aantal transkripte was tussen die drie behandelinge, wat daarop dui dat die groei-regulerende padweë nie in totale onafhanklikheid funksioneer nie, maar wel sekere stappe deel. Na aanleiding van die strukturele ooreenkomste tussen die strigolaktoon (GR24) en KAR1 molekules en die mate van molekulêre kommunikasieoorvleueling word gepostuleer dat hierdie twee molekules dalk aan dieselfde reseptormodule kan bind of stimuleer. Om hierdie rede is twee geendempingsvektors geskep wat daarop gemik is om twee gene, MAX2 en MAX4, in Nicotiana benthamiana uit te doof. Die MAX2 geenproduk is betrokke in die kommunikasie en waarneming van die strigolaktoon en die MAX4 geenproduk is betrokke by die vervaardiging van die hormoon. Oordraagbare geen-kostruksies wat daarop gemik is om enkel- en dubbelstring selfkomplimentêre haarnaald-RNS te vorm, besit die vermoë om getranskribeerde geenprodukte te vernietig. Die pHELLSGATE2 plasmied is ’n binêre vektor wat GATEWAY kloneringstegnologie gebruik, waar λ-faag gebaseerde setelspesifieke rekombinasie eerder as die tradisionele ligeringsreaksie gebruik word. Hierdie konstrukte kan gebruik word om transgeniese plantjies te skep waar die vermoë om strigolaktoon te maak of waar te neem, verloor of onderdruk is. Hierdie transgeniese plantjies kan gebruik word om te bepaal of die plantgroei-stimulerende vermoë van GR24 en rook/KAR1 wel dieselfde padweë gebruik.

abstract: Anti-retroviral drugs and AIDS prevention programs have helped to decrease the rate of new HIV-1 infections in some communities, however, a prophylactic vaccine is still needed to control the epidemic world-wide. Despite over two decades of research, a vaccine against HIV-1 remains elusive, although recent clinical trials have shown promising results. Recent successes have focused on highly conserved, mucosally-targeted antigens within HIV-1 such as the membrane proximal external region (MPER) of the envelope protein, gp41. MPER has been shown to play critical roles in the viral mucosal transmission, though this peptide is not immunogenic on its own. Gag is a structural protein configuring the enveloped virus particles, and has been suggested to constitute a target of the cellular immunity potentially controlling the viral load. It was hypothesized that HIV-1 enveloped virus-like particles (VLPs) consisting of Gag and a deconstructed form of gp41 comprising the MPER, transmembrane, and cytoplasmic domains (dgp41) could be expressed in plants. Plant-optimized HIV-1 genes were constructed and expressed in Nicotiana benthamiana by stable transformation, or transiently using a tobacco mosaic virus-based expression system or a combination of both. Results of biophysical, biochemical and electron microscopy characterization demonstrated that plant cells could support not only the formation of HIV-1 Gag VLPs, but also the accumulation of VLPs that incorporated dgp41. These particles were purified and utilized in mice immunization experiments. Prime-boost strategies combining systemic and mucosal priming with systemic boosting using two different vaccine candidates (VLPs and CTB-MPR - a fusion of MPER and the B-subunit of cholera toxin) were administered to BALB/c mice. Serum antibody responses against both the Gag and gp41 antigens could be elicited in mice systemically primed with VLPs and these responses could be recalled following systemic boosting with VLPs. In addition, mucosal priming with VLPs allowed for a robust boosting response against Gag and gp41 when boosted with either candidate. Functional assays of these antibodies are in progress to test the antibodies' effectiveness in neutralizing and preventing mucosal transmission of HIV-1. This immunogenicity of plant-based Gag/dgp41 VLPs represents an important milestone on the road towards a broadly-efficacious and inexpensive subunit vaccine against HIV-1. / Dissertation/Thesis / Ph.D. Molecular and Cellular Biology 2011

A resistência a doenças em plantas transgênicas tem sido obtida por meio da expressão de genes isolados de bactérias, fungos micoparasitas e plantas. Neste trabalho, relatamos a utilização de um gene do fungo entomopatogênico Metarhizium anisopliae como modo de gerar resistência a doenças fúngicas em plantas. O gene chit1 codifica a quitinase CHIT42 (EC 3.2.1.14), pertencente a uma classe de glicosil-hidrolases capazes de converter quitina em oligômeros de N-acetil-glicosamina (NAcGlc). Quando presentes em tecidos vegetais, supõese que as quitinases ataquem especificamente a parede celular de fungos invasores, provocando danos às hifas e causando a morte por lise das células fúngicas. Deste modo, dois diferentes grupos de plantas transgênicas de Nicotiana tabacum foram produzidos: no primeiro deles, denominado chitplus, os indivíduos possuem o gene chit1 sob o controle do promotor CaMV 35S. O segundo grupo, demoninado chitless, consiste de plantas transformadas com um T-DNA não contendo o gene do fungo. Trinta e quatro plantas transgênicas resistentes à canamicina (17 de cada grupo) foram regeneradas a partir de discos de folhas infectados por Agrobacterium tumefaciens. A produção da quitinase em extratos protéicos de folhas foi analisada por zimogramas em SDS-PAGE contendo glicol-quitina e corados por calcoflúor branco, na forma de um screening dos transgênicos primários. As plantas transgênicas foram testadas, ainda, por meio de ensaios colorimétricos empregando oligômeros sintéticos de NAcGlc como substratos específicos, além de immunoblot e Western blot com soro anti-quitinase. A quantidade de enzima recombinante nas plantas chitplus variou desde nenhuma atividade detectável a elevados níveis de expressão da enzima. A hibridização de Southern blot demonstrou que o número de cópias do gene chit1 integradas no genoma vegetal foi estimado entre uma e quatro. A primeira geração de plantas transgênicas geradas por autofecundação de parentais portadores de duas cópias do transgene foi testada com relação à estabilidade da herança do transgene e em 43 de um total de 67 descendentes, originados de quatro cruzamentos independentes, o padrão de segregação não diferiu das proporções Mendelianas esperadas. Ensaios de resistência, desafiando as plantas transgênicas com o basidiomiceto Rhizoctonia solani foram realizados e uma evidente diminuição da área foliar contendo lesões fúngicas foi observada entre as linhagens transgênicas, embora variações na atividade quitinolítica tenham influenciado o nível de resistência. Nossos resultados sugerem uma relação direta entre a atividade específica de quitinase e ao aumento nos níveis de resistência às lesões causadas pela infecção por R. solani. / Plant resistance in transgenic plants has been obtained by expressing genes isolated from bacteria, mycoparasitic fungi and plants. Here we report the employment of a gene from the entomopathogenic fungus Metarhizium anisopliae as a tool to generate resistance to fungal diseases in plants. The chit1 gene encodes the chitinase CHIT42 (EC 3.2.1.14), belonging to a class of glicosyl-hydrolases able to convert chitin into N-acetyl-glucosamine (GlcNAc) oligomers. When present in plant tissues, chitinases are supposed to disrupt the invading fungal cell wall specifically, causing hyphae damage and leading to cell lysis. Hence two different groups of transgenic Nicotiana tabacum plants were produced. The first group was named chitplus, in which individuals harbour the chit1 gene under the control of the CaMV 35S promoter . The second group, named chitless, carried a T-DNA not containing the fungal gene. Thirty-four kanamicin resistant plants (17 of each group) were regenerated from leaf discs infected with Agrobacterium tumefaciens. Chitinase production in leaf protein extracts was analysed through zymograms in SDS-PAGE containing glycol-chitin and stained by calcofluor white, as a screening of primary transformants. Transgenic plants were also evaluated by colorimetric assays using synthetic GlcNAc oligomers as specific substrates besides immunoblot and Western blot probed with rabbit anti-chitinase sera. The amount of recombinant enzyme in chitplus plants ranged from no detectable chitinase activity to high levels of enzyme expression. Southern blot hybridisation revealed that chit1 copy number inserted into plant genomes varied from one to four. The first self pollinated generation of transgenic lines bearing two copies of the transgene was tested on inheritance stability and in 43 out of 67 descendants, derived from four independent crosses, the segregation pattern was discovered not to differ from the predicted Mendelian ratios. Resistance assays challenging transgenic plants with the basidiomycete Rhizoctonia solani were performed and a clear decrease in the foliar area containing fungal lesions was observed amongst transgenic lines, though variations in chitinase activity also reflected on the resistance level. Our results suggest a direct relationship between chitinase specific activity and the improvement in the resistance to lesions caused by infection.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2016-06-13T16:21:59Z No. of bitstreams: 1 texto completo.pdf: 1075966 bytes, checksum: 047f8bb5c392a3f22d9009ed5f6f71b9 (MD5) / Made available in DSpace on 2016-06-13T16:21:59Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1075966 bytes, checksum: 047f8bb5c392a3f22d9009ed5f6f71b9 (MD5) Previous issue date: 2009-07-31 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Nesse trabalho foi avaliada a importância do transportador de sacarose especificamente em células guarda (CG) e o papel da sacarose sobre os movimentos estomáticos. Utilizou-se plantas de tabaco transformadas com o antisenso do gene do transportador de sacarose sob controle do promotor KST1, específico de GC. As CG das plantas transgênicas apresentaram menores teores de sacarose, maiores nos de amido e um modesto incremento nos de K + . O menor conteúdo de sacarose nas CG das plantas transgênicas esteve associado com menores valores de condutância estomática (g s ). Essa associação sugere a importância da sacarose no simplasto na manutenção de baixos potenciais osmóticos nas CG. Foi observada uma rápida redução nos teores de amido quando os estômatos estavam se abrindo, fato não observado nas plantas não-transformadas. Nas plantas transformadas, com menor g s , foi possível demonstrar uma restrição difusional (estomática) à fotossíntese (A). As plantas transformadas também apresentaram menor taxa de transpiração (E) e menor concentração de CO 2 na câmara sub-estomática, além de maiores valores da razão de composição isotópica (δ 13 C). Entretanto, maiores valores da razão A/E esteve associado com menores valores de A, conseqüentemente, a uma menor taxa de crescimento, porém não a uma menor eficiência baseada nas taxas de crescimento relativas. Os dados de δ 13 C confirmaram a menor g s e reforçam que esse fenótipo se prolongou pelo desenvolvimento das plantas. Por meio de plantas de tabaco com menor g s foi possível demonstrar que o fenótipo de retardamento à seca foi a principal característica desta transformação, proporcionando as plantas transgênicas um menor consumo de água. Os resultados sugerem que a manipulação do transporte de sacarose em CG foi um mecanismo prático e efetivo na aquisição de plantas mais resistentes à seca. / It was evaluated the importance of guard cell (GC) sucrose transporter and the role of sucrose as osmotic on GC. We transformed tobacco plants with antisense gene construct for sucrose transporter driven by KST1, GC specific promoter. Transgenic plants GC have less sucrose, more starch and modest increase in K + contents. Low sucrose contents in GC of transgenic lines were associated with low stomatal conductance (g s ), suggesting the importance of sucrose transporter and symplastic sucrose in maintaining low osmotic potential on GC. It was observed rapid starch disappearance when the guard cells are swelling, fact not observed in control plants. By means of low g s tobacco plants demonstrated diffusional (stomatal) restriction of photosynthesis (A), low transpiration rate (E) and low sub-stomatal CO 2 concentration, high A/E and higher carbon rate composition (δ 13 C). However, higher A/E was associated with lower A, consequently, a slower crop growth rate, but not smaller “efficiency index” as showed by relative growth rate. The δ 13 C data confirms the low conductance, showing that it represents a common stomata behavior over all plant development. By means of low g s tobacco plants, we got desiccation postponement phenotype as principal feature of this transformation, being high water saving plants. These results suggest that manipulation of sucrose transport in GC may be developed as a practical mechanism for drought avoidance and water conservation during irrigation. These results illustrate the importance of fine tuning of sucrose metabolism transport and metabolism in the fitness of stomatal function in contributing to plant survival or growth under unfavorable water conditions.

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2016-09-23T12:09:04Z No. of bitstreams: 1 texto completo.pdf: 703627 bytes, checksum: 193b0cc47fc6514482df37f3b075f580 (MD5) / Made available in DSpace on 2016-09-23T12:09:04Z (GMT). No. of bitstreams: 1 texto completo.pdf: 703627 bytes, checksum: 193b0cc47fc6514482df37f3b075f580 (MD5) Previous issue date: 2000-07-05 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Os processos que regulam a alocação de carbono para os órgãos vegetais e as sementes em desenvolvimento são muito importantes no desenvolvimento das plantas. Na maioria dos vegetais, o nutriente translocado mais importante é a sacarose, e a compreensão da biologia molecular, da bioquímica e da fisiologia de seu transporte é um problema central na biologia vegetal. Recentemente, foi isolado, em um laboratório da Universidade Federal de Viçosa, Viçosa-MG, um cDNA de uma biblioteca de expressão de sementes de soja que codifica um homólogo da proteína de ligação à sacarose (SBP), denominado S-64. Para analisar a função desse homólogo de SBP, tabacos transgênicos foram obtidos, introduzindo-se genes quiméricos que continham a região codificadora do gene s-64, ligado ao promotor 35S-CaMV, na orientação senso ou anti-senso via transformação mediada por Agrobacterium sp. O acúmulo do homólogo de SBP aumentou em tabacos que superexpressaram o gene s-64, assim como a expressão anti-senso do gene s-64 levou ao decréscimo nos níveis da proteína endógena. As plantas anti-senso desenvolveram sintomas característicos de uma inibição do transporte de sacarose à longa distância e apresentaram crescimento e desenvolvimento vegetal reduzidos. Em contraste, as plantas que continham a construção senso revelaram tendência de crescimento mais rápido e aceleração do desenvolvimento floral. O desempenho no desenvolvimento das plantas transgênicas foi correlacionado com a taxa fotossintética, sob condições normais de irradiância. Enquanto a taxa de fotossíntese nas linhagens anti-senso diminuiu, nas linhagens senso ela aumentou. Além disto, tanto a repressão anti-senso quanto a superexpressão senso do homólogo de SBP alteraram o particionamento de carboidratos em folhas maduras. Na geração seguinte, as plantas transgênicas senso e anti-senso revelaram diferentes padrões de expressão do gene s-64. Enquanto nas plantas senso não foi observado aumento no acúmulo da proteína, nas plantas anti-senso uma forte inibição de S-64/SBP persistiu. Além disto, as plantas transgênicas anti-senso R1 tiveram um reduzido crescimento vegetativo e radicular, acompanhado de um florescimento tardio. Seguindo esse padrão, a taxa fotossintética das folhas jovens e maduras diminuiu, o que evidencia uma possível diminuição no carregamento e, ou, descarregamento do floema. Um reduzido peso seco total e radicular, assim como experimentos de enxertia, forneceu evidências de que o gene s-64 está diretamente envolvido no descarregamento do floema. Coletivamente, esses resultados indicam que a proteína S-64 é funcionalmente análoga à proteína de ligação à sacarose e representa um importante componente da via de translocação de sacarose em plantas. / The processes that regulate carbon allocation to various organs in the developing seed is very important in plant development. In most plants, the morg important translocated nutrient is sucrose and, thus, understanding the molecular biology, biochemistry, and physiology of sucrose transport is a central problem in plant biology. A sucrose binding protein (SBP) homologue, designated S-64, was isolated and transgenic tobacco plants were obtained by introducing chimeric genes containing the S-64 coding region linked to the 35S CaMV promoter, either in the sense or antisense orientation, via Agrobacterium tumefaciens- mediated transformation. The accumulation of the SBP homologue was increased in transgenic plants expressing the heterologous sbp gene, whereas those expressing the antisense construct had reduced levels of the protein. The antisense transgenic plants developed symptoms characteristic of an inhibition of sucrose translocation and displayed a reduction in plant growth and development. In contrast, overexpression of the protein accelerated plant growth and the onset of flowering induction. The overall developmental performance of the transgenic plants was correlated with their photosynthetic rate under normal conditions. While photosynthesis in the antisense lines was decreased, in the sense lines photosynthetic rates were increased. Furthermore, both antisense repression and overexpression of the SBP homologue in transgenic lines altered carbohydrate partitioning in mature leaves. On the following generation, both sense and antisense plants revealed different S-64 expression patterns. While the sense plants had no overexpression when compared to control plants, the antisense plants was significantly inhibited by the SBP homologue gene. According to previous results, the antisense plants have a reduced vegetative growth accompanied by a late flowering. The photosynthesis was reduced on young and mature leaves from antisense plants supporting a possible decrease on loading and/or unloading of the phloem. A reduced root growth and dry weight, associated with grafting experiments showed strong evidences that gene is directly involved on phloem unloading. Taken together, these results indicate that S-64 protein is functionally analogous to sucrose binding protein, representing an important component of the sucrose translocation pathway in plants. / Não foi localizado o cpf do autor.

A resistência a doenças em plantas transgênicas tem sido obtida por meio da expressão de genes isolados de bactérias, fungos micoparasitas e plantas. Neste trabalho, relatamos a utilização de um gene do fungo entomopatogênico Metarhizium anisopliae como modo de gerar resistência a doenças fúngicas em plantas. O gene chit1 codifica a quitinase CHIT42 (EC 3.2.1.14), pertencente a uma classe de glicosil-hidrolases capazes de converter quitina em oligômeros de N-acetil-glicosamina (NAcGlc). Quando presentes em tecidos vegetais, supõese que as quitinases ataquem especificamente a parede celular de fungos invasores, provocando danos às hifas e causando a morte por lise das células fúngicas. Deste modo, dois diferentes grupos de plantas transgênicas de Nicotiana tabacum foram produzidos: no primeiro deles, denominado chitplus, os indivíduos possuem o gene chit1 sob o controle do promotor CaMV 35S. O segundo grupo, demoninado chitless, consiste de plantas transformadas com um T-DNA não contendo o gene do fungo. Trinta e quatro plantas transgênicas resistentes à canamicina (17 de cada grupo) foram regeneradas a partir de discos de folhas infectados por Agrobacterium tumefaciens. A produção da quitinase em extratos protéicos de folhas foi analisada por zimogramas em SDS-PAGE contendo glicol-quitina e corados por calcoflúor branco, na forma de um screening dos transgênicos primários. As plantas transgênicas foram testadas, ainda, por meio de ensaios colorimétricos empregando oligômeros sintéticos de NAcGlc como substratos específicos, além de immunoblot e Western blot com soro anti-quitinase. A quantidade de enzima recombinante nas plantas chitplus variou desde nenhuma atividade detectável a elevados níveis de expressão da enzima. A hibridização de Southern blot demonstrou que o número de cópias do gene chit1 integradas no genoma vegetal foi estimado entre uma e quatro. A primeira geração de plantas transgênicas geradas por autofecundação de parentais portadores de duas cópias do transgene foi testada com relação à estabilidade da herança do transgene e em 43 de um total de 67 descendentes, originados de quatro cruzamentos independentes, o padrão de segregação não diferiu das proporções Mendelianas esperadas. Ensaios de resistência, desafiando as plantas transgênicas com o basidiomiceto Rhizoctonia solani foram realizados e uma evidente diminuição da área foliar contendo lesões fúngicas foi observada entre as linhagens transgênicas, embora variações na atividade quitinolítica tenham influenciado o nível de resistência. Nossos resultados sugerem uma relação direta entre a atividade específica de quitinase e ao aumento nos níveis de resistência às lesões causadas pela infecção por R. solani. / Plant resistance in transgenic plants has been obtained by expressing genes isolated from bacteria, mycoparasitic fungi and plants. Here we report the employment of a gene from the entomopathogenic fungus Metarhizium anisopliae as a tool to generate resistance to fungal diseases in plants. The chit1 gene encodes the chitinase CHIT42 (EC 3.2.1.14), belonging to a class of glicosyl-hydrolases able to convert chitin into N-acetyl-glucosamine (GlcNAc) oligomers. When present in plant tissues, chitinases are supposed to disrupt the invading fungal cell wall specifically, causing hyphae damage and leading to cell lysis. Hence two different groups of transgenic Nicotiana tabacum plants were produced. The first group was named chitplus, in which individuals harbour the chit1 gene under the control of the CaMV 35S promoter . The second group, named chitless, carried a T-DNA not containing the fungal gene. Thirty-four kanamicin resistant plants (17 of each group) were regenerated from leaf discs infected with Agrobacterium tumefaciens. Chitinase production in leaf protein extracts was analysed through zymograms in SDS-PAGE containing glycol-chitin and stained by calcofluor white, as a screening of primary transformants. Transgenic plants were also evaluated by colorimetric assays using synthetic GlcNAc oligomers as specific substrates besides immunoblot and Western blot probed with rabbit anti-chitinase sera. The amount of recombinant enzyme in chitplus plants ranged from no detectable chitinase activity to high levels of enzyme expression. Southern blot hybridisation revealed that chit1 copy number inserted into plant genomes varied from one to four. The first self pollinated generation of transgenic lines bearing two copies of the transgene was tested on inheritance stability and in 43 out of 67 descendants, derived from four independent crosses, the segregation pattern was discovered not to differ from the predicted Mendelian ratios. Resistance assays challenging transgenic plants with the basidiomycete Rhizoctonia solani were performed and a clear decrease in the foliar area containing fungal lesions was observed amongst transgenic lines, though variations in chitinase activity also reflected on the resistance level. Our results suggest a direct relationship between chitinase specific activity and the improvement in the resistance to lesions caused by infection.

A resistência a doenças em plantas transgênicas tem sido obtida por meio da expressão de genes isolados de bactérias, fungos micoparasitas e plantas. Neste trabalho, relatamos a utilização de um gene do fungo entomopatogênico Metarhizium anisopliae como modo de gerar resistência a doenças fúngicas em plantas. O gene chit1 codifica a quitinase CHIT42 (EC 3.2.1.14), pertencente a uma classe de glicosil-hidrolases capazes de converter quitina em oligômeros de N-acetil-glicosamina (NAcGlc). Quando presentes em tecidos vegetais, supõese que as quitinases ataquem especificamente a parede celular de fungos invasores, provocando danos às hifas e causando a morte por lise das células fúngicas. Deste modo, dois diferentes grupos de plantas transgênicas de Nicotiana tabacum foram produzidos: no primeiro deles, denominado chitplus, os indivíduos possuem o gene chit1 sob o controle do promotor CaMV 35S. O segundo grupo, demoninado chitless, consiste de plantas transformadas com um T-DNA não contendo o gene do fungo. Trinta e quatro plantas transgênicas resistentes à canamicina (17 de cada grupo) foram regeneradas a partir de discos de folhas infectados por Agrobacterium tumefaciens. A produção da quitinase em extratos protéicos de folhas foi analisada por zimogramas em SDS-PAGE contendo glicol-quitina e corados por calcoflúor branco, na forma de um screening dos transgênicos primários. As plantas transgênicas foram testadas, ainda, por meio de ensaios colorimétricos empregando oligômeros sintéticos de NAcGlc como substratos específicos, além de immunoblot e Western blot com soro anti-quitinase. A quantidade de enzima recombinante nas plantas chitplus variou desde nenhuma atividade detectável a elevados níveis de expressão da enzima. A hibridização de Southern blot demonstrou que o número de cópias do gene chit1 integradas no genoma vegetal foi estimado entre uma e quatro. A primeira geração de plantas transgênicas geradas por autofecundação de parentais portadores de duas cópias do transgene foi testada com relação à estabilidade da herança do transgene e em 43 de um total de 67 descendentes, originados de quatro cruzamentos independentes, o padrão de segregação não diferiu das proporções Mendelianas esperadas. Ensaios de resistência, desafiando as plantas transgênicas com o basidiomiceto Rhizoctonia solani foram realizados e uma evidente diminuição da área foliar contendo lesões fúngicas foi observada entre as linhagens transgênicas, embora variações na atividade quitinolítica tenham influenciado o nível de resistência. Nossos resultados sugerem uma relação direta entre a atividade específica de quitinase e ao aumento nos níveis de resistência às lesões causadas pela infecção por R. solani. / Plant resistance in transgenic plants has been obtained by expressing genes isolated from bacteria, mycoparasitic fungi and plants. Here we report the employment of a gene from the entomopathogenic fungus Metarhizium anisopliae as a tool to generate resistance to fungal diseases in plants. The chit1 gene encodes the chitinase CHIT42 (EC 3.2.1.14), belonging to a class of glicosyl-hydrolases able to convert chitin into N-acetyl-glucosamine (GlcNAc) oligomers. When present in plant tissues, chitinases are supposed to disrupt the invading fungal cell wall specifically, causing hyphae damage and leading to cell lysis. Hence two different groups of transgenic Nicotiana tabacum plants were produced. The first group was named chitplus, in which individuals harbour the chit1 gene under the control of the CaMV 35S promoter . The second group, named chitless, carried a T-DNA not containing the fungal gene. Thirty-four kanamicin resistant plants (17 of each group) were regenerated from leaf discs infected with Agrobacterium tumefaciens. Chitinase production in leaf protein extracts was analysed through zymograms in SDS-PAGE containing glycol-chitin and stained by calcofluor white, as a screening of primary transformants. Transgenic plants were also evaluated by colorimetric assays using synthetic GlcNAc oligomers as specific substrates besides immunoblot and Western blot probed with rabbit anti-chitinase sera. The amount of recombinant enzyme in chitplus plants ranged from no detectable chitinase activity to high levels of enzyme expression. Southern blot hybridisation revealed that chit1 copy number inserted into plant genomes varied from one to four. The first self pollinated generation of transgenic lines bearing two copies of the transgene was tested on inheritance stability and in 43 out of 67 descendants, derived from four independent crosses, the segregation pattern was discovered not to differ from the predicted Mendelian ratios. Resistance assays challenging transgenic plants with the basidiomycete Rhizoctonia solani were performed and a clear decrease in the foliar area containing fungal lesions was observed amongst transgenic lines, though variations in chitinase activity also reflected on the resistance level. Our results suggest a direct relationship between chitinase specific activity and the improvement in the resistance to lesions caused by infection.

Submitted by Gabriela Lopes (gmachadolopesufpel@gmail.com) on 2018-06-13T17:18:47Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Luis Henrique Konzen.pdf: 646860 bytes, checksum: 511c31d83c08144defe344c571319e35 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2018-06-14T20:50:11Z (GMT) No. of bitstreams: 2 Luis Henrique Konzen.pdf: 646860 bytes, checksum: 511c31d83c08144defe344c571319e35 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2018-06-14T20:50:44Z (GMT) No. of bitstreams: 2 Luis Henrique Konzen.pdf: 646860 bytes, checksum: 511c31d83c08144defe344c571319e35 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-06-14T20:50:50Z (GMT). No. of bitstreams: 2 Luis Henrique Konzen.pdf: 646860 bytes, checksum: 511c31d83c08144defe344c571319e35 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-03-02 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / O estabelecimento da lavoura de tabaco se dá através do transplante de mudas, e o passo inicial para promover um estande uniforme de plantas e garantir uma boa produtividade da lavoura de tabaco é a utilização de mudas de boa qualidade, para isso, é necessário a utilização de sementes de altíssima qualidade. Neste sentido, os testes de vigor são muito importantes para obtenção de informações adicionais ao teste de germinação e podem auxiliar na tomada de decisão. Assim, objetivou-se com o presente trabalho determinar metodologias do teste de envelhecimento acelerado para avaliação do vigor em sementes de tabaco. Para o estudo, foram utilizados dez lotes de sementes de tabaco. Inicialmente determinou-se a qualidade inicial das sementes de tabaco através dos testes de germinação, primeira contagem de germinação, índice de velocidade de germinação, emergência de plântulas avaliadas aos 7, 14 e 21 dias após semeadura, índice de velocidade de emergência e envelhecimento acelerado com água conduzido conforme proposto pela AOSA. Após a determinação da qualidade inicial das sementes, estudou se o teste de envelhecimento acelerado nas metodologias: envelhecimento acelerado com água, solução salina saturada (40g de NaCl.100mL-1 de água) e solução salina não saturada (11g de NaCl.100mL-1 de água), submetidas a temperatura de 45 e 41⁰C, por períodos de exposição de 24, 48 e 72 horas. O ensaio foi conduzido em delineamento de blocos ao acaso, as médias obtidas por lote, em cada avaliação, foram comparadas pelo teste de Scott-Knott em nível de probabilidade de 5% e foi realizada análise de correlação linear. De acordo com os resultados obtidos, conclui - se que o teste de envelhecimento acelerado com água conduzido sob temperatura de 45ºC combinado com período de exposição de 24 horas mostra-se adequado para avaliação do vigor de sementes de tabaco. / The establishment of tobacco is performed through seedling transplant, and the initial step to promote a uniform plant stand and ensure a good yield of the tobacco crop is the use of good quality seedlings, for this, the use of seeds of the highest quality is necessary. In this sense, vigor tests are very important to obtain additional information to the standard germination test and can assist in decision-making. Thus, the aim of this work was to determine accelerated aging test methodologies for the evaluation of vigor in tobacco seeds. For the study, ten lots of tobacco seeds were used. The initial quality of the tobacco seeds was determined through the germination test, first germination count, germination speed index, emergence of seedlings at 7, 14 and 21 days after sowing, emergence speed index and the accelerated aging with water conducted as proposed by the AOSA. After the determination of the initial quality of the seeds, the accelerated aging test was carry out in the following methods: accelerated aging with water, saturated saline solution (40g NaCl 100mL-1 water) and unsaturated saline solution (11g NaCl 100mL-1 water), submitted to temperature of 45 and 41°C, for periods of exposure of 24, 48 and 72 hours. The assay was conducted in a randomized block designed, the averages obtained per lot at each evaluation were compared by the Scott-Knott test at a 5% probability level and a linear correlation analysis was performed. According to the results, it is concluded that the accelerated aging test with water conducted under a temperature of 45°C combined with a 24 - hour exposure period is adequate for evaluating the vigor of tobacco seeds.

M.Sc. (Biochemistry) / Plants respond to various stress stimuli by activating a broad-spectrum of defence responses that can be expressed locally at the site of pathogen infection (hypersensitive response-HR) as well as systemically in uninfected tissue (systemic acquired resistance-SAR). The ability to continuously respond to both abiotic and biotic stimuli leads to changes in the plants’ physiology, morphology and development. Therefore, there is a need to define and understand the mechanism of the plant defence system, including the mode of recognition, activation of signalling pathways and subsequent defence. In so doing, a long lasting and effective protection against various pathogens may be established. In the current study, the transcriptome status of cultured cells of Nicotiana tabacum was investigated using annealing control primer (ACP)-based differential display (DD) since it is an improved technology to compare patterns of gene expression in RNA samples, isolated from tissue / cells under different biological conditions, using a novel priming system. Here, ACP-DDRT-PCR was used in combination with a next-generation sequencing technology, namely 454 pyro-sequencing, which is the only technique that generates longer reads which are suitable for de novo assembly and annotation of non-model plants like tobacco of which the genome is not yet published in Genbank. SAR occurs following induction by biotrophic or necrotising pathogens. However, it can also be manifested artificially after chemical treatment. In this study, isonitrosoacetophenone (INAP), a novel compound that was originally isolated from extracts of citrus peel undergoing oxidative stress, was used as a chemical inducer and it was hypothesised that this compound induces defence-related responses in plants. In order to investigate this, tobacco cell suspensions were elicited with 1 mM INAP, followed by ACP-DDRT-PCR and subsequent identification of differentially expressed genes using pyro-sequencing.

Les histones désacétylases de type 2 (HD2) ont été caractérisées chez le tabac comme étant des régulateurs négatifs de la mort cellulaire associée à la réponse hypersensible induite par la cryptogéine, un éliciteur protéique.Les principaux objectifs de cette thèse sont d’accroître nos connaissances générales sur les HD2 et de caractériser leur rôle dans la voie de signalisation induite par la cryptogéine.Nous nous sommes tout d’abord intéressé à l’évolution des HD2 au sein des Viridiplantae et nous avons défini deux groupes de HD2. Nous nous sommes ensuite focalisés sur l’évolution des HD2 dans le genre Nicotiana. Nous avons identifié chez le tabac six gènes codant sept isoformes de HD2. Les quatre isoformes de HD2 de Gr1 seraient redondants fonctionnellement.Pour mieux comprendre le mode d’action des HD2 à l’échelle moléculaire et étant donné que nous n’avons pas pu détecter d’activité enzymatique chez les HD2, nous avons tenté d’identifier les gènes cibles et les partenaires protéiques potentiels des HD2.Des expériences de puces à ADN nous ont permis d’identifier des gènes régulés par la cryptogéine de manière HD2 dépendante, parmi lesquels ERF3 qui pourrait constituer un gène majeur impliqué dans l’initiation de la mort cellulaire.Des expériences de co-immunopurification combinées à des analyses par spectrométrie de masse nous ont permis d’identifier des partenaires protéiques des HD2 dont l’histone H4.De manière générale, les résultats obtenus au cours de cette thèse soulèvent la question de savoir si les HD2 possèdent une activité HDAC intrinsèque et nous ont permis de mieux comprendre le rôle des HD2 dans la signalisation nucléaire induite par la cryptogéine. / Type 2 histone deacetylases (HD2s) have been characterized in Nicotiana tabacum as negative regulators of hypersensitive response (HR)-associated cell death induced by cryptogein, a proteinaceous elicitor secreted by Phytophthora cryptogea.The main objectives of this thesis are to increase our general knowledge about HD2s and to characterize their role in the signaling pathway induced by cryptogein.We first examined the evolution of HD2s in green plants and defined two groups of HD2s. We then focused on the evolution of HD2s in the genus Nicotiana. Six genes coding seven isoforms of HD2s were identified in N. tabacum. The four Gr1 HD2 isoforms are functionally redundant in the response to salt stress.To better understand the mode of action of HD2s at the molecular level, and since we – as well as other research groups – were unable to detect any enzymatic activity from HD2s, we tried to identify target genes and potential protein partners of HD2s.Microarray experiments allowed us to identify genes that are regulated by cryptogein in a HD2-dependant fashion. Among these genes, ERF3, coding an ethylene-responsive factor, could be a major gene involved in the initiation of HR-associated cell death.Co-immunopurification experiments combined with mass spectrometry analyses permitted us to identify protein partners of HD2s such as histone H4.Globally, the results obtained during this thesis call into question whether HD2s do have an HDAC activity themselves, or are associated into complexes containing HDACs from the RDP3/HDA1 family. These results also permitted us to better understand the role of HD2s in the nuclear signaling pathway induced by cryptogein in tobacco.