Desarrollo de métodos de diagnóstico y de plantas transgénicas de papa para el control de infecciones producidas por el virus PVY

El presente trabajo de tesis se propone utilizar las herramientas que provee la ingeniería genética para controlar las infecciones producidas por el virus PVY en plantas de papa, aportando nuevos métodos de diagnóstico para un mejor control de calidad de la semilla, y en combinación con las técnicas de transformación vegetal, modificar cultivares comerciales con el fin de lograr resistencia al virus mediante la estrategia de protección mediada por la cápside Con este fin se establecieron los siguientes objetivos: a) Realizar el clonado molecular del genoma del virus PVY. b) Utilizar clones de cDNA como sondas de hibridación molecular para el diagnóstico. c) Expresar la proteína de cubierta del virus en bacterias para para ser utilizada como antígeno en la producción de anticuerpos policlonales. d) Diseñar nuevas técnicas de diagnóstico basadas en la utilización de anticuerpos. e) Construir genes quiméricos capaces de expresar en plantas la proteína de cubierta del PVY. f) Efectuar la transformación de plantas de papa con las construcciones quiméricas mencionadas en el punto anterior. d) Establecer la existencia de resistencia antiviral en las plantas de papa transgénicas.Tesis digitalizada en SEDICI gracias a la Biblioteca Central de la Facultad de Ciencias Exactas (UNLP).Facultad de Ciencias Exacta

Desarrollo de métodos de diagnóstico y de plantas transgénicas de papa para el control de infecciones producidas por el virus PVY

El presente trabajo de tesis se propone utilizar las herramientas que provee la ingeniería genética para controlar las infecciones producidas por el virus PVY en plantas de papa, aportando nuevos métodos de diagnóstico para un mejor control de calidad de la semilla, y en combinación con las técnicas de transformación vegetal, modificar cultivares comerciales con el fin de lograr resistencia al virus mediante la estrategia de protección mediada por la cápside Con este fin se establecieron los siguientes objetivos: a) Realizar el clonado molecular del genoma del virus PVY. b) Utilizar clones de cDNA como sondas de hibridación molecular para el diagnóstico. c) Expresar la proteína de cubierta del virus en bacterias para para ser utilizada como antígeno en la producción de anticuerpos policlonales. d) Diseñar nuevas técnicas de diagnóstico basadas en la utilización de anticuerpos. e) Construir genes quiméricos capaces de expresar en plantas la proteína de cubierta del PVY. f) Efectuar la transformación de plantas de papa con las construcciones quiméricas mencionadas en el punto anterior. d) Establecer la existencia de resistencia antiviral en las plantas de papa transgénicas.Tesis digitalizada en SEDICI gracias a la Biblioteca Central de la Facultad de Ciencias Exactas (UNLP).Facultad de Ciencias Exacta

Plant-based vaccine for livestock : key points to unleash platform translation in developing countries

Ten years ago the first plant-based vaccine was licensed (DowAgrosciences). It was only 20 years after the first report of a recombinant protein obtained through plant transformation technology. Back then, this vaccine was perceived as the first of an unlimited list of innovative products of a flourishing platform. Unexpectedly, since then, no other veterinary product based on plant molecular pharming (PMP) has reached the market. This review will reflect a transdisciplinary view of the status and challenges that the molecular farming platform faces to become a strategic solution for the agroindustrial sector of developing countries. Recent findings Plant-based veterinary vaccines (PBVV) have the potential to give answers to several challenges that animal health presents today. The urgent need to improve livestock productivity, especially in low and middle-income countries (LMIC), and the current concern about the emergence of antimicrobial resistance associated with animal production, demands new products such as inexpensive vaccines and therapeutics. Based on the translational research scheme, certain barriers that could have limited the development into products of many results obtained in the last 15 years were identified. Summary Unquestionably, the development of innovation in LMIC is a key element in the feasibility of the platform. The emergence of PPP between multiple stakeholders as a strategy to overcome the existing disconnection between academia and industry, could enable the conversion of leading vaccine candidates from the stage of proof of concept into prototypes for industry, and thereby foster ‘productization’ in the field of veterinary vaccines.Inst. de PatobiologíaFil: Perez Aguirreburualde, Maria Sol. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; ArgentinaFil: Petruccelli, Silvana. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos; ArgentinaFil: Bravo Almonacid, Fernando Felix. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Wigdorovitz, Andrés. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

La transformación de cloroplastos

La transformación de cloroplastos presenta numerosas ventajas frente a otras metodologías de transformación vegetal, incluyendo mayor expresión de la proteína recombinante, contención del transgén y la posibilidad de expresar policistrones. Dado que la integración ocurre por recombinación homóloga, es posible dirigirla a sitios predeterminados del genoma plastídico evitando efectos de posición. En nuestro laboratorio hemos desarrollado dos vectores de transformación de cloroplastos de tabaco, que nos permitieron establecer esta técnica de transformación por primera vez en nuestro país.Trabajo galardonado con el Premio Pérez Companc, versión 2004.Academia Nacional de Agronomía y Veterinari

La transformación de cloroplastos

La transformación de cloroplastos presenta numerosas ventajas frente a otras metodologías de transformación vegetal, incluyendo mayor expresión de la proteína recombinante, contención del transgén y la posibilidad de expresar policistrones. Dado que la integración ocurre por recombinación homóloga, es posible dirigirla a sitios predeterminados del genoma plastídico evitando efectos de posición. En nuestro laboratorio hemos desarrollado dos vectores de transformación de cloroplastos de tabaco, que nos permitieron establecer esta técnica de transformación por primera vez en nuestro país.Trabajo galardonado con el Premio Pérez Companc, versión 2004.Academia Nacional de Agronomía y Veterinari

Translocation from the chloroplast stroma into the thylakoid lumen allows expression of recombinant epidermal growth factor in transplastomic tobacco plants

Chloroplast transformation has many potential advantages for the production of recombinant proteins in plants. However, it has been reported that chloroplast expression of many proteins, such as human epidermal growth factor (hEGF), results hindered by post-transcriptional mechanisms. hEGF degradation has been related to the redox potential of the stroma and protein misfolding. To solve this problem, we proposed the redirection of hEGF into the thylakoid lumen where the environment could improve disulfide bonds formation stabilizing the functional conformation of the protein. We generated transplastomic tobacco plants targeting hEGF protein to the thylakoid lumen by adding a transit peptide (Str). Following this approach, we could detect thylakoid lumen-targeted hEGF by western blotting while stromal accumulation of hEGF remained undetectable. Southern blot analysis confirmed the integration of the transgene through homologous recombination into the plastome. Northern blot analysis showed similar levels of egf transcripts in the EGF and StrEGF lines. These results suggest that higher stability of the hEGF peptide in the thylakoid lumen is the primary cause of the increased accumulation of the recombinant protein observed in StrEGF lines. They also highlight the necessity of exploring different sub-organellar destinations to improve the accumulation levels of a specific recombinant protein in plastids.Fil: Morgenfeld, Mauro Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Vater, Catalina Francisca. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Alfano, Edgardo Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Boccardo, Noelia Ayelen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Bravo Almonacid, Fernando Felix. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentin

Resistência extrema a duas estirpes do Potato virus Y (PVY) de batata transgênica, cv. Achat, expressando o gene da capa protéica do PVYO

The coat protein (CP) gene of the potato virus Y strain “o” (PVYO) was introduced into potato, cultivar Achat, via Agrobacterium tumefaciens-mediated transformation. Sixty three putative transgenic lines were challenged against the Brazilian strains PVY-OBR and PVY-NBR. An extremely resistant phenotype, against the two strains, was observed in one line, denominated 1P. No symptoms or positive ELISA results were observed in 16 challenged plants from this line. Another clone, named as 63P, showed a lower level of resistance. Southern blot analysis showed five copies of the CP gene in the extremely resistant line and at least three copies in the other resistant line. The stability of the integrated transgenes in the extreme resistant line was examined during several in vitro multiplications over a period of three years, with no modification in the Southern pattern was observed. The stability of the transgenes, the absence of primary infections and the relatively broad spectrum of resistance suggest that the extremely resistant line obtained in this work can be useful for agricultural purposes.O gene da capa protéica (CP) do Potato virus Y estirpe “o”, foi introduzido em batata cultivar Achat, via Agrobacterium tumefaciens. Sessenta e três linhas possivelmente transgênicas foram desafiadas com as estirpes brasileiras PVY-OBR e PVY-NBR. Uma linha apresentou extrema resistência às duas estirpes inoculadas, e foi denominado clone 1P. Não foram observados sintomas sistêmicos de infecção e as plantas foram negativas em Elisa. Outra linha, denominada clone 63P, mostrou algum nível de resistência. Análises por Southern blot indicaram a presença de pelo menos cinco cópias do gen CP no clone 1P e pelo menos três cópias no clone 63P. A estabilidade do gene introduzido no clone 1P foi avaliada durante três anos, após várias multiplicações in vitro. Não foram observadas mudanças no padrão do Southern blot. A estabilidade do transgene, na ausência de infecções primárias e relativo largo espectro de resistência sugerem que o clone 1P pode ser utilizado para fins comerciais.Fil: Romano, Eduardo. Embrapa Recursos Genéticos; BrasilFil: Ferreira, Adriana T.. Embrapa Hortaliças; BrasilFil: Dusi, André N.. Embrapa Hortaliças; BrasilFil: Proite, Karina. Embrapa Recursos Genéticos; BrasilFil: Buso, Jose A.. Embrapa Hortaliças; BrasilFil: Avila, Antonio C.. Embrapa Hortaliças; BrasilFil: Nishijima, Marta L.. Embrapa Hortaliças; BrasilFil: Nascimento, Adriana S.. Embrapa Hortaliças; BrasilFil: Bravo Almonacid, Fernando Felix. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Mentaberry, Alejandro Nestor. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Monte, Damares. Embrapa Recursos Genéticos; BrasilFil: Campos, Magnolia A.. Embrapa Recursos Genéticos; BrasilFil: Melo, Paulo Eduardo. Embrapa Hortaliças; BrasilFil: Cattony, Monica K.. No especifica;Fil: Torres, Antonio C.. Embrapa Hortaliças; Brasi

Selective pressure against horizontally acquired prokaryotic genes as a driving force of plastid evolution

Altres ajuts: del Consejo Nacional de Investigaciones Científicas y Técnicas- Argentina (CONICET) i del Programa Iberoamericano de Ciencia y Tecnología para el Desarrollo (IBERCAROT).The plastid organelle comprises a high proportion of nucleus-encoded proteins that were acquired from different prokaryotic donors via independent horizontal gene transfers following its primary endosymbiotic origin. What forces drove the targeting of these alien proteins to the plastid remains an unresolved evolutionary question. To better understand this process we screened for suitable candidate proteins to recapitulate their prokaryote-to-eukaryote transition. Here we identify the ancient horizontal transfer of a bacterial polyphenol oxidase (PPO) gene to the nuclear genome of an early land plant ancestor and infer the possible mechanism behind the plastidial localization of the encoded enzyme. Arabidopsis plants expressing PPO versions either lacking or harbouring a plastid-targeting signal allowed examining fitness consequences associated with its subcellular localization. Markedly, a deleterious effect on plant growth was highly correlated with PPO activity only when producing the non-targeted enzyme, suggesting that selection favoured the fixation of plastid-targeted protein versions. Our results reveal a possible evolutionary mechanism of how selection against heterologous genes encoding cytosolic proteins contributed in incrementing plastid proteome complexity from non-endosymbiotic gene sources, a process that may also impact mitochondrial evolution

Expression of the minor isoform pea ferredoxin in tobacco alters photosynthetic electron partitioning and enhances cyclic electron flow

Ferredoxins (Fds) are ferrosulfoproteins that function as low-potential electron carriers in plants. The Fd family is composed of several isoforms that share high sequence homology but differ in functional characteristics. In leaves, at least two isoforms conduct linear and cyclic photosynthetic electron transport around photosystem I, and mounting evidence suggests the existence of at least partial division of duties between these isoforms. To evaluate the contribution of different kinds of Fds to the control of electron fluxes along the photosynthetic electron transport chain, we overexpressed a minor pea (Pisum sativum) Fd isoform (PsFd1) in tobacco (Nicotiana tabacum) plants. The transplastomic OeFd1 plants exhibited variegated leaves and retarded growth and developmental rates. Photosynthetic studies of these plants indicated a reduction in carbon dioxide assimilation rates, photosystem II photochemistry, and linear electron flow. However, the plants showed an increase in nonphotochemical quenching, better control of excitation pressure at photosystem II, and no evidence of photoinhibition, implying a better dynamic regulation to remove excess energy from the photosynthetic electron transport chain. Finally, analysis of P700 redox status during illumination confirmed that the minor pea Fd isoform promotes enhanced cyclic flow around photosystem I. The two novel features of this work are: (1) that Fd levels achieved in transplastomic plants promote an alternative electron partitioning even under greenhouse light growth conditions, a situation that is exacerbated at higher light intensity measurements; and (2) that an alternative, minor Fd isoform has been overexpressed in plants, giving new evidence of labor division among Fd isoforms.Fil: Blanco, Nicolás Ernesto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad de Umea; SueciaFil: Ceccoli, Romina Denis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Dalla Via, Maria Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Voss, Ingo. Universität Osnabrück; AlemaniaFil: Segretin, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones En Ingeniería Genética y Biología Molecular; ArgentinaFil: Bravo Almonacid, Fernando Felix. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones En Ingeniería Genética y Biología Molecular; ArgentinaFil: Melzer, Michael. Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung, Physiologie und Zellbiologie; AlemaniaFil: Hajirezaei, Mohammad Reza. Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung, Physiologie und Zellbiologie; ArgentinaFil: Scheibe, Renate. Universität Osnabrück; AlemaniaFil: Hanke, Guy T.. Universität Osnabrück; Alemani

Improvement of Aroma in Transgenic Potato As a Consequence of Impairing Tuber Browning

Sensory analysis studies are critical in the development of quality enhanced crops, and may be an important component in the public acceptance of genetically modified foods. It has recently been established that odor preferences are shared between humans and mice, suggesting that odor exploration behavior in mice may be used to predict the effect of odors in humans. We have previously found that mice fed diets supplemented with engineered nonbrowning potatoes (-PPO) consumed more potato than mice fed diets supplemented with wild-type potatoes (WT). This prompted us to explore a possible role of potato odor in mice preference for nonbrowning potatoes. Taking advantage of two well established neuroscience paradigms, the “open field test” and the “nose-poking preference test”, we performed experiments where mice exploration behavior was monitored in preference assays on the basis of olfaction alone. No obvious preference was observed towards -PPO or WT lines when fresh potato samples were tested. However, when oxidized samples were tested, mice consistently investigated -PPO potatoes more times and for longer periods than WT potatoes. Congruently, humans discriminated WT from -PPO samples with a considerably better performance when oxidized samples were tested than when fresh samples were tested in blind olfactory experiments. Notably, even though participants ranked all samples with an intermediate level of pleasantness, there was a general consensus that the -PPO samples had a more intense odor and also evoked the sense-impression of a familiar vegetable more often than the WT samples. Taken together, these findings suggest that our previous observations might be influenced, at least in part, by differential odors that are accentuated among the lines once oxidative deterioration takes place. Additionally, our results suggest that nonbrowning potatoes, in addition to their extended shelf life, maintain their odor quality for longer periods of time than WT potatoes. To our knowledge this is the first report on the use of an animal model applied to the sensory analysis of a transgenic crop