EPSPS variability, gene expression, and enzymatic activity in glyphosate-resistant biotypes of Digitaria insularis
Weed resistance to herbicides is a natural phenomenon that exerts selection on individuals in a population. In Brazil, glyphosate resistance was recently detected in Digitaria insularis. The objective of this study was to elucidate mechanisms of weed resistance in this plant, including genetic varia...
- Autores:
-
Galeano Gómez, Esteban
Arrobas Barroso, Arthur
Vasconcelos, Tarsicio
López Rubio, Andrés
Albrecht, AJP
Filho R., Victoria
Carrer, Helaine
- Tipo de recurso:
- Article of investigation
- Fecha de publicación:
- 2016
- Institución:
- Tecnológico de Antioquia
- Repositorio:
- Repositorio Tdea
- Idioma:
- eng
- OAI Identifier:
- oai:dspace.tdea.edu.co:tdea/1197
- Acceso en línea:
- https://dspace.tdea.edu.co/handle/tdea/1197
- Palabra clave:
- Genetic variation
Gene expression
Enzyme activity
http://aims.fao.org/aos/agrovoc/c_15975
http://aims.fao.org/aos/agrovoc/c_27527
http://aims.fao.org/aos/agrovoc/c_2604
Weed resistance
Amino acid substitution
- Rights
- openAccess
- License
- https://creativecommons.org/licenses/by-sa/4.0/
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| dc.title.spa.fl_str_mv |
EPSPS variability, gene expression, and enzymatic activity in glyphosate-resistant biotypes of Digitaria insularis |
| title |
EPSPS variability, gene expression, and enzymatic activity in glyphosate-resistant biotypes of Digitaria insularis |
| spellingShingle |
EPSPS variability, gene expression, and enzymatic activity in glyphosate-resistant biotypes of Digitaria insularis Genetic variation Gene expression Enzyme activity http://aims.fao.org/aos/agrovoc/c_15975 http://aims.fao.org/aos/agrovoc/c_27527 http://aims.fao.org/aos/agrovoc/c_2604 Weed resistance Amino acid substitution |
| title_short |
EPSPS variability, gene expression, and enzymatic activity in glyphosate-resistant biotypes of Digitaria insularis |
| title_full |
EPSPS variability, gene expression, and enzymatic activity in glyphosate-resistant biotypes of Digitaria insularis |
| title_fullStr |
EPSPS variability, gene expression, and enzymatic activity in glyphosate-resistant biotypes of Digitaria insularis |
| title_full_unstemmed |
EPSPS variability, gene expression, and enzymatic activity in glyphosate-resistant biotypes of Digitaria insularis |
| title_sort |
EPSPS variability, gene expression, and enzymatic activity in glyphosate-resistant biotypes of Digitaria insularis |
| dc.creator.fl_str_mv |
Galeano Gómez, Esteban Arrobas Barroso, Arthur Vasconcelos, Tarsicio López Rubio, Andrés Albrecht, AJP Filho R., Victoria Carrer, Helaine |
| dc.contributor.author.none.fl_str_mv |
Galeano Gómez, Esteban Arrobas Barroso, Arthur Vasconcelos, Tarsicio López Rubio, Andrés Albrecht, AJP Filho R., Victoria Carrer, Helaine |
| dc.subject.agrovoc.none.fl_str_mv |
Genetic variation Gene expression Enzyme activity |
| topic |
Genetic variation Gene expression Enzyme activity http://aims.fao.org/aos/agrovoc/c_15975 http://aims.fao.org/aos/agrovoc/c_27527 http://aims.fao.org/aos/agrovoc/c_2604 Weed resistance Amino acid substitution |
| dc.subject.agrovocuri.none.fl_str_mv |
http://aims.fao.org/aos/agrovoc/c_15975 http://aims.fao.org/aos/agrovoc/c_27527 http://aims.fao.org/aos/agrovoc/c_2604 |
| dc.subject.proposal.spa.fl_str_mv |
Weed resistance Amino acid substitution |
| description |
Weed resistance to herbicides is a natural phenomenon that exerts selection on individuals in a population. In Brazil, glyphosate resistance was recently detected in Digitaria insularis. The objective of this study was to elucidate mechanisms of weed resistance in this plant, including genetic variability, allelism, amino acid substitutions, gene expression, and enzymatic activity levels. Most of these have not previously been studied in this species. D. insularis DNA sequences were used to analyze genetic variability. cDNA from resistant and susceptible plants was used to identify mutations, alleles, and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) expression, using real-time quantitative reverse transcription-polymerase chain reaction. In addition, EPSPS activity was measured. We found a decrease in genetic variability between populations related to glyphosate application. Substitutions from proline to threonine and tyrosine to cysteine led to a decrease in EPSPS affinity for the glyphosate. In addition, the EPSPS enzymatic activity was slightly higher in resistant plants, whereas EPSPS gene expression was almost identical in both biotypes, suggesting feedback regulation at different levels. To conclude, our results suggest new molecular mechanisms used by D. insularis to increase glyphosate resistance. |
| publishDate |
2016 |
| dc.date.issued.none.fl_str_mv |
2016 |
| dc.date.accessioned.none.fl_str_mv |
2021-04-29T13:06:27Z |
| dc.date.available.none.fl_str_mv |
2021-04-29T13:06:27Z |
| dc.type.spa.fl_str_mv |
Artículo de revista |
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http://purl.org/coar/version/c_970fb48d4fbd8a85 |
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http://purl.org/coar/resource_type/c_2df8fbb1 |
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Text |
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info:eu-repo/semantics/article |
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http://purl.org/redcol/resource_type/ART |
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info:eu-repo/semantics/publishedVersion |
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1676-5680 |
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https://dspace.tdea.edu.co/handle/tdea/1197 |
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1676-5680 |
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https://dspace.tdea.edu.co/handle/tdea/1197 |
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eng |
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eng |
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15 |
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| dc.relation.ispartofjournal.spa.fl_str_mv |
Genetics and Molecular Research |
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Genetics and Molecular Research . 15(3), pág. 1-15, 2016 |
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http://purl.org/coar/access_right/c_abf2 |
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https://creativecommons.org/licenses/by-sa/4.0/ Atribución-CompartirIgual 4.0 Internacional (CC BY-SA 4.0) http://purl.org/coar/access_right/c_abf2 |
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openAccess |
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15 p. |
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application/pdf |
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Brasil |
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[Ribeirao Preto, SP Brasil]: FUNPEC |
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Brasil |
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10.4238 / gmr.15038730 |
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Tecnológico de Antioquia |
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Galeano Gómez, Estebanda69d5fe-9464-479c-96b8-4c0a529f4037Arrobas Barroso, Arthur8fa11fd9-a177-48aa-a157-107ddf40b352Vasconcelos, Tarsicio91866d5a-20ab-49ab-8037-b8911e0b7207López Rubio, Andrés362936f1-0f76-468f-bdb3-30d6cfe858d5Albrecht, AJP751c4d7e-efe6-444c-a228-94d26da309aaFilho R., Victoriab6136fc5-a0aa-4f33-b565-bb778e6c458bCarrer, Helaine926df20c-63c1-4313-9721-ab87674968bd2021-04-29T13:06:27Z2021-04-29T13:06:27Z20161676-5680https://dspace.tdea.edu.co/handle/tdea/1197Weed resistance to herbicides is a natural phenomenon that exerts selection on individuals in a population. In Brazil, glyphosate resistance was recently detected in Digitaria insularis. The objective of this study was to elucidate mechanisms of weed resistance in this plant, including genetic variability, allelism, amino acid substitutions, gene expression, and enzymatic activity levels. Most of these have not previously been studied in this species. D. insularis DNA sequences were used to analyze genetic variability. cDNA from resistant and susceptible plants was used to identify mutations, alleles, and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) expression, using real-time quantitative reverse transcription-polymerase chain reaction. In addition, EPSPS activity was measured. We found a decrease in genetic variability between populations related to glyphosate application. Substitutions from proline to threonine and tyrosine to cysteine led to a decrease in EPSPS affinity for the glyphosate. In addition, the EPSPS enzymatic activity was slightly higher in resistant plants, whereas EPSPS gene expression was almost identical in both biotypes, suggesting feedback regulation at different levels. To conclude, our results suggest new molecular mechanisms used by D. insularis to increase glyphosate resistance.15 p.application/pdfeng[Ribeirao Preto, SP Brasil]: FUNPECBrasilhttps://creativecommons.org/licenses/by-sa/4.0/info:eu-repo/semantics/openAccessAtribución-CompartirIgual 4.0 Internacional (CC BY-SA 4.0)http://purl.org/coar/access_right/c_abf210.4238 / gmr.15038730EPSPS variability, gene expression, and enzymatic activity in glyphosate-resistant biotypes of Digitaria insularisArtículo de revistahttp://purl.org/coar/resource_type/c_2df8fbb1Textinfo:eu-repo/semantics/articlehttp://purl.org/redcol/resource_type/ARTinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/version/c_970fb48d4fbd8a85Brasil153115Genetics and Molecular ResearchGenetics and Molecular Research . 15(3), pág. 1-15, 2016Genetic variationGene expressionEnzyme activityhttp://aims.fao.org/aos/agrovoc/c_15975http://aims.fao.org/aos/agrovoc/c_27527http://aims.fao.org/aos/agrovoc/c_2604Weed resistanceAmino acid substitutionBaerson SR, Tran M and Brinker R (2000). 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Weed Sci. 62: 29-37. http://dx.doi.org/10.1614/WS-D-13-00066.1Hall T (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/ NT. Nucleic Acids Symp. Ser. 41: 95-98Kahrizi D, Salmanian AH, Afshari A, Moieni A, et al. (2007). Simultaneous substitution of Gly96 to Ala and Ala183 to Thr in 5-enolpyruvylshikimate-3-phosphate synthase gene of E. coli (k12) and transformation of rapeseed (Brassica napus L.) in order to make tolerance to glyphosate. Plant Cell Rep. 26: 95-104. http://dx.doi.org/10.1007/s00299- 006-0208-4Kimura M (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16: 111-120. http://dx.doi.org/10.1007/BF01731581Lanzetta PA, Alvarez LJ, Reinach PS and Candia OA (1979). An improved assay for nanomole amounts of inorganic phosphate. Anal. 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Caracterização anatômica de folha, colmo e rizoma de Digitaria insularis. Planta Daninha 26: 1-8. http://dx.doi.org/10.1590/S0100-83582008000100001Manalil S, Busi R, Renton M and Powles SB (2011). Rapid evolution of herbicide resistance by low herbicide dosages. Weed Sci. 59: 210-217. http://dx.doi.org/10.1614/WS-D-10-00111.1Mondo VHV, Carvalho SJP, Dias ACR and Marcos Filho J (2010). Efeitos da luz e temperatura na germinação de sementes de quatro espécies de plantas daninhas do gênero Digitaria. Rev. Bras. Sementes 32: 131-137. http://dx.doi. org/10.1590/S0101-31222010000100015Nandula VK, Ray JD, Ribeiro DN, Pan Z, et al. (2013). Glyphosate resistance in tall waterhemp (Amaranthus tuberculatus) from Mississippi is due to both altered target-site and nontarget-site mechanisms. Weed Sci. 61: 374-383. http:// dx.doi.org/10.1614/WS-D-12-00155.1Perez-Jones A, Park KW, Polge N, Colquhoun J, et al. (2007). Investigating the mechanisms of glyphosate resistance in Lolium multiflorum. Planta 226: 395-404. http://dx.doi.org/10.1007/s00425-007-0490-6Preston C and Wakelin AM (2008). Resistance to glyphosate from altered herbicide translocation patterns. Pest Manag. Sci. 64: 372-376. http://dx.doi.org/10.1002/ps.1489Salas RA, Dayan FE, Pan Z, Watson SB, et al. (2012). EPSPS gene amplification in glyphosate-resistant Italian ryegrass (Lolium perenne ssp. multiflorum) from Arkansas. Pest Manag. Sci. 68: 1223-1230. http://dx.doi.org/10.1002/ ps.3342Shaner DL, Lindenmeyer RB and Ostlie MH (2012). What have the mechanisms of resistance to glyphosate taught us? Pest Manag. Sci. 68: 3-9. http://dx.doi.org/10.1002/ps.2261Shimono Y, Kurokawa S, Nishida T, Ikeda H, et al. (2013). Phylogeography based on intraspecific sequence variation in chloroplast DNA of Miscanthus sinensis (Poaceae), a native pioneer grass in Japan. Botany 91: 449-456. http:// dx.doi.org/10.1139/cjb-2012-0212Umesha S (2006). Phenylalanine ammonia lyase activity in tomato seedlings and its relationship to bacterial canker disease resistance. Phytoparasitica 34: 68-71. http://dx.doi.org/10.1007/BF02981341Vila-Aiub MM, Neve P and Powles SB (2009). Fitness costs associated with evolved herbicide resistance alleles in plants. New Phytol. 184: 751-767. http://dx.doi.org/10.1111/j.1469-8137.2009.03055.xWang W, Xia H, Yang X, Xu T, et al. (2014). A novel 5-enolpyruvoylshikimate-3-phosphate (EPSP) synthase transgene for glyphosate resistance stimulates growth and fecundity in weedy rice (Oryza sativa) without herbicide. New Phytol. 202: 679-688. http://dx.doi.org/10.1111/nph.12428Woodburn AT (2000). Glyphosate: production, pricing and use worldwide. Pest Manag. Sci. 56: 309-312. http://dx.doi. org/10.1002/(SICI)1526-4998(200004)56:4<309::AID-PS143>3.0.CO;2-CYu Q, Cairns A and Powles S (2007). Glyphosate, paraquat and ACCase multiple herbicide resistance evolved in a Lolium rigidum biotype. 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 incorporada en las Obras Colectivas.

b.	Distribuir copias o fonogramas de las Obras, exhibirlas públicamente, ejecutarlas públicamente y/o ponerlas a disposición pública, incluyéndolas como incorporadas en Obras Colectivas, según corresponda.

c.	Distribuir copias de las Obras Derivadas que se generen, exhibirlas públicamente, ejecutarlas públicamente y/o ponerlas a disposición pública.
Los derechos mencionados anteriormente pueden ser ejercidos en todos los medios y formatos, actualmente conocidos o que se inventen en el futuro. Los derechos antes mencionados incluyen el derecho a realizar dichas modificaciones en la medida que sean técnicamente necesarias para ejercer los derechos en otro medio o formatos, pero de otra manera usted no está autorizado para realizar obras derivadas. Todos los derechos no otorgados expresamente por el Licenciante quedan por este medio reservados, incluyendo pero sin limitarse a aquellos que se mencionan en las secciones 4(d) y 4(e).

4. Restricciones.
La licencia otorgada en la anterior Sección 3 está expresamente sujeta y limitada por las siguientes restricciones:

a.	Usted puede distribuir, exhibir públicamente, ejecutar públicamente, o poner a disposición pública la Obra sólo bajo las condiciones de esta Licencia, y Usted debe incluir una copia de esta licencia o del Identificador Universal de Recursos de la misma con cada copia de la Obra que distribuya, exhiba públicamente, ejecute públicamente o ponga a disposición pública. No es posible ofrecer o imponer ninguna condición sobre la Obra que altere o limite las condiciones de esta Licencia o el ejercicio de los derechos de los destinatarios otorgados en este documento. No es posible sublicenciar la Obra. Usted debe mantener intactos todos los avisos que hagan referencia a esta Licencia y a la cláusula de limitación de garantías. Usted no puede distribuir, exhibir públicamente, ejecutar públicamente, o poner a disposición pública la Obra con alguna medida tecnológica que controle el acceso o la utilización de ella de una forma que sea inconsistente con las condiciones de esta Licencia. Lo anterior se aplica a la Obra incorporada a una Obra Colectiva, pero esto no exige que la Obra Colectiva aparte de la obra misma quede sujeta a las condiciones de esta Licencia. Si Usted crea una Obra Colectiva, previo aviso de cualquier Licenciante debe, en la medida de lo posible, eliminar de la Obra Colectiva cualquier referencia a dicho Licenciante o al Autor Original, según lo solicitado por el Licenciante y conforme lo exige la cláusula 4(c).

b.	Usted no puede ejercer ninguno de los derechos que le han sido otorgados en la Sección 3 precedente de modo que estén principalmente destinados o directamente dirigidos a conseguir un provecho comercial o una compensación monetaria privada. El intercambio de la Obra por otras obras protegidas por derechos de autor, ya sea a través de un sistema para compartir archivos digitales (digital file-sharing) o de cualquier otra manera no será considerado como estar destinado principalmente o dirigido directamente a conseguir un provecho comercial o una compensación monetaria privada, siempre que no se realice un pago mediante una compensación monetaria en relación con el intercambio de obras protegidas por el derecho de autor.

c.	Si usted distribuye, exhibe públicamente, ejecuta públicamente o ejecuta públicamente en forma digital la Obra o cualquier Obra Derivada u Obra Colectiva, Usted debe mantener intacta toda la información de derecho de autor de la Obra y proporcionar, de forma razonable según el medio o manera que Usted esté utilizando: (i) el nombre del Autor Original si está provisto (o seudónimo, si fuere aplicable), y/o (ii) el nombre de la parte o las partes que el Autor Original y/o el Licenciante hubieren designado para la atribución (v.g., un instituto patrocinador, editorial, publicación) en la información de los derechos de autor del Licenciante, términos de servicios o de otras formas razonables; el título de la Obra si está provisto; en la medida de lo razonablemente factible y, si está provisto, el Identificador Uniforme de Recursos (Uniform Resource Identifier) que el Licenciante especifica para ser asociado con la Obra, salvo que tal URI no se refiera a la nota sobre los derechos de autor o a la información sobre el licenciamiento de la Obra; y en el caso de una Obra Derivada, atribuir el crédito identificando el uso de la Obra en la Obra Derivada (v.g., "Traducción Francesa de la Obra del Autor Original," o "Guión Cinematográfico basado en la Obra original del Autor Original"). Tal crédito puede ser implementado de cualquier forma razonable; en el caso, sin embargo, de Obras Derivadas u Obras Colectivas, tal crédito aparecerá, como mínimo, donde aparece el crédito de cualquier otro autor comparable y de una manera, al menos, tan destacada como el crédito de otro autor comparable.

d.	Para evitar toda confusión, el Licenciante aclara que, cuando la obra es una composición musical:

i.	Regalías por interpretación y ejecución bajo licencias generales. El Licenciante se reserva el derecho exclusivo de autorizar la ejecución pública o la ejecución pública digital de la obra y de recolectar, sea individualmente o a través de una sociedad de gestión colectiva de derechos de autor y derechos conexos (por ejemplo, SAYCO), las regalías por la ejecución pública o por la ejecución pública digital de la obra (por ejemplo Webcast) licenciada bajo licencias generales, si la interpretación o ejecución de la obra está primordialmente orientada por o dirigida a la obtención de una ventaja comercial o una compensación monetaria privada.

ii.	Regalías por Fonogramas. El Licenciante se reserva el derecho exclusivo de recolectar, individualmente o a través de una sociedad de gestión colectiva de derechos de autor y derechos conexos (por ejemplo, los consagrados por la SAYCO), una agencia de derechos musicales o algún agente designado, las regalías por cualquier fonograma que Usted cree a partir de la obra (“versión cover”) y distribuya, en los términos del régimen de derechos de autor, si la creación o distribución de esa versión cover está primordialmente destinada o dirigida a obtener una ventaja comercial o una compensación monetaria privada.

e.	Gestión de Derechos de Autor sobre Interpretaciones y Ejecuciones Digitales (WebCasting). Para evitar toda confusión, el Licenciante aclara que, cuando la obra sea un fonograma, el Licenciante se reserva el derecho exclusivo de autorizar la ejecución pública digital de la obra (por ejemplo, webcast) y de recolectar, individualmente o a través de una sociedad de gestión colectiva de derechos de autor y derechos conexos (por ejemplo, ACINPRO), las regalías por la ejecución pública digital de la obra (por ejemplo, webcast), sujeta a las disposiciones aplicables del régimen de Derecho de Autor, si esta ejecución pública digital está primordialmente dirigida a obtener una ventaja comercial o una compensación monetaria privada.

5. Representaciones, Garantías y Limitaciones de Responsabilidad.
A MENOS QUE LAS PARTES LO ACORDARAN DE OTRA FORMA POR ESCRITO, EL LICENCIANTE OFRECE LA OBRA (EN EL ESTADO EN EL QUE SE ENCUENTRA) “TAL CUAL”, SIN BRINDAR GARANTÍAS DE CLASE ALGUNA RESPECTO DE LA OBRA, YA SEA EXPRESA, IMPLÍCITA, LEGAL O CUALQUIERA OTRA, INCLUYENDO, SIN LIMITARSE A ELLAS, GARANTÍAS DE TITULARIDAD, COMERCIABILIDAD, ADAPTABILIDAD O ADECUACIÓN A PROPÓSITO DETERMINADO, AUSENCIA DE INFRACCIÓN, DE AUSENCIA DE DEFECTOS LATENTES O DE OTRO TIPO, O LA PRESENCIA O AUSENCIA DE ERRORES, SEAN O NO DESCUBRIBLES (PUEDAN O NO SER ESTOS DESCUBIERTOS). ALGUNAS JURISDICCIONES NO PERMITEN LA EXCLUSIÓN DE GARANTÍAS IMPLÍCITAS, EN CUYO CASO ESTA EXCLUSIÓN PUEDE NO APLICARSE A USTED.

6. Limitación de responsabilidad.
A MENOS QUE LO EXIJA EXPRESAMENTE LA LEY APLICABLE, EL LICENCIANTE NO SERÁ RESPONSABLE ANTE USTED POR DAÑO ALGUNO, SEA POR RESPONSABILIDAD EXTRACONTRACTUAL, PRECONTRACTUAL O CONTRACTUAL, OBJETIVA O SUBJETIVA, SE TRATE DE DAÑOS MORALES O PATRIMONIALES, DIRECTOS O INDIRECTOS, PREVISTOS O IMPREVISTOS PRODUCIDOS POR EL USO DE ESTA LICENCIA O DE LA OBRA, AUN CUANDO EL LICENCIANTE HAYA SIDO ADVERTIDO DE LA POSIBILIDAD DE DICHOS DAÑOS. ALGUNAS LEYES NO PERMITEN LA EXCLUSIÓN DE CIERTA RESPONSABILIDAD, EN CUYO CASO ESTA EXCLUSIÓN PUEDE NO APLICARSE A USTED.

7. Término.

a.	Esta Licencia y los derechos otorgados en virtud de ella terminarán automáticamente si Usted infringe alguna condición establecida en ella. Sin embargo, los individuos o entidades que han recibido Obras Derivadas o Colectivas de Usted de conformidad con esta Licencia, no verán terminadas sus licencias, siempre que estos individuos o entidades sigan cumpliendo íntegramente las condiciones de estas licencias. Las Secciones 1, 2, 5, 6, 7, y 8 subsistirán a cualquier terminación de esta Licencia.

b.	Sujeta a las condiciones y términos anteriores, la licencia otorgada aquí es perpetua (durante el período de vigencia de los derechos de autor de la obra). No obstante lo anterior, el Licenciante se reserva el derecho a publicar y/o estrenar la Obra bajo condiciones de licencia diferentes o a dejar de distribuirla en los términos de esta Licencia en cualquier momento; en el entendido, sin embargo, que esa elección no servirá para revocar esta licencia o que deba ser otorgada , bajo los términos de esta licencia), y esta licencia continuará en pleno vigor y efecto a menos que sea terminada como se expresa atrás. La Licencia revocada continuará siendo plenamente vigente y efectiva si no se le da término en las condiciones indicadas anteriormente.

8. Varios.

a.	Cada vez que Usted distribuya o ponga a disposición pública la Obra o una Obra Colectiva, el Licenciante ofrecerá al destinatario una licencia en los mismos términos y condiciones que la licencia otorgada a Usted bajo esta Licencia.

b.	Si alguna disposición de esta Licencia resulta invalidada o no exigible, según la legislación vigente, esto no afectará ni la validez ni la aplicabilidad del resto de condiciones de esta Licencia y, sin acción adicional por parte de los sujetos de este acuerdo, aquélla se entenderá reformada lo mínimo necesario para hacer que dicha disposición sea válida y exigible.

c.	Ningún término o disposición de esta Licencia se estimará renunciada y ninguna violación de ella será consentida a menos que esa renuncia o consentimiento sea otorgado por escrito y firmado por la parte que renuncie o consienta.

d.	Esta Licencia refleja el acuerdo pleno entre las partes respecto a la Obra aquí licenciada. No hay arreglos, acuerdos o declaraciones respecto a la Obra que no estén especificados en este documento. El Licenciante no se verá limitado por ninguna disposición adicional que pueda surgir en alguna comunicación emanada de Usted. Esta Licencia no puede ser modificada sin el consentimiento mutuo por escrito del Licenciante y Usted.
 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