Foliar Infiltration of Virus-Derived Small Hairpin RNAs Triggers the RNAi Mechanism against the Cucumber Mosaic Virus
Post-transcriptional gene silencing (PTGS) is an evolutionarily conserved plant defense mechanism against viruses. This paper aimed to evaluate a dsDNA construct (77 bp) as a template for in vitro production of virus-derived artificial small hairpin RNAs (shRNAs) and test for their potential to trig...
- Autores:
-
Villegas Estrada, Bernardo
Sánchez, Manuel Alejandro
Valencia-Jimenez, Arnubio
- Tipo de recurso:
- Article of journal
- Fecha de publicación:
- 2022
- Institución:
- Universidad Francisco de Paula Santander
- Repositorio:
- Repositorio Digital UFPS
- Idioma:
- eng
- OAI Identifier:
- oai:repositorio.ufps.edu.co:ufps/6484
- Acceso en línea:
- https://repositorio.ufps.edu.co/handle/ufps/6484
https://doi.org/10.3390/ijms23094938
- Palabra clave:
- CMV
capsid protein
Nicotiana benthamiana
gene silencing
shRNA
- Rights
- openAccess
- License
- https://creativecommons.org/licenses/by/4.0/
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Foliar Infiltration of Virus-Derived Small Hairpin RNAs Triggers the RNAi Mechanism against the Cucumber Mosaic Virus |
| title |
Foliar Infiltration of Virus-Derived Small Hairpin RNAs Triggers the RNAi Mechanism against the Cucumber Mosaic Virus |
| spellingShingle |
Foliar Infiltration of Virus-Derived Small Hairpin RNAs Triggers the RNAi Mechanism against the Cucumber Mosaic Virus CMV capsid protein Nicotiana benthamiana gene silencing shRNA |
| title_short |
Foliar Infiltration of Virus-Derived Small Hairpin RNAs Triggers the RNAi Mechanism against the Cucumber Mosaic Virus |
| title_full |
Foliar Infiltration of Virus-Derived Small Hairpin RNAs Triggers the RNAi Mechanism against the Cucumber Mosaic Virus |
| title_fullStr |
Foliar Infiltration of Virus-Derived Small Hairpin RNAs Triggers the RNAi Mechanism against the Cucumber Mosaic Virus |
| title_full_unstemmed |
Foliar Infiltration of Virus-Derived Small Hairpin RNAs Triggers the RNAi Mechanism against the Cucumber Mosaic Virus |
| title_sort |
Foliar Infiltration of Virus-Derived Small Hairpin RNAs Triggers the RNAi Mechanism against the Cucumber Mosaic Virus |
| dc.creator.fl_str_mv |
Villegas Estrada, Bernardo Sánchez, Manuel Alejandro Valencia-Jimenez, Arnubio |
| dc.contributor.author.none.fl_str_mv |
Villegas Estrada, Bernardo Sánchez, Manuel Alejandro Valencia-Jimenez, Arnubio |
| dc.subject.proposal.eng.fl_str_mv |
CMV capsid protein Nicotiana benthamiana gene silencing shRNA |
| topic |
CMV capsid protein Nicotiana benthamiana gene silencing shRNA |
| description |
Post-transcriptional gene silencing (PTGS) is an evolutionarily conserved plant defense mechanism against viruses. This paper aimed to evaluate a dsDNA construct (77 bp) as a template for in vitro production of virus-derived artificial small hairpin RNAs (shRNAs) and test for their potential to trigger the RNAi mechanism in Nicotiana benthamiana plants against CMV after their foliar infiltration. This approach allowed for the production of significant amounts of shRNAs (60-mers) quickly and easily. The gene silencing was confirmed using polymerase chain reaction (PCR), immunological-based assays, and real-time PCR (qPCR). The highest levels of gene silencing were recorded for mRNAs coding for replication protein (ORF1a), the viral suppressor of RNA silencing (ORF2b), and the capsid protein (ORF3b), with 98, 94, and 70% of total transcript silencing, respectively. This protocol provides an alternative to producing significant shRNAs that can effectively trigger the RNAi mechanism against CMV |
| publishDate |
2022 |
| dc.date.accessioned.none.fl_str_mv |
2022-05-27T14:04:03Z |
| dc.date.available.none.fl_str_mv |
2022-05-27T14:04:03Z |
| dc.date.issued.none.fl_str_mv |
2022-04-29 |
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Artículo de revista |
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http://purl.org/coar/resource_type/c_2df8fbb1 |
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https://repositorio.ufps.edu.co/handle/ufps/6484 |
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https://doi.org/10.3390/ijms23094938 |
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https://repositorio.ufps.edu.co/handle/ufps/6484 https://doi.org/10.3390/ijms23094938 |
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eng |
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eng |
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International Journal of Molecular Sciences ISSN: 1422-0067, 2022 vol:23 fasc: 9 págs: 4912 - 4938, DOI:10.3390/ijms23094938 |
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Vol. 23 No. 9 (2022) |
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4938 |
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9 (2022) |
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4912 |
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23 |
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Villegas-Estrada, B.; Sánchez, M.A.; Valencia-Jiménez, A. Foliar Infiltration of Virus-Derived Small Hairpin RNAs Triggers the RNAi Mechanism against the Cucumber Mosaic Virus. Int. J. Mol. Sci. 2022, 23, 4938. https://doi.org/ 10.3390/ijms23094938 |
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International Journal of Molecular Sciences |
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info:eu-repo/semantics/openAccess |
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Atribución 4.0 Internacional (CC BY 4.0) |
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https://creativecommons.org/licenses/by/4.0/ Atribución 4.0 Internacional (CC BY 4.0) http://purl.org/coar/access_right/c_abf2 |
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openAccess |
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12 Páginas |
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International Journal of Molecular Sciences I |
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Suiza |
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Villegas Estrada, Bernardo2fad40344d0c530f8c8f7c0f7fae1cf0Sánchez, Manuel Alejandro5c8897da1223ede8de480ac08e6ece74Valencia-Jimenez, Arnubio1f17cc7e800c31852d1714e42f6ae5f76002022-05-27T14:04:03Z2022-05-27T14:04:03Z2022-04-29https://repositorio.ufps.edu.co/handle/ufps/6484https://doi.org/10.3390/ijms23094938Post-transcriptional gene silencing (PTGS) is an evolutionarily conserved plant defense mechanism against viruses. This paper aimed to evaluate a dsDNA construct (77 bp) as a template for in vitro production of virus-derived artificial small hairpin RNAs (shRNAs) and test for their potential to trigger the RNAi mechanism in Nicotiana benthamiana plants against CMV after their foliar infiltration. This approach allowed for the production of significant amounts of shRNAs (60-mers) quickly and easily. The gene silencing was confirmed using polymerase chain reaction (PCR), immunological-based assays, and real-time PCR (qPCR). The highest levels of gene silencing were recorded for mRNAs coding for replication protein (ORF1a), the viral suppressor of RNA silencing (ORF2b), and the capsid protein (ORF3b), with 98, 94, and 70% of total transcript silencing, respectively. This protocol provides an alternative to producing significant shRNAs that can effectively trigger the RNAi mechanism against CMV12 Páginasapplication/pdfengInternational Journal of Molecular Sciences ISuizaInternational Journal of Molecular Sciences ISSN: 1422-0067, 2022 vol:23 fasc: 9 págs: 4912 - 4938, DOI:10.3390/ijms23094938Vol. 23 No. 9 (2022)49389 (2022)491223Villegas-Estrada, B.; Sánchez, M.A.; Valencia-Jiménez, A. Foliar Infiltration of Virus-Derived Small Hairpin RNAs Triggers the RNAi Mechanism against the Cucumber Mosaic Virus. Int. J. Mol. Sci. 2022, 23, 4938. https://doi.org/ 10.3390/ijms23094938International Journal of Molecular SciencesEsta revista esta bajo una licencia Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).https://creativecommons.org/licenses/by/4.0/info:eu-repo/semantics/openAccessAtribución 4.0 Internacional (CC BY 4.0)http://purl.org/coar/access_right/c_abf2https://www.mdpi.com/1422-0067/23/9/4938/htmlFoliar Infiltration of Virus-Derived Small Hairpin RNAs Triggers the RNAi Mechanism against the Cucumber Mosaic VirusArtículo de revistahttp://purl.org/coar/resource_type/c_6501http://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_970fb48d4fbd8a85CMVcapsid proteinNicotiana benthamianagene silencingshRNADoolittle, S.P. A new infectious mosaic disease of cucumber. 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RNAi-mediated resistance to Potato spindle tuber viroid in transgenic tomato expressing a viroid hairpin RNA construct. Mol. Plant Pathol. 2009, 10, 459–469Tabassum, B.; Nasir, I.A.; Khan, A.; Aslam, U.; Tariq, M.; Shahid, N.; Husnain, T. Short hairpin RNA engineering: In planta gene silencing of potato virus Y. Crop. Prot. 2016, 86, 1–8Akbar, S.; Tahir, M.; Wang, M.B.; Liu, Q. Expression Analysis of Hairpin RNA Carrying Sugarcane mosaic virus (SCMV) Derived Sequences and Transgenic Resistance Development in a Model Rice Plant. BioMed Res. Int. 2017, 2017, 1646140.Aslam, U.; Tabassum, B.; Nasir, I.A.; Khan, A.; Husnain, T. A virus-derived short hairpin RNA confers resistance against sugarcane mosaic virus in transgenic sugarcane. Transgenic. Res. 2018, 27, 203–210.Worrall, E.A.; Bravo-Cazar, A.; Nilon, A.T.; Fletcher, S.J.; Robinson, K.E.; Carr, J.P.; Mitter, N. Exogenous application of RNAiinducing double-stranded RNA inhibits aphid-mediated tansmission of a plant virus. Front. 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Methods 2001, 25, 402–408.ORIGINALFoliar Infiltration of Virus-Derived Small Hairpin RNAs.pdfFoliar Infiltration of Virus-Derived Small Hairpin RNAs.pdfapplication/pdf2149638https://repositorio.ufps.edu.co/bitstream/ufps/6484/1/Foliar%20Infiltration%20of%20Virus-Derived%20Small%20Hairpin%20RNAs.pdff866e78d5861bc148d02d890af39f22aMD51open accessLICENSElicense.txtlicense.txttext/plain; charset=utf-814828https://repositorio.ufps.edu.co/bitstream/ufps/6484/2/license.txt2f9959eaf5b71fae44bbf9ec84150c7aMD52open accessTEXTFoliar Infiltration of Virus-Derived Small Hairpin RNAs.pdf.txtFoliar Infiltration of Virus-Derived Small Hairpin RNAs.pdf.txtExtracted texttext/plain51308https://repositorio.ufps.edu.co/bitstream/ufps/6484/3/Foliar%20Infiltration%20of%20Virus-Derived%20Small%20Hairpin%20RNAs.pdf.txtade4037c18488c286e578d4256c9f73bMD53open accessTHUMBNAILFoliar Infiltration of Virus-Derived Small Hairpin RNAs.pdf.jpgFoliar Infiltration of Virus-Derived Small Hairpin RNAs.pdf.jpgGenerated 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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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