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Agrobacterium-Mediated Gene Transient Overexpression and Tobacco Rattle Virus (TRV)-Based Gene Silencing in Cassava

Agrobacterium-mediated transient expression and virus-induced gene silencing (VIGS) are very useful in functional genomics in plants. However, whether these methods are effective in cassava (Manihot esculenta), one of the most important tropical crops, remains elusive. In this study, we used green f...

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Autores principales: Zeng, Hongqiu, Xie, Yanwei, Liu, Guoyin, Wei, Yunxie, Hu, Wei, Shi, Haitao
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6719147/
https://www.ncbi.nlm.nih.gov/pubmed/31443292
http://dx.doi.org/10.3390/ijms20163976
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author Zeng, Hongqiu
Xie, Yanwei
Liu, Guoyin
Wei, Yunxie
Hu, Wei
Shi, Haitao
author_facet Zeng, Hongqiu
Xie, Yanwei
Liu, Guoyin
Wei, Yunxie
Hu, Wei
Shi, Haitao
author_sort Zeng, Hongqiu
collection PubMed
description Agrobacterium-mediated transient expression and virus-induced gene silencing (VIGS) are very useful in functional genomics in plants. However, whether these methods are effective in cassava (Manihot esculenta), one of the most important tropical crops, remains elusive. In this study, we used green fluorescent protein (GFP) and β-glucuronidase (GUS) as reporter genes in a transient expression assay. GFP or GUS could be detected in the infiltrated leaves at 2 days postinfiltration (dpi) and were evidenced by visual GFP and GUS assays, reverse-transcription PCR, and Western blot. In addition, phytoene desaturase (PDS) was used to show the silencing effect in a VIGS system. Both Agrobacterium GV3101 and AGL-1 with tobacco rattle virus (TRV)-MePDS-infiltrated distal leaves showed an albino phenotype at 20 dpi; in particular, the AGL-1-infiltrated plants showed an obvious albino area in the most distal leaves. Moreover, the silencing effect was validated by molecular identification. Notably, compared with the obvious cassava mosaic disease symptom infiltrated by African-cassava-mosaic-virus-based VIGS systems in previous studies, TRV-based VIGS-system-infiltrated cassava plants did not show obvious virus-induced disease symptoms, suggesting a significant advantage. Taken together, these methods could promote functional genomics in cassava.
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spelling pubmed-67191472019-09-10 Agrobacterium-Mediated Gene Transient Overexpression and Tobacco Rattle Virus (TRV)-Based Gene Silencing in Cassava Zeng, Hongqiu Xie, Yanwei Liu, Guoyin Wei, Yunxie Hu, Wei Shi, Haitao Int J Mol Sci Article Agrobacterium-mediated transient expression and virus-induced gene silencing (VIGS) are very useful in functional genomics in plants. However, whether these methods are effective in cassava (Manihot esculenta), one of the most important tropical crops, remains elusive. In this study, we used green fluorescent protein (GFP) and β-glucuronidase (GUS) as reporter genes in a transient expression assay. GFP or GUS could be detected in the infiltrated leaves at 2 days postinfiltration (dpi) and were evidenced by visual GFP and GUS assays, reverse-transcription PCR, and Western blot. In addition, phytoene desaturase (PDS) was used to show the silencing effect in a VIGS system. Both Agrobacterium GV3101 and AGL-1 with tobacco rattle virus (TRV)-MePDS-infiltrated distal leaves showed an albino phenotype at 20 dpi; in particular, the AGL-1-infiltrated plants showed an obvious albino area in the most distal leaves. Moreover, the silencing effect was validated by molecular identification. Notably, compared with the obvious cassava mosaic disease symptom infiltrated by African-cassava-mosaic-virus-based VIGS systems in previous studies, TRV-based VIGS-system-infiltrated cassava plants did not show obvious virus-induced disease symptoms, suggesting a significant advantage. Taken together, these methods could promote functional genomics in cassava. MDPI 2019-08-15 /pmc/articles/PMC6719147/ /pubmed/31443292 http://dx.doi.org/10.3390/ijms20163976 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Zeng, Hongqiu
Xie, Yanwei
Liu, Guoyin
Wei, Yunxie
Hu, Wei
Shi, Haitao
Agrobacterium-Mediated Gene Transient Overexpression and Tobacco Rattle Virus (TRV)-Based Gene Silencing in Cassava
title Agrobacterium-Mediated Gene Transient Overexpression and Tobacco Rattle Virus (TRV)-Based Gene Silencing in Cassava
title_full Agrobacterium-Mediated Gene Transient Overexpression and Tobacco Rattle Virus (TRV)-Based Gene Silencing in Cassava
title_fullStr Agrobacterium-Mediated Gene Transient Overexpression and Tobacco Rattle Virus (TRV)-Based Gene Silencing in Cassava
title_full_unstemmed Agrobacterium-Mediated Gene Transient Overexpression and Tobacco Rattle Virus (TRV)-Based Gene Silencing in Cassava
title_short Agrobacterium-Mediated Gene Transient Overexpression and Tobacco Rattle Virus (TRV)-Based Gene Silencing in Cassava
title_sort agrobacterium-mediated gene transient overexpression and tobacco rattle virus (trv)-based gene silencing in cassava
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6719147/
https://www.ncbi.nlm.nih.gov/pubmed/31443292
http://dx.doi.org/10.3390/ijms20163976
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