A Ligation-Independent Cloning Tobacco Rattle Virus Vector for High-Throughput Virus-Induced Gene Silencing Identifies Roles for NbMADS4-1 and -2 in Floral Development

Arabidopsis thaliana (L.) Heynh. is a model plant species in which to study plant gene functions. Recently developed virus-induced gene silencing (VIGS) offers a rapid and high-throughput technique platform for gene function analysis. In this paper we report optimisation of tobacco rattle virus (TRV)-induced gene silencing in Arabidopsis. The parameters potentially affecting the efficiency of VIGS in Arabidopsis were investigated. These included the concentration and pre-incubation of Agrobacterium inocula (agro-inocula), the concentration of acetosyringone included in agro-inocula, the Agrobacterium inoculation (agro-inoculation) method, the ecotypes and the growth stages of Arabidopsis plants for agro-inoculation, and the growth temperature of agro-inoculated plants. The optimised VIGS procedure involves preparing the agro-inocula with OD600 of 2.0, pre-incubating for 2 h in infiltration buffer containing 200 μm acetosyringone, agro-inoculating by vacuum infiltration, and growth of agro-inoculated plants at 22 −24°C. Following this procedure consistent and highly efficient VIGS was achieved for the genes encoding phytoene desaturase (PDS) and actin in Arabidopsis. The silencing phenotype lasts for at least 6 weeks, and is applicable in at least seven ecotypes, including Col-0, Cvi-0, Sd, Nd-1, Ws-0, Bay-0 and Ler. TRV-induced VIGS was expressed not only in leaves, but also in stems, inflorescences and siliques. However, VIGS was not transmissible through seed to the subsequent generation. The optimised procedure of the TRV-induced gene silencing should facilitate high-throughput functional analysis of genes in Arabidopsis.

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A High Throughput Barley Stripe Mosaic Virus Vector for Virus Induced Gene Silencing in Monocots and Dicots

PLoS ONE ◽

10.1371/journal.pone.0026468 ◽

2011 ◽

Vol 6 (10) ◽

pp. e26468 ◽

Cited By ~ 116

Author(s):

Cheng Yuan ◽

Cui Li ◽

Lijie Yan ◽

Andrew O. Jackson ◽

Zhiyong Liu ◽

...

Keyword(s):

Gene Silencing ◽

Mosaic Virus ◽

High Throughput ◽

Virus Vector ◽

Barley Stripe Mosaic Virus ◽

Virus Induced Gene Silencing

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Efficient Virus-Induced Gene Silencing in Roots Using a Modified Tobacco Rattle Virus Vector

PLANT PHYSIOLOGY ◽

10.1104/pp.104.051466 ◽

2004 ◽

Vol 136 (4) ◽

pp. 3999-4009 ◽

Cited By ~ 87

Author(s):

Tracy Valentine ◽

Jane Shaw ◽

Vivian C. Blok ◽

Mark S. Phillips ◽

Karl J. Oparka ◽

...

Keyword(s):

Gene Silencing ◽

Tobacco Rattle Virus ◽

Virus Vector ◽

Virus Induced Gene Silencing

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Faculty Opinions recommendation of High throughput virus-induced gene silencing implicates heat shock protein 90 in plant disease resistance.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1003609.201391 ◽

2003 ◽

Author(s):

Jen Sheen

Keyword(s):

Disease Resistance ◽

Heat Shock ◽

Heat Shock Protein ◽

Gene Silencing ◽

High Throughput ◽

Plant Disease Resistance ◽

Plant Disease ◽

Heat Shock Protein 90 ◽

Virus Induced Gene Silencing

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Highly Efficient Virus-Induced Gene Silencing in Apple and Soybean by Apple Latent Spherical Virus Vector and Biolistic Inoculation

Methods in Molecular Biology - Virus-Induced Gene Silencing ◽

10.1007/978-1-62703-278-0_13 ◽

2013 ◽

pp. 167-181 ◽

Cited By ~ 12

Author(s):

Noriko Yamagishi ◽

Nobuyuki Yoshikawa

Keyword(s):

Gene Silencing ◽

Virus Vector ◽

Virus Induced Gene Silencing ◽

Apple Latent Spherical Virus ◽

Highly Efficient ◽

Biolistic Inoculation

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A detached petal disc assay and virus-induced gene silencing facilitate the study of Botrytis cinerea resistance in rose flowers

Horticulture Research ◽

10.1038/s41438-019-0219-2 ◽

2019 ◽

Vol 6 (1) ◽

Cited By ~ 2

Author(s):

Xiaoqian Cao ◽

Huijun Yan ◽

Xintong Liu ◽

Dandan Li ◽

Mengjie Sui ◽

...

Keyword(s):

Jasmonic Acid ◽

Gene Silencing ◽

Botrytis Cinerea ◽

High Throughput ◽

Regulatory Protein ◽

Gray Mold ◽

Virus Induced Gene Silencing ◽

Potential Benefits ◽

Rose Petals ◽

Disc Assay

AbstractFresh-cut roses (Rosa hybrida) are one of the most important ornamental crops worldwide, with annual trade in the billions of dollars. Gray mold disease caused by the pathogen Botrytis cinerea is the most serious fungal threat to cut roses, causing extensive postharvest losses. In this study, we optimized a detached petal disc assay (DPDA) for artificial B. cinerea inoculation and quantification of disease symptoms in rose petals. Furthermore, as the identification of rose genes involved in B. cinerea resistance could provide useful genetic and genomic resources, we devised a virus-induced gene silencing (VIGS) procedure for the functional analysis of B. cinerea resistance genes in rose petals. We used RhPR10.1 as a reporter of silencing efficiency and found that the rose cultivar ‘Samantha’ showed the greatest decrease in RhPR10.1 expression among the cultivars tested. To determine whether jasmonic acid and ethylene are required for B. cinerea resistance in rose petals, we used VIGS to silence the expression of RhLOX5 and RhEIN3 (encoding a jasmonic acid biosynthesis pathway protein and an ethylene regulatory protein, respectively) and found that petal susceptibility to B. cinerea was affected. Finally, a VIGS screen of B. cinerea-induced rose transcription factors demonstrated the potential benefits of this method for the high-throughput identification of gene function in B. cinerea resistance. Collectively, our data show that the combination of the DPDA and VIGS is a reliable and high-throughput method for studying B. cinerea resistance in rose.

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

International Journal of Molecular Sciences ◽

10.3390/ijms20163976 ◽

2019 ◽

Vol 20 (16) ◽

pp. 3976 ◽

Cited By ~ 6

Author(s):

Hongqiu Zeng ◽

Yanwei Xie ◽

Guoyin Liu ◽

Yunxie Wei ◽

Wei Hu ◽

...

Keyword(s):

Gene Silencing ◽

Functional Genomics ◽

Transient Expression ◽

Fluorescent Protein ◽

Tobacco Rattle Virus ◽

Mosaic Disease ◽

African Cassava Mosaic Virus ◽

Cassava Mosaic Disease ◽

Virus Induced Gene Silencing ◽

Transient Expression Assay

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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