scholarly journals Peer Review #1 of "Efficient virus-induced gene silencing in Hibiscus hamabo Sieb. et Zucc. using tobacco rattle virus (v0.2)"

2019 ◽  
Keyword(s):  
Gene Silencing ◽  
Peer Review ◽  
Tobacco Rattle Virus ◽  
Virus Induced Gene Silencing

Optimisation of tobacco rattle virus-induced gene silencing in Arabidopsis

2006 ◽  
Vol 33 (4) ◽  
pp. 347 ◽  
Author(s):  
Changchun Wang ◽  
Xinzhong Cai ◽  
Xuemin Wang ◽  
Zhong Zheng
Keyword(s):  
Gene Silencing ◽  
High Throughput ◽  
Function Analysis ◽  
Tobacco Rattle Virus ◽  
Phytoene Desaturase ◽  
Growth Stages ◽  
Virus Induced Gene Silencing ◽  
Gene Functions ◽  
Genes Encoding ◽  
Gene Function Analysis

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.

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

2019 ◽  
Vol 20 (16) ◽  
pp. 3976 ◽  
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.

scholarly journals An Efficient Virus-Induced Gene Silencing System for Functional Genomics Research in Walnut (Juglans regia L.) Fruits

2021 ◽  
Vol 12 ◽  
Author(s):  
Yifan Wang ◽  
Ning Huang ◽  
Niu Ye ◽  
Lingyu Qiu ◽  
Yadong Li ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Gene Function ◽  
Function Analysis ◽  
Juglans Regia ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Tobacco Rattle Virus ◽  
Post Inoculation ◽  
Virus Induced Gene Silencing ◽  
Gene Function Analysis

The Persian walnut (Juglans regia L.) is a leading source of woody oil in warm temperate regions and has high nutritional and medicinal values. It also provides both tree nuts and woody products. Nevertheless, incomplete characterization of the walnut genetic system limits the walnut gene function analysis. This study used the tobacco rattle virus (TRV) vector to construct an infectious pTRV-JrPDS recombinant clone. A co-culture inoculation method utilizing Agrobacterium was screened out from four inoculation methods and optimized to set up an efficient virus-induced gene silencing (VIGS) system for J. regia fruit. The optimized VIGS-TRV system induced complete photobleaching phenotype on the walnut fruits of four cultivars, and the JrPDS transcript levels decreased by up to 88% at 8 days post-inoculation (dpi). While those of browning-related J. regia polyphenol oxidase (PPO) genes JrPPO1 and JrPPO2 decreased by 67 and 80% at 8 dpi, respectively, accompanied by a significant reduction in fruit browning phenotype. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis screening and Western Blot showed that the PPO protein levels were significantly reduced. Moreover, a model of TRV-mediated VIGS system for inoculating J. regia fruit with efficient silence efficiency via co-culture was developed. These results indicate that the VIGS-TRV system is an efficient tool for rapid gene function analysis in J. regia fruits.

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