DNA Interference: a Simple and Efficient Gene-Silencing System for High-Throughput Functional Analysis in the Fern Adiantum

Abstract Background Cassava is an important crop for food security and industry in the least-developed and developing countries. The completion of the cassava genome sequence and identification of large numbers of candidate genes by next-generation sequencing provide extensive resources for cassava molecular breeding and increase the need for rapid and efficient gene function analysis systems in cassava. Several plant virus-induced gene silencing (VIGS) systems have been developed as reverse genetic tools for rapid gene function analysis in cassava. However, these VIGS vectors could cause severe viral symptoms or inefficient gene silencing. Results In this study, we constructed agroinfection-compatible infectious cDNA clones of cassava common mosaic virus isolate CM (CsCMV-CM, genus Potexvirus, family Alphaflexiviridae) that causes systemic infection with mild symptoms in cassava. CsCMV-CM was then modified to a viral vector carrying the Nimble cloning frame, which facilitates the rapid and high-throughput cloning of silencing fragments into the viral genome. The CsCMV-based vector successfully silenced phytoene desaturase (PDS) and magnesium chelatase subunit I (ChlI) in different cassava varieties and Nicotiana benthamiana. The silencing of the ChlI gene could persist for more than two months. Conclusions This CsCMV-based VIGS system provides a new tool for rapid and efficient gene function studies in cassava.

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Optimisation of tobacco rattle virus-induced gene silencing in Arabidopsis

Functional Plant Biology ◽

10.1071/fp05096 ◽

2006 ◽

Vol 33 (4) ◽

pp. 347 ◽

Cited By ~ 46

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.

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A self‐catabolic multifunctional DNAzyme nanosponge for programmable drug delivery and efficient gene silencing

Angewandte Chemie International Edition ◽

10.1002/anie.202101474 ◽

2021 ◽

Author(s):

Jing Wang ◽

Shanshan Yu ◽

Qiong Wu ◽

Xue Gong ◽

Shizhen He ◽

...

Keyword(s):

Drug Delivery ◽

Gene Silencing ◽

Efficient Gene

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High-throughput vectors for efficient gene silencing in plants

Functional Plant Biology ◽

10.1071/fp02033 ◽

2002 ◽

Vol 29 (10) ◽

pp. 1217 ◽

Cited By ~ 99

Author(s):

Chris A. Helliwell ◽

S. Varsha Wesley ◽

Anna J. Wielopolska ◽

Peter M. Waterhouse

Keyword(s):

Gene Silencing ◽

Insertional Mutagenesis ◽

Single Step ◽

Plant Genome ◽

Specific Gene ◽

Single Polymerase Chain Reaction ◽

Efficient Gene ◽

Gene Redundancy ◽

Endogenous Genes ◽

Rapid Generation

A major challenge in the post-genome era of plant biology is to determine the functions of all genes in the plant genome. A straightforward approach to this problem is to reduce or knockout expression of a gene with the hope of seeing a phenotype that is suggestive of its function. Insertional mutagenesis is a useful tool for this type of study but is limited by gene redundancy, lethal knockouts, non-tagged mutants, and the inability to target the inserted element to a specific gene. The efficacy of gene silencing in plants using inverted-repeat transgene constructs that encode a hairpin RNA (hpRNA) has been demonstrated by a number of groups, and has several advantages over insertional mutagenesis. In this paper we describe two improved pHellsgate vectors that facilitate rapid generation of hpRNA-encoding constructs. pHellsgate 4 allows the production of an hpRNA construct in a single step from a single polymerase chain reaction product, while pHellsgate 8 requires a two-step process via an intermediate vector. We show that these vectors are effective at silencing three endogenous genes in Arabidopsis, FLOWERING LOCUS C, PHYTOENE DESATURASE and ETHYLENE INSENSITIVE 2. We also show that a construct of sequences from two genes silences both genes.

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