Extending Functional Genomics: VIGS for Model and Crop Plants

Bernacki, Steven; Tuttle, John R.; Muangsan, Nooduan; Robertson, Dominique

doi:10.1002/9783527622542.ch11

Cited by 2 publications

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“…, 2007). The two modes that are most commonly used are inverted repeat RNAi (IR‐RNAi) (Waterhouse and Helliwell, 2003) and virus‐induced gene silencing (VIGS) (Bernacki et al , 2008). However, both these strategies have limitations for use in high‐throughput mutagenesis on a global scale.…”

Section: Introductionmentioning

confidence: 99%

“…RNAi has been widely exploited as a technique for reverse mutagenesis in plants (Hilson et al, 2004;McGinnis et al, 2005McGinnis et al, , 2007Fu et al, 2007b;Tang et al, 2007). The two modes that are most commonly used are inverted repeat RNAi (IR-RNAi) (Waterhouse and Helliwell, 2003) and virus-induced gene silencing (VIGS) (Bernacki et al, 2008). However, both these strategies have limitations for use in high-throughput mutagenesis on a global scale.…”

Section: Introductionmentioning

confidence: 99%

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Targeted forward mutagenesis by transitive RNAi

Petsch

Scanlon

et al. 2010

The Plant Journal

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SUMMARYA novel technique is described that targets specific populations of transcripts for homology-based gene silencing using transitive RNAi. This approach is designed to target a subset of the transcriptome in order to identify genes involved in a particular localized process, such as photosynthesis. As a proof-of-concept approach, mesophyll cells from Arabidopsis thaliana were laser-microdissected from whole leaves to generate a focused cDNA library that was bi-directionally cloned into a transitive RNAi vector that had been designed to induce silencing of homologous, endogenous genes. Approximately 15% of the transformant plants identified from both sense and antisense libraries exhibited visible phenotypes indicative of photosynthetic defects. Amplification from the genome and sequencing of cDNA inserts identified candidate genes underlying the phenotypes. For 10 of 11 such mutants, re-transformation with an RNAi construct corresponding to the candidate gene recapitulated the original mutant phenotype, and reduction of corresponding endogene transcripts was confirmed. In addition, one of the re-transformed transgenes also silenced transcripts of closely related family members, thereby demonstrating the utility of this approach for mutagenesis of redundant gene functions. Preliminary results using tissue-specific transitive RNAi forward mutagenesis of the Arabidopsis vegetative shoot apical meristem demonstrate the broad applicability of this forward mutagenesis technique for a variety of plant cell types.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Targeted forward mutagenesis by transitive RNAi

Petsch

Scanlon

et al. 2010

The Plant Journal

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“…Double-stranded RNA (dsRNA), typically generated from either an inverted repeat of the target sequence (IR-RNAi) or a viral-vector-derived RNA (VIGS), acts as the trigger for posttranscriptional gene silencing (PTGS) of homologous sequences (Becker and Lange 2010;Bernacki et al 2008;Waterhouse and Helliwell 2003;Watson et al 2005). Although IR-RNAi and virus induced gene silencing (VIGS) function to efficiently downregulate gene expression of a particular target sequence/s, both methods are of limited use for high-throughput mutagenesis on a global scale.…”

Section: Rnai As a Forward Mutagenesis Toolmentioning

confidence: 99%

Mutagenesis by Transitive RNAi

Petsch

Scanlon

et al. 2011

RNA Technologies

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Transitive RNAi is a posttranscriptional mechanism of gene silencing that is based on the phenomenon of "transitivity." This term refers to the spreading of silencing outside of the initial target sequence and is associated with transgeneinduced posttranscriptional gene silencing (PTGS). Transitive RNAi is triggered by placing an inverted repeat sequence immediately 3¢ of the sense transgene that is to be targeted. Placement of the inverted repeat in this region is thought to increase the efficiency by which RDR6 initiates copying of the transgene to generate doublestranded RNA (dsRNA). In a proof-of-concept approach, we showed that select subsets of genes can be manipulated with transitive RNAi in a high-throughput forward mutagenesis approach (Plant J 61:873-882, 2010). Laser microdissection of

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Extending Functional Genomics: VIGS for Model and Crop Plants

Cited by 2 publications

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Targeted forward mutagenesis by transitive RNAi

Targeted forward mutagenesis by transitive RNAi

Mutagenesis by Transitive RNAi

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