Abstract

Virus-induced gene silencing (VIGS) is a transient loss-of-function assay that involves threesteps: engineering the genome of a viral vector to include a fragment of host gene that is targeted tobe silenced, infecting the plant hosts and suppressing the target gene expression by posttranscriptionalgene silencing (PTGS). VIGS is a well-established reverse genetics technology forassessment of gene functions in plants. However, the efficiency of this technology may be low insome plant species, and this often limits the application of the technique to more permissive modelhosts. Aiming at increasing VIGS efficiency in functional studies, particularly in key crop speciessuch as tomato (Solanum lycopersicum), we tested an innovative approach that consisted in: a)enhancement of the target gene cleavage efficiency by exploiting the artificial microRNA(amiRNA) technology; and b) validation of a bioinformatic method for selecting the most suitablegene fragments for induction of gene silencing.The recently developed amiRNA technology modifies an endogenous gene silencingmechanism that processes natural miRNA precursors to small silencing RNAs targeting specifictranscripts for degradation. Based on natural miRNA structures, amiRNAs are commonly designedto contain mismatches at specific nucleotides with respect to their target sites, thus increasingeffectiveness of target gene cleavage as compared to RNA silencing processes guided by otherperfectly matching small RNAs.The WMD3 software (wmd3.weigelworld.org) was used for both identification of putativeamiRNA sequences and selection of suitable gene regions. In WMD3, an algorithm generates insilico all possible amiRNAs putatively able to anneal to full-length target mRNA. We selected andcompared cDNA fragments (110-120 nt) from gene regions with either high or low content ofputative amiRNAs, inserted point mutations to express amiRNA-like small RNAs from the viralvectors, and cloned these cDNAs into tobacco rattle virus (TRV)-based VIGS vectors. The variableVIGS effects of such vectors were analyzed on two tomato reporter genes, phytoene desaturase(PDS) and magnesium chelatase (ChlI or SU), whose VIGS phenotypes consist in leaf bleachingand yellowing, respectively, and therefore could be visually assessed. VIGS efficiency by severaldifferent inserts was compared by evaluating intensity of VIGS phenotype, target mRNA levels andaccumulation of VIGS-target specific small interfering RNAs. Overall, our results clearly indicatedthat: i) VIGS efficiency increased when gene sequences inserted in TRV vectors included amiRNAlikepoint mutations; ii) VIGS efficiency was significantly reduced when cDNA fragments fromgene regions with low amiRNA content were expressed in TRV vectors; and iii) WMD3 wasproved an effective bioinformatic tool to select proper target gene sequences in VIGS experiment.Our results are discussed in the light of their beneficial contri

Autore Pugliese

• Cillo Fabrizio , Bubici Giovanni Nicola , Stavolone Livia

Tutti gli autori

• Carluccio A.V.; Bubici G.; Stavolone L.; Cillo F.

Titolo volume/Rivista

Non Disponibile

Anno di pubblicazione

2014

ISSN

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ISBN

978-88-904570-4-3

Numero di citazioni Wos

Nessuna citazione

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Numero di citazioni Scopus

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Settori ERC

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Codici ASJC

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