Improvement Of Virus-Induced Gene Silencing Efficiency By Artificial miRNA-Based Constructs In Tomato

Virus-induced gene silencing (VIGS) is a transient loss-of-function assay that involves three steps: engineering the genome of a viral vector to include a fragment of host gene that is targeted to be silenced, infecting the plant hosts and suppressing the target gene expression by posttranscriptional gene silencing (PTGS). VIGS is a well-established reverse genetics technology for assessment of gene functions in plants. However, the efficiency of this technology may be low in some plant species, and this often limits the application of the technique to more permissive model hosts. Aiming at increasing VIGS efficiency in functional studies, particularly in key crop species such 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 suitable gene fragments for induction of gene silencing. The recently developed amiRNA technology modifies an endogenous gene silencing mechanism that processes natural miRNA precursors to small silencing RNAs targeting specific transcripts for degradation. Based on natural miRNA structures, amiRNAs are commonly designed to contain mismatches at specific nucleotides with respect to their target sites, thus increasing effectiveness of target gene cleavage as compared to RNA silencing processes guided by other perfectly matching small RNAs. The WMD3 software (wmd3.weigelworld.org) was used for both identification of putative amiRNA sequences and selection of suitable gene regions. In WMD3, an algorithm generates in silico all possible amiRNAs putatively able to anneal to full-length target mRNA. We selected and compared cDNA fragments (110-120 nt) from gene regions with either high or low content of putative amiRNAs, inserted point mutations to express amiRNA-like small RNAs from the viral vectors, and cloned these cDNAs into tobacco rattle virus (TRV)-based VIGS vectors. The variable VIGS 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 bleaching and yellowing, respectively, and therefore could be visually assessed. VIGS efficiency by several different inserts was compared by evaluating intensity of VIGS phenotype, target mRNA levels and accumulation of VIGS-target specific small interfering RNAs. Overall, our results clearly indicated that: i) VIGS efficiency increased when gene sequences inserted in TRV vectors included amiRNAlike point mutations; ii) VIGS efficiency was significantly reduced when cDNA fragments from gene regions with low amiRNA content were expressed in TRV vectors; and iii) WMD3 was proved an effective bioinformatic tool to select proper target gene sequences in VIGS experiment. Our results are discussed in the light of their beneficial contribution to the functional analysis of genes of interest in tomato and other crops.