Abstract
BackgroundLocalized introduction and transient expression of T-DNA constructs mediated by agro-infiltration of leaf tissues has been largely used in dicot plants for analyzing the transitivity and the cell-to cell movement of the RNAi signal. In cereals, however, the morphology of the leaf and particularly the structure of the leaf epidermis, prevent infiltration of a bacterial suspension in cells by simple pressure, a method otherwise successful in dicots leaves. This study aimed at establishing a rapid method for the functional analysis of rice genes based on the triggering of RNA interference (RNAi) following Agrobacterium-mediated transient transformation of leaves.ResultsUsing an agro-infection protocol combining a wound treatment and a surfactant, we were able to obtain in a reliable manner transient expression of a T-DNA-borne uidA gene in leaf cells of japonica and indica rice cultivars. Using this protocol to transiently inhibit gene expression in leaf cells, we introduced hairpin RNA (hpRNA) T-DNA constructs containing gene specific tags of the phytoene desaturase (OsPDS) and of the SLENDER 1 (OsSLR1) genes previously proven to trigger RNAi of target genes in stable transformants. SiRNA accumulation was observed in the agro-infected leaf area for both constructs indicating successful triggering of the silencing signal. Accumulation of secondary siRNA was observed in both stably and transiently transformed leaf tissues expressing the HpRNA OsSLR1 construct. Gene silencing signalling was investigated in monitoring the parallel time course of OsPDS-derived mRNA and siRNA accumulation in the agro-infiltrated leaf area and adjacent systemic sectors. The sensitive RT-Q-PCR method evidenced a consistent, parallel decrease of OsPDS transcripts in both the agroinfiltred and adjacent tissues, with a time lag for the latter.ConclusionsThese results indicate that the method is efficient at inducing gene silencing in the agro-infected leaf area. The transfer of low amounts of siRNA, probably occurring passively through the symplastic pathway from the agro-infected area, seemed sufficient to trigger degradation of target transcripts in the adjacent tissues. This method is therefore well suited to study the cell-to-cell movement of the silencing signal in a monocot plant and further test the functionality of natural and artificial miRNA expression constructs.Electronic supplementary materialThe online version of this article (doi:10.1186/1939-8433-5-23) contains supplementary material, which is available to authorized users.
Highlights
Localized introduction and transient expression of T-DNA constructs mediated by agro-infiltration of leaf tissues has been largely used in dicot plants for analyzing the transitivity and the cell-to cell movement of the RNA interference (RNAi) signal
Transient assays by agro-infiltration have been increasingly used as a simple and rapid method for assaying gene function (Wroblewski et al 2005; Small 2007). Because they provide a rapid, versatile and convenient way for achieving a very high level of gene expression in a distinct and defined zone of the leaf, these Agrobacterium-mediated transient expression systems have been largely used for analyzing the induction of the RNA silencing process, thereby validating gene function through down regulation of gene expression as illustrated recently in grapevine (Bertazzon et al 2012)- and analyzing the mechanism of transitivity and movement of RNA silencing in leaves (e.g. Schöb et al 1997, Voinnet 2001)
Agroinfection of rice leaf tissues with an A. tumefaciens suspension To establish an efficient method for Agrobacterium infection of rice leaves, we tested different A. tumefaciens strains (GV2200, EHA105, AGL1, LBA4404, and GV3101) carrying the same binary plasmid as well as different infiltration media and temperature, and assessed the efficiency of T-DNA transfer by expression of the carried uidA gene by GUS histochemical assays
Summary
Localized introduction and transient expression of T-DNA constructs mediated by agro-infiltration of leaf tissues has been largely used in dicot plants for analyzing the transitivity and the cell-to cell movement of the RNAi signal. Bombardment-mediated introduction of dsRNA corresponding to endogenous genes or transgenes in cells of maize, barley and wheat leaves proved to induce interference with gene function but restricted to the single cell where the delivery is achieved (Schweizer et al 2000) This stresses the need to establish a rapid, versatile and convenient protocol for achieving a very high level of gene expression in planta e.g. in a distinct and defined zone of the cereal leaf
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