An in planta, Agrobacterium-mediated transient gene expression method for inducing gene silencing in rice (Oryza sativa L.) leaves

Abstract

Background: 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. 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. Results: Using 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. Conclusions: These 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.

Cucurbit yellow stunting disorder virus and watermelon chlorotic stunt virus induced gene silencing in tobacco plants

“…Although the transformed plant cells are limited to the spotted region, RNA silencing signals could be systemically transmitted, as supposed by Eckardt (2002). Andrieu et al (2012) mentioned that, the results of their work 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.…”

When plant virology met Agrobacterium: the rise of the deconstructed clones

“…Progress into this line of research has recently been demonstrated by Andrieu et al . (), who developed a protocol for the agroinfection of rice leaves for gene silencing purposes. They point out that inefficient agroinfection in cereal leaves is largely due to the physiological properties of the leaves.…”

“…BeYDV replicons have also been proposed as efficient vehicles of targeted genome engineering thanks to the transient expression of LSL replicons containing zinc finger nucleases or CRISPR/ CAS elements for highly efficient targeting of transgenes or mutagenesis (Baltes et al, 2014). Another geminivirus, beet curly top virus (BCTV), has been used to develop a replicating expression system (Kim et al, 2007).…”

Recent Advancements in Gene Expression and Enabling Technologies in Crop Plants

“…In these plants, poor Agrobacterium infiltration by simple pressure has been attributed to differences in leaf architecture and structure of the epidermis (Andrieu et al 2012). Recently, Agrobacterium-mediated transient expression has been achieved in rice leaf tissue which facilitated a rapid analysis of rice genes based on RNA-interference (Andrieu et al 2012). In this case, the use of a surfactant (Silwet L-77) improved Agrobacterium infiltration and enhanced transgene expression.…”