RNA Silencing Mediated by Direct Repeats in Maize: A Potential Tool for Functional Genomics

Abstract

It has been shown in tobacco and Arabidopsis that transgenes with multiple direct repeats induce RNA silencing at high frequency. In this study, we tried to establish a direct repeat-induced RNA silencing system in maize and evaluate whether it can be developed as a high throughput tool for functional genomics. Our results showed that the construct phC4, which carries four direct repeats of a chloramphenicol acetyl-transferase (CAT) gene, was able to induce silencing of itself with high efficiency in maize. Using a transient expression system, we further demonstrated that construct phC3G with a beta-glucuronidase (GUS) gene located downstream of three direct repeats of CAT gene silenced not only itself in maize calli but also an "endogenous" GUS gene, which was stably expressed in maize calli. Most importantly, when constructs with the maize iojap (ij) gene inserted in either sense or antisense orientation into the downstream of four direct repeats of CAT gene were transformed into maize plants, co-suppression of endogenous and transgenic ij genes was detected in majority of transgenic maize plants. Our co-suppression results suggest that with improvements, this new approach has the potential to become an efficient research tool for high throughput functional genomics.

RNA Silencing in Plants: Mechanisms, Technologies and Applications in Horticultural Crops

“…A more recent study showed that transgenes with 3 or 4-copy intrinsic direct repeats (Fig. 1-D ) induce potent gene silencing with almost 100% efficiency in both Arabidopsis and maize [82, 83]. Direct-repeat transgenes are not expected to produce dsRNA directly, so how they induce potent gene silencing remains unclear.…”

“…The silencing is associated with the accumulation of siRNAs, and has therefore been suggested to be PTGS, although the exact size of the siRNAs has not been determined. The intrinsic direct repeats appear to be capable of inducing transitive silencing: when a sequence with no homology to the direct repeats is transcriptionally fused at the 3’ end, the transgene can induce silencing against both the target of the direct repeats and that of the non-homologous fusion sequence [82, 83]. This suggests that siRNAs derived from the direct repeats can induce secondary siRNA production from the downstream sequence.…”