Dataset Information

Silencing of aphid genes by dsRNA feeding from plants.

ABSTRACT:

Background

RNA interference (RNAi) is a valuable reverse genetics tool to study gene function in various organisms, including hemipteran insects such as aphids. Previous work has shown that RNAi-mediated knockdown of pea aphid (Acyrthosiphon pisum) genes can be achieved through direct injection of double-stranded RNA (dsRNA) or small-interfering RNAs (siRNA) into the pea aphid hemolymph or by feeding these insects on artificial diets containing the small RNAs.

Methodology/principal findings

In this study, we have developed the plant-mediated RNAi technology for aphids to allow for gene silencing in the aphid natural environment and minimize handling of these insects during experiments. The green peach aphid M. persicae was selected because it has a broad plant host range that includes the model plants Nicotiana benthamiana and Arabidopsis thaliana for which transgenic materials can relatively quickly be generated. We targeted M. persicae Rack1, which is predominantly expressed in the gut, and M. persicae C002 (MpC002), which is predominantly expressed in the salivary glands. The aphids were fed on N. benthamiana leaf disks transiently producing dsRNA corresponding to these genes and on A. thaliana plants stably producing the dsRNAs. MpC002 and Rack-1 expression were knocked down by up to 60% on transgenic N. benthamiana and A. thaliana. Moreover, silenced M. persicae produced less progeny consistent with these genes having essential functions.

Conclusions/significance

Similar levels of gene silencing were achieved in our plant-mediated RNAi approach and published silencing methods for aphids. Furthermore, the N. benthamiana leaf disk assay can be developed into a screen to assess which genes are essential for aphid survival on plants. Our results also demonstrate the feasibility of the plant-mediated RNAi approach for aphid control.

SUBMITTER: Pitino M

PROVIDER: S-EPMC3187792 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:RNA interference (RNAi) is a post-translational regulatory mechanism that controls gene expression in plants. This process can be artificially induced by double-stranded RNA (dsRNA) molecules with sequence homology to target mRNAs. Exogenously applied dsRNA on leaves has been shown to silence virulence genes of fungi and viruses, conferring protection to plants. Coupling dsRNA to nanoparticles has been demonstrated to prolong the silencing effect. The ability of exogenous dsRNA to silence endogenous genes in plants is currently under debate, mainly due to the difficulty in delivering dsRNA into plant tissues and organs. Our study aims to develop a method based on the exogenous application of dsRNA on tomato flowers for silencing endogenous genes controlling ovary growth. Two methods of dsRNA delivery into tomato flower buds (i.e., pedicel soaking and injection) were compared to test their efficacy in silencing the tomato Aux/IAA9 (SlIAA9) gene, which encodes for a known repressor of ovary growth. We examined the silencing effect of dsRNA alone and coupled to layered double hydroxide (LDHs) nanoparticles. We found that injection into the pedicel led to the silencing of SlIAA9 and the efficacy of the method was confirmed by choosing a different ovary growth repressor gene (SlAGAMOUS-like 6; SlAGL6). The coupling of dsRNA to LDHs increased the silencing effect in the case of SlIAA9. Silencing of the two repressors caused an increase in ovary size only when flower buds were treated with dsRNA coupled to LDHs. RNA-Seq of small RNAs showed that induction of RNAi was caused by the processing of injected dsRNA. In this work, we demonstrate for the first time that exogenous dsRNA coupled to LDHs can induce post-transcriptional gene silencing in the young tomato ovary by injection into the flower pedicel. This method represents a silencing tool for the study of the molecular changes occurring during the early stages of ovary/fruit growth as a consequence of downregulation of target genes, without the need to produce transgenic plants stably expressing RNAi constructs.