Abstract
The potato/tomato psyllid, Bactericera cockerelli (B. cockerelli), is an important plant pest and the vector of the phloem-limited bacterium Candidatus Liberibacter psyllaurous (solanacearum), which is associated with the zebra chip disease of potatoes. Previously, we reported induction of RNA interference effects in B. cockerelli via in vitro-prepared dsRNA/siRNAs after intrathoracic injection, and after feeding of artificial diets containing these effector RNAs. In order to deliver RNAi effectors via plant hosts and to rapidly identify effective target sequences in plant-feeding B. cockerelli, here we developed a plant virus vector-based in planta system for evaluating candidate sequences. We show that recombinant Tobacco mosaic virus (TMV) containing B. cockerelli sequences can efficiently infect and generate small interfering RNAs in tomato (Solanum lycopersicum), tomatillo (Physalis philadelphica) and tobacco (Nicotiana tabacum) plants, and more importantly delivery of interfering sequences via TMV induces RNAi effects, as measured by actin and V-ATPase mRNA reductions, in B. cockerelli feeding on these plants. RNAi effects were primarily detected in the B. cockerelli guts. In contrast to our results with TMV, recombinant Potato virus X (PVX) and Tobacco rattle virus (TRV) did not give robust infections in all plants and did not induce detectable RNAi effects in B. cockerelli. The greatest RNA interference effects were observed when B. cockerelli nymphs were allowed to feed on leaf discs collected from inoculated or lower expanded leaves from corresponding TMV-infected plants. Tomatillo plants infected with recombinant TMV containing B. cockerelli actin or V-ATPase sequences also showed phenotypic effects resulting in decreased B. cockerelli progeny production as compared to plants infected by recombinant TMV containing GFP. These results showed that RNAi effects can be achieved in plants against the phloem feeder, B. cockerelli, and the TMV-plant system will provide a faster and more convenient method for screening of suitable RNAi target sequences in planta.
Highlights
RNA interference (RNAi) is a natural gene regulation and antiviral defense system of eukaryotes
We show that B. cockerelli fed on plants infected with recombinant Tobacco mosaic virus (TMV) containing B. cockerelli BC- ATPase and/or BC-Actin sequences exhibit RNAi effects as measured by reduction in target mRNA levels and the number of progeny produced on these plants
In order to rapidly and efficiently synthesize the Double-stranded RNAs (dsRNA)/small interfering RNAs (siRNAs) in planta, virus induced gene silencing (VIGS) vectors including TMV, Potato virus X (PVX) and Tobacco rattle virus (TRV) were tested in different plant species such as tomato, tomatillo and Turkish tobacco plants as they are all favorable hosts for B. cockerelli
Summary
RNA interference (RNAi) is a natural gene regulation and antiviral defense system of eukaryotes. The RNA-induced silencing complex (RISC) incorporates one strand (the guide strand) from the siRNAs and this serves to sequester the complementary sequence (by base-pairing) within the specific target RNA. The Argonaute protein, which is a component of RISC, degrades the target RNA. RNAi can be manipulated to suppress endogenous gene expression through siRNA-mediated degradation of mRNA transcripts [1,2], and is a powerful reverse genetics tool to study gene function in a variety of organisms. In plants, RNAi-based technologies have been used for generation of plants which are resistant toward bacterial, fungal, nematode or virus-induced plant diseases [3,4,5,6], and even showed promise for targeting parasitic weeds such as Triphysaria versicolor [7]
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