dsRNA Uptake in Plant Pests and Pathogens: Insights into RNAi-Based Insect and Fungal Control Technology

Efforts to develop more environmentally friendly alternatives to traditional broad-spectrum pesticides in agriculture have recently turned to RNA interference (RNAi) technology. With the built-in, sequence-specific knockdown of gene targets following delivery of double-stranded RNA (dsRNA), RNAi offers the promise of controlling pests and pathogens without adversely affecting non-target species. Significant advances in the efficacy of this technology have been observed in a wide range of species, including many insect pests and fungal pathogens. Two different dsRNA application methods are being developed. First, host induced gene silencing (HIGS) harnesses dsRNA production through the thoughtful and precise engineering of transgenic plants and second, spray induced gene silencing (SIGS) that uses surface applications of a topically applied dsRNA molecule. Regardless of the dsRNA delivery method, one aspect that is critical to the success of RNAi is the ability of the target organism to internalize the dsRNA and take advantage of the host RNAi cellular machinery. The efficiency of dsRNA uptake mechanisms varies across species, and in some uptake is negligible, rendering them effectively resistant to this new generation of control technologies. If RNAi-based methods of control are to be used widely, it is critically important to understand the mechanisms underpinning dsRNA uptake. Understanding dsRNA uptake mechanisms will also provide insight into the design and formulation of dsRNAs for improved delivery and provide clues into the development of potential host resistance to these technologies.

In silico identification of off-target pesticidal dsRNA binding in honey bees (Apis mellifera)

Background Pesticidal RNAs that silence critical gene function have great potential in pest management, but the benefits of this technology must be weighed against non-target organism risks. Methods Published studies that developed pesticidal double stranded RNAs (dsRNAs) were collated into a database. The target gene sequences for these pesticidal RNAs were determined, and the degree of similarity with sequences in the honey bee genome were evaluated statistically. Results We identified 101 insecticidal RNAs sharing high sequence similarity with genomic regions in honey bees. The likelihood that off-target sequences were similar increased with the number of nucleotides in the dsRNA molecule. The similarities of non-target genes to the pesticidal RNA was unaffected by taxonomic relatedness of the target insect to honey bees, contrary to previous assertions. Gene groups active during honey bee development had disproportionately high sequence similarity with pesticidal RNAs relative to other areas of the genome. Discussion Although sequence similarity does not itself guarantee a significant phenotypic effect in honey bees by the primary dsRNA, in silico screening may help to identify appropriate experimental endpoints within a risk assessment framework for pesticidal RNAi.

Double-stranded RNA viruses infecting Epichloe festucae

Some isolates of Epichloë festucae are asymptomatically infected by Epichloë festucae virus 1 (EfV1), a member of the family Totiviridae. This virus has a genome composed by a 5109 bp molecule of double stranded RNA (dsRNA). In addition, a 3 kbp dsRNA molecule which could be the genome of another virus (EfV2) was frequently found in isolates of the fungal endophyte. In a survey of two populations of E. festucae it was found that 73% of the isolates were infected by one or both viruses. Although both viruses are very efficiently transmitted to conidia, a barrier to virus transmission occurs during ascospore formation. Keywords: Epichloë festucae, virus, dsRNA, Totivirus

Three different M1 RNA-containing viruslike particle types in Saccharomyces cerevisiae: in vitro M1 double-stranded RNA synthesis

Killer strains of Saccharomyces cerevisiae bear at least two different double-stranded RNAs (dsRNAs) encapsidated in 39-nm viruslike particles (VLPs) of which the major coat protein is coded by the larger RNA (L-A dsRNA). The smaller dsRNA (M1 or M2) encodes an extracellular protein toxin (K1 or K2 toxin). Based on their densities on CsCl gradients, L-A- and M1-containing particles can be separated. Using this method, we detected a new type of M1 dsRNA-containing VLP (M1-H VLP, for heavy) that has a higher density than those previously reported (M1-L VLP, for light). M1-H and M1-L VLPs are present together in the same strains and in all those we tested. M1-H, M1-L, and L-A VLPs all have the same types of proteins in the same approximate proportions, but whereas L-A VLPs and M1-L VLPs have one dsRNA molecule per particle, M1-H VLPs contain two M1 dsRNA molecules per particle. Their RNA polymerase produces mainly plus single strands that are all extruded in the case of M1-H particles but are partially retained inside the M1-L particles to be used later for dsRNA synthesis. We show that M1-H VLPs are formed in vitro from the M1-L VLPs. We also show that the peak of M1 dsRNA synthesis is in fractions lighter than M1-L VLPs, presumably those carrying only a single plus M1 strand. We suggest that VLPs carrying two M1 dsRNAs (each 1.8 kilobases) can exist because the particle is designed to carry one L-A dsRNA (4.5 kilobases).

Three different M1 RNA-containing viruslike particle types in Saccharomyces cerevisiae: in vitro M1 double-stranded RNA synthesis.

Killer strains of Saccharomyces cerevisiae bear at least two different double-stranded RNAs (dsRNAs) encapsidated in 39-nm viruslike particles (VLPs) of which the major coat protein is coded by the larger RNA (L-A dsRNA). The smaller dsRNA (M1 or M2) encodes an extracellular protein toxin (K1 or K2 toxin). Based on their densities on CsCl gradients, L-A- and M1-containing particles can be separated. Using this method, we detected a new type of M1 dsRNA-containing VLP (M1-H VLP, for heavy) that has a higher density than those previously reported (M1-L VLP, for light). M1-H and M1-L VLPs are present together in the same strains and in all those we tested. M1-H, M1-L, and L-A VLPs all have the same types of proteins in the same approximate proportions, but whereas L-A VLPs and M1-L VLPs have one dsRNA molecule per particle, M1-H VLPs contain two M1 dsRNA molecules per particle. Their RNA polymerase produces mainly plus single strands that are all extruded in the case of M1-H particles but are partially retained inside the M1-L particles to be used later for dsRNA synthesis. We show that M1-H VLPs are formed in vitro from the M1-L VLPs. We also show that the peak of M1 dsRNA synthesis is in fractions lighter than M1-L VLPs, presumably those carrying only a single plus M1 strand. We suggest that VLPs carrying two M1 dsRNAs (each 1.8 kilobases) can exist because the particle is designed to carry one L-A dsRNA (4.5 kilobases).