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Two Studies Identify Potential RNAi Targets in Key Crop Pests

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Two research groups this month reported on the identification of promising targets for RNAi-based control of two key agricultural pests: Peregrinus maidis, also known as the corn planthopper, and Sitobion avenae, or the grain aphid.

P. maidis is a significant problem for the agriculture industry, destroying a variety of crops including corn, sorghum, and pearl millet. While the insect directly damages plants by feeding on their vascular tissue, it also causes indirect damage through the transmission of viruses, and is the only known vector of Maize mosaic rhabdovirus and Maize stripe tenuivirus.

Efforts to control P. maidis typically relied on insecticide use, but "indiscriminate usage has resulted in resistance leading to a resurgence of the insect, and has led to serious environmental pollution," a team of Kansas State University investigators wrote in a paper appearing in PLoS One.

As such, there has been growing interest in developing insect-resistant plant varieties, yet "few sources of genetic resistance have been reported" for those crops that are especially impacted by the pest.

To address this, the KSU scientists turned to RNAi, focusing on V-ATPase — a class of evolutionarily conserved enzymes with a variety of cellular functions including nutrient uptake and ion balance in the gut system, with various subunits previously identified as essential.

To determine if RNAi could be used to knock down this target in P. maidis, the KSU group first tried feeding third-stage nymphs dsRNA against the V-ATPase subunits V-ATPase B or V-ATPase D and negative control dsRNA. A similar experiment was conducted by injecting the different dsRNAs into both adult insects and nymphs.

The nymphs fed V-ATPase D dsRNA showed a "significant reduction" in target transcript abundance after six days of feeding, which corresponded to an increase in mortality. The researchers were also able to successfully feed dsRNA against another V-ATPase to brown planthoppers, triggering reductions in target transcripts but with no apparent effect on mortality.

Meanwhile, P. maidis nymphs injected with either the V-ATPase D or V-ATPase B dsRNA lost the ability to deliver eggs as adult females — an effect that was found to be the result of underdeveloped reproductive organs.

"When newly emerged female and male adults were injected with dsRNA-V-ATPase B or D, then paired and allowed to mate, females oviposited fewer eggs" than individuals injected with control dsRNA, the researchers wrote. "Similarly, deformed ovaries and oocytes were observed in females."

Having successfully established dsRNA injection- and ingestion-based RNAi in P. maidis, the KSU team expects that the gene-silencing method may begin to be used to develop plants resistant to the pest, as well as investigate the details of virus-vector interactions.

Like P. maidis, S. avenae has been a major cause of crop yield losses by both feeding on plants and by acting as a vector for plant viruses.

RNAi-based methods of controlling various aphid species have been attempted, and in 2009 a group from the University of Manitoba reported that feeding dsRNA targeting V-ATPase to pea aphids led to significant mortality. Still, RNAi had not been demonstrated in S. avenae.

Looking to fill this gap, researchers from the Chinese Academy of Agricultural Sciences conducted a de novo transcriptome assembly and gene-expression analysis of the alimentary canals of S. avenae before and after feeding on wheat plants, they wrote in BMC Genomics.

The transcriptome profiling generated 30,427 unigenes with an average length of 664 base pairs, according to the paper. A comparison of the transcriptomes of alimentary canals of pre- and post- feeding grain aphids indicated that 5,490 unigenes were differentially expressed, among which diverse genes and/or pathways were identified and annotated.

Sixteen highly expressed genes were selected as targets in a dsRNA feeding assay, five of which caused higher mortality and developmental stunting when downregulated. Importantly, three of the five targets had no identified orthologs, which is key given that gene-silencing strategies suitable for crop plants must be highly specific in order to avoid biosafety issues, the researchers noted.

Additionally, the lethal effect from silencing the five target genes was achieved at a relatively low concentration of dsRNA — 7 nanograms per microliter — will help "maintain the minimal risk of non-specific effects," they added.

The scientists also examined whether a systemic RNAi effect, like the kind observed in certain insects such as Hyalophora cecropia, can be observed in S. avenae by feeding the pests fluorescently labeled dsRNA.

Indeed, the fluorescence signal was "observed first in the mouthparts, and then centralized in the midgut, and finally it spread through the whole body," they wrote in the BMC Genomics paper. Additionally, BLAST analysis of the transcriptome data from the experiments against the public databases "revealed the presence of some RNAi core machinery elements such as Argonaute-2B, Dicer-1, SID-1, and also TAR RNA binding protein."

However, they cautioned that the mechanisms behind the fluorescence spread still need to be studied.

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