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Monsanto Team Reports on Study of Environmental RNAi in Agricultural Pests

NEW YORK (GenomeWeb) – While environmental RNAi (eRNAi) — sequence-specific knockdown of gene expression via ingested double-stranded RNA — has been demonstrated in nematodes and certain insect species, the mechanisms underlying this process and its variability remain largely unknown.

Research published last month by researchers from Monsanto, however, has added some color to the picture, suggesting that the stability of long dsRNAs in the gut lumens of insects plays a key role in their sensitivity to eRNAi.

Additionally, the level of gene-silencing siRNAs, which are processed from dsRNAs, that target a specific insect transcript is likely to be an important factor for a measureable RNAi effect in those insects that lack a silencing amplification mechanism, the Monsanto team found.

One of the first observations of eRNAi was made in Caenorhabditis elegans, which took up dsRNAs in solution or in bacteria expressing the molecules. Yet the capacity for this brand of gene silencing is variable even among nematodes of the same genus.

Meanwhile, a number of insect species have also proved susceptible to eRNAi under laboratory conditions, "but the degree of susceptibility varies dramatically and often very large doses of synthesized dsRNA must be ingested to induce any measurable effect on target gene expression," the Monsanto team wrote in its paper, which appeared in RNA. Still, such doses are far higher than what are ordinarily produced by plants and therefore are not likely to be present under natural feeding conditions.  

Some insects, such as the Western corn rootworm (WCR), are highly responsive to eRNAi at doses that would occur in plants naturally. As such, Monsanto has a number of RNAi-based pest-control products under development. It's most advanced is Smart Stax Pro, a transgenic line of corn that expresses widely used toxic Bt proteins along with dsRNA designed to silence a gene essential to the insects and could reach the market by 2017.

The company also recently advanced to formal product development a topically applied, RNAi-based pesticide against another coleopteran, the Colorado potato beetle (CPB).

Previous studies indicate that long dsRNA, in the range of 50 to 60 base pairs, are required for eRNAi in the corn rootworm, and that small RNAs are either not taken up efficiently or are not stable enough for oral delivery.

Building upon these findings, the Monsanto investigators conducted molecular analyses of the uptake and processing of plant endogenous dsRNAs in the WCR and CPB, as well as two lepidopteran insects, the fall armyworm and the corn earworm, which are recalcitrant to eRNAi.

In line with earlier observations, dsRNA smaller than 50 to 60 base pairs in length were insufficient to engage eRNAi in WCR or CPB, they wrote in RNA. "We demonstrated that only long dsRNAs were selectively accumulated in insects receptive to eRNAi while abundant plant small RNAs and smaller RNA fragments were not taken up or were unstable in the gut lumen environment."

The scientists also found that plant-derived long dsRNAs were processed by WCR and CPB larvae primarily into 21 nucleotide-long siRNAs by their endogenous RNAi machinery — an event that occurs in dsRNA-receptive gut epithelial cells.

"Thus, while only long dsRNA can be efficiently taken up from gut lumen, it is possible that both long dsRNA and processed siRNAs are transported to other insect organs and tissues," they stated.

The Monsanto group further noted that the magnitude of accumulated plant-derived RNAs in WCRs was high, with up to 12 percent of all siRNAs found in the larvae to have originated from host corn roots on which the pests feed.

In lepidopteran larvae, no accumulation of host plant-derived siRNAs was observed.

Surprisingly, a genome-wide transcriptome analysis of WCR fed an artificial diet including total RNA from host and non-host plants did not reveal evidence of significant gene regulation by endogenous plant dsRNA at the level of transcript accumulation.

Small RNA sequencing revealed multiple plant-derived siRNAs in WCR with perfect or near-perfect complementarity to the insect's genes, but the overall number of plant-derived siRNAs per gene was low, "suggesting very limited if any direct effect of exogenous siRNAs on WCR gene expression," they wrote.

Therefore, robust gene suppression may depend on the presence of a high number of siRNAs of perfect complementarity to a target gene in insects such as WCR, which lack a mechanism for silencing signal amplification such as the one in C. elegans.

"In summary, our data demonstrates that several insects with similar feeding behavior and consuming a common plant food source manifest a dramatically different capability to take up exogenous dsRNA," the scientists concluded. "Unlike coleopterans examined in this study, the recalcitrant lepidopterans showed enhanced dsRNA degradation in the gut lumen and a lack of stable accumulation of plant-derived sRNAs, indicating a significant variation in the potential ability to participate in eRNAi."