NEW YORK (GenomeWeb) – Researchers from Monsanto and Miami University this month reported on a novel in vivo assay that can be used to identify genes related to environmental RNAi in the Western corn rootworm (WCR), a key agricultural pest and target of an RNAi-incorporating strain of corn under development by the company.
According to the scientists, the assay system can be used to quickly survey candidate WCR genes for their involvement in the RNAi process and has the potential to be adapted for genome-wide RNAi screening to provide new insights into how the gene-silencing pathway functions in the insect.
The system also has the potential to be modified for use with other insects, Miami University researcher and senior author of the study Yoshinori Tomoyasu said this week.
As one of the biggest causes of crop loss in the US, the WCR has long been a focus for Monsanto and other ag-bio firms, which have developed transgenic corn varieties that express so-called Bt proteins, which are derived from the bacterium Bacillus thuringiensis and are toxic to the pest.
However, in recent years there has been evidence of WCR resistance to Bt proteins and chemical insecticides, prompting interest in new approaches to its control. For Monsanto, this has led to the creation of Smart Stax Pro, a corn strain the expresses two distinct Bt traits and dsRNAs that silence the WCR version of a gene called Snf7 that is involved in intracellular transport. And because WCR are capable of environmental RNAi, wherein ingested dsRNA triggers systemic gene silencing, Snf7 silencing can be achieved as the insects feed on corn roots and root hairs.
Smart Stax Pro is set for launch by 2017.
As part of Monsanto's efforts with Smart Stax Pro and the other RNAi-based products moving through its pipeline, company researchers teamed up Tomoyasu, who has long studied systemic RNAi in insects, to uncover the molecular mechanisms behind environmental RNAi in WCR.
Systemic RNAi is characterized by three main processes: cellular uptake of dsRNA from the extracellular environment; the spreading of a gene-silencing signal between cells; and, in some cases, intestinal dsRNA uptake, i.e. environmental RNAi.
The bulk of the research into systemic RNAi has been conducted in C. elegans, which has led to the identification of several genes critical to the process including sid-1, which has been linked to intracellular dsRNA transport. And while systemic RNAi is known to exist in insects as well, "the molecules and pathways involved in systemic RNAi in insects remain largely unknown," Tomoyasu and the Monsanto team wrote in their paper, which appeared in PLOS One.
Given WCR's robust environmental RNAi response and the estimated $1 billion in losses it causes North American farmers each year, the need for insights into the mechanisms that underlie environmental RNAi in the insect is particularly important. Such information will allow for determining the rate limiting steps involved with dsRNA toxicity in WCR and potential dsRNA resistance mechanisms, the study's authors noted.
To develop their WCR assay, the scientists tweaked an in vivo assay Tomoyasu previously developed to screen for genes involved in the systemic RNAi pathway of the red flour beetle, which isn't capable of environmental RNAi. The adapted system consists of two RNAi feeding experiments — one in which dsRNAs for a candidate gene involved in environmental RNAi are fed to insect larvae for two to three days; and a second in which larvae are fed with dsRNAs for a marker gene.
"The marker gene would be a gene that has a visual and/or measurable function in the insect, in which the effect can be easily observed and measured upon knockdown," the team wrote in PLOS One. "If the candidate gene in the first step is essential for RNAi — including environmental RNAi — the messenger RNA levels of the marker gene will not be altered by the second RNAi, hence no changes in phenotype will be detected."
But if a phenotypic change is detected due to knockdown of the marker gene, it can be assumed that the candidate gene is not involved in RNAi.
Using the system, Tomoyasu and his Monsanto collaborators identified two genes for use as markers in their WCR assay system: ebony and lacasse 2 (lac2). Silencing of these genes resulted in visible pigmentation defects in the insects but do not cause immediate lethality.
They then examined the optimal length and concentration of dsRNA for the assay system. Noting in their paper that dsRNA length is known to affect the efficiency of system RNAi, the researchers tested different length dsRNAs for lac2 and ebony, finding that ones longer than 100 base pairs were required for easily distinguishable phenotypes.
In terms of dose dependency, a concentration of at least 500 nanograms per milliliter was required to trigger a scorable pigmentation phenotype using ebony or lac2 as markers in the assay system.
The investigators also looked at evolutionarily conserved genes known to be important in the RNAi pathway to serve as positive controls for their assay. They ultimately selected Argonaute 2 and Dicer-2, which when silenced led to significant reductions in their mRNA levels but did not cause any noticeable abnormalities and did not affect pigmentation.
In developing the assay, the researchers encountered competition between the dsRNAs they tested — not a surprising finding given that mixtures of RNAi molecules are known to sometime vie for the various components of the gene-silencing system. Notably, they discovered that both dsRNA concentration as well as length impacted competition, with longer molecules outmatching shorter ones.
"It's known that … multiple RNAi [molecules against different targets] compete with each other," Tomoyasu explained to Gene Silencing News. "But usually that competition has something to do with concentration of double-stranded RNA, so we were surprised to actually see that competition happened in regards to the length."
Given that Dicer cleaves dsRNA into similarly sized siRNAs, Tomoyasu said that this finding suggests that competition between different dsRNAs may be happening earlier in the RNAi process than previously thought, perhaps during cellular uptake.
To test their assay, the investigators focused on two WCR sid-1 homologs. The involvement of these so-called sid-1-like (sil) genes has been a matter of debate. Some point to their established role in systemic RNAi in nematodes and their absence in Drosophila, which lack a robust systemic RNAi response, as evidence of their importance in the process. Yet mosquito species do not possess sil genes but exhibit systemic RNAi, while lepidopterans have multiple sil genes but don't show such responses.
With the system, Tomoyasu and the Monsanto group showed that both WCR sil genes they tested are involved in environmental RNAi in the insect. However, the suppression of the marker gene RNAi by sil RNAi was not robust, which suggests that the genes are only partially involved in the environmental RNAi processes.
It is possible that the two sil genes act redundantly in the environmental RNAi processes, rescuing the marker gene RNAi suppression, the researchers pointed out, but modifications to the assay will be required to explore that hypothesis.
Going forward, the researchers anticipate using their assay system to evaluate additional candidate genes whose orthologs have been implicated in systemic RNAi in other organisms.
"Surveying various sets of genes in WCR with the assay system … followed by functional analyses for the genes identified through the assay, [would help approach] the molecular basis of WCR environmental RNAi," they concluded in the PLOS One paper. "Detailed knowledge of the molecules and mechanisms responsible for environmental RNAi will help determine an efficient way of utilizing RNAi for insect pest management."