NEW YORK (GenomeWeb) – Researchers from the Max Planck Institute of Molecular Plant Physiology this week published a report describing a method to express long double-stranded RNAs, designed to silence essential genes in a key agricultural pest, only in a specific part of plants where the RNAi molecules cannot be processed into less effective siRNAs.
Using the method, the investigators could fully protect the crop from Colorado potato beetles (CPB), opening the door for a new strategy to control an insect that has developed resistance to all major insecticide classes.
Although RNAi is widely explored for its therapeutic potential, it has steadily been gaining ground as a technology with agricultural applications. Already, a number of RNAi-modified food plants have cleared regulatory hurdles in the US, and just this month the US Department of Agriculture deregulated two apple varieties that use the gene-silencing technology to suppress an enzyme that causes the fruit to brown.
Meanwhile, another kind of application of the technology involving so-called plant-incorporated protectants (PIPs) is following close behind. This approach involves modifying a plant to express dsRNAs targeting an essential pest gene. When the pest feeds on the plant, it also ingests the RNAi molecules, triggering gene silencing and killing the insect.
However, it has been shown that dsRNAs of at least 60 base pairs are required for a robust RNAi effect in insects. But plants' endogenous RNAi system processes most long dsRNAs into siRNAs, resulting in weak gene silencing and incomplete pest lethality.
"This has been a real constraint in the development of pesticidal RNAi," Jonathan Lundgren, a US Department of Agriculture entomologist who is not involved with the Max Planck team, said. As a result, the first generation of RNAi PIP crops have been combining the gene-silencing technology with other forms of pest control.
One such example is Monsanto's Smart Stax Pro, a strain of corn that expresses dsRNA targeting a gene essential to the Western corn rootworm, along with two widely used toxic proteins derived from the bacterium Bacillus thuringiensis.
Looking to address this issue, Max Planck's Jiang Zhang and colleagues looked to chloroplasts, a type of plastid responsible for photosynthesis and derived from formerly free-living cyanobacteria. Because these cyanobacteria lack an RNAi pathway, they hypothesized that dsRNA expressed from the chloroplast genome would not be diced into siRNAs and would retain its gene-silencing potential.
For their experiments, they targeted the CPB, which consumes the leaves of potatoes and other solanaceous plants. The insect has no known natural enemies in many parts of the world and, given its resistance to existing pesticides, is responsible for significant damage to these crops worldwide.
As reported in Science, they first tested this approach in tobacco, transforming the plant's plastid genome with three different types of dsRNA constructs — one in which the dsRNA is generated via transcription from dual promoters (ptDP); one in which the dsRNA is produced from two convergent promoters but with each strand flanked by sequences forming stem-loop secondary structures for stability (ptSL); and one in which hairpin RNAs are produced by transcription of two transgene copies arranged as an inverted repeat (ptHP).
All three types of expression constructs produced substantial amounts of long dsRNAs in the plants' leaves, but the scientists selected ptDP for subsequent testing since it performed equally well with ptSL and because ptHP, although robust, in some cases produced shorter than expected transcripts.
Zhang and his collaborators then sought to identify the best target genes for CPB control, selecting two essential ones: ACT, which encodes the cytoskeletal protein beta-actin, and SHR, which encodes a component of cellular machinery known as endosomal sorting complex required for vesicle transport. Notably, SHR — also known as Snf7 — is the gene targeted by the RNAi component in Monsanto's corn rootworm product.
The investigators designed ptDP cassettes to express dsRNAs against either ACT, SHR, or both and introduced them into the plastid genome of potato plants.
Although all of the transgenic plants were phenotypically indistinguishable from wild-type plants in regards to growth and tuber production, Northern blot analysis revealed that accumulation levels of ACT dsRNA were significantly higher than levels of SHR or ACT/SHR dsRNA.
Zhang told GenomeWeb that the reasons behind this effect are unclear, but that he suspects the higher GC content of the ACT dsRNA may have given these molecules greater stability than their counterparts.
To test the pest-control potential of their approach, the researchers provided detached leaves from the transgenic plants to first-instar CPB larvae and found that it resulted in high mortality, with the ACT dsRNA-expressing plants causing 100 percent mortality within five days — an effect confirmed to be the result of RNAi gene silencing.
In contrast, the SHR plants led to only 70 percent mortality in CPB larvae after nine days, which correlates to the lower levels of dsRNA accumulation in the plant. "Consequently, testing other fragments of the SHR gene seems an appropriate future strategy to improve the insecticidal efficiency of SHR dsRNA-expressing transplastomic plants," the Max Planck team wrote.
To assess the potential for CPB resistance to the transgenic plants, the scientists studied the leaf area consumed by both larvae and adult insects, finding little to no evidence of visible consumption due to the cessation of larval feeding after 24 hours.
Whole plants were also exposed to second-instar larvae, which are generally less sensitive to insecticides than first-instar larvae.
They found 17 percent survival after five days "presumably due to the initial larval growth and development on wild-type leaves," according to the Science paper. "However, the larvae grew very poorly after transfer to the transplastomic plants, and the damage they caused to the leaves was very small."
Further, under normal conditions CPB larvae hatch and feed on the same plant and therefore would not be expected to have a wild-type diet prior to feeding on the RNAi-expressing plant in field conditions.
Overall, the results point to the potential of using dsRNA to control an agriculture pest when the target of transgenesis is shifted away from the nucleus and onto the plastid, the researchers concluded.
Developing the approach for commercial applications, however, would likely require the involvement of a commercial partner given the extent of additional development and testing that would be needed, Zhang noted.
Whether any company would be interested, however, is unclear.
At least one company, Monsanto, is actively pursuing an RNAi-based method of CPB control, and earlier this year announced that this program had moved out of the discovery stage and into formal development.
Unlike its corn rootworm product, Monsanto's CPB effort involves a topically applied dsRNA treatment that is sprayed on like standard pesticides. As Robert McCarroll, the firm's vice president of global chemistry technology, told GenomeWeb earlier this year, developing transgenic traits is expensive and therefore reserved for large-acre row crops such as corn.
But for those larger crops, the Max Planck team's method may have a future.
"For pesticidal RNAi to take off, it's going to have to be very effective at killing the target insects," Lundgren said. "If this raises that level of effectiveness, it may be a future direction that is pursued more avidly."