Researchers at the Martin Luther University Halle-Wittenberg (MLU) have developed RNA-based active agents that appear to reliably protect plants against cucumber mosaic virus (CMV), the most common virus in agriculture and horticulture. Developed to help fight the virus by directing the plant’s natural defenses in the right direction the new molecules, known as efficient double-stranded RNA (edsRNA), demonstrate a broad spectrum effect supporting the plant’s immune system in combating the virus. The team’s laboratory experiments found that 80–100% of treated plants survived CMV infection with a high viral load.
Research lead Sven-Erik Behrens, PhD, at the Institute of Biochemistry and Biotechnology at MLU, and colleagues, reported their findings in Nucleic Acids Research, in a paper titled, “A new level of RNA-based plant protection – dsRNAs designed from functionally characterized siRNAs highly effective against cucumber mosaic virus.” In their paper, the team concluded, “Overall, the results of this study significantly expand the potential for more efficient use of RNA in biological crop protection.”
Virus-induced plant diseases remain a major problem in agriculture that is “recently exacerbated by global trade and climate change,” the authors wrote. Pesticides represent the most common methods for controlling viral infections, but such agents may have non-specific effects on other organisms and can be harmful to humans. “Urgently needed alternative crop protection methods should not only be environmentally sustainable, but also specific, i.e., effective only against a specific target pathogen, and adaptable to the evolution of the pathogen,” the team noted. “RNA-mediated crop protection increasingly becomes a viable alternative to agrochemicals that threaten biodiversity and human health.”
Cucumber mosaic virus is a particularly devastating virus for crops. About 90 species of aphids transmit the virus, which affects more than 1,200 plant species, including numerous agricultural crops such as squash, cucumbers, cereals, and medicinal and aromatic plants. Infected plants are easily identified by a characteristic mosaic pattern on their leaves. Once infected, the plants fail to thrive and their fruits cannot be sold. There are currently no approved agents against CMV.
When a virus infects a plant it uses the plant’s cells as a host. The virus multiplies via its genetic material in the form of RNA molecules in the plant cells. Once injected, these foreign RNA molecules trigger an initial response from the plant’s immune system. Special enzyme scissors recognize and cut the viral RNA molecules. This process produces small interfering RNAs (siRNAs), which spread throughout the plant and trigger a second step of the immune response. The siRNA molecules bind to special protein complexes and guide them to the RNA molecules of the virus. Once there, the proteins begin to break down the harmful RNA molecules of the virus by converting them into harmless, degradable fragments.
“In general, this defense process is not very effective,” said Behrens. “A viral infection produces many different siRNA molecules, but only a few have a protective effect.” Through their newly reported work, the team developed a method to identify functionally effective siRNA molecules, termed esiRNAs, that are highly efficient in the process. “Using an in vitro screen that reliably identifies esiRNAs from siRNA pools, we identified esiRNAs against cucumber mosaic virus …,” the authors explained.
They were able to combine several of these esiRNA molecules into efficient double-stranded RNA molecules (edsRNAs) that could be applied to plants. These edsRNAs act as a package that, soon after entering the plant cells, is broken down into a large number of highly effective siRNA molecules that attack the virus at different sites which can significantly increase the protective effect. “RNA viruses such as the cucumber mosaic virus are dangerous because they can evolve rapidly,” Behrens said. “In addition, the genetic material of this virus is made up of three separate parts, which can get mixed up, further increasing the chance of new mutations. To achieve maximum protection against the virus, our active ingredients target different parts of the genome.”
The team conducted numerous laboratory experiments on the model plant Nicotania benthamiana and was able to show that edsRNA-based active agents reliably protected against cucumber mosaic virus. “The plants in our experiments were infected with a very high viral load: all of our untreated plants died,” reported Behrens. In contrast, 80–100% of the treated plants survived. “… optimal protection was achieved with newly designed multivalent ‘effective dsRNAs’ (edsRNAs), which contain the sequences of several esiRNAs and are preferentially processed into precisely these esiRNAs,” the authors stated. “The esiRNA components can attack one or more target RNAs at different sites, be active in different silencing complexes, and provide cross-protection against different viral variants—important properties for combating rapidly mutating pathogens such as CMV.”
The team has in addition optimized the process of screening for efficient siRNAs and can adapt the procedure to target new viral mutations within two to four weeks. “Time is an important factor: when a new virus variant emerges, we can very quickly modify the active agent accordingly,” Behrens said. The approach may also be applied to other pathogens and pests. “The success of our approach, eNA screen followed by edsRNA design from the identified esiRNAs, promises similar success for other plant pathogens, most of which have significantly less plasticity than CMV,” the authors stated.
Until now, the substances have been administered manually in the laboratory, either by injection or by rubbing them into the plant leaves. The team is working with pharmacist and drug delivery specialist Karsten Mäder, PhD, a professor at MLU to make the RNA-based substances more durable and easier to apply to plants. For example, they could be sprayed on.
At the same time, the researchers are planning field trials to test the RNA-based substances under real conditions. “It will now be important to further test and improve esiRNA and edsRNA actives in combination with suitable formulations in agricultural applications, i.e., greenhouse or field trials,” the investigators wrote in their report. They are also talking to companies about future industrial production. In addition, potential new crop protection products still have to go through an approval process, so it will be some time before a product to combat cucumber mosaic virus enters the market. “However, we are convinced that our approach is feasible. The first crop protection product with an RNA-based active ingredient was recently approved in the USA,” said Behrens.