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Genes in silence

Molecular tool based on the interfering RNA technique may aid in combating agricultural pests

The agricultural sector has a new biotech tool to combat the agricultural pests causing enormous crop losses worldwide. The American multinationals Monsanto (recently acquired by the German corporation Bayer) and DowDuPont obtained approval last year for a transgenic seed made using the technique of gene silencing by RNA (ribonucleic acid) interference, or simply RNAi, for commercial use in the United States. Developed as an insecticide, it was created to control the American corn rootworm (Diabrotica virgifera), the larval phase of a beetle that is the principal threat to US corn plantations. It’s the first time that RNAi molecules are being used in the fight against field pests.

In Brazil, Tropical Melhoramento & Genética (TMG), a company based in Paraná and specializing in genetic improvement of soybean and cotton, hopes to have an RNAi molecule ready this year for use against the neotropical brown stink bug (Euschistus heros), a significant oilseed pest. The technology is being developed by researchers at the University of Campinas (UNICAMP). The Brazilian Agricultural Research Corporation (EMBRAPA) has also created a transgenic bean with the RNAi technique in order to control a virus known as golden mosaic, which attacks this bean variety. Although already approved since 2011 by the National Biosafety Technical Commission (CTNBIO) of the Brazilian Ministry of Science, Technology, Innovation and Communications (MCTIC), the product has not yet been commercialized.

Created in the lab, RNAi molecules can neutralize the action of a target gene in any organism—hence the term gene silencing. Its mechanism of action is similar to that which occurs naturally in all living things when fighting viral attacks. When this occurs, the body’s defenses try to neutralize the attacker’s action by silencing its genes. Similarly, RNAi technology causes the cell to attack the product of its own gene, as if it were destroying viral genetic material, in a process similar to that of an autoimmune response. In agricultural use, RNAi is programmed to inactivate specific genes in pests and pathogens that are associated with processes essential to their survival.

The discovery that the RNA interference process could be used to benefit the human body earned researchers Andrew Fire and Craig Mello of the Carnegie Institution of Washington the 2006 Nobel Prize in Physiology or Medicine. The finding was promising because it revealed a new way to assume control of the cell, opening up a wide range of applications. Since then, the tool has been applied in basic research to study gene function, and is seen as promising in the field of medicine for the treatment of genetic diseases [see Pesquisa FAPESP issue no. 133].

To understand how this new technology works one must review the processes of cell biology, specifically the mechanism of gene expression. Genes are segments of the DNA molecule that control the metabolic functions of cells, primarily through the production of proteins. “Our genetic information is all stored in our DNA. To give life and utility to this information, the DNA must initially be transcribed into RNA. Then, this messenger RNA is translated into proteins. This is the basic process of gene expression,” explains biologist Alexandre Garcia, TMG’s research manager.

As the name makes clear, the synthetic RNAi molecule interferes in the process of translating messenger RNA into protein, fragmenting it. It intercepts and destroys the cellular information carried by RNA within the cell before it can be processed and originate proteins. Thus, the process of gene expression that depended on the data contained within that RNA will no longer occur.

Pioneering corn
The transgenic corn launched by Monsanto and DowDuPont has been dubbed DvSnf7 internally, and its trade name is SmartStax. The alphabet soup moniker is formed from the initials of the caterpillar Diabrotica virgifera and the name of the target gene, Snf7. When the insect feeds on the plant, the RNAi molecule enters its organism and silences the Snf7 gene, preventing the production of a protein that is vital for the larvae’s tissue function. This results in death for the insect, known as “the US$1 billion beetle” because of the enormous damage it’s done to American agriculture. Brazilian chemist Renata Bolognesi joined the Monsanto team that created transgenic corn.

“The application of RNAi technology in agriculture is very promising,” says Henrique Marques-Souza, an agricultural engineer at the Institute of Biology (IB) at UNICAMP and leader of a national research group on gene silencing. He specialized in the subject during his doctoral studies at the University of Cologne, in Germany, completed in 2007, and during a postdoctoral fellowship at the University of California, Berkeley, USA, conducted between 2008 and 2010. When he returned to Brazil, he began teaching at UNICAMP, where he created the Brazilian Laboratory on Silencing Technologies (BLAST) and launched his research on gene silencing for agriculture.

In 2012, Marques-Souza and fellow agricultural engineer Antonio Vargas de Oliveira Figueira, from the Center for Nuclear Energy in Agriculture of the University of São Paulo (CENA-USP), set up a sequence database for the tomato leafminer (Tuta absoluta) and performed gene silencing assays for several of the insect’s genes. “This work gave our group visibility. Today, we can develop RNAi technology for virtually any pest on any crop,” the UNICAMP researcher states. Last year, the technique was licensed to TMG by Inova, UNICAMP’s innovation agency.

The biggest challenge to creating an insecticide using RNAi for topical application is the molecule’s reduced stability in the environment

The first product the company will develop using the new tool will be aimed at controlling the neotropical soybean bug, one of the greatest threats to Brazilian soybean crops. “We hope to have selected the best RNA molecules by the end of the year. All the work will be done experimentally and through controlled studies,” says TMG’s Garcia, noting that the road to commercializing these molecules is long, and depends both on the technology used to promote their application and the outlook for regulatory approvals.

Marques-Souza observes that there are two ways to get RNAi molecules into contact with pests: by creating transgenic plants, such as those of Monsanto and DowDuPont with the DvSnf7 corn, or by developing insecticidal products. This is the method TMG has chosen, with plans to create a product for topical application, such as by spraying. “In addition to reducing the time it takes to produce a transgenic plant, topical RNAi can be considered a natural product because it is a biological molecule that doesn’t cause genetic changes in the crop or in the target organism,” explains Marques-Souza.

However, creating an insecticide that uses RNAi isn’t trivial, and there are several obstacles to be overcome. “These molecules degrade rapidly in the natural environment, which makes it difficult to create a product for crop application. That’s why there are already studies that seek to employ nanotechnology resources to protect the RNAi molecule [see Pesquisa FAPESP issue no. 266] and increase its stability,” notes biologist Fernando Luís Cônsoli, from the Department of Entomology and Acarology of the Luiz de Queiroz College of Agriculture (ESALQ-USP), in Piracicaba, São Paulo.

The nanoencapsulation of RNAi molecules is the thesis research project of biologist Cyro von Zuben, an IB-UNICAMP doctoral student supervised by Marques-Souza. By the end of the year, working together with São Paulo State’s Institute for Technological Research (IPT) and the National Nanotechnology Laboratory (LNNANO) of the Brazilian Center for Research in Energy and Materials (CNPEM), tests with clay nanoparticles should begin with several target species under study in the BLAST laboratory. In Australia, one research group recently published an article describing the use of nanoparticles that guarantee the integrity and delivery of RNAi for 30 days.

Cisioli also warns that the process of selecting RNAi molecules must consider the risk of the technology affecting the plant itself or nontarget organisms, including humans. According to Marques-Souza, this is a genuine concern, but one that can be overcome. “When selecting a target gene to attack a pest organism, we have to be careful to choose a gene sequence that is not complementary to a plant gene or any other organism that feeds on it. If this precaution is taken, gene silencing will only occur in the target organism,” he says. “The assurance that RNAi will have no effect on humans will only come with further study.”

Alexandre Garcia adds, “The technology is based on RNA sequences. All living things have sequences in common, but each species has numerous unique sequences that differentiate them from other species.” Today, with the amount of information and genome databases available, it is possible to find sequences that are unique to the target species and to design molecules that will act only on those sequences. “In this way, the whole process becomes specific and unique to the target organism,” he adds.

CTNBIO standard
An important step in regulating the use of topical RNAi in Brazil was taken at the beginning of the year when CTNBIO published a regulation defining the technology as a method that does not use transgenics, and classifying it as Plant Breeding Innovation (TIMP). Thus it will not necessarily be subject to the same approval criteria as those required for genetically modified organisms.

“The process for developing a transgenic plant and going through all the global regulatory mechanisms to approve its commercial cultivation is extremely expensive and time-consuming, and could exceed $100 million and 15 years,” says Garcia. “In the case of the neotropical brown stink bug, the use of RNAi technology as a topical application product is more promising. This technology should be available to Brazilian farmers within a few years.”

RNAi for control of Tuta absoluta in tomato cultivation (no. 11/12869-6); Grant Mechanism Regular Research Grant; Principal Investigator Antônio Vargas de Oliveira Figueira (USP); Investment R$153,800.39.

Scientific articles
PARTHASARATHY, R. et al. Physiological and cellular responses caused by RNAi-mediated suppression of Snf7 orthologue in western corn rootworm (Diabrotica virgifera virgifera) larvae. PLOS ONE. Online. Jan. 18, 2013.
CAMARGO, R. A. et al. RNA interference as a gene silencing tool to control Tuta absoluta in tomato (Solanum lycopersicum). PeerJ. Online. Dec. 15, 2016.