Forum for Science, Industry and Business

Sponsored by:     3M 
Search our Site:

 

Yellow Biotechnology: Using plants to silence insect genes in a high-throughput manner

02.02.2012
Using virus vector-mediated RNAi enables scientists to rapidly study the function of insect genes

Yellow Biotechnology refers to biotechnology with insects − analogous to the green (plants) and red (animals) biotechnology. Active ingredients or genes in insects are characterized and used for research or application in agriculture and medicine.


Nicotine-resistant larvae of the Tobacco hornworm Manduca sexta have become a new tool for investigating unknown gene functions in Lepidoptera – thanks to a novel RNAi-based procedure. Courtesy of Jan-Peter Kasper

Scientists at the Max Planck Institute for Chemical Ecology in Jena, Germany, are now using a procedure which brings forward ecological research on insects: They study gene functions in moth larvae by manipulating genes using the RNA interference technology (RNAi). RNAi is induced by feeding larvae with plants that have been treated with viral vectors. This method called “plant virus based dsRNA producing system” (VDPS) increases sample throughput compared to the use of genetically transformed plants.

Natural toxins against herbivores

More than 200,000 insects species are herbivores. They depend on plants for food and have adapted their metabolism accordingly in the course of evolution to render plant defenses, such as the toxins plants produce to fend off herbivores, ineffective. The operating instructions of these detoxification processes are coded in different genes. Insects have evolved an enormous diversity of adaptation mechanisms; they colonize most habitats on this planet – which makes them interesting research objects in ecological studies. Which insect species attack which plants species? Which toxins or signaling substances are involved? Has the insect species adapted to one specific plant species or is it a food generalist? Interesting for agriculture: Which genes allow particular pest insects, such as the pollen beetle Meligethes aeneus or the Western corn rootworm Diabrotica virgifera virgifera, to be so destructive to crop plants? Knowing these detoxification genes and switching them off with the consequence that plant toxins are no longer effective, is currently a research subject in plant breeding. First success stories have already been reported – thanks to the use of RNAi technology.

Scientists at the Max Planck Institute for Chemical Ecology examined a well-known plant toxin: nicotine. Plants of the species Nicotiana attenuata (coyote tobacco) produce nicotine as a defensive substance against herbivores. However, it does not have any toxic effects on their worst enemy: larvae of the tobacco hornworm Manduca sexta. The insect is resistant against this alkaloid; genes that encode nicotine-catabolizing enzymes may be responsible for its resistance. These so called CYP genes are involved in the formation of cytochrome P450 enzymes; the expression of some of these genes is increased as soon as the insect larvae are exposed to nicotine in their food. Ian Baldwin and his team identified the DNA sequences of CYP genes in Manduca sexta and were able to switch off these genes using RNAi technology, but expressed in the plant.

Using plants to silence insect genes

RNA interference (RNAi) is triggered by the production of double-stranded RNA (dsRNA) comprising about 300 base pairs in the cells of tobacco plants. If larvae feed on these plant, the RNA is released in the insect gut. In the experiments, the dsRNA harbored the sequence of the insect gene,CYP6B46, a special cytochrome P450 oxidoreductase specific for Manduca sexta larvae. In a next step, the dsRNA was enzymatically broken down into smaller RNA segments; a special enzyme complex called RISC (RNA-induced silencing complex), which carries several of these RNA segments, specifically binds to the messenger RNA (mRNA) of the CYP6B46 gene and disassembles the mRNA in such a way that the cytochrome P450 enzyme cannot be produced anymore. “We were impressed by the high specificity of these RNAi experiments. The analysis of mRNA transcripts of closely related CYP6 genes revealed that only the CYP6B46 gene was silenced. This means that there was no collateral damage from the procedure: the gene silencing worked on only one targeted gene,” says Ian Baldwin.

The use of additional CYP RNAi probes revealed further interesting results: Young caterpillars which had ingested dsRNA of the CYP4M3 gene gained significantly less weight within 14 days in comparison to larvae reared on control plants – very likely a consequence of the nicotine and its toxic effect which had been restored by switching off the CYP gene. The RNAi experiments had been conducted using plant viral vectors. Unlike genetically transformed tobacco plants in which CYP dsRNA is produced constitutively, the virus vector-based technique provides dsRNA transiently produced in wildtype tobacco plants. Both methods worked well but the “plant virus-based dsRNA producing system” (VDPS) allows for a throughput of RNAi samples that is four times faster. Many unknown functions of different insect genes involved in the adaptation of insects to their environment can now be analyzed using the VDPS technique.
However, it is still unclear how the individual steps in the RNAi mechanism – from producing dsRNA in the plant cell via their uptake in the insect gut to the silencing of the detoxification genes – are accomplished to induce a maximum effect. One experiment provided some interesting information: If the enzymatic step which dices dsRNA into small fragments is inhibited in the experimental plants, the amount of transcripts of the detoxification gene was reduced even further. Therefore the plant mediated RNAi procedure may be more effective, if the caterpillars ingest complete dsRNA instead of smaller diced RNA segments. [JWK, AO]

Original Publication:
Kumar, P., Pandit, S.S., Baldwin, I.T.: Tobacco Rattle Virus vector: A rapid and transient means of silencing Manduca sexta genes by plant mediated RNA interference. PLoS ONE, DOI: 10.1371/journal.pone.0031347

Further Information:
Prof. Dr. Ian T. Baldwin, MPI for Chemical Ecology, Jena
Tel.: 03641 - 57 1100, baldwin@ice.mpg.de
Picture Material:
Angela Overmeyer M.A., Tel. 03641 - 57 2110, overmeyer@ice.mpg.de
or Download via http://www.ice.mpg.de/ext/735.html

Dr. Jan-Wolfhard Kellmann | Max-Planck-Institut
Further information:
http://www.ice.mpg.de

More articles from Life Sciences:

nachricht Protein linked to cancer acts as a viscous glue in cell division
08.07.2020 | Rensselaer Polytechnic Institute

nachricht Enzymes as double agents: new mechanism discovered in protein modification
08.07.2020 | Westfälische Wilhelms-Universität Münster

All articles from Life Sciences >>>

The most recent press releases about innovation >>>

Die letzten 5 Focus-News des innovations-reports im Überblick:

Im Focus: Excitation of robust materials

Kiel physics team observed extremely fast electronic changes in real time in a special material class

In physics, they are currently the subject of intensive research; in electronics, they could enable completely new functions. So-called topological materials...

Im Focus: Electrons in the fast lane

Solar cells based on perovskite compounds could soon make electricity generation from sunlight even more efficient and cheaper. The laboratory efficiency of these perovskite solar cells already exceeds that of the well-known silicon solar cells. An international team led by Stefan Weber from the Max Planck Institute for Polymer Research (MPI-P) in Mainz has found microscopic structures in perovskite crystals that can guide the charge transport in the solar cell. Clever alignment of these "electron highways" could make perovskite solar cells even more powerful.

Solar cells convert sunlight into electricity. During this process, the electrons of the material inside the cell absorb the energy of the light....

Im Focus: The lightest electromagnetic shielding material in the world

Empa researchers have succeeded in applying aerogels to microelectronics: Aerogels based on cellulose nanofibers can effectively shield electromagnetic radiation over a wide frequency range – and they are unrivalled in terms of weight.

Electric motors and electronic devices generate electromagnetic fields that sometimes have to be shielded in order not to affect neighboring electronic...

Im Focus: Gentle wall contact – the right scenario for a fusion power plant

Quasi-continuous power exhaust developed as a wall-friendly method on ASDEX Upgrade

A promising operating mode for the plasma of a future power plant has been developed at the ASDEX Upgrade fusion device at Max Planck Institute for Plasma...

Im Focus: ILA Goes Digital – Automation & Production Technology for Adaptable Aircraft Production

Live event – July 1, 2020 - 11:00 to 11:45 (CET)
"Automation in Aerospace Industry @ Fraunhofer IFAM"

The Fraunhofer Institute for Manufacturing Technology and Advanced Materials IFAM l Stade is presenting its forward-looking R&D portfolio for the first time at...

All Focus news of the innovation-report >>>

Anzeige

Anzeige

VideoLinks
Industry & Economy
Event News

Contact Tracing Apps against COVID-19: German National Academy Leopoldina hosts international virtual panel discussion

07.07.2020 | Event News

International conference QuApps shows status quo of quantum technology

02.07.2020 | Event News

Dresden Nexus Conference 2020: Same Time, Virtual Format, Registration Opened

19.05.2020 | Event News

 
Latest News

Shock-dissipating fractal cubes could forge high-tech armor

08.07.2020 | Materials Sciences

Scientists use nanoparticle-delivered gene therapy to inhibit blinding eye disease in rodents

08.07.2020 | Health and Medicine

'Growing' active sites on quantum dots for robust H2 photogeneration

08.07.2020 | Physics and Astronomy

VideoLinks
Science & Research
Overview of more VideoLinks >>>