Projects / Programmes
USING RNA INTERFERENCE AND SYSTEMS BIOLOGY APPROACHES FOR VALIDATION OF INSECTICIDE TARGETS IN COLORADO POTATO BEETLE GUTS
| Code |
Science |
Field |
Subfield |
| 4.06.05 |
Biotechnical sciences |
Biotechnology |
Plant biotechnology |
| Code |
Science |
Field |
| T490 |
Technological sciences |
Biotechnology |
| Code |
Science |
Field |
| 4.04 |
Agricultural and Veterinary Sciences |
Agricultural biotechnology |
Colorado potato beetle, Leptinotarsa decemlineata, dsRNA insecticides, RNAi, omics technologies, bioinformatics, RNA-seq, Next Generation Sequencing
Researchers (1)
| no. |
Code |
Name and surname |
Research area |
Role |
Period |
No. of publicationsNo. of publications |
| 1. |
29617 |
PhD Marko Petek |
Biotechnology |
Head |
2016 - 2017 |
0 |
Organisations (1)
| no. |
Code |
Research organisation |
City |
Registration number |
No. of publicationsNo. of publications |
| 1. |
0105 |
National Institute of Biology |
Ljubljana |
5055784 |
0 |
Abstract
The Colorado potato beetle (CPB) is an important agricultural pest. It is well known for its ability to rapidly evolve resistance to insecticides and is predicted to expand to the north of Europe due to the prospects of climate change. In the last decade double-stranded RNAs (dsRNAs) were shown to have the potential to become environmentally-acceptable insecticidal agents. These molecules activate the RNA interference (RNAi) mechanism that mediates a sequence-specific suppression of gene expression, also called gene silencing.
In CPB, gene silencing can be effectively triggered and spread systemically by ingestion of dsRNA. If the targeted gene silencing disrupts an essential life-sustaining process in CPB, the dsRNAs can act as an insecticide. The advantages of RNAi insecticides include high specificity for target species, low probability of resistance development and short environmental degradation period into nontoxic compounds. Crucial for the efficiency as well as the environmental safety of dsRNA insecticides is the selection of target genes.
This project's main objective is to identify and validate gene targets for CPB specific RNAi insecticides. To achieve that, the methodology for identification and validation of RNAi insecticide efficiency will be established at NIB. This will include a candidate gene selection procedure, establishment of dsRNA production methodology and the evaluation of dsRNAs' insecticidal potential in insect feeding trials. Additionally, a Next Generation Sequencing (NGS) RNA-seq analysis will be performed to explore the global changes induced by the RNAi insecticide on the CPB gut transcriptome. To explore how a RNAi insecticide changes the bacterial community structure in CPB guts, larval gut contents will be examined by NGS to obtain bacterial metagenomic profiles.
Available statistical tools will be used for evaluation of RNAi insecticide efficiency and already developed NGS pipelines for the analysis of RNA-seq data will be adopted, whereas for metagenomics data, a dedicated analysis pipeline will be established at NIB.
The project will contribute to science by:
- exploring the potential of selected candidate genes as new targets for RNAi insecticides,
- validating computationally-predicted gene functions in an agricultural pest,
- following the expression of selected target genes in different CPB tissues and life stages,
- studying the global transcriptional patterns by RNA-seq in CPB guts after RNAi insecticidal treatment,
- investigating the effect of an RNAi insecticide on CPB's gut metagenome.
In future, new discoveries based on this knowledge will lead to more sustainable agricultural practices. The developed gene silencing methodology could be adapted for other organisms and impact scientific disciplines beyond crop protection. Due to the prospects of RNAi technology in agriculture and biotechnology, this project can represent an opportunity for establishment of new companies that would commercialize the RNAi technologies.
This postdoctoral project will also be an opportunity to establish closer connections with other scientists working in the RNAi field and to join consortia for international project grant applications.
Significance for science
There are not many global transcriptomic studies of dsRNA responses in insects; most studies examined only the expression of target gene and sometimes also a handful of genes with high nucleotide similarity to the target. Even less common are the studies examining the effect of dsRNAs with no target gene in the treated organism i.e. non-specific response to dsRNA. We performed an RNA-seq to examine the effect of dietary intake of dsEGFP on the CBP gut tissue which is contributing to close this knowledge gap. With more similar studies in other insects we might be able to discover new players involved in the uptake of dsRNA and induction of RNAi mechanism. An even more important impact is expected from the results of the metagenomics study that examined dsRNA's affect on abundance of bacterial clades in the CPB gut. Confirmation of these results in other insects and ecological systems could open a new frontier of interaction studies for examples studies examining if spraying dsRNAs can alter plant epiphyte or soil microbiomes.
Significance for the country
We have established protocols at NIB that can be used immediately to test dsRNAs activity against herbivorous insects. Moreover, this gene silencing methodology can be adapted to test other RNAi-susceptible organisms, e.g. arthropods and nematodes, to either confirm specific gene functions or develop new, more sustainable pest or pathogen protection strategies. Biotechnologically produced compounds and bio-based green chemicals are among the current priorities of the Slovenia's Smart Specialisation Strategy. Due to the prospects of dsRNA production in agriculture and biotechnology, the results and expertise gained in this project is useful for companies that want to develop and commercialize new products based on RNAi. Procedures for the detection and quantification of dsRNA can be adapted and used for the purposes of environmental monitoring of dsRNA, which will become important when plant protection products using dsRNA appear on the market. This project also enabled the participation of Slovenian scientists in the COST Action dedicated to RNAi for crop protection. Through that the project helped establish close connections and collaborations with other research groups in the EU working in the perspective field of biotechnology.
Most important scientific results
Final report
Most important socioeconomically and culturally relevant results
Interim report,
final report
