Inside the Beetle: Understanding the Molecular Effects of RNAi-Based Crop Protection
RNA interference (RNAi) could provide new ways of controlling agricultural pests by targeting specific genes. But observing whether an insect survives is only part of the picture. My PhD research investigates what happens inside cabbage stem flea beetles after RNAi treatment, using long-read RNA sequencing to characterize the gene activity and transcript structure.
By PhD fellow Mathias Helmer Eskildsen
Protecting crops against insect pests is essential for maintaining agriculture production. At the same time, there is growing interest in developing more biosustainable crop protection methods that target pests while reducing unwanted effects on other organisms. RNAi uses small RNA molecules or long double-stranded RNA molecules to interfere with the expression of specific genes. If the targeted gene performs an essential biological function, interfering with its expression will affect development, physiology or survival of the pest.
But how does an insect respond when one of these genes is targeted?
One of the insects studied in my PhD project is the cabbage stem flea beetle (Psylliodes chrysocephala), an important pest of oilseed rape. We investigated small interfering RNAs (siRNAs) designed to target essential genes in the beetle.
One of these genes was Rpt4, a regulatory component of the 26S proteasome. The proteasome is involved in protein degradation and is essential for maintaining cellular function. Treatment with Rpt4-targeting siRNA led to significant mortality 4 days post-treatment. This showed a clear biological effect, but mortality alone does not tell us if the beetle died due to the target protein being silenced or if other processes were also affected. To what degree does the insect e.g. activate stress responses? Are other cellular processes altered? And are there changes in the RNA molecules produced from individual genes?
To address these questions, we turned to transcriptomics.
I used Oxford Nanopore Technologies (ONT) long-read sequencing. Unlike conventional short-read sequencing, which reads RNA converted into DNA (cDNA) including many short fragments that subsequently have to be reconstructed computationally, long-read sequencing can capture much longer sections of individual transcripts. Furthermore, ONT allows direct sequencing of RNA molecules in a single read without prior conversion into cDNA. This is useful because a single gene can produce several different RNA molecules through processes such as alternative splicing. These transcript variants can have different biological functions compared to the “clean script”, but they can be difficult to identify using short sequencing reads.
By analysing beetles treated with Rpt4-targeting siRNA and comparing them with beetles receiving a scrambled control sequence, we can investigate how RNAi treatment affects both gene expression and the structure of the transcripts being produced.
Thousands of previously undescribed transcripts
The long-read data also provided an opportunity to improve our understanding of the cabbage stem flea beetle transcriptome itself. The analysis identified more than 38,000 transcript isoforms originating from 26,078 genes. More than 10,000 of these genes were classified as novel relative to the existing annotation.
This is important because molecular analyses depend heavily on the quality of the available reference genome and its annotation. If genes or transcript variants are missing from the reference, biologically important responses may remain undetected.
The long-read data therefore provides two types of information at the same time. They allow us to investigate how the beetle responds to RNAi treatment, while also improving the molecular map that we use to interpret that response.
Connecting molecular responses with biological effects
The next step is to connect these different levels of information and understanding the sequence of events from exposure to siRNA, through changes in gene expression and transcript structure, to the physiological effects observed in the insect.
This is relevant not only for understanding whether an RNAi treatment works, but also for understanding how it works. Detailed molecular information can help identify the biological pathways affected by treatments and distinguish the intended RNAi response from broader physiological responses.
For emerging RNA based plant protection products, this type of mechanistic information can contribute to a more complete understanding of biological effects and provide knowledge relevant to their future assessment.
By combining insect bioassays with long-read transcriptomics, our work moves beyond observing the final outcome of RNAi treatment and begins to reveal the molecular processes leading to it.
Ultimately, understanding what happens inside the insect may help us better understand both the opportunities and the biological consequences of RNAi based crop protection.