But when foreign DNA is introduced into a host plant, its natural defences kick into to repress, or silence, the expression of that genetic material so it does not get converted into unfamiliar proteins or other molecules.
This silencing is a process known as DNA methylation, a biological process used by cells to control gene activity.
It presents a multimillion-dollar problem to scientists. Finding solutions means understanding how DNA methylation starts and how gene silencing is triggered in plants.
Scientists at the University of Missouri, Columbia have now established a new understanding of the problem. Their research results have substantial implications for reducing the cost and time needed for producing improved, transgenic crops.
“(Scientists) have known for decades that some regions of a plant genome express really well and make proteins (such as genes),” said Keith Slotkin, associate professor with the division of biological sciences and member of the Donald Danforth Plant Science Center. “Other regions do not express well. When we try to engineer gene expression with transgenes, they often get stuck in this not-expressed-well category, or somewhere between. We wanted our added traits and transgenes to express well for generations, but this lack of good expression leads to significant expense and time added into the product development pipeline.”
He said he hopes his group’s research can shorten the development time between concept and crop.
Slotkin said in a news release that, no matter what new trait a plant biologist works on, they are going to have to fight against the tidal wave of gene silencing.
A key event in the initiation of transgene silencing by the plant is the recruitment of a protein called ‘RNA Polymerase V (Pol V)’. Pol V is present across the genome and its job is to basically keep an eye on different regions of genetic material that need silencing. Once identified, DNA methylation starts.
However, the researchers have discovered that it is not only the presence of Pol V that triggers gene silencing but also the work of small RNAs critical to plant growth that drive Pol V to the focus gene or the newly introduced transgene.
Slotkin said that plants are silencing transgenes because they look like things called transposable elements, or TEs. A transposable element is a DNA sequence that can change its position within a genome.
“TEs are evolutionarily ancient but it is important that the plant identify which sequences are TEs and target them for silencing,” he said. “We know that these same TE silencing pathways are working to silence the transgenes so the future goal will be to design transgenes that the plant does not see as TEs but sees as genes.”
In the process of understanding how gene silencing actually starts, the research team needed to do everything in the first generation of the transgenic plants to watch the beginnings of the process. The study plant species they used was Arabidopsis thaliana.
“If you have one in 1,000 seeds that obtained the transgene, you can select for this one seed by using an antibiotic-resistant or herbicide-resistant gene within the transgene,” he said. “This gene would lie next to the gene that you are trying to express in the transgene design (two genes total, yours plus the resistance gene). Then you can use the herbicide to kill the 999 plants, leaving the one plant as the only one still growing. This is easy and routine.”
He said the tough part is that, once you have 10, 20 or hundreds of these plants that you know had each received the resistance gene, you need to determine which of them have a full-length transgene insertion that expresses well both now and into the future.
“That is a lot of work in the lab, because you are screening for a rare event in which the plant’s normal response to silence TEs, which has evolved over millions of years, has been successfully tricked and is not being triggered by the insertion of the new transgene. Sometimes this is just one in 1,000 and it takes a lot of effort to find that single one.”
In the lab, the team planted thousands of seeds that may have integrated a transgene. But if only a few plants showed results the experiment needed to be repeated to accumulate enough transgene tissue in order to measure DNA methylation.
While it is a monumental amount of work to grow and regrow enough plants, Slotkin said it is all necessary in order to be able to investigate the first generation of transgene silencing.
He said that the goal is not to remove Pol V or any of the components of the silencing machinery from the genome. While the transgene will express itself in a desired way, any removal of the Pol V mechanism that evolved to restrain transposable elements could jump around and mutate the rest of the genome.
“Crop production requires uniformity, and these new mutations just can’t be tolerated,” said Slotkin. “The goal is to engineer transgenes that don’t trigger this process in normal plants. We are going to make transgenes that are resistant to Pol V and (able to) express at their highest potential.”
The research was published in the journal Nature Plants.
