Crop improvement frequently entails the transfer of genetic material from one organism to another in order to generate a desirable characteristic. Crops having these so-called “transgenes” include disease-resistant cotton and golden rice enriched with beta-carotene.
Plants, on the other hand, have a natural defensive response when foreign DNA is introduced into a host organism: they suppress or quiet the production of the new genetic material. The global agricultural improvement business is dealing with a multibillion-dollar problem known as “silencing,” a process that involves DNA methylation.
Keith Slotkin, Ph.D., member of the Donald Danforth Plant Science Center and associate professor, University of Missouri Columbia’s Division of Biological Sciences, has established a new understanding of how DNA methylation begins in the first place, or how the silencing of new and foreign genetic material is triggered in plants.
These findings were recently published in the scientific journal Nature Plants as An siRNA-guided ARGONAUTE protein leads RNA Polymerase V to induce DNA methylation.
The work of the Slotkin laboratory’s graduate students, postdoctoral associates, and technicians, all of whom are represented as authors, spans four years and has significant implications for decreasing the cost and effort required to produce transgenic crops.
“Gene silencing is a key bottleneck that is inhibiting plant improvement… no matter what new trait a plant biologist works on, they are going to have to fight against the tidal wave of gene silencing,” said Slotkin.
In most cases, breeders must start with thousands of plants to find the handful that expresses rather than repress the desired feature. This research helps crop breeders to avoid trait silencing from the start by understanding how and why DNA methylation happens.
Gene silencing is a key bottleneck that is inhibiting plant improvement… no matter what new trait a plant biologist works on, they are going to have to fight against the tidal wave of gene silencing.
Keith Slotkin
“One day we could start with three plants instead of thousands. All of the time and money that is usually put into producing a crop is slimmed down,” said Slotkin.
Another significant accomplishment of Slotkin and his team’s research was the development of a new model for gene silencing. According to one popular notion, a crucial protein called ‘RNA Polymerase V’ is found throughout the genome and scans different areas of genetic material for regions that need to be silenced.
The DNA methylation process begins once RNA Polymerase V finds a gene region to mute. However, the authors showed that the presence of short RNAs (which are essential for plant growth and development) recruits RNA Polymerase V to the gene or transgene, rather than the presence of RNA Polymerase V itself, which causes gene silencing.
“Our model is saying that small RNAs are driving RNA Polymerase V to the new location in the first place. If we get rid of the small RNA machinery, RNA Polymerase V doesn’t know where to go,” Slotkin described.
Furthermore, the Slotkin team’s techniques were just as valuable as their conclusions, and the researchers overcame a number of key technological challenges in the process. Gene silencing, for example, is usually examined as a cycle rather than as a single event.
As a result, the researchers had to pay close attention to the commencement of the process in the first generation of transgenic plants. “That’s a huge challenge,” Slotkin notes.
“We plant thousands of seeds that may have integrated a transgene. Sometimes we only get five plants back because they did not transform well. This isn’t enough, as we want a lot of tissue off of them in order to measure DNA methylation… and these experiments require biological replicates, so more tissue is needed, and the experiment needs to be done again.”
While growing and regrowing enough plants for such an experiment is a huge undertaking, “it is all worth it to be able to investigate the first generation of transgene silencing,” remarked Slotkin.
Meredith Sigman, a former graduate student at the Danforth Center, is the paper’s first author, while Andrea McCue, a former postdoctoral assistant in the Slotkin lab, is the paper’s last author.
The entire Slotkin research team is still looking into how gene silencing starts. They go into the fundamentals of how DNA methylation is initiated and how it might help overcome barriers in transgenic plant production.
“We are at a crop research institute like the Danforth Center to make the plant improvement process easier for everyone,” concluded Slotkin.
