Transgenes are fragments of DNA that are foreign to the genome that have been artificially integrated. Inserting foreign DNA into plants is the basis behind the success of modern molecular crop improvement. Though transgenesis often improves plant fitness through beneficial trait delivery, (such as herbicide and disease resistance) and delivering the reagents necessary for gene editing, plant cells regularly silence transgenes which hampers the speed of crop improvement progress. In this work, I addressed both design and molecular factors of transgenes that are important in early transgene silencing.

Transgene silencing has been historically investigated well after the process was triggered. Therefore, I investigated methods of early activation of transgene silencing, with the goal of avoiding early activation of transgene silencing. In Chapter 2, I used silencing-prone transgenes to understand the importance of the quality of integration. Transgenes are typically integrated into the genome in a multi-copy, often fragmented manner. I found that high quality integrations can outweigh poorly designed transgenes, even over several generations. This approach of identification is time-consuming and laborious, so in Chapter 3 and 4 I investigated the pathway of silencing to prevent transgenes from transitioning from early silencing to late stage silencing. I identified both highly important and non-important factors by way of gene editing technologies. Together these findings demonstrate approaches to mitigate the likelihood of transgene silencing.

Transgene silencing requires transcription, but to this point, it has been unclear what aspects of transcription are relevant for silencing. In Chapter 6, I found that two portions of a transgene promoter can independently trigger transgene silencing. In transgenes with the same predicted transcript, through a combination of machine learning and novel long-read RNA sequencing in Chapter 7, I found that the promoter can change the types of transcripts that are produced. And in Chapter 5, I determined that other than the standard mRNA polymerase RNA Polymerase II, RNA Polymerase III transcripts can also be recognized in the initiation of silencing. These transcriptional findings provide the potential roles of the promoter for importance in the initiation of silencing.

Overall, this research advances the understanding of transgene silencing. From genetic considerations to early transcriptional identifiers, this work contributes to new hypotheses of the molecular progression of transgene silencing as well as a deeper understanding of evading silencing to promote quicker plant improvement processes. My thesis tells the future generation of plant genome engineers to focus on the quality of the integration event and the promoter of the transgene transcript.

Select Publications

Liu P., Panda K., Edwards S.A., Swanson R., Yi H., Pandesha P., Hung Y.-H., Klaas G., Ye X., Collins M.V., Renken K.N., Gilbertson L.A., Veena V., Hancock C.N., Slotkin R.K. Transposase-assisted target-site integration for efficient plant genome engineering (2024) Nature, 631 (8021), pp. 593 - 600 DOI: 10.1038/s41586-024-07613-8

Edwards S.A., Slotkin R.K. Broken up but still living together: how ARGONAUTE’s retention of cleaved fragments explains its role during chromatin modification (2023) Genes and Development, 37 (3-4), pp. 69 - 71. DOI: 10.1101/gad.350424.123

Doctoral Program Committee

• Dr. R. Keith Slotkin, Chair
• Dr. Jim Birchler
• Dr. J. Chris Pires
• Dr. Blake C. Meyers