Abstract

Plant transposable elements and transgenes are continuously targeted for transcriptional repression via the pathway of RNA-directed DNA methylation (RdDM). Although perpetual RdDM of the endogenous genomic loci has been well studied in the field, much less is known about the de novo RdDM pathway, the pioneering round of DNA methylation responsible for triggering and initiating silencing of new transgenes. Despite being two distinct pathways, both perpetual and de novo mechanisms of RdDM require the production of small interfering RNAs (siRNAs) and the key proteins of the ARGONAUTE4 (AGO4) clade. However, the order and likely mechanism of recruitment of AGO4 to its chromatin targets is different between the two RdDM pathways. Here, the functional role of AGO4 and its catalytic activity was investigated during siRNA loading, recruitment, and establishment of DNA methylation during the pioneering round of RdDM. The data in this thesis demonstrated that the AGO4-clade proteins are essential for de novo RdDM, but AGO4’s catalytic activity is not necessary. The data here also challenged the prevailing paradigm that RdDM targeting is primarily driven by siRNA accumulation and AGO4 loading. Together, the data demonstrated that AGO4's defining role in de novo RdDM is as a molecular scaffold that couples siRNA target recognition with chromatin engagement and assembly of the de novo silencing machinery.

Committee Chair

Richard Slotkin

Committee Members

Craig Pikaard; Dmitri Nusinow; Hani Zaher; Kevin Cox

Degree

Doctor of Philosophy (PhD)

Author's Department

Biology & Biomedical Sciences (Plant & Microbial Biosciences)

Author's School

Graduate School of Arts and Sciences

Document Type

Dissertation

Date of Award

8-11-2026

Language

English (en)

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