Genome-scale mapping of plant transcription factor effector domains reveals conserved regulatory principles across eukaryotes

Eukaryotic species diverged over a billion years ago, yet our molecular view of gene regulation remains heavily skewed toward opisthokont model systems, particularly yeast and humans. Whether the protein features governing transcriptional regulation are conserved across eukaryotes or have diversified within individual supergroups remains unresolved. To provide a distinct evolutionary reference point, we used a massively parallel reporter assay in plant cells to identify transcriptional effector domains and define their sequence constraints, measuring ∼26,000 protein fragments tiling nearly all Arabidopsis transcription factors (1,859 proteins). Comparison with the activity of the same sequences in yeast revealed that activation is governed by a mixture of deeply conserved biochemical features and lineage-dependent functional differences. Likewise, plant repression domains are governed by flexible sequence grammars extending beyond conserved repression motifs. Together, these results reveal both broadly conserved and clade-specific principles governing eukaryotic transcriptional regulation.