The PagDMG6341-PagWD40-PagPOLD4 Module Coordinates Base Excision Repair in '84K' Poplar (Populus alba × P....

Base excision repair (BER) is a critical pathway for repairing damaged DNA bases in cells; however, the mechanisms of protein recruitment and interaction in this pathway remain largely unexplored in higher plants. In this study, we used '84K' poplar (Populus alba × P. glandulosa) as the experimental system and applied a low concentration of 5-aminouracil (5-AU) to induce DNA base lesions. Through transcriptome analysis and weighted gene co-expression network analysis (WGCNA), we identified two key BER-responsive genes: the DNA glycosylase family gene PagDMG6341 and the DNA polymerase δ subunit PagPOLD4. PagDMG6341 was significantly upregulated during the arrest phase of 5-AU treatment, whereas PagPOLD4 expression peaked during the subsequent release phase. RNA interference (RNAi) lines for each gene resulted in impaired growth and increased susceptibility to 5-AU in '84K' poplar, supporting their functional roles in DNA repair and development. To further investigate their potential interaction network, we performed yeast two-hybrid (Y2H) screening, AlphaFold3-based structural modelling, confirmatory Y2H, bimolecular fluorescence complementation (BiFC) assays, and luciferase complementation imaging (LCI) assays. These experiments demonstrated that a Transducin/WD40-repeat-like scaffold protein (PagWD40) interacts independently with both PagDMG6341 and PagPOLD4. The yeast three-hybrid (Y3H) assay further showed that PagWD40 functions as a molecular scaffold, linking PagDMG6341 and PagPOLD4 to form a functional complex. This study reveals a new mechanism in which PagWD40 functions as a scaffold protein linking a DNA glycosylase with DNA polymerase δ in the plant BER pathway, thereby providing new insights into the organisation of plant DNA damage repair networks.