Rad53 Orchestrates Divergent Pathways Governing Genotoxic Resistance, Morphogenesis, and Virulence in Candida albicans: Mechanistic Decoupling through Overexpression Analysis

The DNA damage response (DDR), an evolutionarily conserved surveillance mechanism enabling cellular detection of genotoxic lesions and cell cycle arrest for repair, coordinates genomic stability through key effector kinases. In Candida albicans, our prior work characterized Rad53-mediated transcriptional reprogramming based on the RAD53 deletion strain, establishing its canonical DDR functions. However, Rad53 activation dynamically orchestrates cell cycle synchronization and repair coordination during genotoxic challenge. Investigation of cellular modifications resulting from increased functionality of Rad53 is essential to gain a deeper understanding of the regulatory response of Rad53. To this end, we overexpressed Rad53 revealing its dual regulatory capacity in both stress adaptation and developmental plasticity. Global transcriptomic profiling demonstrated that RAD53 overexpression enhances methyl methanesulfonate (MMS) resistance while paradoxically stimulating filamentous growth, accompanied by upregulation of DNA repair/replication genes (RFA3, POL30) and non-canonical DDR targets including cell wall remodeling factors. Comparative analysis of RAD53 deletion and overexpression transcriptomes identified enriched transcription factors, notably Sfl1, Yox1, and Rfx1. In particular, Rfx1 mediated the resistance to genotoxic stress resulting from RAD53 overexpression or deletion. Surprisingly, Rad53 overexpression substantially hindered hyphal formation and virulence, whereas its kinase-dead mutation restored this deficiency. Notably, Rad53's role in mediating morphogenesis was independent of Rfx1. This multi-modal functionality positions Rad53 as a critical node interfacing genomic stability, developmental plasticity, and pathogenicity in C. albicans. Our study enhances the understanding of the DDR pathway in C. albicans, providing a potential target for treatment of this fungal pathogen.