Tracking Arabidopsis PCNA1 and PCNA2 with a Human PCNA Nanobody-Based Chromobody

Background Proliferating Cell Nuclear Antigen (PCNA) is a crucial protein in eukaryotes that recruits nuclear factors during DNA replication and repair, but also cytoplasmic proteins involved in apoptosis and cell signaling. In the model plant Arabidopsis thaliana, two highly similar PCNA isoforms, AtPCNA1 and AtPCNA2, have been identifi ed. However their functions remain unexplored, probably because loss-of-function mutants, as observed in other model systems, are lethal. The use of nanobodies -single-domain antibody fragments- for targeted protein degradation and mislocalization represents a promising complementary alternative for studying gene function. However, these approaches are still poorly used in plant research. Results To explore the applicability of this strategy in plants, we designed and expressed a modifi ed human PCNA chromobody (PCNA Cb) consisting of a commercial anti-human PCNA nanobody-fl uorescent protein fusion, in Arabidopsis to test its capacity to track the dynamics of PCNA isoforms. We fi rst demonstrated that AtPCNA1 and AtPCNA2 assemble in a heteromeric complex and exhibit a highly overlapping localization during the cell cycle. Co-immunoprecipitation assays and microscopic analyses confi rmed that PCNA Cb binds to AtPCNA1 and AtPCNA2 in vivo and fully colocalizes with them throughout the cell cycle in living cells. Conclusions Our results demonstrate that a human-derived PCNA chromobody can be used to monitor endogenous Arabidopsis PCNA proteins. While further optimization is required to enhance binding effi ciency, this tool offers a promising platform for future studies aimed at elucidating PCNA function in plants.