Protocol for SUMO fusion-based functional rescue of SUMOylation-defective protein in Drosophila

Here, we present a protocol for the functional rescue of small ubiquitin-like modifier (SUMO)ylation-defective proteins using a SUMO fusion-based gain-of-function approach. Using Panx involved in the Piwi-piRNA-mediated genome defense in Drosophila, we describe the restoration of transcriptional silencing by stable fusion of a non-cleavable SUMO variant. We then detail procedures for the quantitative evaluation of transcriptional repression. This approach enables causal analysis of SUMO-dependent protein function beyond conventional loss-of-function strategies. For complete details of this protocol, please refer to Wu et al.¹.