Abstract: Ubiquitination is a common protein modification, mostly targeting protein substrates for degradation. As E3 ligases serve critical roles in substrate recognition, their biological functions are of special interest. Through a targeted reverse genetic screen, we discovered a novel E3, SNIPER8 (snc1-influencing plant E3 ligase reverse genetic, 8), that negatively regulates immunity in Arabidopsis. Overexpression of SNIPER8 suppressed the phenotypes of the autoimmune mutant snc1, which carries a gain-of-function mutation in a TIR-type NLR (Toll/ Interleukin-1 Receptor-like nucleotide-binding leucine-rich repeat receptor). Conversely, knocking out sniper8 enhanced the dwarfism of snc1. SNIPER8 and its three paralogs function redundantly, and knocking out all four genes in wild-type background yielded strong autoimmunity. To search for the substrates of SNIPER8, a suppressor screen with the sniper8 quadruple mutant was carried out, which yielded many loss-of-function alleles of SNC1. As the transcriptional corepressor TPR1 (Topless-related protein 1) was known to be required for SNC1- mediated immunity, its relationship with SNIPER8 was examined. SNIPER8 was found to directly interact with TPR1 and negatively regulate its protein levels. In addition, SNIPER8 overexpression can suppress TPR1-mediated autoimmunity, whereas the autoimmunity of sniper8 quadruple mutant fully depends on both the SNC1 locus and three redundant TPL/TPR genes. Taken together, SNIPER8 is a negative regulatory E3 ligase in plant immunity that promotes TPR1 turnover. This regulation is essential for maintaining immune homeostasis and preventing autoimmune activation.

Key words: E3 ligase, plant immunity, snc1-influencing plant E3 ligase reverse genetic (SNIPER8), suppressor of npr1-1, constitutive 1 (SNC1), Topless-related protein 1 (TPR1), ubiquitination