to constitutive of signal

34 N-terminal (Nt) acetylation (NTA) is an ample and irreversible co-translational 35 protein modification catalyzed by ribosome-associated Nt-acetyltransferases (NATs). 36 NTA on specific proteins can act as a degradation signal (called an Ac/N-degron) for 37 proteolysis in yeast and mammals. However, in plants, the biological relevance of NTA 38 remains largely unexplored. In this study, we reveal that Arabidopsis thaliana SIGMA 39 FACTOR-BINDING PROTEIN 1 (SIB1), a transcription co-regulator and a positive 40 regulator of salicylic acid-primed cell death, undergoes an absolute NTA on the 41 initiator methionine; Nt-acetyltransferase B (NatB) partly contributes to this 42 modification. While NTA results in destabilization of certain target proteins, our 43 genetic and biochemical analyses revealed that plant NatB-involved NTA instead 44 renders SIB1 more stable. Given that the ubiquitin/proteasome system stimulates SIB1 45 degradation, it seems that the NTA-conferred stability ensures the timely expression of 46 SIB1-dependent genes, mostly related to immune responses. Taking our findings 47 together, here we report a non-canonical NTA-driven protein stabilization in land 48 plants. internal standard. The SA-responsive gene WRKY70 was used as a positive control. The 700 data represent the means of three independent biological replicates and error bars 701 indicate standard deviation. Lower case letters indicate statistically significant 702 differences between the mean values ( P < 0.05, one-way ANOVA with post-hoc 703 Tukey’s HSD test). C, The expression levels of the GFP-tagged native and modified 704 SIB1 proteins. Fusion proteins were detected by immunoblot assay using αGFP detected in the naa20 mutant . D, The transcript levels of Nb Naa20 and SIB1-GFP in si20 or EV. si20 : Nb Naa20- silenced N. benthamiana plants. EV: N. benthamiana plants infiltrated with the VIGS empty vector, which serves as a negative control. The SIB1-GFP was amplified by using SIB1 -specific forward primer and GFP specific reverse primer. Nb ACT was used as an internal standard. The data represent the means of three independent biological replicates and error bars indicate standard deviation. Lower case letters indicate statistically significant differences between the mean values ( P < 0.05, one-way ANOVA with post-hoc Tukey’s HSD test). E, Nb Naa20 was silenced in N. benthamiana by the VIGS approach. The expression levels of the SIB1-GFP fusion protein was detected using αGFP antibody. Coomassie brilliant blue staining (CBB) was used as a loading control. and modified SIB1 protein. Proteins isolated from transgenic plants were enriched by IP and subjected to MS analysis. The data represent two independent biological replicates ( N.D. , not detectable). (P < 0.05, one-way ANOVA with post-hoc Tukey’s HSD test). E, The relative transcript levels of immune-related genes in 20-d-old plants were analyzed using RT-qPCR. ACT2 was used as an internal standard. The data represent the means of three independent biological replicates and error bars indicate standard deviation. Lowercase letters indicate statistically significant differences between mean values ( P < 0.05, one-way ANOVA with post-hoc Tukey’s HSD test).