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Abstract Most angiosperm plants recognise the 22-residue flagellin (flg22) epitope in bacterial flagellin via homologs of cell surface receptor FLS2 (flagellin sensitive-2) and mount pattern-triggered immune responses. However, flg22 is buried within the flagellin protein indicating that proteases might be required for flg22 release. Here, we demonstrate the extracellular subtilase SBT5.2 not only releases flg22, but also inactivates the immunogenicity of flagellin and flg22 by cleaving within the flg22 epitope, consistent with previous reports that flg22 is unstable in the apoplast. The prolonged lifetime of flg22 in sbt5.2 mutant plants results in increased bacterial immunity in priming assays, indicating that SBT5.2 counterbalances flagellin immunogenicity to provide spatial-temporal control and restrict costly immune responses and that bacteria take advantage of the host proteolytic machinery to avoid detection by flagellin having a protease-sensitive flg22 epitope.

Abstract The family Brassicaceae is a source of important crop species, including Brassica napus (oilseed rape), Brassica oleracea, and B. rapa, that is used globally for oil production or as a food source (e.g., pak choi or turnip). However, despite advances in recent years, including genome sequencing, a lack of established tools tailored to the study of Brassica crop species has impeded efforts to understand their molecular processes in greater detail. Here, we describe the use of a simple Agrobacterium‐mediated transient expression system adapted to B. rapa and B. napus that could facilitate study of molecular and biochemical events in these species. We also demonstrate the use of this method to characterize the N‐degron pathway of protein degradation in B. rapa. The N‐degron pathway is a subset of the ubiquitin‐proteasome system and represents a mechanism through which proteins may be targeted for degradation based on the identity of their N‐terminal amino acid residue. Interestingly, N‐degron‐mediated processes in plants have been implicated in the regulation of traits with potential agronomic importance, including the responses to pathogens and to abiotic stresses such as flooding tolerance. The stability of transiently expressed N‐degron reporter proteins in B. rapa indicates that its N‐degron pathway is highly conserved with that of Arabidopsis thaliana. These findings highlight the utility of Agrobacterium‐mediated transient expression in B. rapa and B. napus and establish a framework to investigate the N‐degron pathway and its roles in regulating agronomical traits in these species. Significance statement We describe an Agrobacterium‐mediated transient expression system applicable to Brassica crops and demonstrate its utility by identifying the destabilizing residues of the N‐degron pathway in B. rapa. As the N‐degron pathway functions as an integrator of environmental signals, this study could facilitate efforts to improve the robustness of Brassica crops.