A secreted citrus protease cleaves an outer membrane protein of the Huanglongbing pathogen

Abstract Plants secrete a variety of proteases as a defense response during infection by microbial pathogens. However, the relationship between their catalytic activities and antimicrobial functions remains largely unknown. Particularly, few biologically relevant substrates of these proteases have been identified. Huanglongbing (HLB) has been a major threat to the citrus industry worldwide. The HLB-associated bacterium, Candidatus Liberibacter asiaticus (Las), was previously shown to deploy an inhibitor of papain-like cysteine proteases (PLCPs) to promote disease in citrus. In this study, we identified an outer membrane protein (OMP) of Las, LasOMP1, as a substrate of the citrus PLCP CsRD21a. LasOMP1 is one of the most highly expressed genes in Las. CsRD21a cleaves LasOMP1 and produces cleaved peptide products, which could be detected in vitro and in HLB-diseased citrus plants. We found that CsRD21a targets the N-terminal portion of LasOMP1, potentially at an extracellular loop region. Importantly, transgenic sweet orange overexpressing CsRD21a showed reduced Las titers and delayed disease symptoms, highlighting that engineering this protease is a promising strategy to enhance HLB resistance in citrus. Together, our work reveals a pathogen-derived substrate of plant PLCPs and highlights bacterial OMPs as direct targets of plant defense. Significance Statement Huanglongbing (HLB) is the most devastating disease of citrus with no resistance having been identified in commercial cultivars. Previous work implicated papain-like cysteine proteases (PLCPs) as an important hub of defense in citrus; however, their precise role in HLB tolerance remained unclear. Here, we identify and characterize an outer membrane protein (OMP) from the HLB-associated bacterium as a substrate of the citrus PLCP RD21a. We demonstrate a specific cleavage of the bacterial OMP by citrus RD21a, which may impair pathogen growth and/or activate plant immunity. Importantly, overexpression of RD21a enhances HLB tolerance in sweet oranges. This work identifies OMPs as substrates of plant PLCPs and provides insights into protease defense functions.