Two Liberibacter Proteins Combine to Suppress Critical Innate Immune Defenses in Citrus

We adopted a systems-based approach to determine the role of two Candidatus Liberibacter asiaticus (CLas) proteins, LasP(235) and Effector 3, in Huanglongbing (HLB) pathogenesis. While a published work suggests the involvement of these CLas proteins HLB pathogenesis, the exact structure-based mechanism of their action has not been elucidated. We conducted the following experiments to determine the structure-based mechanisms of action. First, we immunoprecipitated the interacting citrus protein partners of LasP(235) and Effector 3 from the healthy and CLas-infected Hamlin extracts and identified them by Liquid Chromatography with tandem mass spectrometry (LC–MS/MS). Second, we performed a split green fluorescent protein (GFP) assay in tobacco to validate that the interactions observed in vitro are also retained in planta. The notable in planta citrus targets of LasP(235) and Effector 3 include citrus innate immune proteins. Third, in vitro and in planta studies were performed to show that LasP(235) and Effector 3 interact with and inhibit the functions of multiple citrus proteins belonging to the innate immune pathways. These inhibitory interactions led to a high level of reactive oxygen species, blocking of bactericidal lipid transfer protein (LTP), and induction of premature programed cell death (PCD), all of which are beneficial to CLas lifecycle and HLB pathogenesis. Finally, we performed molecular dynamics simulations to visualize the interactions of LasP(235) and Effector 3, respectively, with LTP and Kunitz protease inhibitor. This led to the design of an LTP mimic, which sequestered and blocked LasP(235)and rescued the bactericidal activity of LTP thereby proving that LasP(235), indeed, participates in HLB pathogenesis.