AUTHOR=Basu Supratim , Huynh Loan , Zhang Shujian , Rabara Roel , Nguyen Hau , Velásquez Guzmán Jeanette , Hao Guixia , Miles Godfrey , Shi Qingchun , Stover Ed , Gupta Goutam 

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

JOURNAL=Frontiers in Plant Science

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2022.869178

DOI=10.3389/fpls.2022.869178

ISSN=1664-462X

ABSTRACT=<p>We adopted a systems-based approach to determine the role of two <italic>Candidatus</italic> Liberibacter asiaticus (<italic>C</italic>Las) proteins, <italic>LasP</italic><sub><italic>235</italic></sub> and Effector 3, in Huanglongbing (HLB) pathogenesis. While a published work suggests the involvement of these <italic>C</italic>Las 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 <italic>LasP</italic><sub>235</sub> and Effector 3 from the healthy and <italic>C</italic>Las-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 <italic>in vitro</italic> are also retained <italic>in planta</italic>. The notable <italic>in planta</italic> citrus targets of <italic>LasP</italic><sub><italic>235</italic></sub> and Effector 3 include citrus innate immune proteins. Third, <italic>in vitro</italic> and <italic>in planta</italic> studies were performed to show that <italic>LasP</italic><sub><italic>235</italic></sub> 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 <italic>C</italic>Las lifecycle and HLB pathogenesis. Finally, we performed molecular dynamics simulations to visualize the interactions of <italic>LasP</italic><sub><italic>235</italic></sub> and Effector 3, respectively, with LTP and Kunitz protease inhibitor. This led to the design of an LTP mimic, which sequestered and blocked <italic>LasP</italic><sub><italic>235</italic></sub>and rescued the bactericidal activity of LTP thereby proving that <italic>LasP</italic><sub><italic>235</italic></sub>, indeed, participates in HLB pathogenesis.</p>