Identification of Candidatus Liberibacter asiaticus effector interactome by proximity labeling coupled with proteomics in citrus

PROJECT SUMMARYInstructions:The summary is limited to 250 words. The names and affiliated organizations of all Project Directors/Principal Investigators (PD/PI) should be listed in addition tothe title of the project. The summary should be a self-contained specific description of the activity to be undertaken and should focus on: overall project goal(s)and supporting objectives; plans to accomplish project goal(s); and relevance of the project to the goals of the program. The importance of a concise informativeProject Summary cannot be overemphasized.  Title:​Identification of ​Candidatus ​Liberibacter asiaticus effector interactome by proximity labelingcoupled with proteomics in citrus PD:​ Park Chan Ho  Institution: ​ Soil Culture Solutions LLC (d/b/a Soilcea) CO-PD: ​ Wang Zhiyong  Institution:​Carnegie Institution for Science CO-PD:​Lagos Yianni Institution:​Soil Culture Solutions LLC (d/b/a Soilcea)  Huanglongbing disease ("HLB") has killed millions of citrus trees and reduced average yieldin Florida by 33% costing the citrus industry billions in lost revenue.CRISPR is the most powerful tool for rapid breeding of new HLB-resistant citrus trees buthas to be preceded by identifying specific gene-editing targets. The identification of target genes hasbeen challenging because of poor understanding of the HLB pathogenesis mechanism and technicallimitations of HLB-citrus protein-interaction screening methods. A powerful new tool to addressthese limitations is TurboID an engineered biotin ligase that can label its proximal proteins when it isexpressed in cells.The proposed project addresses this opportunity by ​employing TurboID-mediated proximitylabeling to identify citrus proteins that interact with ​effector proteins that are critical for HLBbacterial infection.We willthen use the discovered proteins as gene-editing targets for ​CRISPR precisionbreeding ​to develop citrus varieties that disrupt the effector-citrus interactions leading to geneticresistance to HLB.To that end we will perform two objectives. The first objective is generating effector-TurboIDexpressing transgenic citrus plants. We will use HLB bacterium Sec-dependent effector ("SDE")proteins proven to cause HLB-disease symptoms including SDE1 and SDE15.The second objective is identifying SDE-interacting citrus proteins by TurboID coupled withmass spectrometry. We will produce a high-confidence list of SDE-interacting citrus proteins throughdata analysis.We anticipate this project will identify gene-editing targets that we will then use to developnon-GMO HLB-resistant citrus trees that will be sold through citrus nurseries to citrus growers.