SBIR-STTR Award

Huanglongbing disease ("HLB") has killed millions of citrus trees and reduced average yield in 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 but has to be preceded by identifying specific gene-editing targets. The identification of target genes has been challenging because of poor understanding of the HLB pathogenesis mechanism and technical limitations of HLB-citrus protein-interaction screening methods. A powerful new tool to address these limitations is Turbo ID an engineered biotin ligase that can label its proximal proteins when it is expressed in cells. The proposed project addresses this opportunity by "employing Turbo ID-mediated proximity labeling to identify citrus proteins that interact with "effector proteins that are critical for HLB bacterial infection. We will then use the discovered proteins as gene-editing targets for "CRISPR precision breeding" to develop citrus varieties that disrupt the effector-citrus interactions leading to genetic resistance to HLB. To that end we will perform two objectives. The first objective is generating effector-TurboI Dexpressing 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 Turbo ID coupled with mass spectrometry. We will produce a high-confidence list of SDE-interacting citrus proteins through data analysis. We anticipate this project will identify gene-editing targets that we will then use to develop non-GMO HLB-resistant citrus trees that will be sold through citrus nurseries to citrus growers.

Huanglongbing disease("HLB") caused by the bacterium Candidatus Liberibacterasiaticus ("CLas") has killed millions of citrus trees and reduced average yield in Florida by 50%costing billions in lost revenue. CRISPR is the most powerful tool for rapid breeding of new HLB-resistant citrus trees but has to be preceded by identifying specific gene-editing targets. The identification of target genes has been challenging because of poor understanding of the HLB pathogenes is mechanism and technical limitations of HLB-citrus protein-interaction screening methods. A powerful new tool to address these limitations is TurboID an engineered biotinligase that can label its proximal proteins when it is expressed in cells. The proposed project addressed this opportunity by employing TurboID-mediated proximity labeling to identify citrus proteins that interact with effect or proteins that are critical for HLB CLas infection. During Phase I research we produced a high-confidence list of CLas Sec-dependent effector ("SDE")-interacting citrus proteins by using TurboID coupled with mass spectrometry. In Phase II we will use the discovered proteins as gene-editing targets for CRISPR precision breeding to develop citrus varieties that disrupt the effector-citrus interactions leading to genetic resistance to HLB. To that end we will perform three objectives. The first is CRISPR-editing of promising SDE-interacting genes and generating non-transgenic CRISPR-edited citrus varieties. The second is testing CRISPR-edited plants for resistance to HLB through graft inoculation and proteomics analysis. The third is to commence field trials. We anticipate this project will develop non-GMOHLB-resistant citrus trees that will be sold to citrus growers.