University of Florida

We Propose to Generate Non-transgenic HLB Resistant Citrus Varieties via Genome Editing of Citrus Susceptibility (S) Genes to HLB Using CRISPR/CAS9 Technology. Our Central Hypothesis Is That Candidatus Liberibacter Asiaticus (las), Which Utilizes a Set of Bacterial Secreted Effector Proteins to Manipulate Key Disease S Gene(s), Can Be Eliminated by Modification of Key S Gene(s). We Propose to Characterize the Interaction Between Las Effectors and Key S Genes or Their Products. Multiple Candidate S Genes Based on Interactions With Key Effectors Have Been Identified in Our Preliminary Study. We Will Generate Mutations in the Candidate S Genes Using CRISPR-CAS9 and Screen the Mutants for Resistance to HLB. the Confirmed S Gene(s) Will Be Modified in Additional Selected Citrus Scion and Rootstock Varieties. in Parallel, We Will Construct Mutations in the Key S Genes to Pinpoint the Critical Residues for Interactions With Las Effectors, Which Will Assist in Directing Plant Modifications and Validation of Identified Resistant Lines. This Proposal Addresses One of the Six Areas of Highest Priority Determined by the Citrus Disease Sub-committee (CDS): Development of Tolerance or Resistance to HLB in Cultivars Commercially Important in All Citrus Production Regions. the Strategy to Be Employed and the Objectives Are Developed Based on Input From Citrus Industry. Citrus Grower Sponsored Projects Established the Foundation of This Application, E.g., Adoption of Crispr Technology on Citrus. the Non-transgenic Plants Generated Here May Not Be Regulated by Usda and Could Be Commercialized Immediately, If Proven HLB Resistant and With Acceptable Horticultural Traits.