Genetic and Physiological Characteristics of Edited Citrus and Their Impact on HLB Tolerance

Huanglongbing (HLB) disease, caused by Liberibacte asiaticus (Las), severely impacts citrus production, and currently, there is no cure. Developing HLB-resistant or tolerant cultivars is crucial, with modifying defense-related genes being a promising approach to managing HLB. NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1) is a positive regulator of systemic acquired resistance (SAR), which enhances resistance to pathogens, whereas NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 3 (NPR3) is a negative regulator of SAR. To unambiguously address the role of in HLB, we introduced mutations into the gene in sweet orange ( L. Osbeck) through genome editing and assessed their effects on morphology, physiology, and resistance/tolerance to HLB. Several genome-edited 'Hamlin' sweet orange trees harboring frameshift-inducing insertions or deletions were identified. After confirming the genome editing using Sanger sequencing, selected lines were grafted onto C-146 trifoliate hybrid rootstocks for clonal propagation. The progenies were then infected with Las using a no-choice Asian Citrus Psyllid (ACP) feeding assay. Evaluation of the genetic and physiological characteristics of -edited citrus trees under greenhouse conditions revealed that the edited trees exhibited greater vigor than the wild-type trees, despite the lack of significant differences in Las titers. Although further field evaluation is needed, our findings indicate that contributes to HLB-caused tree deterioration and demonstrate that editing can enhance tolerance to HLB.