Genetic and physiological characteristics of CsNPR3 edited citrus and their impact on HLB tolerance

Huanglongbing (HLB) disease, caused by <i>Candidatus</i> Liberibacte<i>r</i> asiaticus (<i>Ca</i>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 <i>CsNPR3</i> in HLB, we introduced mutations into the <i>CsNPR3</i> gene in sweet orange (<i>Citrus sinensis</i> 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 <i>Ca</i>Las using a no-choice Asian Citrus Psyllid (ACP) feeding assay. Evaluation of the genetic and physiological characteristics of <i>CsNPR3</i>-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 <i>Ca</i>Las titers. Although further field evaluation is needed, our findings indicate that <i>CsNPR3</i> contributes to HLB-caused tree deterioration and demonstrate that editing <i>CsNPR3</i> can enhance tolerance to HLB.