Single-nucleus transcriptomics reveals the cellular immune responses to <i>Candidatus</i> Liberibacter asiaticus in rough lemon

Citrus Huanglongbing (HLB) is the most destructive disease in citriculture, mainly caused by <i>Candidatus</i> Liberibacter asiaticus (<i>C</i>Las). However, the immune response of citrus to <i>C</i>Las at the cellular level remains to be elucidated. In this study, the first single-cell atlas of rough lemon (<i>Citrus jambhiri</i> Lush.) root apexes were generated using single-nucleus RNA sequencing at 20 weeks postinoculation with <i>C</i>Las. According to gene expression patterns, the single-cell atlas was partitioned into 20 transcriptionally distinct clusters, and five cell types were identified within these clusters. A significant number of defense-related genes were co-upregulated across the five cell types following <i>C</i>Las infection, whereas genes involved in signal transduction pathways, such as tubulin beta-6 chain (<i>TUBB1</i>) and the phospholipase D alpha 1 (<i>PLD1</i>), were concurrently downregulated. Based on pseudotime trajectory analysis, the key pathways and genes involved in the coordination of cell differentiation and resistance in citrus under <i>C</i>Las infection were characterized. Following <i>C</i>Las infection, the development of phloem cells was significantly delayed, and the differentiation of cambium cells into xylem cells was evident. The expression of genes associated with lignin synthesis was significantly upregulated in these cells. The reduction in phloem cell differentiation and the enhanced differentiation of cambium cells into defense-related xylem cells may represent the primary vascular immune mechanisms exhibited by citrus plants in response to <i>C</i>Las infection. Additionally, DNA-binding one zinc finger transcription factor <i>DOF2.4</i> was found to potentially serve dual roles in regulating vascular cell development and inducing plant resistance against <i>C</i>Las. In conclusion, this study collectively provides insights into the cellular innate immunity responses of citrus to <i>C</i>Las infection. These findings hold significant implications for the sustainable development of citriculture amidst the ongoing global HLB epidemic, and offer novel insights into vascular immunity and plant defense responses.