Genome-wide dissection of <i>PP2</i> genes reveals <i>CsPP2-3/5/18</i> as key regulators of phloem protein deposition and bacterial immunity in <i>Citrus sinensis</i>

Citrus Huanglongbing (HLB), caused by the phloem-restricted bacterium <i>Candidatus</i> Liberibacter asiaticus (<i>C</i>Las), is a devastating disease threatening global citrus production. <i>C</i>Las infection triggers excessive accumulation of phloem proteins (PPs) that obstruct sieve pores, a dual-edged process potentially restricting pathogen spread while impairing phloem transport. Despite its pathophysiological significance, systematic identification and functional characterization of PPs in citrus, particularly their roles in <i>C</i>Las defense, remain unclear. Here, we performed a genome-wide analysis of the <i>PP2</i> gene family in the HLB-susceptible sweet orange (<i>Citrus sinensis</i>) and identified 26 <i>CsPP2</i> genes. Phylogenetic and structural analyses uncovered evolutionary divergence and regulatory complexity among CsPP2 family members. Using promoter-driven <i>GUS</i> gene expression assays in transgenic hairy roots, we identified three phloem-specific paralogs, <i>CsPP2-3</i>, <i>CsPP2-5</i>, and <i>CsPP2-18</i>, and delineated core regulatory regions conferring tissue specificity. Overexpression of each gene significantly enhanced phloem protein deposition. Notably, although virus-induced silencing of individual <i>CsPP2s</i> did not affect resistance to <i>Xanthomonas citri</i> subsp. <i>citri</i>, overexpression of any of the three genes substantially enhanced resistance against this apoplastic pathogen, demonstrating functional redundancy. However, the three paralogs exhibited marked functional divergence in response to <i>C</i>Las: <i>CsPP2-3</i> and <i>CsPP2-18</i> conferred enhanced resistance, whereas <i>CsPP2-5</i> increased susceptibility. Distinct defense-related gene expression profiles further supported their specialized immune roles. Our study provides the first systematic identification of <i>PP2</i> genes in citrus and reveals the functional differentiation of <i>CsPP2-3/5/18</i> as key regulators of phloem-mediated defense. These findings provide crucial insights into phloem defense regulatory networks and identify novel genetic targets for HLB resistance breeding.