Comparative genomics of a poinsettia-associated phytoplasma and functional validation of its SAP11-homologous effectors that induce plant branching

Phytoplasmas are insect-transmitted plant pathogens that manipulate host development through secreted effector proteins. While they are notorious for causing agricultural losses, in the ornamental plant poinsettia (<i>Euphorbia pulcherrima</i>), phytoplasma infection is uniquely harnessed to induce the commercially desirable free-branching trait. However, the effectors responsible for this phenotype have remained unknown. To address this question, we sequenced and analysed the 705,138 bp genome of '<i>Candidatus</i> Phytoplasma pruni' PR2021, a strain associated with the high-branching cultivar Princettia Pink. Comparative genomics confirmed its species assignment and revealed an unusual effector repertoire. PR2021 lacks most previously described effectors but encodes two distinct SAP11 homologues, a family of effectors known to induce plant branching. Genomic context analysis showed that one homologue is located within a potential mobile unit (PMU) and is nearly identical to the SAP11 of the distantly related '<i>Ca</i>. P. asteris', while the other is located outside PMU regions and is divergent in protein sequence (39.0% identity) and predicted structure. Functional assays using <i>Agrobacterium</i>-mediated transient expression in <i>Nicotiana benthamiana</i> demonstrated that each homologue independently induced significant branching, whereas co-expression did not enhance the phenotype, indicating overlapping functions. These findings establish a direct connection between poinsettia branching and SAP11-homologous effectors, providing the first experimental evidence linking phytoplasma effector activity to this horticulturally important trait. This work expands understanding of phytoplasma effector diversity and mobility, while offering a functional framework for developing pathogen-free strategies to modulate ornamental plant architecture.