Pei, S.-C. | Search bioRxiv

bioRxiv2025

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 (Euphorbia pulcherrima), 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 analyzed the genome of Candidatus 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 homologs, a family of effectors known to induce plant branching. Genomic context analysis showed that one homolog is located within a potential mobile unit (PMU) and is nearly identical to the SAP11 of the distantly related Ca. P. asteris, while the other is located outside PMU regions and is divergent in sequence and predicted structure. Functional assays using Agrobacterium-mediated transient expression in Nicotiana benthamiana demonstrated that each homolog 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. IMPACT STATEMENTPhytoplasmas are uncultivated bacterial pathogens that reprogram host development through secreted effectors. While they are notorious for causing agricultural losses, phytoplasma infection is uniquely harnessed to induce the desirable free-branching trait in poinsettia, although the molecular basis has remained unresolved. Through analysis of the complete genome of Candidatus Phytoplasma pruni PR2021, a strain associated with a high-branching cultivar, we identified two SAP11-homologous effectors with contrasting genomic and evolutionary contexts. One appears vertically inherited and divergent from previously characterized homologs, whereas the other is embedded in a potential mobile unit and likely acquired through horizontal transfer. Importantly, both homologs induce branching despite substantial sequence divergence. Taken together, this work advances understanding of phytoplasma genome evolution and effector diversity, while providing experimental evidence that links effector function to host developmental manipulation. Beyond its horticultural relevance, it illustrates how horizontal gene transfer and lineage-specific retention shape phytoplasma effector complements, offering a foundation for future efforts to dissect and re-engineer effector-host interactions. DATA SUMMARYAll genome assemblies analyzed in this study were obtained from the National Center for Biotechnology Information (NCBI) Genome Database. The accession numbers are provided in Table S1.

genomics↗

bioRxiv2024

Delineating bacterial genera based on gene content analysis: a case study of the Mycoplasmatales-Entomoplasmatales clade within the class Mollicutes

Genome-based analysis allows for large-scale classification of diverse bacteria and has been widely adopted for delineating species. Unfortunately, for higher taxonomic ranks such as genus, establishing a generally accepted approach based on genome analysis is challenging. While core-genome phylogenies depict the evolutionary relationships among species, determining the correspondence between clades and genera may not be straightforward. For genotypic divergence, percentage of conserved proteins (POCP) and genome-wide average amino acid identity (AAI) are commonly used, but often do not provide a clear threshold for classification. In this work, we investigated the utility of global comparisons and data visualization in identifying clusters of species based on their overall gene content, and rationalized that such patterns can be integrated with phylogeny and other information such as phenotypes for improving taxonomy. As a proof of concept, we selected 177 representative genome sequences from the Mycoplasmatales-Entomoplasmatales clade within the class Mollicutes for a case study. We found that the clustering patterns corresponded to the current understanding of these organisms, namely the split into three above-genus groups: Hominis, Pneumoniae, and Spiroplasma-Entomoplasmataceae-Mycoides (SEM). However, at the genus level, several important issues were found. For example, recent taxonomic revisions that split the Hominis group into three genera and Entomoplasmataceae into five genera are problematic, as those newly described or emended genera lack clear differentiations in gene content from one another. Moreover, several cases of mis-classification were identified. These findings demonstrated the utility of this approach and the potential application for other bacteria. Impact statementTaxonomy provides a foundation for communication that involves biological entities. As such, robust classification and standardized nomenclature are critical. In recent years, genome analysis has been widely adopted for delineating species, but generally accepted approaches for delineating higher taxonomic ranks such as genus are lacking. In this work, we demonstrated that the comparison of overall gene content among species provides an intuitive method for identifying groups of similar organisms that can correspond to genera. Moreover, several critical issues were identified when we applied the method to evaluate recent taxonomic revisions that affected many pathogens with biomedical or economic importance. These findings serve as a cautionary tale against the over-reliance of core-genome centered approaches in taxonomy. Data SummaryThe 183 genome assemblies analyzed in this study were obtained from the National Center for Biotechnology Information (NCBI) Genome Database. The accession numbers are provided in Table S1.

genomics↗