Effector Innovation in Genome-Reduced Phytoplasmas and Other Host-Dependent Mollicutes

Abstract Obligate host-associated bacteria with reduced genomes, such as phytoplasmas, face strong evolutionary constraints, including metabolic dependence on hosts, limited horizontal gene transfer (HGT), and frequent population bottlenecks. Despite these limitations, phytoplasmas, which are parasitic, insect-transmitted plant pathogens maintain a diverse arsenal of secreted effectors that manipulate both plant and insect hosts to promote infection and transmission. These effectors can suppress immunity and reprogram plant development, including witch’s broom and leaf-like flowers, through ubiquitin-independent degradation of key transcription factors. However, how phytoplasmas diversify and maintain these effectors in the absence of frequent genetic exchange remains unclear. To address this, we analysed the effectoromes of 239 phytoplasma genomes and identified a diverse set of secreted proteins, which we designated as Phytoplasma-Associated Molecular Effectors (PhAMEs). We found that PhAMEs targeting evolutionarily conserved and structurally constrained surfaces of host proteins are widespread across phytoplasmas. These effectors adopt compact, efficient folds. They often function as molecular scaffolds with dual interaction surfaces capable of linking host proteins or integrating signalling pathways. Such scaffolding PhAMEs have evolved multiple times independently, providing clear evidence of convergent evolution. Despite severe genomic constrains imposed by genome reduction and limited HGT, gene duplications, interface variations, domain fusions, and repeat expansions have helped the shaping effector fold and diversity. While the overall effector repertoire of phytoplasmas appeared largely unique, some PhAME domains share similarities with proteins from other mollicutes and pathogens. Collectively, our findings shed light on how genome-reduced bacteria innovate molecular functions and offer insights into phytoplasma biology, effector evolution, and host-pathogen dynamics. They also lay the groundwork for protein engineering approaches aimed at discovering or designing novel biomolecules with biotechnological potential. Competing Interest Statement Two patents based on the SAP05-mediated ubiquitin-independent degradation have been published (International Publication Numbers: WO2022/129621 and WO2024/256685). F.G.M., V.T., S.T.M. and S.A.H. have also filed patent applications related to the work described in this manuscript. Footnotes ↵Funding: This work was supported by the UK Research and Innovation (UKRI) Engineering and Physical Sciences Research Council (grant number: EP/X024415/1) and the UKRI BBSRC (Grant BBS/E/J/000PR9797) with additional support from the John Innes Centre (JIC) Knowledge Exchange (KEC) Innovation Funds, John Innes Foundation and the Gatsby Charitable Foundation. Competing Interests: Two patents based on the SAP05-mediated ubiquitin-independent degradation have been published (International Publication Numbers: WO2022/129621 and WO2024/256685). F.G.M., V.T., S.T.M. and S.A.H. have also filed patent applications related to the work described in this manuscript.