Poplar CLE peptides promoting ectomycorrhizal symbiosis identified through genome-wide analysis of responsive small secreted peptides

Plant small secreted peptides (SSPs) are involved in numerous developmental processes and adaptive plant responses. These include root development, immunity, and symbiotic relationships in herbaceous plants; three processes crucial for establishing functional ectomycorrhizal associations in trees. While fungal SSPs involved in ectomycorrhizal establishment have been identified, the role of plant SSPs remains largely unexplored. Although thousands of SSPs have been predicted in plant genomes, their small size and high sequence divergence hinder accurate automated annotation. To address this issue, we combined de novo gene prediction with a family-specific motif search to identify 1,053 SSPs from 21 symbiosis-related families in the genomes of two ectomycorrhizal (ECM) tree species: poplar (Populus trichocarpa) and English oak (Quercus robur). Nearly half of these SSPs, which included signaling, antimicrobial, and peptidase inhibitor peptides, were transcriptionally regulated during ectomycorrhizal symbiosis with various fungal partners, implying that SSPs involved in ECM symbiosis support a diversity of functions. Five ectomycorrhizal-responsive CLAVATA3/EMBRYO SURROUNDING REGION-related (CLE) peptides from poplar enhanced ectomycorrhizal root formation in functional assays. These peptides, which belong to CLE clades associated with meristematic activity, are phylogenetically distinct from CLEs involved in the autoregulation of arbuscular mycorrhizal and rhizobial symbioses, indicating that poplar co-opted a distinct set of SSPs for ECM development. The activity of these peptides did not increase lateral root number but inhibited adventitious and lateral root growth, suggesting their role in promoting ectomycorrhizal root organogenesis. Our results expand the understanding of host tree contributions to ectomycorrhizal development and identify a set of candidate SSPs for future functional studies, thereby highlighting a previously uncharacterized layer of regulation in tree-fungi mutualism.