EveD, a vesicle-associated rhizobial candidate effector shaping nodulation competitiveness and host genotype responses

Bacterial extracellular vesicles (EVs) are an ancestral secretion route that delivers cargoes to host cells. Although rhizobial EVs transport Nod factors and other bacterial components, their role in protein delivery during symbiosis remains unexplored. Here we characterise EveD, a large soluble repeat-containing protein of Sinorhizobium fredii HH103. Its export is independent of the type III secretion system, and EveD is recovered only in EV-containing fractions. eveD expression is not induced by genistein or soybean seed exudates and does not follow the canonical NodD1-TtsI pathway, but partially depends on the symbiotic regulator SyrM. When expressed in plant cells, EveD localises mainly to the cytoplasm, associates with endoplasmic reticulum-like structures and occasionally occurs in the nucleus. The eveD mutant shows increased nodule occupancy relative to the wild type in Glycine max cv. Williams 82. EveD modulates early responses in wild Glycine soja accessions in a genotype-dependent manner, constraining pseudonodule organogenesis in JL2 while promoting it in CH2. These findings identify EveD as an EV-associated rhizobial protein contributing to bacterial competitiveness and genotype-dependent symbiotic responses, and highlight EVs as an additional route for protein export during legume symbiosis.