Jimenez-Guerrero, I. | bioRxiv Science

bioRxiv2026

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.

microbiology↗

bioRxiv2023

The type VI secretion system of Sinorhizobium fredii USDA257 is required for a successful symbiosis with Glycine max cv Pekin

For agriculture, the symbiosis carried out by rhizobia with legumes stands out as crucial for both economic and environmental reasons. In this process, the bacteria colonize the roots of the plants, inducing the formation of plant organs called nodules. Within these structures, rhizobia fix the environmental nitrogen into ammonia reducing the demand for this essential element required for plant growth. Various bacterial secretion systems (TXSS, Type X Secretion System) are involved in the establishment of this symbiosis, with the T3SS being the most extensively studied. The T6SS is a nanoweapon present in 25% of gram-negative bacteria, commonly used against other gram-negative bacteria, though some of them use it to manipulate eukaryotic cells. Interestingly, although T6SS is widely distributed among rhizobia, whether it has a specific role in symbiosis with legumes remains elusive. Sinorhizobium fredii USDA257 is a fast-growing rhizobium with the capacity to nodulate a great variety of legume plants. This strain harbors a single T6SS cluster, containing the genes encoding all the structural components of the system and two genes encoding potential effectors that could target the cell wall of the plants and/or be acting as a toxin/antitoxin system. We have demonstrated that this system is active and can be induced in poor culture media. In addition, we have seen by fluorescence microscopy that the T6SS is active in nodules. Competition assays between USDA257 and different preys have shown that USDA257 cannot kill any of them using its T6SS under tested conditions. By constract, nodulation assays demonstrated that USDA257 utilizes this protein secretion system to enhance nodulation and competitiveness with its host Glycine max cv Pekin.

microbiology↗