The rhizobial type <scp>III</scp> effectors <scp>ErnA</scp> and Sup3 hijack the <scp>SUMOylation</scp> pathway to trigger nodule formation in <i>Aeschynomene</i> species

Rhizobial type III effectors (T3Es) play a crucial role in the symbiotic relationship between rhizobia and legumes by manipulating host cellular processes to promote nodule formation. Previously, we identified two T3Es, ErnA and Sup3, that trigger nodulation in Aeschynomene spp. in the absence of Nod factors. Here, we further investigate the mode of action of these T3Es during root nodule symbiosis. We employed protein interaction assays, in vitro binding and enzymatic activity assays, mutational analyses, and functional nodulation tests to dissect the roles of ErnA and Sup3 and their interactions with the host Small Ubiquitin-like MOdifier (SUMO) pathway (SUMOylation). We demonstrate that ErnA contains a SUMO-interacting motif (SIM) at its C terminus, which promotes its interaction with SUMO proteins in vitro and in plant nuclei. Additionally, we show that Sup3 possesses a C-terminal SUMO protease domain, which not only interacts with SUMO proteins in vitro and in the nucleus but also exhibits SUMO protease activity. Deletion of the SIM in ErnA or mutation of the catalytic site in Sup3 abolished their ability to trigger nodulation in Aeschynomene indica. These findings suggest that type III secretion system-dependent symbiosis is regulated by posttranslational modification through SUMOylation and that ErnA and Sup3 modulate this SUMOylation pathway to trigger nodulation.