A LjBAK1-associated E3 ubiquitin ligase, LjPUB7, negatively regulates early symbiosis by targeting NFRs.

Nod factor receptors (NFRs) are essential for initiating symbiotic signaling in legumes, mediating rhizobial infection and nodule development. Tight regulation of NFR levels is crucial to prevent inappropriate immune responses and maintain cellular homeostasis. Co-expression of LjNFR1 and LjNFR5 triggers cell death in Nicotiana benthamiana, which is specifically inhibited by LjBAK1-mediated ubiquitination and subsequent degradation, suggesting the existence of a LjBAK1-E3 ligase complex for NFR protein turnover. Further analysis identified LjPUB7, a plant U-box E3 ubiquitin ligase in Lotus japonicus, as a regulator of early symbiotic interactions. LjPUB7 interacts with both LjBAK1 and NFRs, and directly ubiquitinates NFRs. Loss-of-function Ljpub7 mutants display increased infection thread formation, enhanced nodule development, and elevated expression of early nodulation genes. These findings reveal that LjPUB7 negatively regulates early rhizobial infection by targeting NFR1 and NFR5 for ubiquitination and degradation, thereby providing insights into the fine-tuned control of symbiotic signaling in legumes.