Lotus japonicus CIP73 functions as a transcriptional repressor to inhibit Nodule Inception gene expression in nodule symbiosis

Abstract

Background and aims

Many legumes establish symbiotic relationships with soil bacteria to form specialized organs called nodules, where bacteria reside and convert nitrogen gas into ammonium for host plants. Lotus japonicus CIP73 (CCaMK-interacting protein of approximately 73 kD), a member of the large ubiquitin superfamily, is phosphorylated by CCaMK and plays a role in regulating nodule formation. However, the biochemical function of CIP73 in regulating nodulation has remained unclear.

Methods

We evaluated the molecular mechanism of CIP73 in regulating nodulation through genetics, biochemistry, and molecular biology in both laboratory and greenhouse settings.

Results

Our study demonstrates that CIP73 functions as a transcriptional repressor that directly binds to the NIN promoter to suppress its expression in L. japonicus. The N-terminus of CIP73 is essential for its role in repressing NIN expression, with CIP73 binding to a 31 bp CIP73 binding site (CBS31) in the NIN promoter. Importantly, our findings reveal that the inhibition of NIN expression by CIP73 negatively impacts the number of nodules locally, while ectopic expression of NIN systemically suppresses nodule organogenesis in the cip73 mutants.

Conclusion

Our research suggests that CIP73 functions as a transcriptional repressor that directly binds to the NIN promoter, ensuring precise spatiotemporal expression and creating a finely tuned mechanism to regulate nodule organogenesis.