ESN3, a nodule-specific small peptide essential for symbiotic nitrogen fixation in Medicago truncatula.

Symbiotic interactions between legumes and rhizobia rely on the symbiotic interface, the symbiosome membrane. However, the regulatory mechanisms for symbiosome membrane maintenance remain largely elusive. Methods include forward genetics screenings of mutants, transcript-based cloning and genetic complementation, yeast two-hybrid library screening and Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 gene editing. We characterized Early Senescent Nodule 3 (ESN3), a key regulator of symbiotic nitrogen fixation (SNF) encoding a nodule-specific, 54 aa-long small peptide, which localizes to the symbiosome membrane in Medicago truncatula. We identified SYPTAXIN132 as an ESN3-interacting protein. We showed that syp132a mutants exhibited premature nodule senescence and upregulation of senescence-associated gene expression, similar to the esn3 mutant. Our proteomic studies revealed significant alterations of a large number of membrane proteins in the symbiosome fraction of the esn3 mutant, consistent with a key role of ESN3 in symbiosome maintenance. Furthermore, our data indicated that the expression of ESN3, but not SYP132A, is responsive to gibberellic acid (GA) treatments and DELLA proteins are positive regulators of ESN3 gene expression. In this study, we demonstrated that ESN3 encodes a novel nodule-specific small peptide, playing a key role in SNF. In conjunction with the syntaxin SYP132A, GA signaling plays a negative role in SNF at a later stage of nodule development.