Widely-conserved transcription factors regulate symbiosis-specific NCR genes

Abstract Symbiotic nitrogen fixation by rhizobium bacteria within the cells of legume root nodules alleviates the need for nitrogen fertilizers. Nitrogen fixation requires the endosymbionts to differentiate into bacteroids and this can be reversible or terminal. The latter is controlled by the plant, is more beneficial and has evolved in a large clade of the legume family. The plant effectors of terminal differentiation are Nodule-specific Cysteine-Rich NCR peptides, which are absent in legumes such as soybean where there is no terminal differentiation of rhizobia. It was assumed that NCRs coevolved with specific transcription factors, but our work demonstrates that expression of NCR genes does not require NCR-specific transcription factors. Introduction of the Medicago truncatula NCR169 gene under its own promoter into soybean roots resulted in nodule-specific expression of NCR169, leading to bacteroid changes associated with terminal differentiation. We identified two AT-Hook Motif Nuclear Localized (AHL) transcription factors from both M. truncatula and soybean nodules that bound to AT-rich sequences in the NCR169 promoter inducing its expression. Whereas mutation of NCR169 arrested bacteroid development at a late stage, the absence of MtAHL1 or MtAHL2 completely blocked bacteroid differentiation indicating that they also regulate other NCR genes required for development of nitrogen-fixing nodules. Regulation of NCRs by orthologous transcription factors in non-IRLC legumes opens the possibility of increasing the efficiency of nitrogen fixation in legumes lacking NCRs.