miR169c‐NFYA‐C‐ENOD40 modulates nitrogen inhibitory effects in soybean nodulation.

Summary: Legume crops contribute a great portion of clean nitrogen (N) to agro‐ecosystems through symbiotic N2 fixation in the nodule; however, the nodulation is always inhibited by high N availability which is known as the N inhibitory effect through largely unknown mechanisms.We functionally investigated miR169c‐GmNFYA‐C‐GmENOD40 under multiple N conditions in soybean (Glycine max) (ENOD, Early Nodulin; NFYA, Nuclear Factor‐Y Subunit A). We elucidated their regulatory roles in soybean nodulation through analyzing expression patterns, micro–messenger RNA (miRNA–mRNA) interactions, phenotypes of transgenic soybean plants and genetic interactions.We found that miR169c expression was induced by high N, whereas its target GmNFYA‐C was preferentially expressed in nodules and induced by rhizobium inoculation. Overexpression of miR169c inhibited nodulation through targeting 3′‐UTR of GmNFYA‐C, whereas knockout miR169c through CRISPR‐cas9 promoted nodulation. However, overexpression of GmNFYA‐C promoted soybean nodulation through facilitating rhizobium infection and increasing the expression of symbiotic signaling gene GmENOD40. Besides, GmNFYA‐C directly induced the expression of GmENOD40. In addition, overexpression of GmNFYA‐C without the target site of miR169c partially attenuated the inhibitory effect of high N on soybean nodulation.We discovered a new regulatory pathway involving the miR169c‐NFYA‐C‐ENOD40 module that regulates soybean nodulation in response to N availability. This pathway provides substantial new insights into the mechanisms underlying the N inhibitory effect on nodulation. [ABSTRACT FROM AUTHOR]