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.

Root‐associated microbes are critical for plant growth and nutrient acquisition. However, scant information exists on optimizing communities of beneficial root‐associated microbes or the mechanisms underlying their interactions with host plants. In this report, we demonstrate that root‐associated microbes are critical influencers of host plant growth and nutrient acquisition. Three synthetic communities (SynComs) were constructed based on functional screening of 1,893 microbial strains isolated from root‐associated compartments of soybean plants. Functional assemblage of SynComs promoted significant plant growth and nutrient acquisition under both N/P nutrient deficiency and sufficiency conditions. Field trials further revealed that application of SynComs stably and significantly promoted plant growth, facilitated N and P acquisition, and subsequently increased soybean yield. Among the tested communities, SynCom1 exhibited the greatest promotion effect, with yield increases of up to 36.1% observed in two field sites. Further RNA‐seq implied that SynCom application systemically regulates N and P signaling networks at the transcriptional level, which leads to increased representation of important growth pathways, especially those related to auxin responses. Overall, this study details a promising strategy for constructing SynComs based on functional screening, which are capable of enhancing nutrient acquisition and crop yield through the activities of beneficial root‐associated microbes.

Rhizosphere microorganisms play important roles in plant health and nutrition, and interactions among plants and microorganisms are important for establishment of root microbiomes. As yet, plant-microbe and microbe-microbe interactions in the rhizosphere remain largely mysterious. In this study, rhizosphere fungal community structure was first studied in a field experiment with two soybean cultivars contrasting in nodulation grown in two rhizobium inoculation treatments. Following this, recombinant inbred lines (RILs) contrasting in markers across three QTLs for biological nitrogen fixation (BNF) were evaluated for effects of genotype and rhizobium inoculation to the rhizosphere fungal community as assessed using ITS1 amplicon sequencing. The soybean plants tested herein not only hosted rhizosphere fungal communities that were distinct from bulk soils, but also specifically recruited and enriched Cladosporium from bulk soils. The resulting rhizosphere fungal communities varied among soybean genotypes, as well as, between rhizobium inoculation treatments. Besides, Cladosporium were mostly enriched in the rhizospheres of soybean genotypes carrying two or three favorable BNF QTLs, suggesting a close association between soybean traits associated with nodulation and those affecting the rhizosphere fungal community. This inference was bolstered by the observation that introduction of exogenous rhizobia significantly altered rhizosphere fungal communities to the point that these communities could be distinguished based on the combination of soybean genotype and whether exogenous rhizobia was applied. Interestingly, grouping of host plants by BNF QTLs also distinguished fungal community responses to rhizobium inoculation. Taken together, these results reveal that complex crosskingdom interactions exist among host plants, symbiotic N 2 fixing bacteria and fungal communities in the soybean rhizosphere.