Rhizobia–legume symbiosis is an important type of plant–microbe mutualism; however, the establishment of this association is complicated and can be affected by many factors. The soybean rhizosphere has a specific microbial community, yet whether these organisms affect rhizobial nodulation has not been well investigated. Here, we analyzed the compositions and relationships of soybean rhizocompartment microbiota in three types of soil. First, we found that the rhizosphere community composition of soybean varied significantly in different soils, and the association network between rhizobia and other rhizosphere bacteria was examined. Second, we found that some rhizosphere microbes were correlated with the composition of bradyrhizobia and sinorhizobia in nodules. We cultivated 278 candidate Bacillus isolates from alkaline soil. Finally, interaction and nodulation assays showed that the Bacillus cereus group specifically promotes and suppresses the growth of sinorhizobia and bradyrhizobia, respectively, and alleviates the effects of saline–alkali conditions on the nodulation of sinorhizobia as well as affecting its colonization in nodules. Our findings demonstrate a crucial role of the bacterial microbiota in shaping rhizobia–host interactions in soybean, and provide a framework for improving the symbiotic efficiency of this system of mutualism through the use of synthetic bacterial communities.

MicroRNAs are noncoding RNAs that act as master regulators to modulate various biological processes by posttranscriptionally repressing their target genes. Repression of their target mRNA(s) can modulate signaling cascades and subsequent cellular events. Recently, a role for miR172 in soybean (Glycine max) nodulation has been described; however, the molecular mechanism through which miR172 acts to regulate nodulation has yet to be explored. Here, we demonstrate that soybean miR172c modulates both rhizobium infection and nodule organogenesis. miR172c was induced in soybean roots inoculated with either compatible Bradyrhizobium japonicum or lipooligosaccharide Nod factor and was highly upregulated during nodule development. Reduced activity and overexpression of miR172c caused dramatic changes in nodule initiation and nodule number. We show that soybean miR172c regulates nodule formation by repressing its target gene, Nodule Number Control1, which encodes a protein that directly targets the promoter of the early nodulin gene, ENOD40. Interestingly, transcriptional levels of miR172c were regulated by both Nod Factor Receptor1α/5α-mediated activation and by autoregulation of nodulation-mediated inhibition. Thus, we established a direct link between miR172c and the Nod factor signaling pathway in addition to adding a new layer to the precise nodulation regulation mechanism of soybean.

Plant root architecture is highly plastic during development and can adapt to many environmental stresses. The proper distribution of roots within the soil under various conditions such as salinity, water deficit, and nutrient deficiency greatly affects plant survival. Salinity profoundly affects the root system architecture of Arabidopsis (Arabidopsis thaliana). However, despite the inhibitory effects of salinity on root length and the number of roots, very little is known concerning influence of salinity on root growth direction and the underlying mechanisms. Here we show that salt modulates root growth direction by reducing the gravity response. Exposure to salt stress causes rapid degradation of amyloplasts in root columella cells of Arabidopsis. The altered root growth direction in response to salt was found to be correlated with PIN-FORMED2 (PIN2) messenger RNA abundance and expression and localization of the protein. Furthermore, responsiveness to gravity of salt overly sensitive (sos) mutants is substantially reduced, indicating that salt-induced altered gravitropism of root growth is mediated by ion disequilibrium. Mutation of SOS genes also leads to reduced amyloplast degradation in root tip columella cells and the defects in PIN2 gene expression in response to salt stress. These results indicate that the SOS pathway may mediate the decrease of PIN2 messenger RNA in salinity-induced modification of gravitropic response in Arabidopsis roots. Our findings provide new insights into the development of a root system necessary for plant adaptation to high salinity and implicate an important role of the SOS signaling pathway in this process.

Legume root nodules convert atmospheric nitrogen gas into ammonium through symbiosis with a prokaryotic microsymbiont broadly called rhizobia. Auxin signaling is required for determinant nodule development; however, the molecular mechanism of auxin-mediated nodule formation remains largely unknown. Here, we show in soybean (Glycine max) that the microRNA miR167 acts as a positive regulator of lateral root organs, namely nodules and lateral roots. miR167c expression was up-regulated in the vasculature, pericycle, and cortex of soybean roots following inoculation with Bradyrhizobium japonicum strain USDA110 (the microsymbiont). It was found to positively regulate nodule numbers directly by repressing the target genes GmARF8a and GmARF8b (homologous genes of Arabidopsis [Arabidopsis thaliana] AtARF8 that encode auxin response factors). Moreover, the expression of miR167 and its targets was up- and down-regulated by auxin, respectively. The miR167-GmARF8 module also positively regulated nodulation efficiency under low microsymbiont density, a condition often associated with environmental stress. The regulatory role of miR167 on nodule initiation was dependent on the Nod factor receptor GmNFR1α, and it acts upstream of the nodulation-associated genes NODULE INCEPTION, NODULATION SIGNALING PATHWAY1, EARLY NODULIN40-1, NF-YA1 (previously known as HAEM ACTIVATOR PROTEIN2-1), and NF-YA2. miR167 also promoted lateral root numbers. Collectively, our findings establish a key role for the miR167-GmARF8 module in auxin-mediated nodule and lateral root formation in soybean.

The plant architecture has significant effects on grain yield of various crops, including soybean (Glycine max), but the knowledge on optimization of plant architecture in order to increase yield potential is still limited. Recently, CRISPR/Cas9 system has revolutionized genome editing, and has been widely utilized to edit the genomes of a diverse range of crop plants. In the present study, we employed the CRISPR/Cas9 system to mutate four genes encoding SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors of the SPL9 family in soybean. These four GmSPL9 genes are negatively regulated by GmmiR156b, a target for the improvement of soybean plant architecture and yields. The soybean Williams 82 was transformed with the binary CRISPR/Cas9 plasmid, assembled with four sgRNA expression cassettes driven by the Arabidopsis thaliana U3 or U6 promoter, targeting different sites of these four SPL9 genes via Agrobacterium tumefaciens-mediated transformation. A 1-bp deletion was detected in one target site of the GmSPL9a and one target site of the GmSPL9b, respectively, by DNA sequencing analysis of two T0-generation plants. T2-generation spl9a and spl9b homozygous single mutants exhibited no obvious phenotype changes; but the T2 double homozygous mutant spl9a/spl9b possessed shorter plastochron length. In T4 generation, higher-order mutant plants carrying various combinations of mutations showed increased node number on the main stem and branch number, consequently increased total node number per plants at different levels. In addition, the expression levels of the examined GmSPL9 genes were higher in the spl9b-1 single mutant than wild-type plants, which might suggest a feedback regulation on the expression of the investigated GmSPL9 genes in soybean. Our results showed that CRISPR/Cas9-mediated targeted mutagenesis of four GmSPL9 genes in different combinations altered plant architecture in soybean. The findings demonstrated that GmSPL9a, GmSPL9b, GmSPL9c and GmSPL9 function as redundant transcription factors in regulating plant architecture in soybean.