The NIN transcription factor coordinates CEP and CLE signaling peptides that regulate nodulation antagonistically.

Legumes tightly regulate nodule number to balance the cost of supporting symbiotic rhizobia with the benefits of nitrogen fixation. C-terminally Encoded Peptides (CEPs) and CLAVATA3-like (CLE) peptides positively and negatively regulate nodulation, respectively, through independent systemic pathways, but how these regulations are coordinated remains unknown. Here, we show that rhizobia, Nod Factors, and cytokinins induce a symbiosis-specific CEP gene, MtCEP7, which positively regulates rhizobial infection. Via grafting and split root studies, we reveal that MtCEP7 increases nodule number systemically through the MtCRA2 receptor. MtCEP7 and MtCLE13 expression in rhizobia-inoculated roots rely on the MtCRE1 cytokinin receptor and on the MtNIN transcription factor. MtNIN binds and transactivates MtCEP7 and MtCLE13, and a NIN Binding Site (NBS) identified within the proximal MtCEP7 promoter is required for its symbiotic activation. Overall, these results demonstrate that a cytokinin-MtCRE1-MtNIN regulatory module coordinates the expression of two antagonistic, symbiosis-related, peptide hormones from different families to fine-tune nodule number.

The Rhizobium leguminosarum biovar trifolii ANU794 induces novel developmental responses on the subterranean clover cultivar Woogenellup.

The clover-nodulating Rhizobium leguminosarum bv. trifolii ANU794 initiates normal root-nodule development with abnormally low efficiency on the Trifolium subterraneum cv. Woogenellup. The cellular and developmental responses of Woogenellup roots to the site- and dose-defined inoculation of green fluorescent protein (gfp)-labeled cells of ANU843 (nodulation proficient) and ANU794 was investigated using light, fluorescence, and confocal microscopy. Strain ANU794-gfp induced three primordia types and four developmental responses at the inoculation site: true or aberrant nodules (on 5 and 25% of plants, respectively), hybrid structures (20% of plants), or lateral roots (50% of plants). The novel hybrid structures possessed nodule and lateral root-like features and unusual vascular patterning. Strain ANU794-gfp induces lateral root formation by stimulating pericycle cell divisions at all nearby protoxylem poles. Only true nodules induced by ANU794-gfp contained intracellular bacteria. In contrast, strain ANU843-gfp induced nodules only and lateral root formation was suppressed at spot inoculation sites. Primordium types were distinguishable by the emission spectrum characteristics of phenolic UV-absorbing and fluorescent compounds that accumulate in primordium cells. Hybrid primordia contained (at least) two fluorescent cell populations, suggesting that they are chimeric. The results suggest that ANU794 may produce both nodule- and lateral root-generating signals simultaneously.