Integrated roles of BclA and DD-carboxypeptidase 1 in Bradyrhizobium differentiation within NCR-producing and NCR-lacking root nodules

Legume plants host nitrogen-fixing endosymbiotic Rhizobium bacteria in root nodules. In Medicago truncatula, the bacteria undergo an irreversible (terminal) differentiation mediated by hitherto unidentified plant factors. We demonstrated that these factors are nodule-specific cysteine-rich (NCR) peptides that are targeted to the bacteria and enter the bacterial membrane and cytosol. Obstruction of NCR transport in the dnf1-1 signal peptidase mutant correlated with the absence of terminal bacterial differentiation. On the contrary, ectopic expression of NCRs in legumes devoid of NCRs or challenge of cultured rhizobia with peptides provoked symptoms of terminal differentiation. Because NCRs resemble antimicrobial peptides, our findings reveal a previously unknown innovation of the host plant, which adopts effectors of the innate immune system for symbiosis to manipulate the cell fate of endosymbiotic bacteria.

Leguminous plants (such as peas and soybeans) and rhizobial soil bacteria are symbiotic partners that communicate through molecular signaling pathways, resulting in the formation of nodules on legume roots and occasionally stems that house nitrogen-fixing bacteria. Nodule formation has been assumed to be exclusively initiated by the binding of bacterial, host-specific lipochito-oligosaccharidic Nod factors, encoded by the nodABC genes, to kinase-like receptors of the plant. Here we show by complete genome sequencing of two symbiotic, photosynthetic, Bradyrhizobium strains, BTAi1 and ORS278, that canonical nodABC genes and typical lipochito-oligosaccharidic Nod factors are not required for symbiosis in some legumes. Mutational analyses indicated that these unique rhizobia use an alternative pathway to initiate symbioses, where a purine derivative may play a key role in triggering nodule formation.

Symbiosis between legumes and Rhizobium bacteria leads to the formation of root nodules where bacteria in the infected plant cells are converted into nitrogen-fixing bacteroids. Nodules with a persistent meristem are indeterminate, whereas nodules without meristem are determinate. The symbiotic plant cells in both nodule types are polyploid because of several cycles of endoreduplication (genome replication without mitosis and cytokinesis) and grow consequently to extreme sizes. Here we demonstrate that differentiation of bacteroids in indeterminate nodules of Medicago and related legumes from the galegoid clade shows remarkable similarity to host cell differentiation. During bacteroid maturation, repeated DNA replication without cytokinesis results in extensive amplification of the entire bacterial genome and elongation of bacteria. This finding reveals a positive correlation in prokaryotes between DNA content and cell size, similar to that in eukaryotes. These polyploid bacteroids are metabolically functional but display increased membrane permeability and are nonviable, because they lose their ability to resume growth. In contrast, bacteroids in determinate nodules of the nongalegoid legumes lotus and bean are comparable to free-living bacteria in their genomic DNA content, cell size, and viability. Using recombinant Rhizobium strains nodulating both legume types, we show that bacteroid differentiation is controlled by the host plant. Plant factors present in nodules of galegoid legumes but absent from nodules of nongalegoid legumes block bacterial cell division and trigger endoreduplication cycles, thereby forcing the endosymbionts toward a terminally differentiated state. Hence, Medicago and related legumes have evolved a mechanism to dominate the symbiosis.