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Molecular inventions underlying the evolution of the nitrogen-fixing root nodule symbiosis

Final Report Summary - EVOLVINGNODULES (Molecular inventions underlying the evolution of the nitrogen-fixing root nodule symbiosis)

The root nodule symbiosis with nitrogen-fixing bacteria (RNS) enables plants to overcome nitrogen limitations. Legumes such as soybean, beans, peas and lentils form RNS and offer, together with their protein-rich seeds, great potential for sustainable agriculture and healthy human nutrition. RNS is restricted to a single clade of flowering plants comprising only four orders, the Fabales, Fagales, Cucurbitales and Rosales (FaFaCuRo). This clade comprises legumes and actinorhizal trees such as alder, but also important fruits and vegetables that are unable to form RNS such as melons, squash and apple, peaches and strawberries. EvolvingNodules aimed to identify the key genetic differences between plants that are capable of forming RNS and those that are not.
The phylogenetic distribution pattern of RNS calls for specific trait acquisitions and genetic changes in FaFaCuRo’s common ancestor. Via comparative analysis, EvolvingNodules identified the unifying symbiotic trait unique to this clade: the ability to take up bacteria into living plant cells via the formation of intracellular bacteria-containing tube-shaped structures (infection threads) within root cortical cells (Parniske, 2018). Furthermore, we identified the acquisition of a novel cis-regulatory element in the promoter of the transcription factor gene NODULE INCEPTION (NIN) as a key molecular event enabling this ancestral trait. NIN is indispensable for nodule and infection thread development. This cis-element, called PACE, (Predisposition-Associated Cis-Element) was exclusively found in the promoter of the NIN gene of FaFaCuRo member species. PACE determines expression of NIN in cortical cells at early stages of bacterial infection. PACE acquisition placed NIN gene expression under the control of an ancient signal transduction pathway required for arbuscular mycorrhiza symbiosis. Our data pinpoint the acquisition of PACE as a key event in the evolution of RNS that connected NIN expression control to symbiotic signal transduction and thus enabled the development of cortical infection threads (Cathebras, Gong et al., manuscript in revision).
Despite of its obvious benefits for plant nutrition, the phylogenetic distribution of RNS among the FaFaCuRo clade is scattered and patchy. At the start of this project, the reasons for this uneven distribution were unclear. Doctoral candidate Max Griesmann in collaboration with the Plant Genome and Systems Biology team led by Klaus F. X. Mayer at the Helmholtz Zentrum Munich, performed large scale comparative phylogenomic comparisons across angiosperm genomes, including novel FaFaCuRo genome sequences obtained through collaboration with an international consortium including the BGI, Shenzhen, China. He discovered a diversity of loss-of-function mutations within the NIN genes of multiple, phylogenetically scattered non-nodulating FaFaCuRo member species indicating that these mutations arose independently from each other. Given the indispensable role of NIN in RNS development of model legumes, the loss of a functional NIN gene is a likely cause for the absence of RNS in those species. The discovery that RNS was lost several times independently revealed an unexpected selection pressure against this symbiosis. Hence, RNS appears to have provided, at least in the course of evolutionary timescales, competitive disadvantages that led to multiple independent losses of this symbiosis (Griesmann et al. 2018, Science).
Overall, our data position the NIN gene at the centre stage of the evolutionary events leading to both the origin of RNS and its subsequent loss. EvolvingNodules thus provided an essential building block for installing RNS in crop plants inside and outside the FaFaCuRo clade, with the long-term vision to reduce the dependency on chemical nitrogen fertilizer and its associated climate and environmental footprint.