Skip to main content

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

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

Nitrogen is one of the major nutrients required for crop production. One group of closely related plants belonging to the Fabales, Fagales, Cucurbitales and Rosales (FaFaCuRo) evolved to become independent of soil-bound nitrogen by engaging in symbiosis with bacteria that convert atmospheric nitrogen to plant-usable ammonium and are hosted within specialized organs, the root nodules. Soybean forms such nodules and, due to the resulting nitrogen-rich seeds, has become major player in global food production, ecology and nitrogen cycles. Current models state that this symbiosis evolved several times independently and predict a genetic predisposition acquired by a common ancestor of the FaFaCuRo clade.

We are taking a combination of phylogenomic and genetic approaches to identify the switches that facilitated the evolution of nodulation. Whole genome comparison of multiple nodulating and non-nodulating species will reveal genetic patterns specific for the nodulating species. Once candidates are identified, they can be tested for their relevance in nodulation by generating targeted knockout mutants using gene-editing technologies such as CRISPR/Cas. Ultimately we aim to transfer the root nodule symbiosis to closely related sister genera that are presently non-nodulating.

Among the postulated independent origins of nodulation, one occurred within the Rosaceae resulting in ancestral, lateral root-related actinorhiza nodules with Frankia bacteria. However, most genera of the Rosaceae including economically valuable targets such as apple and strawberry are non-nodulating. Thus, by retracing small evolutionary steps, we will take a huge leap towards nitrogen-fertilizer independent crops for sustainable agriculture.

Within the Rosaceae nodulation evolved relatively recently (30 mya) in the common ancestor of the subfamily Dryadoideae, including the genus Dryas. This genus is particularly interesting for comparative genomic and genetic analysis because it comprises nodulating (D. drummondii) and non-nodulating species (D. octopetala). This offers an important genetic resource for targeted proof-of-concept tests of candidate loci. We established hairy root transformation for Dryas and successfully performed initial experiments to analyse gene expression using promoter:reporter constructs.

In collaboration with the BGI, Shenzhen, China, we obtained genome sequences of two Dryas species, D. drummondii and D. octopetala. In collaboration with the Helmholtz Zentrum München, Germany, a phylogenomics pipeline was developed including orthology inference as the basis for identifying patterns such as presence and absence of genes across a larger number of genomes. Based on previous genome comparisons we have identified two potential players, one at the level of bacteria perception mediated by plant receptor kinases and the other at the level of transcriptional regulation of symbiosis related genes. We identifed a transcription factor-DNA interaction, which appears to be restricted to the FaFaCuRo clade and may have played a key role in the evolution of the root nodule symbiosis.

Symbiosis receptor-like kinase (SYMRK) is not only required for root nodule symbiosis with rhizobia or Frankia bacteria (Markmann et al., 2008, PLoS Biology), but also for the fungal arbuscular mycorrhiza (AM) symbiosis (Stracke et al., 2002, Nature). We could demonstrate that SYMRK of the model legume L. japonicus interacts with the Nod factor receptor NFR5 (Antolín-Llovera et al., 2014, Current Biology; Ried et al., 2014, eLIFE) and that overexpression of SYMRK or NFR5 results in the spontaneous activation of nodule organogenesis (Ried et al., 2014, eLIFE). We could narrow down SYMRK sequences that are specifically required for nodulation but not for AM, thus pinpointing domains that appear to be associated with the evolution of nodulation. We thereby determined an important player that must be present in a plant species in order to engineer a root nodule symbiosis competent plant.