The actinorhizal plant Datisca glomerata : interpreting its symbiotic adaptations by omics-based comparisons with model and non-model organisms

Nitrogen is the element that most often limits plant growth and development. Common agricultural practices rely on the application of large quantities of industrially-produced nitrogen fertilizer, which poses a worldwide environmental threat. Sustainable agriculture encourages the use of biologically fixed nitrogen. However, access to this is still limited to a restricted group of dicotyledonous plants that share among them the ability to form a root nodule symbiosis. After an intricate molecular dialogue, these plants accommodate in the cells of a newly root-derived organ - the nodule - a class of bacteria that produce the nitrogenase enzyme by which they are able to reduce di-nitrogen from air to bioavailable ammonia. This mutualism allows the plant access to nitrogen in exchange for carbon. This thesis focuses particularly on the actinorhizal symbioses established between the North American plant Datisca glomerata (Datiscaceae, Cucurbitales) and Frankia actinobacteria from cluster II (Frankiaceae, Frankiales). The main aim of this thesis was to improve our understanding about the genetic basis underlying the evolution of root nodule symbioses. Genome-wide comparative analysis indicated that the loss or fragmentation of genes coding for Nodule Inception (NIN) and/or Rhizobium-directed Polar Growth was a major event for the loss of nodulation in close relatives of plants that are able to form a root nodule symbiosis. To acquire more information about the requirements in plant adaptations to meet a symbiosis with Frankia cluster II strains, the nodule transcriptome of D. glomerata was compared with that of Ceanothus thyrsiflorus (Rhamnaceae, Rosales). This study suggested that cluster II Frankia strains use lipochitooligosaccharide Nod factors to signal to their host plants. In addition, it suggested that the nitrogen metabolism likely differs between these symbioses: while transcript profiles from nodules of D. glomerata supports pathways for arginine catabolism, which was previously suggested, those from nodules of C. thyrsiflorus support pathways for asparagine biosynthesis. Since nodules of both plants house Frankia strains from cluster II, the differences in nitrogen metabolism are most likely a feature of the host plant and not of the bacterial symbiont. As part of an approach to establish D. glomerata as a model organism for actinorhizal Cucurbitales, the effects of phytohormones towards expression of genes putatively involved in signaling for nodule development were investigated. In D. glomerata, similarly to legume plants, the phytohormones cytokinin and auxin were proposed to play a central role in nodule development as they exert a positive effect on the expression of NIN as well as on that of genes whose promoters are presumably transactivated by NIN. Furthermore, transporter proteins expressed in nodules of D. glomerata and of Casuarina glauca (Casuarinaceae, Fagales), which probably act in supplying C-metabolites to intracellular Frankia, were characterized for their substrate specificity. Results indicated that citrate, and not malate, might be the C-metabolite supplied to both Candidatus Frankia datiscae Dg1 and Frankia casuarinae CcI3 strains in symbiosis. To explore the option of D. glomerata-mediated control towards its microsymbiont, a nodule-specific defensin-like peptide was characterized (DgDef1). Whereas DgDef1 acts as an antimicrobial peptide against Gram-negative strains in a range compatible with a role in symbiosis, no differentiation was shown in assays with the Gram-positive Streptomyces coelicolor. Nonetheless, DgDef1 induced changes in membrane integrity of the legume symbiont Sinorhizobium meliloti 1021 as well as in its transcription profile, e.g., on transcription of genes associated with dicarboxylate uptake. Thus, a role for DgDef1 in acting against ineffective microsymbionts is suggested. Phylogenetic analysis suggested that actinorhizal nodule-specific defensins and legume nodule-specific cysteine-rich peptides share a common origin, which in an evolutionary scenario of symbiont shift leads to the hypothesis that these peptides have been lost in most legumes lineages. Collectively, the data presented in this thesis support the idea that root nodule symbioses share more mechanisms than previously assumed, e.g., in the defense against ineffective microsymbionts (“bacterial cheaters”), supporting the new paradigm that the common ancestor of legumes and actinorhizal plants had evolved a symbiosis that was later lost in most lineages.At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript. Paper 5: Manuscript.