Understanding how a cytokinin-signaling transcription factor can both positively and negatively control symbiotic nodulation – CytoSYM

When grown in nitrogen limited conditions, legume plants establish a symbiotic association with soil nitrogen fixing bacteria collectively named rhizobia. This symbiosis leads to the formation on the roots of the legume plant of new organs termed root nodules. In these nodules, bacteria find a favorable environment to fix atmospheric nitrogen and supply it in a reduced form to their host. Cytokinins are key regulators of nodulation. The aim of the CytoSYM project is to understand how a cytokinin-signaling transcription factor, RRB3, can function as a positive but also as a negative regulator in the early stages of nodulation. The proposed project comprises 3 Work Packages (WP). The WP1 will identify by combined single cell RNAseq (rrb3 mutant vs wild-type) and ChIPseq approaches the genes directly controlled by RRB3 in the different cell types during the response to rhizobia. To determine whether RRB3 transcriptional function involves modulating chromatin accessibility, chromatin compaction will be followed in response to rhizobium by ATACseq and histone acetylation by ChiPseq comparatively in the WT and in rrb3 mutant plants. WP2 will study the role of target genes selected from WP1 in the control of nodulation using loss-of-function approaches, in particular to identify new actors with positive or negative regulatory roles.This WP2 will also focus on two already known key players in nodulation to explain the dual role of RRB3 in the regulation of nodulation: NIN which is a transcription factor essential for the initiation of nodules, and TML2 which is a F-Box protein inhibiting nodulation. Finally, the WP3 aims to identify the partners of RRB3 capable of modulating its transcription activity by an immunoprecipitation approach associated with mass spectrometry. The role of these partners in the initiation of nodulation will then be investigated, in particular by loss-of-function approaches. Ultimately, this project will provide a better understanding of how a transcription factor, RRB3, can exert opposite regulations of nodulation over time, depending on the root tissues, and in relation to chromatin or transcriptional interactors modulating its activity, and to reveal the spatiotemporal dynamics of gene expression and chromatin remodeling during the early stages of nitrogen-fixing symbiotic nodulation in legumes.