Sophia Muller

The nitrogen-fixing symbiosis between rhizobia and legumes has long attracted interest due to its agricultural relevance and potential for sustainable crop production. Efforts to transfer this trait to non-nodulating species require a comprehensive understanding of the molecular and developmental events underlying nodule formation. Of particular importance is the controlled initiation of nodulation, which shares regulatory features with lateral root development and arbuscular mycorrhizal (AM) symbiosis, yet ultimately diverges to produce a distinct organ. During nodule initiation, pericycle cells are the first to re-enter the cell cycle, but unlike in lateral root development, these cells do not contribute directly to organ formation; instead, the bulk of the nodule originates from the inner cortical cell layers. Understanding how pericycle activation is coordinated and redirected represents a central knowledge gap in the field.

To shed light on this specialization within the nodulation program, this thesis first investigates the hormonal regulation of auxin and ethylene, two phytohormones central to the earliest stages of nodule initiation. In Chapter 2, we focus on auxin, which is essential for triggering the first cell divisions during primordium formation. Using spatiotemporal analyses, we characterize auxin biosynthesis and transport throughout early nodulation stages. Chapter 3 examines ethylene, a gaseous hormone with a predominantly inhibitory role in nodulation. Remarkably, we find that different isoforms of the ethylene biosynthesis genes MtACS3 and MtACS10 are oppositely regulated upon rhizobial inoculation, generating a spatial shift of ethylene production from the inner to the outer root tissues without altering overall ACC concentrations. These findings demonstrate that high spatial resolution is critical, as the regulatory dynamics observed in either chapter would not have been detectable in bulk tissue analyses.

To resolve early nodulation processes at higher resolution, we developed and optimized single-cell and single-nucleus RNA-seq workflows for roots and nodules in Chapter 4. Comparison of isolation protocols and high-throughput platforms identifies critical factors affecting RNA quality, cell viability, and transcriptome coverage. Optimized protoplast-based approaches provide higher mapping rates and broader cell-type representation, while integration with nuclei-based methods offers access to complementary information and underrepresented populations. Leveraging these optimized approaches, Chapter 5 identifies transcription factors that may confer pericycle competence during nodule initiation. Integration of bulk time-course RNA-seq with our root single-cell atlas highlights ~600 pericycle-enriched, nodulation-responsive genes, and lead us to focus on three transcription factors—MtbZIP43, MtDREB3, and MtSCL14. These genes display distinct but overlapping expression patterns in the pericycle and vasculature and are dynamically induced after inoculation. RNA interference lines show no strong phenotypes, whereas overexpression reduces nodule numbers.

Finally, Chapter 6 correlates these findings with each other and puts them in a broader context, showing how the combined hormonal, transcriptional, and methodological insights advance our understanding of the spatial regulation underlying symbiotic organogenesis.