Deciphering the transcriptional and molecular morpho-dynamic landscape controlling intracellular uptake of rhizobia

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The Rhizobium-legume symbiosis is one of the most advanced intracellular plant-microbe interaction and mainly involves mutualistic recognition, infection via infection threads (ITs) and rhizobial release into the host cells in root nodules. During these processes, extensive and coordinated changes in gene expression patterns and molecular morpho-dynamics, such as plasma membrane proliferation and formation of the peribacteriod membrane (PBM) occur. Understanding the transcriptional, molecular and cellular patterns at this critical stage of nodulation is essential to elucidate molecular regulatory networks that control rhizobia-host cell interaction and subsequent rhizobia-legume symbiotic nitrogen fixation. However, the transcriptional and molecular morpho-dynamic landscape controlling intracellular uptake of rhizobia remains largely uncharacterized. Previously, the Ott lab found that SYMREM1 physically interacts with Nod factor receptors and is required for IT growth and rhizobia release, indicating that SYMREM1 plays a specific role in rhizobial uptake at the earlier stage of nodulation of M. truncatula. The Li lab has identified GmSYMREM1 as the functional ortholog of MtSYMREM1 in G. max and validated the interaction between GmSYMREM1 and NFR1α/5α. The collaborative work between the Ott and Li lab has also shown that the induction of GmSYMREM1 by rhizobia is regulated by miR172-NNC1 module. These data suggest that both Medicago and soybean which are indeterminate and determinate nodulators, respectively, may share a conserved mechanism by which SYMREM1-mediated plasma membrane nanodomain controls rhizobial infection and release into the host cells during legumes-rhizobial symbiosis. This joint proposal will:1) expand and finalize our joint analysis on the transcriptional regulation of SYMREM1 genes; 2) use complementary approaches such as ChIP-seq and promoter evolution to identify our future targets; 3) adopt the approaches like single cell sequencing to map the epigenetic dynamics that modulate cell specification during nodulation; 4) use these promoters not only for engineering synthetic modules that allow precision targeting of proteins to the PBM but also unravel the transcription factor complex that regulates these modules; and 5) address morpho-dynamic processes with ultrastructural resolution using correlative microscopy and identify key factors that mediate bacterial release. This project will greatly advance our knowledge on mechanisms controlling intracellular uptake of rhizobia. We will uncover and distinguish general molecular mechanisms of PBM targeting of proteins, rhizobial release and host cell differentiation in symbiotic nodules in indeterminate and determinate nodulators.