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

根瘤菌-豆科植物共生是一种最先进的胞内植物-微生物互作方式，主要涉及相互识别、通过侵染线(ITS)侵染以及根瘤菌释放到根瘤中的宿主细胞中。在这些过程中，基因表达模式和分子形态动力学发生了广泛和协调的变化，如质膜增殖和细菌周膜的形成。了解在结瘤这一关键阶段的转录、分子和细胞模式对于阐明控制根瘤菌-寄主细胞相互作用和随后的根瘤菌-豆科共生固氮的分子调控网络至关重要。然而，控制根瘤菌细胞内摄取的转录和分子形态动态格局在很大程度上仍未确定。此前，OTT实验室发现SYMREM1与结瘤因子受体物理上相互作用，是IT生长和根瘤菌释放所必需的，表明SYMREM1在根瘤菌结瘤的早期阶段对根瘤菌的吸收起着特定的作用。LI实验室已鉴定GmSYMREM1为MtSYMREM1在G.max中的功能同源基因，并验证了GmSYMREM1与NFR1α/5α之间的相互作用。OTT和LI实验室的合作工作也表明，根瘤菌对GmSYMREM1的诱导受miR172-NNC1模块的调控。这些数据表明，紫花苜蓿和大豆分别是不确定的和确定的结瘤植物，它们可能具有共同的保守机制，即SYMREM1介导的质膜纳米结构域在豆科植物与根瘤菌共生过程中控制根瘤菌的侵染并释放到宿主细胞中。这项联合建议将：1)扩大并最终完成我们对SYMREM1基因转录调控的联合分析；2)使用互补的方法，如芯片序列和启动子进化，来确定我们未来的靶点；3)采用单细胞测序等方法，绘制在结瘤过程中调节细胞规格的表观遗传动态图；4)不仅将这些启动子用于工程合成模块，使蛋白质能够精确靶向PBM，而且还能解开调节这些模块的转录因子复合体；以及5)使用相关显微镜，以超微结构的分辨率解决形态动态过程，并确定影响细菌释放的关键因素。该项目将极大地促进我们对根瘤菌细胞内摄取的控制机制的了解。我们将揭示和区分PBM靶向蛋白质、根瘤菌释放和寄主细胞分化的一般分子机制，在不确定和确定结瘤中共生根瘤。

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.