Plant Hormones Differentially Control the Sub-Cellular Localization of Plasma Membrane Microdomains during the Early Stage of Soybean Nodulation

Phytohormones regulate the mutualistic symbiotic interaction between legumes and rhizobia, nitrogen-fixing soil bacteria, notably by controlling the formation of the infection thread in the root hair (RH). At the cellular level, the formation of the infection thread is promoted by the translocation of plasma membrane microdomains at the tip of the RH. We hypothesize that phytohormones regulate the translocation of plasma membrane microdomains to regulate infection thread formation. Accordingly, we treated with hormone and hormone inhibitors transgenic soybean roots expressing fusions between the Green Fluorescent Protein (GFP) and GmFWL1 or GmFLOT2/4, two microdomain-associated proteins translocated at the tip of the soybean RH in response to rhizobia. Auxin and cytokinin treatments are suffcient to trigger or inhibit the translocation of GmFWL1 and GmFLOT2/4 to the RH tip independently of the presence of rhizobia, respectively. Unexpectedly, the application of salicylic acid, a phytohormone regulating the plant defense system, also promotes the translocation of GmFWL1 and GmFLOT2/4 to the RH tip regardless of the presence of rhizobia. These results suggest that phytohormones are playing a central role in controlling the early stages of rhizobia infection by regulating the translocation of plasma membrane microdomains. They also support the concept of crosstalk of phytohormones to control nodulation

Unleashing the potential of the root hair cell as a single plant cell type model in root systems biology

Plant root is an organ composed of multiple cell types with different functions.This multicellular complexity limits our understanding of root biology because -omics studies performed at the level of the entire root reflect the average responses of all cells composing the organ. To overcome this difficulty and allow a more comprehensive understanding of root cell biology, an approach is needed that would focus on one single cell type in the plant root. Because of its biological functions (i.e., uptake of water and various nutrients; primary site of infection by nitrogen-fixing bacteria in legumes), the root hair cell is an attractive single cell model to study root cell response to various stresses and treatments. To fully study their biology, we have recently optimized procedures in obtaining root hair cell samples. We culture the plants using an ultrasound aeroponic system maximizing root hair cell density on the entire root systems and allowing the homogeneous teatment of the root system. We then isolate the root hair cells in liquid nitrogen. Isolated root hair yields could be up to 800 to 1000 mg of plant cells from 60 root systems. Using soy bean as a model, the purity of the root hair was assessed by comparing the expression level of genes previously identified as soy bean root hair specific between prepartions of isolated root hair cells and stripped roots, roots devoid in root hairs. Enlarging our tests to include other plant species, our results support the isolation of large quantities of highly purified root hair cells which is compatible with a systems biology approach

Analysis of the functional conservation of genes in a plant single cell type: the root hair cell

The evolution of plant species is tightly associated with major changes in their genome such as their allopolyploidy and autopolyploidy. These paleopolyploid events and recent genome duplications have contributed to the large sizes of the plant genomes and to the abundance of duplicated genes. This increases genomic content and, as a consequence, provides material for genetic mutations, drift, and selection. Therefore, genome duplication creates new possibilities of molecular evolution. Several studies focusing on the evolution of duplicated genes during plant development and in response to environmental stresses has been conducted. However, the cellular complexity of the plant organs used in these studies represents a difficulty to precisely characterize the molecular and functional conservation and divergence of duplicated genes. In this dissertation, taking advantage of publicly available genomic and transcriptomic sequences and functional genomic datasets, we aimed to precisely delineate the conservation and divergence of plant gene transcription and protein function. This analysis has been conducted with an unprecedented level of resolution using a single plant cell type, the root hair cell which emerged around 400 million years ago (mya). The rationale of the selection of this single plant cell type to conduct the projects is the following: the molecular response of a plant tissue or organ, which is often selected when conducting plant molecular studies, is a reflection of the average molecular reponses of the different cell types composing the tissue/organ. This cellular complexity is a limitation in our understanding of the molecular evolution of plant genes. This concept will be largely discussed in the introduction of this dissertation (Chapter 1). In chapter two, we described the development of an innovative plant culture system, the ultrasound aeroponic system, to access the root hair cells. This innovative plant culture system not only provides easy access to isolated root hair cells but also facilitates root hair observation and isolation, as well as the generation of transgenic root hair cells to enhance functional genomic studies. Finally, the ultrasound aeroponic system is compatible with the application of biotic and abiotic treatments on the plant root system. This is important because it opens avenues to precisely understand the adaptation of different plant species to environmental stresses and the evolution of these responses. In chapter 3, mining the Arabidopsis (Arabidopsis thaliana) and soybean (Glycine max) genome sequence and root hair transcriptomes, we performed a comparative analysis to reveal the molecular evolution of plant genes at the single cell type level. Our analysis revealed that the transcriptional activity of plant genes and the mechanisms controlling their expression in root hair cells are highly conserved between plant species. Focusing on nodulation, a biological process initiated by the symbiotic interaction between nitrogen-fixing bacteria (Rhizobia) and the legume root hair cell, we also performed a comparative genomic and transcriptomic analysis of the major regulators of the nodulation process and their homologs. This analysis is reported in Chapter 4. This study revealed the level of conservation and divergence of the nodulation-related genes across various legume species. In chapter 5, we further examined the molecular and functional conservation of genes and proteins by performing a comparative functional analysis of two regulators of the nodulation process: GmFWL1 and its interaction partner, a flotillin protein. Both proteins are microdomain-associated proteins. In response to rhizobial infection, these proteins translocate to the root hair cell tip where rhizobial recognition and invasion occur prior to the initiation of the infection process. Similar localization patterns of the Medicago (Medicago truncatula) flotillin ortholog in response to rhizobial infection (i.e., translocation at the tip of the root hair cell) suggests the conservation of the soybean and Medicago flotillin cellular functions and, more broadly, the role of plasma membrane microdomains during the nodulation process. This dissertation describes the use of a plant single cell type, the root hair cell, to study the conservation of plant genes expression and function. This work will expand our knowledge in plant evolutionary biology

Gene Silencing of \u3ci\u3eArgonaute5\u3c/i\u3e Negatively Affects the Establishment of the Legume-Rhizobia Symbiosis

The establishment of the symbiosis between legumes and nitrogen-fixing rhizobia is finely regulated at the transcriptional, posttranscriptional and posttranslational levels. Argonaute5 (AGO5), a protein involved in RNA silencing, can bind both viral RNAs and microRNAs to control plant-microbe interactions and plant physiology. For instance, AGO5 regulates the systemic resistance of Arabidopsis against Potato Virus X as well as the pigmentation of soybean (Glycine max) seeds. Here, we show that AGO5 is also playing a central role in legume nodulation based on its preferential expression in common bean (Phaseolus vulgaris) and soybean roots and nodules. We also report that the expression of AGO5 is induced after 1 h of inoculation with rhizobia. Down-regulation of AGO5 gene in P. vulgaris and G. max causes diminished root hair curling, reduces nodule formation and interferes with the induction of three critical symbiotic genes: Nuclear Factor Y-B (NF-YB), Nodule Inception (NIN) and Flotillin2 (FLOT2). Our findings provide evidence that the common bean and soybean AGO5 genes play an essential role in the establishment of the symbiosis with rhizobia

Comprehensive Comparative Genomic and Transcriptomic Analyses of the Legume Genes Controlling the Nodulation Process

Nitrogen is one of the most essential plant nutrients and one of the major factors limiting crop productivity. Having the goal to perform a more sustainable agriculture, there is a need to maximize biological nitrogen fixation, a feature of legumes. To enhance our understanding of the molecular mechanisms controlling the interaction between legumes and rhizobia, the symbiotic partner fixing and assimilating the atmospheric nitrogen for the plant, researchers took advantage of genetic and genomic resources developed across different legume models (e.g., Medicago truncatula, Lotus japonicus, Glycine max, and Phaseolus vulgaris) to identify key regulatory protein coding genes of the nodulation process. In this study, we are presenting the results of a comprehensive comparative genomic analysis to highlight orthologous and paralogous relationships between the legume genes controlling nodulation. Mining large transcriptomic datasets, we also identified several orthologous and paralogous genes characterized by the induction of their expression during nodulation across legume plant species. This comprehensive study prompts new insights into the evolution of the nodulation process in legume plant and will benefit the scientific community interested in the transfer of function algenomic information between species

Plant Hormones Differentially Control the Sub-Cellular Localization of Plasma Membrane Microdomains during the Early Stage of Soybean Nodulation

Phytohormones regulate the mutualistic symbiotic interaction between legumes and rhizobia, nitrogen-fixing soil bacteria, notably by controlling the formation of the infection thread in the root hair (RH). At the cellular level, the formation of the infection thread is promoted by the translocation of plasma membrane microdomains at the tip of the RH. We hypothesize that phytohormones regulate the translocation of plasma membrane microdomains to regulate infection thread formation. Accordingly, we treated with hormone and hormone inhibitors transgenic soybean roots expressing fusions between the Green Fluorescent Protein (GFP) and GmFWL1 or GmFLOT2/4, two microdomain-associated proteins translocated at the tip of the soybean RH in response to rhizobia. Auxin and cytokinin treatments are suffcient to trigger or inhibit the translocation of GmFWL1 and GmFLOT2/4 to the RH tip independently of the presence of rhizobia, respectively. Unexpectedly, the application of salicylic acid, a phytohormone regulating the plant defense system, also promotes the translocation of GmFWL1 and GmFLOT2/4 to the RH tip regardless of the presence of rhizobia. These results suggest that phytohormones are playing a central role in controlling the early stages of rhizobia infection by regulating the translocation of plasma membrane microdomains. They also support the concept of crosstalk of phytohormones to control nodulation