Microbes, plants, and humans are globally interconnected through dynamic symbiotic relationships that influence ecosystem balance, agricultural productivity, and human health. Among these interactions, endosymbiosis, characterized by microorganisms residing within the cells of other species, represents a critical relationship that requires sustained communication across kingdoms. This thesis explores the connections between microbes, plants, and humans and examines recently developed approaches to the study of endosymbiosis and their implications for plant productivity and human nutrition. Starting with a discussion of the new methods by which scientists are beginning to target crop genetic engineering for certain beneficial effects on the human gut microbiome and moving to a discussion of new available RNA-sequencing technologies and their applications in plant-microbe endosymbiosis, I present unique avenues to symbiosis research that overcome barriers to prior investigations. Applying single-nuclei and spatial transcriptomic technologies to the arbuscular mycorrhizal symbiosis, I demonstrate the utility of such approaches in the identification of symbiosis-responsive cell populations from both species and present hundreds of new targets for further functional characterization and genetic engineering of an improved symbiosis. Lastly, I analyze the effects of such genetic modifications on symbiotic interactions in agriculturally-relevant plant species, contributing to the development of strategies to enhance symbiosis in agricultural settings. In essence, this thesis presents fundamental discoveries about previously unknown aspects of cross-species communication during symbiosis and offers insights into the latest methodologies applied to analyze symbiosis at a cellular level.

The plant cell wall is a complex and integral component of the cell mediating the cellular response to both external and internal stimuli. As the first physical barrier to the environment external to the cell, the cell wall must be able to withstand pressures or attacks while also sensing more subtle environmental perturbations. As the physical support structure and shaper of the cell, the wall must be able to dynamically adapt to necessary growth or developmental conditions as the cell matures, including adapting to stress conditions. Recent transcriptomic and targeted proteomic and metabolomic studies have implicated cell wall-related biosynthetic and modification genes and enzymes in biotic and abiotic stress responses. Targeted manipulation of expression of cell wall-modifying enzymes confers either stress tolerance or susceptibility to transgenic plants. Research coupling the cell wall-related transcriptomic response to chemical wall analyses in field-grown Sorghum bicolor exposed to drought stress shows that while a large and consistent transcriptomic response occurs, large compositional shifts in the cell wall do not occur, the only exception being in the younger leaves of a drought-tolerant genotype, which had increases in matrix monosaccharides. Further, the cell wall response to drought stresses in these S. bicolor leaves did not negatively impact sugar yield, in one genotype increasing sugar yield from non-structural polysaccharides like starch.

Changes in the cell wall-related transcriptomes and cell wall compositions of field-grown, drought stressed S. bicolor leaves and roots frequently implicated changes in pectin biosynthesis and modification. This is consistent with drought and in fact several biotic and abiotic stress studies in both eudicotyledons and commelinid grasses. As a heterogeneous polysaccharide containing domains with distinct branching, architecture, and chemistry, pectin mediates cell wall hydration status, extensibility of the expanding or maturing wall, and even acts as a signaling molecule. Recent studies have demonstrated that targeted pectin modification through reverse genetics confers either tolerance or susceptibility to a variety of biotic and abiotic stresses. Despite its significance in the wall, pectin biosynthesis is still poorly understood, with less than twenty-five of the more than sixty putative transferases necessary for its unique linkages described. One of the most heterogeneous domains of pectin, rhamnogalacturonan I (RG-I), is defined by its repeating -(1,2)-α-L-Rhap-(1,4)-β-D-GalpA- backbone. This backbone can be substituted on the rhamnosyl residues at the O3 or O4 positions with four potential side chains, consisting of either linear arabinan, linear galactan, type I arabinogalactans, or type II arabinogalactans. However, structures other than these four canonical side chains have been described previously. Though galactans comprise the bulk of RG-I, the full biosynthetic pathway is still unknown. To date, two different RG-I galactosyltransferases have been described, both involved in linear (1,4)-β-D-galactan elongation. An additional RG-I galactosyltransferase, GT47F, has been identified that is crucial to normal development in Nicotiana benthamiana and embryo and vegetative development in Arabidopsis thaliana. The silencing of GT47F results in dwarfed N. benthamiana and A. thaliana, with misshapen cells and mildly chlorotic leaves in N. benthamiana. RG-I composition from silenced N. benthamiana plants demonstrated a smaller RG-I with less galactan per RG-I moiety. Although monoclonal antibody binding to isolated pectin from these lines did not demonstrate a difference in linear (1,4)-β-D-galactan or the unsubstituted RG-I backbone, an increase in branched (1,6)-β-galactans was observed in silenced N. benthamiana lines. T-DNA lines in which the third exon is interrupted, and which likely abolish all activity, are homozygous embryo-lethal in A. thaliana. N. benthamiana microsomes over-expressing a YFP-tagged GT47F demonstrated binding of UDP-galactose in microscale thermophoresis experiments. Taken together, GT47F likely encodes a developmentally-required galactosyltransferase that contributes to RG-I galactosylation. Absence of this galactosylation results in increased branched galactan epitopes and smaller molecular weight of RG-I.

Plants form a variety of endosymbiotic relationships with bacteria and fungi that promote plant growth and fitness through reciprocal nutrient exchange. Central to the development and function of endosymbiotic relationships is the synthesis of a specialized host-derived membrane, which compartmentalizes the endosymbiont inside plant cells, and creates a dynamic interface for the exchange of nutrients and information. The development of these interfaces is dependent upon bidirectional signaling between the plant and microorganism, and is highly coordinated with the morphological differentiation of the endosymbiont within plant cells. Previous studies using glycan-directed monoclonal antibodies indicated that glycoproteins, glycolipids, and pectic polysaccharides localize to periarbuscular and symbiosome membranes. Among these epitopes were arabinogalactan proteins (AGPs) and glycosyl inositolphophorylceramides (GIPCs), which have potent signaling properties in plants. These epitopes appeared to be developmentally regulated, which led to the hypothesis that a membrane-associated glycocalyx of glycoproteins and glycolipids might be important for mediating interactions through these membrane interfaces. However, AGPs have not been well documented outside of Arabidosis thaliana and are intrinsically disordered, which make them difficult to identify and study in plants species capable of forming endosymbiosis. Here, we have developed a new bioinformatic search tool that identifies AGP-encoding genes based on the noncontiguous hydroxyproline motifs that direct AGP glycosylation. We used this tool to identify all putative AGP-encoding genes in the Medicago truncatula genome, which were cross-referenced to transcriptomic studies of roots engaged in symbiosis with Sinohizobium meliloti and the arbuscular mycorrhizal (AM) fungus Rhizohagus irregularis. Using this approach we identified a small three-member family of tandemly duplicated SYMBIOSIS-ASSOCIATED ARABINOGALACTAN PEPTIDES (SAPs) that were differentially expressed in root nodules and AM colonized roots. SAPs localized to symbiotic membranes and knockdown of SAP expression using RNAi-mediated gene silencing impaired the growth and differentiation of Sinorhizobium meliloti and Rhizphagus irregularis within these compartments. In parallel we also identified a glycosyltransferase gene highly expressed in root nodules and AM colonized roots as GIPC GLUCOSAMINE TRANSFERASE 1 (GINT1), and showed that the corresponding GINT1 enzyme functions in the synthesis of HexN(Ac) decorated GIPCs in planta. Silencing of GINT1 using RNAi impaired the development of symbiotic membranes, which resulted in the senescence of symbiosomes and arbuscules. Taken together these results provide genetic evidence to support that reprogramming of the membrane-associated glycocalyx with specific AGPs and GIPCs is necessary for endosymbiosis.

Large-scale, sustainable production of lignocellulosic bioenergy from plant biomass will likely depend on a variety of dedicated bioenergy crops. Compared to their genetic and genomic diversity, polysaccharide composition varies little from one species to the next. Genetic engineering can be used to dramatically improve plant biomass for biofuel production. Engineering strategies that act independently of genetic context are particularly important, since they can improve the polysaccharide composition in a variety of species. Cellulose is the most abundant polysaccharide in all plants and the source of the six-carbon sugar glucose that is, ultimately, converted by genetically engineered microbes into liquid fuels and a variety of useful commodity chemicals. The ideal bioenergy crop would be as cellulose-dense as possible, and overexpression of plant and fungal enzymes can dramatically increase cellulose production and make simpler to break down. The second most abundant polysaccharide in bioenergy crops is xylan, composed of the five-carbon sugar xylose. Xylan associates tightly with cellulose microfibrils and, via its side chains, forms covalent linkages with other components of the cell wall, making xylan a significant contributor to the difficulty of biomass deconstruction. Xylan degrades and releases toxic compounds during the pretreatment of lignocellulosic biomass. These compounds and xylose itself are significantly detrimental to the microbial fermentation of glucose. Thus, a reduction in the amount of xylan and its complexity are additional characteristics that should be engineered into the ideal bioenergy crop. From a plant genetic engineering perspective, reducing the biosynthesis of a polysaccharide is significantly more challenging than increasing it, as a loss-of-function mutation in a specific gene would typically be required. Bioenergy crops often have multiple, functionally redundant, copies of a gene in their genome. Even with the advancement of genome editing technologies like CRISPR, homologous enzymes may have diverged in DNA sequence, making the task of knocking out each copy very laborious and time-consuming. I developed a protein-level genetic engineering approach to significantly reduce xylan content by overexpressing a catalytically dead or diminished (i.e. antimorphic) version of the xylan biosynthetic enzyme Irregular xylem10 (IRX10) in the model plant Arabidopsis thaliana. This work additionally provides experimental evidence supporting the hypothesis that xylan biosynthesis requires a complex of proteins in the Golgi, a complex in which IRX10 is the agent of catalysis. The journal publication of this work has been adapted in Chapter 1. High-level expression of the antimorph “out-competed” the native enzyme for its place in the complex, thereby generating xylan synthase complexes with little to no catalytic activity. A major advantage to this strategy is that sufficiently high expression would likely have the same effect on any redundant IRX10 homologs the plant may express. Additionally, the amino acid residues mutated to create the antimorphic protein are fully conserved in all land plants, so the technology could be translated to a variety of bioenergy crops with relative ease and the US Patent Application regarding it is the subject of Chapter 2. Continuing my study of dominant genetic engineering strategies for improving the qualities of biomass crops, I conducted an intensive study of carbohydrate active enzymes and proteins that have been heterologously expressed in plants to modify polysaccharides in muro. This invited review, for future publication in Frontiers in Plant Science, is covered in Chapter 3.

One of the largest and longest standing societal challenges is acquiring enough food to support the human population. Our current era has the added complexities of the population nearing 9 billion, a globalized industrial agricultural production system, and a clearer understanding of how environmentally harmful and unsustainable that system is. While these issues are multifaceted, agriculture is fundamentally coupled with plant and microbial biology and there has been a diversity of avenues pursued by researchers seeking potential solutions and disruptive innovations to address our need to provide healthy food for the world without destroying it in the process. Modern agricultural practices currently require high energetic costs through extensive irrigation, application of synthetic fertilizers and pesticides, and the use of large, mechanized equipment. The lack of biodiversity in large-scale monoculture systems also presents issues in crop nutritional quality, disease pressure, and soil health. Recent advancements in our understanding of plant root systems and the relationships they form with soil microbiota have demonstrated a wealth of potential applications regarding these issues as well as in further illuminating the complex processes at play in plant health and development in an ecological context. It has become increasingly clear that plant roots are essential components to local and global nutrient cycling and drive much of the microbial activity in soils. In order to ensure a future with sustainable, climate-resilient agriculture which enables nutritious diets, we must continue to explore the wealth of knowledge underground. This dissertation seeks to clarify some of the socioeconomic reasons why we grow food the way we do and the limitations of material available for study, as well as underlying biochemical and genetic mechanisms which influence plant root biology and their relationships with microorganisms. The results of this research contribute to the growing body of knowledge in root biology and a paradigm shift in how we understand plants and agriculture to be connected to the wider ecosystem.Chapter 1 seeks to understand the historical and contemporary contexts which influence the scale, scope, and direction of research in agricultural and plant sciences. Analyzing the socioeconomic institution of modern Science, from the conditions leading to the Scientific Revolution in the 16th century to modern day, reveals the conserved influence of western European colonial-imperialism on global agricultural production. From its inception, the institution of Science is shown to be integral to the expansion and maintenance of Western imperial powers materially and socially, by driving critical revenue generation via the enabling and adoption of cash crop agriculture and through controlling the value and direction of intellectual pursuits. From the 19th century we see Science increasingly entangled with emerging capitalist corporate and state interests, which further entrench practices that began with cash crop agriculture and prove to be detrimental to environmental health while distancing crop production from nutritional needs. I describe how these relationships ultimately limit the resources we have available for research in the modern day, hindering our abilities to address the myriad socio-scientific challenges of this century. This clarity is important when making the critical and strategic assessments necessary to direct on-going and future research and teaching efforts. Chapter 2 is an effort to detail that plant roots are essential to plant health and adaptation as well as important contributors to numerous ecological and biogeochemical processes. Despite this, they have been comparatively understudied aspects of plant biology. Recent developments have indicated that a better understanding of root systems can reveal breeding and engineering applications towards plants more well-suited for low-input agricultural systems and frequent stresses. I highlight four co-authored publications concerning the study of root systems: Detailing the limitations and considerations of root system study as well as demonstrations of progress to address these challenges, metabarcoding combined with genomics and data analytics programs to inform the basis of rhizosphere microbiome heritability in Sorghum bicolor, constitutive promoters to fine tune gene expression in synthetic biology with demonstrated function in root systems, and utilizing root system imaging methods and software to inform the effect of a bacterial metabolite on plant-bacterial associations in Arabidopsis thaliana. These studies hope to provide examples of and inspire further efforts to understand root systems, thus enabling improvements in plant performance under new and changing agricultural practices better suited for the world we live in today. Chapter 3 seeks to further illuminate the underlying mechanisms of pectin biosynthesis. Pectin is an abundant component of plant cell walls demonstrated to be important in cell to cell adhesion, plant development, and a variety of signaling pathways. Understanding has so far remained limited due to the wide diversity and redundancy of enzymes involved in the process. The pectin component rhamnogalacturonan I (RG-I) is biosynthesized in part by rhamnosyltransferases (RRTs) in the GT106 family. While it is known that RRTs are expressed in a range of tissues, few studies have demonstrated their function outside of RG-I biosynthesis in seed coat mucilage. In this study we show Lotus japonicus RRT1 contributes to the addition of rhamnose monosaccharide residues to RG-I in root tissues. Mutants contained ~19% less rhamnose in their roots and pectin from aboveground tissues had less galactose and more xylose. RG-I in root tissue from Ljrrt1 also had a larger molecular weight and altered structure compared to wild type (WT) Gifu plants, but this was not due to transcriptional differences in other GTs responsible for RG-I biosynthesis. Mutants exhibited altered root morphology, impacted stem and root growth, and impairment of nodule formation when inoculated with Mesorhizobium loti. These findings constitute the first demonstration of RRT function in vascular plants outside of seed coat mucilage and contribute to the increasingly nuanced understanding of RG-I in cell wall biosynthesis and intersecting processes. Chapter 4 is an effort to characterize an unknown subclass of plant GTPase-related signaling proteins which appear to influence symbiotic relationships formed in root systems. Plants possess a unique class of heterotrimeric G? subunits called extra-large GTPases (XLGs) which contribute to numerous developmental and stress responses. XLGs have an uncharacterized N-terminal domain, a G?-like C-terminal domain, and overlapping and distinct functions compared to conventional G? subunits. In this study, we identified homologs of XLG3 in Lotus japonicus responsive to rhizobial and mycorrhizal symbiosis. However, these proteins were approximately one-third the size of conventional XLGs and only aligned to the N-terminal domain, containing a putative NLS and the cysteine-rich domain of unknown function. Multiple sequence alignment and phylogenetic analysis determined SXLGs did not share domains with other mono- or heterotrimeric G-protein classes and exhibited a pattern of duplication and neofunctionalization typical of genes involved in symbiotic signaling pathways. Transient expression of LjSXLGs in tobacco demonstrated their potential for localization to the plasma membrane, nucleus, and nucleolus. Analysis of L. japonicus sxlg2 mutants revealed transient impairment of immature nodule formation in a destructive experimental setup and inhibition of infection events in a nutrient-limited non-destructive experimental setup, with no observed difference in nodule maturation rate. Additionally, sxlg2 mutants showed a potential impairment of the root growth response in N-limited conditions. SXLGs present an ideal opportunity to better understand the evolution, function, and structure of XLGs and are another example of G-protein involvement in symbiotic relationships. Ultimately, this research supports growing efforts to develop more resilient and sustainable agricultural practices through a focus on root systems biology by providing assessments of the technical and methodological resources in the field, demonstrating dynamics of pectin biosynthesis in root tissue, and uncovering new elements of plant symbiotic and G-protein related signaling pathways. These findings will promote further mechanistic and evolutionary discoveries in aspects of root biology that remain filled with questions and unknowns, but also notable potential in the development of roots more amenable to regenerative agricultural approaches, maximizing benefits from microbial associations, and utilization in the biosynthesis of valuable products and biofuels. The situating of historical and contemporary socioeconomic contexts that have heavily influenced the progression of agriculture and plant sciences over the past half a millennium is a critical addition to our ability to wholly assess the materials, practices, and technologies available for further research. This clarity is essential if we truly wish to address the systemic challenges of our era. Technical solutions have limited ability to resolve complex socio-scientific issues without understanding the broader social contexts they were born from and operate in. In the same way that systems biology has begun to permeate many scientific disciplines, our perspectives must shift to accommodate the nuance and complexity of how interconnected this world is