Biology subjects

Harrison, M. J.

Publications and source records attributed to Harrison, M. J..

6 recordsLinked to original sources

bioRxiv2025

Mutation of M. truncatula SOBIR1 affects rhizobial specificity and arbuscular mycorrhizal colonization

Legume plants form symbiotic interactions with bacteria (rhizobia sp.) to obtain fixed nitrogen and arbuscular mycorrhizal fungi to obtain phosphorus and other nutrients. These interactions require the plant to distinguish beneficial from pathogenic organisms and trigger the plants innate immune system. We identified mutants in the Medicago truncatula SOBIR1 gene, known to be involved in innate immune response in multiple plants. We examined nodulation with multiple strains and species of Sinorhizobium and examined mycorrhizal interactions with the arbuscular mycorrhizal fungus Glomus versiforme (recently renamed Diversispora epigaea). Plants containing mutations in SOBIR1 exhibit normal nodulation with strains of S. meliloti, but fewer nodules when inoculated with S. medicae strains. The S. medicae nodules show evidence of accumulation of polyphenolic compounds, and abnormal arrangement of the rhizobia within the nodules. In contrast, when inoculated with Glomus versiforme. M. truncatula sobir1 mutant plants have increased mycorrhizal colonization and arbuscule number compared to the wild type. We localized a tagged SOBIR1 protein to the periarbuscular membrane interface. Together the data suggest the immune kinase SOBIR1 is involved in both nodulation and mycorrhizal interactions, but the effects of mutation differ.

plant biology↗

bioRxiv2025

Yeast-2-Hybrid-Seq and Bifluorescence Complementation Resources for assessing Protein:Protein Interactions in Arbuscular Mycorrhizal Roots: CKL2 as a Case Study

Reverse genetics, facilitated by CRISPR technologies and comprehensive sequence-indexed insertion mutant collections, has advanced the identification of plants genes essential for arbuscular mycorrhizal (AM) symbiosis. However, a mutant phenotype alone is generally insufficient to reveal the specific role of the protein in AM symbiosis and in many cases, identifying interacting partner proteins is useful. To enable identification of protein:protein interactions during AM symbiosis, we established a Medicago truncatula -Diversispora epigaea yeast-two-hybrid (Y2H) library which, through Y2H-seq screening, can provide a rank-ordered list of candidate interactors of a protein of interest. We also developed a vector system to facilitate bimolecular fluorescence complementation assays (BIFC) in mycorrhizal roots so that protein interactions can be assessed in their native cell types and sub-cellular locations. We demonstrate the utility of a Y2H-seq screen coupled with BIFC in mycorrhizal roots, with a search for proteins that interact with CYCLIN DEPENDENT LIKE KINASE 2 (CKL2), a kinase essential for AM symbiosis. The Y2H-seq screen identified three 14-3-3 proteins as the highest ranked CKL2 interacting proteins. BIFC assays in mycorrhizal roots provided evidence for a CKL2:14-3-3 interaction at the periarbuscular membrane (PAM) in colonized root cells. Down-regulation of 14-3-3 by RNA interference provides initial evidence for a function in AM symbiosis. Thus, CKL2 may utilize 14-3-3 proteins to direct signaling from the PAM. The Y2H and BIFC resources will accelerate understanding of protein functions during AM symbiosis.

plant biology↗

bioRxiv2024

Near-Zero Photon Bioimaging

Enhancing the reliability and reproducibility of optical microscopy by reducing specimen irradiance continues to be an important biotechnology target. As irradiance levels are reduced, however, the particle nature of light is heightened, giving rise to Poisson noise, or photon sparsity that restricts only a few (0.5%) image pixels to comprise a photon. Photon-sparsity can be addressed by collecting more than 200 photons per pixel; this, however, requires extended acquisition durations and, thus, suboptimal imaging rates. Here, we introduce near-zero photon imaging, a method that operates at kHz rates and 10,000-fold lower irradiance than modern microscopy. To achieve this performance, we deployed a judiciously designed epi-fluorescence microscope enabling ultralow background and artificial intelligence that learns to reconstruct biological images from as low as 0.01 photons per pixel. We demonstrate that near-zero photon imaging captures the structure of both multicellular and subcellular targets with high fidelity, including features represented by nearly zero photons. Beyond optical microscopy, the near-zero photon imaging paradigm can be applied in remote sensing, covert applications, and biological or biomedical imaging that utilize damaging or quantum light.

biophysics↗

bioRxiv2023

Microenvironmental Stiffness Induces Metabolic Reprogramming in Glioblastoma

The mechanical properties of solid tumors influence tumor cell phenotype and ability to invade into surrounding tissues. Using bioengineered scaffolds to provide a matrix microenvironment for patient-derived glioblastoma (GBM) spheroids, this study demonstrates that a soft, brain-like matrix induces GBM cells to shift to a glycolysis-weighted metabolic state which supports invasive behavior. We first show that orthotopic murine GBM tumors are stiffer than peri-tumoral brain tissues, but tumor stiffness is heterogenous where tumor edges are softer than the tumor core. Then, we developed three-dimensional scaffolds with {micro}-compressive moduli resembling either stiffer, tumor core or softer, peri-tumoral brain tissue. We demonstrate that the softer matrix microenvironment induces a shift in GBM cell metabolism toward glycolysis which manifests in lower proliferation rate and increased migration activities. Finally, we show that these mechanical cues are transduced from the matrix via CD44 and integrin receptors to induce metabolic and phenotypic changes in cancer cells.

cancer biology↗

bioRxiv2022

Characterization and visualization of global metabolomic responses of Brachypodium distachyon to environmental changes

Plant responses to environmental change are mediated via changes in cellular metabolomes. However, <5% of signals obtained from tandem liquid chromatography mass spectrometry (LC-MS/MS) can be identified, limiting our understanding of how different metabolite classes change under biotic/abiotic stress. To address this challenge, we performed untargeted LC-MS/MS of leaves, roots and other organs of Brachypodium distachyon, a model Poaceae species, under 17 different organ-condition combinations, including copper deficiency, heat stress, low phosphate and arbuscular mycorrhizal symbiosis (AMS). We used a combination of information theory-based metrics and machine learning-based identification of metabolite structural classes to assess metabolomic changes. Both leaf and root metabolomes were significantly affected by the growth medium. Leaf metabolomes were more diverse than root metabolomes, but the latter were more specialized and more responsive to environmental change. We also found that one week of copper deficiency shielded the root metabolome, but not the leaf metabolome, from perturbation due to heat stress. Using a recently published deep learning based method for metabolite class predictions, we analyzed the responsiveness of each metabolite class to environmental change, which revealed significant perturbations of various lipid classes and phenylpropanoids such as cinnamic acids and flavonoids. Co-accumulation analysis further identified condition-specific metabolic biomarkers. Finally, to make these results publicly accessible, we developed a novel visualization platform on the Bioanalytical Resource website, where significantly perturbed metabolic classes can be readily visualized. Overall, our study illustrates how emerging chemoinformatic methods can be applied to reveal novel insights into the dynamic plant metabolome and plant stress adaptation.

plant biology↗

bioRxiv2020

Constitutive overexpression of RAM1 increases arbuscule density during arbuscular mycorrhizal symbiosis in Brachypodium distachyon

Arbuscular mycorrhizal (AM) symbiosis is a mutually beneficial association of plants and fungi of the sub-phylum Glomeromycotina. The endosymbiotic AM fungi colonize the inner cortical cells of the roots, where they form branched hyphae called arbuscules that function in nutrient exchange with the plant. To support arbuscule development and subsequently bidirectional nutrient exchange, the root cortical cells undergo substantial transcriptional re-programming. REDUCED ARBUSCULAR MYCORRHIZA 1 (RAM1), studied in several dicot plant species, is a major regulator of this cortical cell transcriptional program. Here, we generated ram1 mutants and RAM1 overexpressors in a monocot, Brachypodium distachyon. The AM phenotypes of two ram1 lines revealed that RAM1 is only partly required to enable arbuscule development in B. distachyon. Transgenic lines constitutively overexpressing BdRAM1 showed constitutive expression of AM-inducible genes even in the shoots. Following inoculation with AM fungi, BdRAM1-overexpressing roots showed higher arbuscule densities relative to controls, indicating the potential to manipulate the relative proportion of symbiotic interfaces via modulation of RAM1. However, the overexpressors also show altered expression of hormone biosynthesis genes and aberrant growth patterns including stunted bushy shoots and poor seed set. While these phenotypes possibly provide additional clues about BdRAM1s scope of influence, they also indicate that directed approaches to increase the density of symbiotic interfaces will require a more focused, potentially cell-type specific manipulation of transcription factor gene expression.

plant biology↗