Biology subjects

Wudick, M. M.

Publications and source records attributed to Wudick, M. M..

8 recordsLinked to original sources

bioRxiv2026

Cell identities along the proximal-distal and micropylar-chalazal axes in the Arabidopsis heart-stage seed

* Seeds are complex reproductive organs consisting of diverse maternal and filial tissues. During development, the embryo and specialized tissues for nutrient storage required for seed germination and early seedling establishment emerge. * To explore the cellular diversity and differentiation of seeds, we performed single cell RNA-sequencing on heart stage Arabidopsis seeds and identified 20,097 cells that were grouped into [≥]21 distinct cell clusters. 20 of the 21 clusters were spatially assigned by combining bioinformatic analysis, imaging reporter fusion marker lines, and spatial transcriptomics. * Our analysis revealed a high degree of differentiation of epidermal cell and inner cell layers along the rotational and axial seed axes, highlighting the importance of cell position and ontogenesis. We identified unexpected spatial domains, including a cluster marked by abscission zone-specific transcripts, and a nucellar cluster shaped by developmentally programmed cell death. Surprisingly, embryo and endosperm showed similarities in transcript profiles despite distinct and complementary functions. * In summary, our findings establish seeds as a transcriptionally complex organ with high cell type heterogeneity and provide a basis for investigating the differentiation of diverse cell layers and spatial transcript profiles.

cell biology↗

bioRxiv2025

A critical role of sux cistron-mediated sucrose uptake for virulence of the rice blight pathogen Xanthomonas oryzae pv. oryzae

The virulence of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight (BB) of rice, critically depends on the activation of SWEET sucrose uniporters of the host. To date, the role of SWEET-released sucrose for virulence remains unclear. We here identified the sux locus of Xoo, consisting of a LacI-type repressor (SuxR), an outer membrane TonB-like porin (SuxA), an inner membrane MFS H+-symporter (SuxC), and a cytosolic sucrose hydrolase (SuxB). Structural and functional analyses demonstrate that SuxB has exclusive sucrose hydrolase activity. Mutant analyses show that the transporter SuxC and the sucrose hydrolase are necessary for growth of bacteria on sucrose, while SuxA is not essential, likely due to the ability of other porins to transport sucrose across the outer membrane. Consistent with a role of SuxR as a sucrose repressor, transcriptome studies show sucrose-dependent regulation of the suxA/suxB genes. Besides a role of sucrose for reproduction, we found that sucrose promotes motility, EPS production, biofilm formation, and virulence. Notably, the SuxC sucrose H+-symporter and the sucrose hydrolase SuxB were required for full virulence of Xoo on indica and japonica rice varieties. Our findings indicate that pathogen-induced sucrose efflux via SWEETs provides sucrose to Xoo, that Xoo uses the sux gene cluster to acquire and utilize sucrose, and that sucrose promotes bacterial fitness and xylem colonization. Significance StatementUnderstanding disease mechanisms is critical for developing strategies to protect plants against infections. Bacterial blight, a major threat to global rice production, is caused by Xanthomonas oryzae pv. oryzae strains that hijack rice SWEET sucrose uniporters. Xanthomonas oryzae pv. oryzae deploys a set of molecular "keys", TAL effectors, to trigger host sucrose release. Sugars could serve as nutrients for the bacteria or prime host defense. Here, we provide evidence that the pathogens ability to attack and grow depends on sucrose uptake. We explore how crucial virulence functions, such as motility, EPS production, and biofilm formation, depend on the activity of the sucrose utilization system, supporting a key role of sucrose for the nutrition of the bacteria in the xylem.

microbiology↗

bioRxiv2025

Cryo-EM structure of plant urea transporter DUR3 reveals essential role of C-terminal domain in tetramer assembly and insights into proton-coupled transport

Nitrogen pollution, caused by the overuse of nitrogen-fertilizers such as urea in agricultural production, has become a serious problem that threatens both the environment and human health. Improving the nitrogen use efficiency of crops is an important approach to reduce fertilizer use. DUR3, a high-affinity urea transporter, enables plants to acquire urea from natural or agricultural soils with low urea concentrations and has been proposed as a promising target for engineering crops to improve nitrogen-fertilizer use efficiency. Despite extensive studies for more than 20 years, the structure, substrate recognition and transport mechanism of DUR3 have remained unknown. Here we report the cryo-EM structure of maize DUR3 (ZmDUR3) at 3.41 [A] resolution. ZmDUR3 adopts an amino acid-polyamine-organocation (APC) superfamily fold and is structurally distinct from animal facilitative urea transporters. Our structure provides important insights into urea recognition by DUR3 and suggests that a pair of acidic residues is potentially involved in proton coupling during urea transport. Furthermore, our structural and functional studies indicate that DUR3 assembles into a tetramer and tetramer formation is important for its function. Our results pave the way for engineering plant DUR3 protein for applications in sustainable agricultural production.

plant biology↗

bioRxiv2023

A monochromatically excitable green-red dual-fluorophore fusion incorporating a new large Stokes shift fluorescent protein

Genetically encoded sensors enable quantitative imaging of analytes in live cells. State-of-the-art sensors are commonly constructed by combining ligand-binding domains with one or more sensitized fluorescent protein (FP) domains. Sensors based on a single FP are susceptible to artifacts caused by differing expression levels or sensor distribution in vivo. Hence, our lab developed dual-FP Matryoshka technology introduced by a single cassette that contains a stable large Stokes shift (LSS) reference FP nested within a reporter FP (cpEGFP), allowing simple construction of intensiometric sensors with the capacity for ratiometric quantification. The first-generation Green-Orange (GO) Matryoshka cassette established proof of concept but required custom optical setups to maximize achievable dynamic range. Here, we present a genetically encoded calcium sensor that employs optimized second-generation Green-Apple (GA) Matryoshka technology that incorporates a newly designed red LSSmApple fluorophore. LSSmApple provides improved excitation spectrum overlap with cpEGFP, allowing for monochromatic co-excitation with blue light. The exceptionally large Stokes shift of LSSmApple results in improved emission spectrum separation from cpEGFP, which minimizes fluorophore bleed-through and facilitates imaging using standard dichroics and red fluorescent protein (RFP) emission filters. We developed an image analysis pipeline for yeast (Saccharomyces cerevisiae) timelapse imaging that utilizes LSSmApple to segment and track cells for high-throughput quantitative analysis. In summary, we engineered a new fluorescent protein, constructed a genetically encoded calcium indicator (GA-MatryoshCaMP6s), and performed calcium imaging in yeast as a demonstration.

bioengineering↗

bioRxiv2023

Rice Yellow Mottle Virus resistance by genome editing of the Oryza sativa L. ssp. japonica nucleoporin gene OsCPR5.1 but not OsCPR5.2

Rice yellow mottle virus (RYMV) causes one of the most devastating rice diseases in Africa. Management of RYMV is challenging. Genetic resistance provides the most effective and environment-friendly control. The recessive resistance locus rymv2 (OsCPR5.1) had been identified in African rice (O. glaberrima), however, introgression into O. sativa ssp. japonica and indica remains challenging due to crossing barriers. Here, we evaluated whether CRISPR/Cas9 genome editing of the two rice nucleoporin paralogs OsCPR5.1 (RYMV2) and OsCPR5.2 can be used to introduce RYMV resistance into the japonica variety Kitaake. Both paralogs had been shown to complement the defects of the Arabidopsis atcpr5 mutant, indicating partial redundancy. Despite striking sequence and structural similarities between the two paralogs, only oscpr5.1 loss-of-function mutants were fully resistant, while loss-of-function oscpr5.2 mutants remained susceptible, intimating that OsCPR5.1 plays a specific role in RYMV susceptibility. Notably, edited lines with short in-frame deletions or replacements in the N-terminal domain (predicted to be unstructured) of OsCPR5.1 were hypersusceptible to RYMV. In contrast to mutations in the single Arabidopsis AtCPR5 gene, which caused severely dwarfed plants, oscpr5.1 and oscpr5.2 single knockout mutants show neither substantial growth defects nor symptoms indicative of programmed cell death, possibly reflecting functional redundancy of the isoforms regarding other important functions. The specific editing of OsCPR5.1, while maintaining OsCPR5.2 activity, provides a promising strategy for generating RYMV-resistance in elite Oryza sativa lines as well as for effective stacking with other RYMV resistance genes or other traits.

plant biology↗

bioRxiv2022

OzTracs: Optical Osmolality Reporters Engineered from Mechanosensitive Ion Channels

Interactions between physical forces and membrane proteins underpin many forms of environmental sensation and acclimation. Microbes survive sudden osmotic stresses with the help of mechanically gated ion channels and osmolyte transporters. Plant mechanosensitive ion channels have been shown to function in defense signaling. Here, we engineered genetically encoded osmolality sensors (OzTracs) by fusing green fluorescent protein spectral variants to the mechanosensitive ion channels MscL from E. coli or MSL10 from A. thaliana. When expressed in yeast cells, OzTrac sensors reported osmolality changes as a proportional change in emission ratio of the two fluorescent protein domains. Live-cell imaging revealed accumulation of fluorescent sensors in internal aggregates presumably derived from the endomembrane system. Thus, OzTrac sensors serve as osmolality-dependent reporters through an indirect mechanism, such as effects on molecular crowding or fluorophore solvation.

cell biology↗

bioRxiv2020

Unique and distinct identities and functions of leaf phloem cells revealed by single cell transcriptomics

AO_SCPLOWBSTRACTC_SCPLOWThe leaf vasculature plays a key role in solute translocation. Veins consist of at least seven distinct cell types, with specific roles in transport, metabolism, and signaling. Little is known about the vascular cells in leaves, in particular the phloem parenchyma (PP). PP effluxes sucrose into the apoplasm as a basis for phloem loading; yet PP has only been characterized microscopically. Here, we enriched vascular cells from Arabidopsis leaves to generate a single-cell transcriptome atlas of leaf vasculature. We identified [≥]19 cell clusters, encompassing epidermis, guard cells, hydathodes, mesophyll, and all vascular cell types, and used metabolic pathway analysis to define their roles. Clusters comprising PP cells were enriched for transporters, including SWEET11 and SWEET12 sucrose and UmamiT amino acid efflux carriers. PP development occurs independently from APL, a transcription factor required for phloem differentiation. PP cells have a unique pattern of amino acid metabolism activity distinct from companion cells (CC), explaining differential distribution/metabolism of amino acids in veins. The kinship relation of the vascular clusters is strikingly similar to the vein morphology, except for a clear separation of CC from the other vascular cells including PP. In summary, our scRNA-seq analysis provides a wide range of information into the leaf vasculature and the role and relationship of the leaf cell types.

plant biology↗

bioRxiv2020

Noxious effects of cell surface display glutamate sensors on plant growth and development

Plants use electrical and chemical signals for systemic communication. Herbivory, for instance, appears to trigger local apoplasmic glutamate accumulation, systemic electrical signals and calcium waves that travel to report insect damage to neighboring leaves and initiate defense. To monitor extra- and intracellular glutamate concentrations in plants, we generated Arabidopsis lines expressing genetically encoded fluorescent glutamate sensors. In contrast to cytosolically localized sensors, extracellularly displayed variants inhibited plant growth and proper development. Phenotypic analyses of high-affinity display sensor lines revealed that root meristem development, particularly the quiescent center (QC), number of lateral roots, vegetative growth and floral architecture were impacted. Notably, the severity of the phenotypes was positively correlated with the affinity of the display sensors, intimating that their ability to sequester glutamate at the surface of the plasma membrane was responsible for the defects. Root growth defects were suppressed by supplementing culture media with low levels of glutamate. Together, the data indicate that sequestration of glutamate at the cell surface either disrupts the supply of glutamate to meristematic cells and/or impairs localized glutamatergic signaling important for developmental processes. SIGNIFICANCE STATEMENTGlutamate is an important signaling molecule in animals and plants. The affinity-dependent effects of surface-displayed glutamate sensors on plant development intimate that glutamate levels in the vicinity of the plasma membrane play important roles in signaling in plants.

plant biology↗