Biology subjects

Demirer, G. S.

Publications and source records attributed to Demirer, G. S..

16 recordsLinked to original sources

bioRxiv2026

CRISPR-Associated Transposases Enable Programmable DNA Integration in Plants

Programmable DNA integration is a major challenge in plant genome engineering. CRISPR-associated transposases (CAST) catalyze efficient RNA-guided DNA integration without double-strand breaks, yet their activity has not been established in plants. Here, we reconstituted and engineered a Type I-F CAST for programmable DNA integration in plant cells. We validated expression of the wild-type Pseudoalteromonas CAST (PseCAST) machinery in plants and established targeted episomal integration in Arabidopsis thaliana protoplasts and chromosomal integration at a transgenic locus in Nicotiana benthamiana. The evolved PseCAST system, evoCAST, showed chromosomal integration efficiencies of 2.7%, representing a 6-fold improvement over wild-type PseCAST. evoCAST also enabled the insertion of cis-regulatory elements into a synthetic landing pad with 8% efficiency. evoCAST was subsequently retargeted to six endogenous genomic loci, demonstrating programmable integration across diverse chromosomal contexts. Finally, a cofactor screen identified the chromatin-associated factor AtHMGB2 as an enhancer of evoCAST-mediated integration activity in plants. These results establish CAST as a functional platform for programmable DNA insertion in plants and provide a foundation for developing targeted genome-engineering technologies for crop biotechnology.

bioengineering↗

bioRxiv2026

Sentinel Plants Enable Aboveground Detection of Belowground Soil Microbial Activity

Rhizosphere microbial processes play a central role in soil function and plant health yet remain difficult to monitor noninvasively. Engineered sentinel plants that use bacterial-to-plant communication channels are promising. However, no such efforts have thus far enabled a detectable aboveground response in the sentinel plant. Here, we optimize a previously described synthetic bacteria-to-plant communication channel based on the p-coumaroyl-homoserine lactone (pC-HSL) signaling molecule in plants to function as aboveground sentinels of belowground microbial activities. Arabidopsis thaliana sentinel plants harboring this optimized circuit detect root-applied pC-HSL at concentrations as low as 30 nM in roots and 3 M in leaves, demonstrating long-distance signal transmission from below ground to aboveground tissues. Moreover, sentinel plants report pC-HSL production by engineered Escherichia coli and Pseudomonas putida colonizing plant roots in both plate and soil assays. These results establish an engineered plant platform that converts rhizosphere microbial activity into a visible aboveground signal, enabling a minimally invasive platform for monitoring rhizosphere microbial gene expression and for precision agriculture and soil management.

bioengineering↗

bioRxiv2026

Sentinel plants enable quantitative monitoring of bioavailable nitrate in soils and microbial environments

Microbial transformations of nitrogen in soils strongly influence plant nutrition and ecosystem function, yet these processes remain difficult to monitor. Existing approaches rely largely on destructive soil sampling and laboratory analysis, limiting the ability to track nitrate dynamics in situ. Here, we engineer "sentinel plants," genetically encoded plant biosensors that convert nitrate perception into a quantitative signal reporting plant-accessible nitrate. The sensor uses a synthetic nitrate-responsive promoter coupled to a ratiometric luciferase reporter, enabling high-dynamic-range measurements. Sentinel plants exhibit a dose-dependent, reversible nitrate response with high specificity over alternative nitrogen sources. In agricultural soils from multiple California field sites, sensor output closely tracked analytically measured nitrate concentrations and resolved nitrate amendments without destructive extraction. Beyond environmental sensing, sentinel plants enabled screening of nitrogen-fixing microbial communities and the detection of microbially generated nitrate in both liquid culture and soil systems. Using this platform, we identified a minimal three-member microbial consortium capable of converting atmospheric nitrogen into nitrate via sequential nitrogen fixation and nitrification. This consortium increased tissue nitrate accumulation and plant fresh weight, demonstrating that sentinel plants can both monitor nitrate availability and identify microbial communities that enhance plant growth. Significance StatementNitrogen availability in soils fluctuates across space and time, yet most measurements rely on extracting soil samples and analyzing them in the laboratory. Such measurements provide only snapshots of nitrogen status and do not necessarily reflect the nitrogen that plants themselves experience. Here, we engineer plants that act as living nitrate sensors by converting nitrate perception into a measurable optical signal. Because these sensors operate within intact plants, they report nitrate availability as integrated through plant uptake and physiology rather than through chemical extraction alone. Using this platform, we tracked nitrate levels in agricultural soils and identified a minimal microbial consortium capable of converting atmospheric nitrogen into plant-available nitrate. This plant-based sensing strategy enables direct monitoring of nitrogen dynamics in soils and microbial environments, providing a platform for identifying microbial communities that enhance nitrogen availability for crops.

bioengineering↗

bioRxiv2025

Extracellular contractile injection systems for high efficiency protein delivery to plants

Protein delivery into plant cells remains a major barrier in plant biotechnology. Extracellular contractile injection systems (eCIS) are phage tail-like nanomachines evolved by bacteria to engage eukaryotic host cells and deliver protein effectors. The Photorhabdus virulence cassette (PVC) is a well-characterized eCIS that naturally targets insect hosts. Here, we retargeted PVCs to plants by engineering their tail fibers to recognize the plant receptor FLAGELLIN SENSITIVE2 (FLS2). We designed and characterized a library of FLS2-targeting PVC variants and evaluated their ability to deliver native and non-native protein cargoes, including a fluorescent protein, Cre recombinase, and R2 retroelement genome editor. Engineered PVCs delivered Cre into wild-type Arabidopsis thaliana protoplasts and Nicotiana benthamiana leaf cells with efficiencies of up to 15%. Delivery was FLS2-dependent and scaled with receptor abundance, reaching more than 75% of leaf cells in FLS2-overexpressing lines. We further demonstrated PVC-mediated delivery of R2, resulting in targeted insertion of an mCherry reporter into the plant genome. This work establishes PVCs as receptor-mediated protein-delivery vehicles for plant cells and provides a foundation for targeted, DNA-free genome engineering in plant biotechnology.

bioengineering↗

bioRxiv2025

Optimized R2 Retroelement Complexes Enable Precise and Efficient DNA Insertion into Plant Genomes

Precise, targeted insertion of multi-kilobase DNA sequences into plant genomes is critical for studying gene function, ensuring robust transgene expression, and stacking traits in crops, but remains challenging. Existing targeted insertion methods in plants relying on programmable nucleases are inefficient and can generate unwanted mutations. Newer technologies based on prime editors, transposases, and site-specific recombinases extend capabilities but remain constrained with low efficiencies, off-target integration, silencing, or limited DNA payload size. R2 non-long terminal repeat (non-LTR) retrotransposons integrate via target-primed reverse transcription (TPRT) specifically targeting the 25S ribosomal DNA multicopy site and enabling double-strand-break-free installation of gene-sized DNA sequences. We adapted the avian Taeniopygia guttata R2 protein (R2Tg) for targeted DNA insertion into plant genomes through engineering of R2Tg expression cassettes and RNA payloads carrying intron-disrupted mCherry and RUBY retrotransposition reporters with length-optimized rDNA homology arms. These efforts, together with optimized construct delivery formats and incubation temperatures, define R2 editor design rules enabling efficient DNA integration and functional protein expression from the 25S rDNA locus. In Nicotiana benthamiana leaves, Arabidopsis thaliana protoplasts, and Solanum lycopersicum seedlings, the optimized R2Tg editor system achieved targeted insertion with efficiencies up to 24% payloads ranging in size from 2kb to 5kb. This work establishes a compact R2Tg ribonucleoprotein platform for targeted DNA insertion into plant genomes, targeting a multicopy genomic safe harbor site to enable efficient multi-kilobase gene addition

bioengineering↗

bioRxiv2025

A 3D-Printed Capillary Tube Holder for High-Throughput Chemotaxis Assays

Bacterial chemotaxis is an important behavior to study to understand spatial segregation of species in mixed communities and the assembly of host microbiomes. This is particularly relevant in the rhizosphere, where chemoattraction towards root exudates is an important determinate of plant colonization. However, current methods to screen chemoeffectors are limited in their throughput, creating a barrier to generating comprehensive datasets describing chemotactic profiles for species of interest. Here, we describe a novel, 3D-printed capillary tube holder approach, which enables up to 384 simultaneous capillary tube chemotaxis assays. We optimized and benchmarked our assay using Escherichia coli k12 and Bacillus subtilis 3610 and their known chemoattractants: serine and aspartate. We then tested the threshold concentration of these chemoattractants in our assay and found we could detect chemoattraction towards concentrations spanning multiple orders of magnitudes. In this paper, we describe our novel high-throughput chemotaxis assay in detail and provide the necessary files for 3D printing the capillary tube holder.

microbiology↗

bioRxiv2025

Vacuum and sonication treatment enables efficient transient gene expression in various monocot and eudicot plant seedlings

Transient gene expression in intact plants is essential for rapidly addressing biological questions, where the current toolkit can be improved for higher efficiencies and broader plant species range. Here, we introduce VAST (Vacuum and Sonication-Assisted Transient expression): a transient transformation method that substantially enhances gene expression efficiency, reproducibility, and versatility across diverse monocot and eudicot intact seedlings. By systematically optimizing plant growth conditions and incorporating vacuum infiltration and sonication pre-treatments prior to seedling co-culture with Agrobacterium tumefaciens, we significantly improved transient gene expression efficiency while minimizing tissue damage compared to existing methods in Arabidopsis thaliana. We further demonstrated the broad applicability of VAST by successfully transforming key model and crop species, including tomato, Brassica rapa, Medicago sativa, Setaria italica (foxtail millet), switchgrass, and wheat. We also demonstrated a case study using the VAST-mediated transient transformation, where a cross-species analyses of nitrate-responsive gene expression highlighted both the conserved and diverged biological responses between the two model crops, A. thaliana and S. italica. VASTs simplicity, versatility, and efficiency provide a powerful tool for functional genomics, synthetic biology, and biotechnology research, opening new avenues for the rapid exploration of gene function, regulation, and editing in diverse plant systems.

plant biology↗

bioRxiv2023

Conservation and divergence of regulatory architecture in nitrate-responsive plant gene circuits

Nitrogen is an essential element for all life processes in plants. As such, plant roots dynamically respond to nitrogen availability below-ground by executing a signaling and transcriptional cascade resulting in altered plant growth, optimized for nutrient uptake. The NIN-LIKE PROTEIN 7 (NLP7) transcription factor senses nitrogen and along with its closely related paralog NLP6, partially coordinates these transcriptional responses. While post-translational regulation of NLP6/7 is well established, its upstream transcriptional regulation remains understudied in Arabidopsis and other plant species. Here, we dissect a previously identified sub-circuit upstream of NLP6/7 in Arabidopsis and which was predicted to contain multiple multi-node feedforward loops suggestive of an optimized design principle of nitrogen transcriptional regulation. This sub-circuit comprises AUXIN RESPONSE FACTOR 18 (ARF18), ARF9, DEHYDRATION RESPONSE ELEMENT BINDING-PROTEIN 26 (DREB26), A NAC-DOMAIN CONTAINING PROTEIN 32 (ANAC032), NLP6 and NLP7 and their regulation of NITRITE REDUCTASE 1 (NIR1). Conservation and divergence of this circuit and their influence on N-dependent root system architecture are similarly assessed in Solanum lycopersicum. The specific binding sites of these factors within their respective promoters and their putative cis-regulatory architecture are identified. The direct or indirect nature of these interactions are validated in planta. The resulting models were genetically validated in varying concentrations of available nitrate by measuring the transcriptional output of the network revealing rewiring of nitrogen regulation across distinct plant lineages. Significance StatementNitrogen is a critical nutrient for plant growth and yield. While external N has facilitated modern agriculture, over-application of N-containing fertilizers has drastic ecological and environmental consequences. Here, we experimentally validate a six gene regulatory circuit with extensive feedforward loops identified to act upstream of the critical NIN-LIKE PROTEIN 6/7 transcription factors which regulates a nitrogen metabolic enzyme. Our results indicate conservation and divergence in these circuits between Arabidopsis and tomato despite the similar role of NLP7 in N-dependent changes in root system architecture. The resulting network models complement existing knowledge of NLP7 regulation by providing a framework for targeted transcriptional engineering to increase plant nitrogen use efficiency.

plant biology↗

bioRxiv2022

The Phosphate Deprivation Response is Mediated by an Interaction between Brassinosteroid Signaling and Zinc in Tomato

Phosphate is a necessary macronutrient for basic biological processes, plant growth, and agriculture. Plants modulate their root system architecture and cellular processes to adapt to phosphate deprivation albeit with a growth penalty. Excess application of phosphate fertilizer, on the other hand, leads to eutrophication and has a negative environmental impact. Moreover, phosphate mined from rock reserves is a finite and non-recyclable resource and its levels are nearing complete depletion. Here, we show that Solanum pennellii, a wild relative of tomato, is partially insensitive to phosphate deprivation. Furthermore, it mounts a constitutive response under phosphate sufficiency. We demonstrate that activated brassinosteroid signaling through a tomato BZR1 ortholog gives rise to the same constitutive phosphate deficiency response, which is dependent on zinc over-accumulation. Collectively, these results reveal an additional strategy by which plants can adapt to phosphate starvation.

plant biology↗

bioRxiv2021

Carbon nanotube biocompatibility in plants is determined by their surface chemistry

Agriculture faces significant global challenges including climate change and an increasing food demand due to a growing population. Addressing these challenges will require the adoption of transformative innovations into biotechnology practice, such as nanotechnology. Recently, nanomaterials have emerged as unmatched tools for their use as biosensors, or as biomolecule delivery vehicles. Despite their increasingly prolific use, plant-nanomaterial interactions remain poorly characterized, drawing into question the breadth of their utility and their broader environmental compatibility. Herein, we characterize Arabidopsis thaliana transcriptional response to single walled carbon nanotubes (SWNTs) with two different surface chemistries commonly used for biosensing and nucleic acid delivery: oligonucleotide adsorbed-pristine SWNTs, and polyethyleneimine-SWNTs loaded with plasmid DNA (PEI-SWNTs), both introduced by leaf infiltration. We observed that SWNTs elicit a mild stress response almost undistinguishable from the infiltration process, indicating that these nanomaterials are well-tolerated by the plant. However, PEI-SWNTs induce a much larger transcriptional reprogramming that involves stress, immunity, and senescence responses. PEI-SWNT-induced transcriptional profile is very similar to that of mutant plants displaying a constitutive immune response or treated with stress-priming agrochemicals. We selected molecular markers from our transcriptomic analysis and identified PEI as the main cause of this reaction. We show that PEI-SWNT response is concentration-dependent and, when persistent over time, leads to cell death. We probed a panel of PEI variant-functionalized SWNTs across two plant species and identified biocompatible SWNT surface functionalizations. Our results highlight the importance of nanoparticle surface chemistry on their biocompatibility and will facilitate the use of functionalized nanomaterials for agricultural improvement. Significance statementNanomaterials can be used in agriculture as biosensors to monitor plant health, as fertilizers or growth regulators, and as delivery vehicles for genome engineering reagents to improve crops. However, the interactions between nanoparticles and plant cells are not well understood. Here, we characterize the plant transcriptomic response to single-walled carbon nanotubes (SWNTs) commonly used for sensing and nucleic acid delivery. While SWNTs themselves are well tolerated by plants, SWNTs surface-functionalized with positively charged polymers become toxic and produce cell death. We identify molecular markers of this toxic response to create biocompatible SWNT formulations. These results highlight the significance of nanoparticle surface chemistry, perhaps more than the nanoparticles themselves, on downstream interactions of nanoparticles with the environment.

plant biology↗

bioRxiv2021

A ratiometric dual color luciferase reporter for fast characterization of transcriptional regulatory elements

Plant synthetic biology requires precise characterization of genetic elements to construct complex genetic circuits that can improve plant traits or confer them new characteristics. Transcriptional reporter assays are essential to quantify the effect of gene expression regulator elements. Therefore, transcriptional reporter systems are a key tool in understanding control of gene expression in biology. In this work we construct and characterize a dual-color luciferase ratiometric reporter system that possesses several advantages over currently used reporters. It is ratiometric, reducing variability and increasing consistency between experiments; it is fast, as both reporters can be measured at the same time in a single reaction, and it is cheaper to perform than current dual-luciferase reporter assays. We have validated our system quantifying the transcriptional capability of a panel of promoters and terminators commonly used in synthetic biology with a broad range of expression magnitudes, and in a biologically relevant system, nitrate response.

synthetic biology↗

bioRxiv2021

Nanoparticle Cellular Internalization is Not Required for RNA Delivery to Mature Plant Leaves

Rapidly growing interest in nanoparticle-mediated delivery of DNA and RNA to plants requires a better understanding of how nanoparticles and their cargoes translocate in plant tissues and into plant cells. However, little is known about how the size and shape of nanoparticles influences transport in plants and use of their cargoes, limiting development and deployment of nanotechnology in plant systems. Here, we employ non-biolistically delivered DNA-modified gold nanoparticles (AuNP) spanning various sizes (5 - 20 nm) and shapes (spheres and rods) to systematically investigate their transport following infiltration into Nicotiana benthamiana (Nb) leaves. Generally, smaller AuNPs demonstrate more rapid, higher, and longer-lasting levels of association with plant cell walls compared to larger AuNPs. We observe internalization of rod-shaped but not spherical AuNPs into plant cells, yet surprisingly, 10 nm spherical AuNP functionalized with small-interfering RNA (siRNA) are most efficient at siRNA delivery and inducing gene silencing in mature plant leaves. These results indicate the importance of nanoparticle size in efficient biomolecule delivery, and, counterintuitively, demonstrate that efficient cargo delivery is possible and potentially optimal in the absence of nanoparticle cellular internalization. Our results highlight nanoparticle features of importance for transport within plant tissues, providing a mechanistic overview of how nanoparticles can be designed to achieve efficacious bio-cargo delivery for future developments in plant nanobiotechnology.

bioengineering↗

bioRxiv2021

Gold nanocluster mediated delivery of siRNA to intact plant cells for efficient gene knockdown

RNA interference (RNAi), which involves the delivery of small interfering RNA molecules (siRNA), has been used to validate target genes in plants, to understand and control cellular metabolic pathways, and as a green alternative for crop pest tolerance. Conventional siRNA delivery methods such as viruses and Agrobacterium-mediated delivery exhibit limitations in host plant species range and their use can result in uncontrolled DNA integration into the plant host genome. Here, we synthesize polyethyleneimine functionalized gold nanoclusters (PEI-AuNCs) to mediate siRNA delivery into intact plant cells and show these constructs enable efficient gene knockdown. We demonstrate that functionalized AuNCs protect siRNA from RNase degradation and are small enough (~2 nm) to bypass the plant cell wall which exhibits a size exclusion limit of 5-20 nm. These AuNCs in turn enable up to 76.5 {+/-} 5.9% GFP mRNA knockdown efficiency with no cellular toxicity. Our data suggest this simple and biocompatible platform for passive delivery of siRNA into intact plant cells could have broad applications in plant biotechnology.

bioengineering↗

bioRxiv2019

Nanotubes effectively deliver siRNA to intact plant cells and protect siRNA against nuclease degradation

Post-transcriptional gene silencing (PTGS) is a powerful tool to understand and control plant metabolic pathways, which is central to plant biotechnology. PTGS is commonly accomplished through delivery of small interfering RNA (siRNA) into cells. While siRNA delivery has been optimized for mammalian systems, it remains a significant challenge for plants due to the plant cell wall. Standard plant siRNA delivery methods (Agrobacterium and viruses) involve coding siRNA into DNA vectors, and are only tractable for certain plant species. Herein, we develop a nanotube-based platform for direct delivery of siRNA, and show high silencing efficiency in intact plant cells. We demonstrate that nanotubes successfully deliver siRNA and silence endogenous genes owing to effective intracellular delivery and nanotube-induced protection of siRNA from nuclease degradation. This study establishes that nanotubes, which are below the size exclusion limit of the plant cell wall, could enable a myriad of plant biotechnology applications that rely on RNA delivery.

plant biology↗

bioRxiv2019

DNA Nanostructures Coordinate Gene Silencing in Mature Plants

Plant bioengineering may generate high yielding and stress-resistant crops amidst a changing climate and a growing global population (1-3). However, delivery of biomolecules to plants relies on Agrobacterium infection (4) or biolistic particle delivery (5), the former of which is only amenable to DNA delivery. The difficulty in delivering functional biomolecules such as RNA to plant cells is due to the plant cell wall which is absent in mammalian cells and poses the dominant physical barrier to exogenous biomolecule delivery in plants. DNA nanostructure-mediated biomolecule delivery is an effective strategy to deliver cargoes across the lipid bilayer of mammalian cells, however, nanoparticle-mediated delivery remains unexplored for passive biomolecule delivery across the cell wall in plants. Herein, we report a systematic assessment of different DNA nanostructures for their ability to internalize into cells of mature plants, deliver small interfering RNAs (siRNAs), and effectively silence a constitutively-expressed gene in Nicotiana benthamiana leaves. We show that nanostructure internalization into plant cells and the corresponding gene silencing efficiency depends on the DNA nanostructure size, shape, compactness, stiffness, and location of the siRNA attachment locus on the nanostructure. We further confirm that the internalization efficiency of DNA nanostructures correlates with their respective gene silencing efficiencies, but that the endogenous gene silencing pathway depends on the siRNA attachment locus. Our work establishes the feasibility of biomolecule delivery to plants with DNA nanostructures, and details both the design parameters of importance for plant cell internalization, and also assesses the impact of DNA nanostructure geometry for gene silencing mechanisms.

plant biology↗

bioRxiv2017

Nanoparticle-Guided Biomolecule Delivery for Transgene Expression and Gene Silencing in Mature Plants

Genetic engineering of plants is at the core of sustainability efforts, natural product synthesis, and agricultural crop engineering. The plant cell wall is a barrier that limits the ease and throughput with which exogenous biomolecules can be delivered to plants. Current delivery methods either suffer from host range limitations, low transformation efficiencies, tissue damage, or unavoidable DNA integration into the host genome. Here, we demonstrate efficient diffusion-based biomolecule delivery into tissues and organs of intact plants of several species with a suite of pristine and chemically-functionalized high aspect ratio nanomaterials. Efficient DNA delivery and strong protein expression without transgene integration is accomplished in Nicotiana benthamiana (Nb), Eruca sativa (arugula), Triticum aestivum (wheat) and Gossypium hirsutum (cotton) leaves and arugula protoplasts. We also demonstrate a second nanoparticle-based strategy in which small interfering RNA (siRNA) is delivered to Nb leaves and silence a gene with 95% efficiency. We find that nanomaterials not only facilitate biomolecule transport into plant cells but also protect polynucleotides from nuclease degradation. Our work provides a tool for species-independent and passive delivery of genetic material, without transgene integration, into plant cells for diverse biotechnology applications.

plant biology↗