Genetic engineering of plants has enhanced crop productivity in the face of climate change and a growing global population by conferring desirable genetic traits to agricultural crops. Efficient ...genetic transformation in plants remains a challenge due to the cell wall, a barrier to exogenous biomolecule delivery. Conventional delivery methods are inefficient, damaging to tissue, or are only effective in a limited number of plant species. Nanoparticles are promising materials for biomolecule delivery, owing to their ability to traverse plant cell walls without external force and highly tunable physicochemical properties for diverse cargo conjugation and broad host range applicability. With the advent of engineered nuclease biotechnologies, we discuss the potential of nanoparticles as an optimal platform to deliver biomolecules to plants for genetic engineering.
Plant biotechnology is key to ensuring food and energy security; however, biomolecule delivery and progeny regeneration continue to be key challenges in plant genetic engineering.
Conventional biomolecule delivery methods in plants have critical drawbacks, such as low efficiency, narrow species range, limited cargo types, and tissue damage.
Advances in nanotechnology have created opportunities to overcome limitations in conventional methods: nanoparticles are promising for species-independent passive delivery of DNA, RNA, and proteins.
The advent of nuclease-based genome editing (e.g., CRISPR-Cas9) has ushered in a new era of precise genetic engineering that, among other impacts, has enabled the development of genetically engineered crops without harsh regulatory restrictions.
The potential of nanoparticles to overcome limitations in conventional delivery makes them excellent candidates for delivery of nuclease-based genome editing cargo, thus making nanoparticle delivery a critical technology for the advancement of plant genetic engineering.
Delivery of biomolecules to plants relies on Agrobacterium infection or biolistic particle delivery, the former of which is amenable only 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 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 without external mechanical aid remains unexplored for 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 siRNAs, and effectively silence a constitutively expressed gene in Nicotiana benthamiana leaves. We show that nanostructure internalization into plant cells and 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 both details the design parameters of importance for plant cell internalization and also assesses the impact of DNA nanostructure geometry for gene silencing mechanisms.
Genetic engineering of plants is at the core of sustainability efforts, natural product synthesis and crop engineering. The plant cell wall is a barrier that limits the ease and throughput of ...exogenous biomolecule delivery 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 intact plants of several species with 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 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.
Exogenous biomolecule delivery into plants is difficult because the plant cell wall poses a dominant transport barrier, thereby limiting the efficiency of plant genetic engineering. Traditional DNA ...delivery methods for plants suffer from host-species limitations, low transformation efficiencies, tissue damage, or unavoidable and uncontrolled DNA integration into the host genome. We have demonstrated efficient plasmid DNA delivery into intact plants of several species with functionalized high-aspect-ratio carbon nanotube (CNT) nanoparticles (NPs), enabling efficient DNA delivery into a variety of non-model plant species (arugula, wheat, and cotton) and resulting in high protein expression levels without transgene integration. Herein, we provide a protocol that can be implemented by plant biologists and adapted to produce functionalized single-walled CNTs (SWNTs) with surface chemistries optimized for delivery of plasmid DNA in a plant species-independent manner. This protocol describes how to prepare, construct, and optimize polyethylenimine (PEI)-functionalized SWNTs and perform plasmid DNA loading. The authors also provide guidance on material characterization, gene expression evaluation, and storage conditions. The entire protocol, from the covalent functionalization of SWNTs to expression quantification, can be completed in 5 d.
RNA interference, which involves the delivery of small interfering RNA (siRNA), has been used to validate target genes, to understand and control cellular metabolic pathways, and to use as a “green” ...alternative to confer pest tolerance in crops. Conventional siRNA delivery methods such as viruses and Agrobacterium-mediated delivery exhibit plant species range limitations and uncontrolled DNA integration into the plant genome. Here, we synthesize polyethylenimine-functionalized gold nanoclusters (PEI-AuNCs) to mediate siRNA delivery into intact plants and show that these nanoclusters enable efficient gene knockdown. We further demonstrate that PEI-AuNCs protect siRNA from RNase degradation while the complex is small enough to bypass the plant cell wall. Consequently, AuNCs enable gene knockdown with efficiencies of up 76.5 ± 5.9% and 76.1 ± 9.5% for GFP and ROQ1, respectively, with no observable toxicity. Our data suggest that AuNCs can deliver siRNA into intact plant cells for broad applications in plant biotechnology.
Optical nanoscale technologies often implement covalent or noncovalent strategies for the modification of nanoparticles, whereby both functionalizations are leveraged for multimodal applications but ...can affect the intrinsic fluorescence of nanoparticles. Specifically, single‐walled carbon nanotubes (SWCNTs) can enable real‐time imaging and cellular delivery; however, the introduction of covalent SWCNT sidewall functionalizations often attenuates SWCNT fluorescence. Recent advances in SWCNT covalent functionalization chemistries preserve the SWCNT's pristine graphitic lattice and intrinsic fluorescence, and here, such covalently functionalized SWCNTs maintain intrinsic fluorescence‐based molecular recognition of neurotransmitter and protein analytes. The covalently modified SWCNT nanosensor preserves its fluorescence response towards its analyte for certain nanosensors, presumably dependent on the intermolecular interactions between SWCNTs or the steric hindrance introduced by the covalent functionalization that hinders noncovalent interactions with the SWCNT surface. These SWCNT nanosensors are further functionalized via their covalent handles with a targeting ligand, biotin, to self‐assemble on passivated microscopy slides, and these dual‐functionalized SWCNT materials are explored for future use in multiplexed sensing and imaging applications.
Recent advances in covalent functionalization of single‐walled carbon nanotubes are explored to create intrinsically near‐infrared fluorescent carbon nanotube nanosensors with multimodal applications. Simultaneous covalent and noncovalent functionalizations on single‐walled carbon nanotube (SWCNT) surfaces interact through steric hindrance and intermolecular interactions. The ability to dual functionalize SWCNT nanoparticles has potential applications in diverse biotechnology applications including biomolecule imaging applications described herein.
Posttranscriptional 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. Standard plant siRNA delivery methods (
and viruses) involve coding siRNA into DNA vectors and are only tractable for certain plant species. Here, 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 could enable a myriad of plant biotechnology applications that rely on RNA delivery to intact cells.
Rapidly growing interest in the 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 influence transport in plants and the delivery efficiency of their cargoes, limiting the development of nanotechnology in plant systems. In this study we employed non-biolistically delivered DNA-modified gold nanoparticles (AuNPs) of various sizes (5-20 nm) and shapes (spheres and rods) to systematically investigate their transport following infiltration into Nicotiana benthamiana leaves. Generally, smaller AuNPs demonstrated more rapid, higher and longer-lasting levels of association with plant cell walls compared with larger AuNPs. We observed internalization of rod-shaped but not spherical AuNPs into plant cells, yet, surprisingly, 10 nm spherical AuNPs functionalized with small-interfering RNA (siRNA) were the 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. Overall, 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 biocargo delivery for future developments in plant nanobiotechnology.
Nanoparticles for protein delivery in planta Wang, Jeffrey W.; Cunningham, Francis J.; Goh, Natalie S. ...
Current opinion in plant biology,
04/2021, Letnik:
60
Journal Article
Recenzirano
Odprti dostop
Delivery of proteins into walled plant cells remains a challenge with few tractable solutions. Recent advances in biomacromolecule delivery using nanotechnology may evince methods to be exploited for ...protein delivery. While protein delivery remains no small feat, even in mammalian systems, the ability for nanoparticles to penetrate the cell wall and be decorated with a plethora of functional moieties makes them ideal protein vehicles in plants. As advances in protein biotechnology accelerate, so does the need for commensurate delivery systems. However, the road to nanoparticle-mediated protein delivery is fraught with challenges in regard to cell wall penetration, intracellular delivery, endosomal escape, and nanoparticle chemistry and design. The dearth of literature surrounding protein delivery in walled plant cells hints at the challenge of this problem but also indicates vast opportunity for innovations in plant-tailored nanotechnology.
Nanomaterials have emerged as an invaluable tool for the delivery of biomolecules such as DNA and RNA, with various applications in genetic engineering and post-transcriptional genetic manipulation. ...Alongside this development, there has been an increasing use of polymer-based techniques, such as polyethylenimine (PEI), to electrostatically load polynucleotide cargoes onto nanomaterial carriers. However, there remains a need to assess nanomaterial properties, conjugation conditions, and biocompatibility of these nanomaterial–polymer constructs, particularly for use in plant systems. In this work, we develop mechanisms to optimize DNA loading on single-walled carbon nanotubes (SWNTs) with a library of polymer-SWNT constructs and assess DNA loading ability, polydispersity, and both chemical and colloidal stability. Counterintuitively, we demonstrate that polymer hydrolysis from nanomaterial surfaces can occur depending on polymer properties and attachment chemistries, and we describe mitigation strategies against construct degradation. Given the growing interest in delivery applications in plant systems, we also assess the stress response of plants to polymer-based nanomaterials and provide recommendations for future design of nanomaterial-based polynucleotide delivery strategies.
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