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1. Nanoparticle cellular internalization is not required for RNA delivery to mature plant leaves

   https://doi.org/10.1038/s41565-021-01018-8  

   Huan Zhang, Natalie S. ( April 2022 , Nature nanotechnology)

   Pulizzi, Fabio (Ed.)

   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. 

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2. Dynamic Construction, Perception, and Remodeling of Plant Cell Walls

   https://doi.org/10.1146/annurev-arplant-081519-035846  

   Anderson, Charles T. ; Kieber, Joseph J. ( April 2020 , Annual Review of Plant Biology)

   null (Ed.)

   Plant cell walls are dynamic structures that are synthesized by plants to provide durable coverings for the delicate cells they encase. They are made of polysaccharides, proteins, and other biomolecules and have evolved to withstand large amounts of physical force and to resist external attack by herbivores and pathogens but can in many cases expand, contract, and undergo controlled degradation and reconstruction to facilitate developmental transitions and regulate plant physiology and reproduction. Recent advances in genetics, microscopy, biochemistry, structural biology, and physical characterization methods have revealed a diverse set of mechanisms by which plant cells dynamically monitor and regulate the composition and architecture of their cell walls, but much remains to be discovered about how the nanoscale assembly of these remarkable structures underpins the majestic forms and vital ecological functions achieved by plants. 

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3. Sonoporation: Past, Present, and Future

   https://doi.org/10.1002/admt.202100885  

   Rich, Joseph ; Tian, Zhenhua ; Huang, Tony Jun ( September 2021 , Advanced Materials Technologies)

   A surge of research in intracellular delivery technologies is underway with the increased innovations in cell‐based therapies and cell reprogramming. Particularly, physical cell membrane permeabilization techniques are highlighted as the leading technologies because of their unique features, including versatility, independence of cargo properties, and high‐throughput delivery that is critical for providing the desired cell quantity for cell‐based therapies. Amongst the physical permeabilization methods, sonoporation holds great promise and demonstrates to deliver a variety of functional cargos, such as biomolecular drugs, proteins, and plasmids, to various cells including cancer, immune, and stem cells. However, traditional bubble‐based sonoporation methods usually require special contrast agents. Bubble‐based sonoporation methods also have high chances of inducing irreversible damage to critical cell components, lowering the cell viability, and reducing the effectiveness of delivered cargos. To overcome these limitations, several novel non‐bubble‐based sonoporation mechanisms are under development. This review will cover both the bubble‐based and non‐bubble‐based sonoporation mechanisms being employed for intracellular delivery, the technologies being investigated to overcome the limitations of traditional platforms, as well as perspectives on the future sonoporation mechanisms, technologies, and applications.

    

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4. Biochemical and Genetic Analysis Identify CSLD3 as a beta-1,4-glucan Synthase that Functions during Plant Cell Wall Synthesis.

   https://doi.org/doi: 10.1105/tpc.19.00637  

   *Yang J., *Bak G. ( May 2020 , The plant cell)

   In plants, changes in cell size and shape during development fundamentally depend on the ability to synthesize and modify cell wall polysaccharides. The main classes of cell wall polysaccharides produced by terrestrial plants are cellulose, hemicelluloses, and pectins. Members of the cellulose synthase (CESA) and cellulose synthase-like (CSL) families encode glycosyltransferases that synthesize the β-1,4-linked glycan backbones of cellulose and most hemicellulosic polysaccharides that comprise plant cell walls. Cellulose microfibrils are the major load-bearing component in plant cell walls and are assembled from individual β-1,4-glucan polymers synthesized by CESA proteins that are organized into multimeric complexes called CESA complexes, in the plant plasma membrane. During distinct modes of polarized cell wall deposition, such as in the tip growth that occurs during the formation of root hairs and pollen tubes or de novo formation of cell plates during plant cytokinesis, newly synthesized cell wall polysaccharides are deposited in a restricted region of the cell. These processes require the activity of members of the CESA-like D subfamily. However, while these CSLD polysaccharide synthases are essential, the nature of the polysaccharides they synthesize has remained elusive. Here, we use a combination of genetic rescue experiments with CSLD-CESA chimeric proteins, in vitro biochemical reconstitution, and supporting computational modeling and simulation, to demonstrate that Arabidopsis (Arabidopsis thaliana) CSLD3 is a UDP-glucose-dependent β-1,4-glucan synthase that forms protein complexes displaying similar ultrastructural features to those formed by CESA6. 

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5. Delivered complementation in planta (DCIP) enables measurement of peptide-mediated protein delivery efficiency in plants

   https://doi.org/10.1038/s42003-023-05191-5  

   Wang, Jeffrey W. ; Squire, Henry J. ; Goh, Natalie S. ; Ni, Heyuan Michael ; Lien, Edward ; Wong, Cerise ; González-Grandío, Eduardo ; Landry, Markita P. ( August 2023 , Communications Biology)

   Using a fluorescence complementation assay, Delivered Complementation in Planta (DCIP), we demonstrate cell-penetrating peptide-mediated cytosolic delivery of peptides and recombinant proteins in Nicotiana benthamiana. We show that DCIP enables quantitative measurement of protein delivery efficiency and enables functional screening of cell-penetrating peptides for in-planta protein delivery. Finally, we demonstrate that DCIP detects cell-penetrating peptide-mediated delivery of recombinantly expressed proteins such as mCherry and Lifeact into intact leaves. We also demonstrate delivery of a recombinant plant transcription factor, WUSCHEL (AtWUS), into N. benthamiana. RT-qPCR analysis of AtWUS delivery in Arabidopsis seedlings also suggests delivered WUS can recapitulate transcriptional changes induced by overexpression of AtWUS. Taken together, our findings demonstrate that DCIP offers a new and powerful tool for interrogating cytosolic delivery of proteins in plants and highlights future avenues for engineering plant physiology.

    

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