Extracellular contractile injection systems for high efficiency protein delivery to plants

Extracellular contractile injection systems for high efficiency protein delivery to plants | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Extracellular contractile injection systems for high efficiency protein delivery to plants Gozde Demirer, Mark Legendre, Carlos Heredia, Clair Colee This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7412028/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Efficient delivery of functional proteins into plant cells remains a major barrier in plant biotechnology. Extracellular contractile injection systems (eCISs) are phage tail-like nanomachines evolved by bacteria to interface with eukaryotic host cells and deliver protein effectors. The Photorhabdus virulence cassette (PVC), a well-characterized eCIS, naturally targets insect hosts but can be reprogrammed for protein cargo delivery in mammalian systems. Here, we adapted PVCs for targeted delivery to plants by engineering their tail fibers to recognize a natural plant immune receptor, FLAGELLIN SENSITIVE2 (FLS2). We designed a library of FLS2-binding PVC variants and demonstrated efficient loading and delivery of non-native cargoes, including a fluorescent reporter protein and the Cre recombinase. We showed that engineered PVCs can deliver these proteins to Arabidopsis thaliana protoplasts and Nicotiana benthamiana leaf cells with efficiencies up to 40%. We elucidated that the delivery efficiency is correlated with receptor surface density, demonstrating that receptor selection and expression level are key parameters for optimization. This work establishes PVCs as novel, programmable protein delivery nanoparticles for plants, capable of targeting plant membrane receptors and effectively delivering diverse functional proteins. By enabling precise, DNA-free delivery of gene editing proteins, plant-targeted PVCs provide the framework for next-generation genome engineering strategies with broad potential in agricultural nanobiotechnology. Biological sciences/Biotechnology/Nanobiotechnology/Nanoparticles Biological sciences/Biotechnology/Biomaterials/Drug delivery Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Advancements in plant biotechnology continue to highlight genetic engineering strategies as a sustainable path to improved crop traits. However, inefficient biomolecule delivery across the rigid cell wall of many species remains a major barrier to plant genetic engineering. Nanotechnology has emerged as a more ubiquitous, higher efficiency, and less destructive alternative to the traditional delivery approaches of Agrobacterium and biolistics, enabling delivery of diverse biomolecular cargoes to various plant species 1 . While nanomaterials have been developed for efficient nucleic acid delivery to plants 2–5 , nanoparticle-mediated protein delivery remains largely unexplored in plants 6 . The most developed nanomolecular tools for protein delivery to plants to date rely on peptide-based ionic complexes, such as cell-penetrating peptides, to translocate protein cargoes across cell membranes, but these tools remain low in efficiency and are untargeted 7–14 . Protein nanoparticles emerge as an alternative platform with great potential for protein delivery applications 15,16 . In nature, many bacteria have evolved specialized protein nanomachines to translocate effector proteins into eukaryotic host cells. Among these, contractile injection systems (CISs) are phage tail-like protein nanosyringes consisting of a rigid inner tube terminated by a sharp spike protein, encased in a contractile sheath anchored to a baseplate 17–20 . Upon recognition of specific host receptors, sheath contraction drives the inner tube through the target membrane, delivering loaded protein payloads directly into the host cytosol 21,22 . Contractile injection systems can be anchored to bacterial membranes for delivery requiring direct cell-to-cell contact 23 , or they can be released as extracellular complexes following suicidal lysis of the producing bacteria. These extracellular contractile injection systems (eCISs) function independently of their bacterial hosts, freely interacting with target cells across all kingdoms of life to deliver protein cargoes for a wide range of purposes 24–26 . Structural and biochemical studies have revealed conserved mechanisms for selective cargo loading 27–29 and demonstrated tail fiber reprogramming to alter host specificity 30,31 . Recent engineering advances have enabled eCIS-mediated delivery of diverse functional proteins, including genome editing machinery, into non-native mammalian hosts for therapeutic purposes 27,32 . The Photorhabdus virulence cassette (PVC) is one of the most well-characterized eCIS subfamilies 29,33 . Naturally, PVCs deliver protein toxins into insect host cells, but recent work has retargeted them to deliver heterologous proteins into mammalian cells 32,34 . However, PVCs or any other protein nanoparticles have not been applied to plant systems yet, where targeted protein delivery could address longstanding challenges in plant genetic transformation and enable precise delivery of genome editing machinery into desired cell types, while avoiding the introduction of foreign DNA and unwanted transgenes. Here, we harnessed PVC extracellular contractile injection systems for targeted protein delivery to plants by reprogramming tail fibers to recognize natural plant membrane receptors. We demonstrated efficient loading and delivery of diverse functional protein cargoes, including a fluorescent reporter protein and a recombinase, into both plant cells in culture and intact leaf cells with combined delivery and activity efficiencies up to 40%. This work establishes PVC eCISs as a versatile protein delivery platform in plants, offering a technological breakthrough for targeted protein delivery in agriculture. Results Retargeting PVC eCISs to the plant cell membrane Originating as eCISs produced by the endosymbiotic bacterium Photorhabdus asymbiotica , the linear Photorhabdus virulence cassette (PVC) gene cluster consists of 16 structural genes ( pvc1-16 ) responsible for the successful expression and self-assembly of the functional nanoparticle 35 . Immediately downstream of these structural genes are two payload constructs ( Pnf and Pdp1 ) that encode its native toxin effector protein cargoes. In addition to these two payload constructs are four accessory genes necessary for cargo loading. Combined, these structural and payload genes constitute the PVCpnf gene locus ( Fig. 1A ). E. coli has previously been engineered to recombinantly express the PVCpnf gene locus from P. asymbiotica ATCC 43949 32 . In our studies, we used an expression system that reconstitutes the PVCpnf locus into two independent expression vectors for modularity to create diverse libraries, harboring either the structural components of the locus (pStructural) or its cargo components (pCargo). PVC generation and isolation was achieved using previously described methods 35 , where co-transformation of a pStructural and pCargo variants resulted in the production of unique PVC variants in E. coli ( Fig. 1A ). To retarget PVC nanoparticles to the plant cell membrane, we focused on the engineering of the tail fiber binding domain. PVC’s homologous tail fiber binding domain has been identified and engineered to retarget PVC tropism to mammalian hosts 32 . We tested whether a similar strategy could be employed to produce functional PVCs that target plant cell surface protein receptors, thus enabling targeted delivery of protein cargoes to plant cells. We used AlphaFold to predict the structure of the distal tip of the tail fiber produced by the pvc13 gene of the PVCpnf locus ( Fig 1B ). The tail fiber exists as a Pvc13 homotrimer, and the structural prediction of this supramolecular complex revealed a helical tail fiber body region—structurally similar to the short tail fibers of bacteriophages—and a terminal globular region previously identified as the Pvc13 binding domain 32 . We produced a small library of engineered PVCs with binding regions that target a ubiquitous and abundant plant cell surface receptor, FLAGELLIN SENSITIVE2 (FLS2). The FLS2 receptor is a Microbe-Associated Molecular Pattern recognition receptor that binds and detects the conserved 22-amino acid fragment of bacterial flagellin known as flg22 36 . We chose to target PVCs to the FLS2 receptor due to its naturally high expression levels in the leaf tissue of most plant species 37 . The PVC library was designed to present the flg22 epitope at various extensions from the tail fiber body using 0x, 2x, and 4x (GGSGG) n=0,2,4 flexible linkers, accounting for any potential steric obstacles to receptor binding ( Fig. 1B ). AlphaFold simulations confirmed that all members of the PVC library maintained the expected helical structure of the tail fiber body without disturbing the disordered nature of the flg22 epitope. We also used ChimeraX to align the predicted AlphaFold tail fiber structures of the PVC library to the binding region of the FLS2 receptor ( Fig. 1C and Supplementary Fig. 1A, B ). The resulting alignment verified proper binding of the flg22 epitope to the receptor despite fusion to the larger tail fiber body structure, suggesting the potential functionality of the nanoparticles. After expression and purification in E. Coli , negative-stain transmission electron microscopy (TEM) confirmed proper self-assembly of all PVC library members and demonstrated limited particle instability that would result in spontaneous sheath contraction in various buffers ( Fig. 1D and Supplementary Fig. 2A ). Generated PVCs showed similar morphology and length distributions independent of their altered tail fiber domains, suggesting that tail fiber modification did not impact sheath structure or loading mechanisms ( Supplementary Fig. 1C ). Finally, denaturing gel electrophoresis revealed successful incorporation of the expected PVC structural proteins into each member of the library ( Fig. 1E ). Together, these results confirm the successful design, generation, and isolation of recombinant PVC nanoparticles. To test whether FLS2 receptor can be targeted with flg22-displaying PVC particles for protein delivery into plants, we generated the 0xlinker PVC variant carrying the native insect toxin proteins Pnf and Pdp1 and delivered them to Arabidopsis thaliana protoplasts ( Supplementary Fig. 3 ). Compared to the native insect-targeting tail fiber domain, flg22-displaying PVC particles caused ~4 times more cell lethality, indicating successful targeting and toxin protein delivery into plant cells ( Supplementary Fig. 3 ). Validating PVC-mediated delivery of novel protein cargoes to plant cells While the exact molecular mechanism of PVC cargo loading is currently unknown, N-terminal protein sequences naturally present on the PnfandPdp1proteins shuttle the effector cargoes to the lumen of the PVC sheath 27,28 . We designed a cargo protein engineering strategy that fuses the Pnf packaging domain to the N-terminus of a novel cargo protein and a HiBiT tag to the protein’s C-terminus ( Fig. 2A ). This dual-tagging strategy allows for loading of novel cargoes as well as their subsequent chemiluminescent detection via HiBiT luminescence complementation. We demonstrated the feasibility of this engineering strategy using an mScarlet fluorescent protein as a non-native cargo for loading into PVCs, generating a 39.8 kDa functional cargo protein. Denaturing western blot analysis against the HiBiT tag on the engineered mScarlet revealed successful loading of the protein into PVCs with a total cargo loading capacity of approximately 4.5 ng mScarlet per 1 mg of PVC particle ( Fig. 2B and Supplementary Fig. 2B for standard curves), confirming that non-native cargoes can be encoded for shuttling into the FLS2-targeting PVC library for downstream studies. Using these mScarlet-loaded particles, we tested whether FLS2-targeting PVCs were capable of delivering the fluorescent protein to plant cells. For this, A. thaliana mesophyll protoplasts were incubated overnight following challenge with 1 mg/mL mScarlet-loaded PVCs ( Fig. 2C ), where successful PVC binding and delivery were expected to yield nuclear-localized mScarlet signal. When the 0xlinker FLS2-binding PVC variant was loaded with the mScarlet protein cargo and applied, clear nuclear mScarlet signal could be detected in protoplasts in response to PVC activity after 18 h of incubation ( Fig. 2D ). These initial mScarlet delivery results validated the flg22-binding epitope for PVC retargeting, and ability to deliver non-native cargo into plants, which encouraged further probing to increase delivery efficiency and cargo range. Interrogating PVC activity using a Cre-based reporter system After confirming fluorescent protein delivery with PVCs, we next investigated the capacity for our engineered particles to load and deliver genome-editing protein cargoes. The Cre recombinase is a well-studied nuclease effector with a molecular weight below that of the native PVC Pnfcargo, making it an ideal PVC cargo candidate. A similar cargo engineering strategy as in Fig. 2A was used to modify the Cre recombinase for PVC loading and cargo detection. Fusion of the PVC packaging domain to the N-terminus of Cre was expected to shuttle the enzyme into the PVC lumen. Addition of a plant-optimized NLS would further improve Cre activity in plant cells and a C-terminal HiBiT tag allowed for chemiluminescent detection of loaded Cre following PVC preparation ( Fig. 3A ). Denaturing western blot analysis confirmed loading of the Cre cargo into the PVC sheath lumen at an efficiency of approximately 10 ng Cre cargo for every 1 mg of PVC nanoparticle ( Fig. 3B and Supplementary Fig. 2C for standard curves). Upon producing Cre-loaded nanoparticles for our entire library of FLS2-binding PVC variants, we next developed an in-planta assay for detecting intracellular Cre activity. The molecular reporter for this assay was a Cre flip-excision (FLEX) reporter 38 : an anti-parallel YFP coding DNA sequence flanked on either side by two orthogonal pairs of LoxP sites ( Fig. 3C ). In response to intracellular Cre activity, the reporter irreversibly inverts to produce nuclear-localized YFP signal. Two versions of the FLEX reporter were produced: a traditional FLEX reporter and a FLEX-GV reporter flanked by geminiviral components ( Fig. 3D ) that is expected to produce high copy numbers within the cell, thus increasing reporter sensitivity and signal output. FLEX reporter was expressed in plant cells along with a constitutive Cre expression plasmid, where intracellular Cre production should produce positive reporter signal. FLEX reporters were validated in Arabidopsis thaliana protoplasts via PEG transfection ( Fig. 3E ) and Nicotiana benthamiana leaf tissue via Agrobacteria infiltration ( Fig. 3F ). Both constructs showed minimal leakage in negative controls and full coverage when co-delivered with a Cre-expression vector. We observed increased signal output intensities under the high copy FLEX-GV reporter compared to the FLEX switch, as indicated by brighter and larger nuclear YFP signals ( Fig. 3E, F ). Together, these results confirm the design and implementation of two Cre-responsive FLEX reporters for use in planta . These reporters provide a powerful tool for assessing intracellular activity of an exogenously applied Cre recombinase cargo. After confirming Cre loading into PVCs and proper function of FLEX constructs as a tool to report the delivery of Cre protein cargoes, we next attempted to deliver Cre-loaded PVCs into FLEX reporter-expressing plant cells. Initially screening for PVC activity in A. thaliana mesophyll protoplasts, cells were PEG-transfected with either of the FLEX switches prior to Cre-loaded PVC challenge and subsequent quantification using confocal microscopy ( Fig. 4A ). PVC activity saturated at relatively low concentrations when applied to mammalian cells 32 . This trend was confirmed in our protoplast assays, where PVC activity saturated at concentrations as low as 100 ng/µL PVC particles ( Supplementary Fig. 4A-C ). Additionally, while Cre delivery events saturated beyond 24 hours of incubation, maximal reporter signal intensity occurred at 48 hours during peak FLEX reporter expression ( Supplementary Fig. 4D, E ). Together, these results informed optimal assay conditions for screening the FLS2-binding PVC library. Given that the cell wall is enzymatically removed from protoplast cells, steric hindrance at receptor binding sites was assumed to be negligible, providing all members of the FLS2-binding PVC library comparable access to membrane receptors. This was confirmed after screening PVC activity in protoplasts using the FLEX reporter, where similar levels of reporter activity, hence Cre delivery, were registered across the entire PVC library of 0, 2, and 4x linker variants ( Fig. 4B ). The average percentage of cells expressing YFP was ~5%, reflecting both Cre delivery and PVC activity efficiency ( Fig. 4B, C ). While PVC activity did not depend on the linker length, we confirmed that the flg22 binding epitope was, in fact, required for PVC activity in plant cells, where native insect receptor-binding tail fiber domain did not result in PVC activity in negative control samples ( Supplementary Fig. 5 ). The FLEX-GV switch outperformed the FLEX reporter, registering overall efficiencies of 16%, 10%, and 15% with 0, 2, 4x linker PVCs, respectively ( Fig. 4B, D ). The lower-copy FLEX switch was not sufficiently sensitive to detect differences in PVC activity across the nanoparticle library, but the higher-copy FLEX-GV reporter identified the 0xlinker variant as the highest performing PVC variant with activities as high as 16% in protoplasts. A similar workflow was employed in N. benthamiana leaves, where leaves were Agro-transformed with one of the FLEX switches prior to Cre-loaded PVC challenge ( Fig. 5A ). Cre-driven YFP expression was low and inconsistent across biological replicates when probed using the low copy number FLEX switch ( Fig. 5B, C ), likely due to insufficient reporter sensitivity. However, when the FLEX-GV reporter was deployed, YFP expression recovered to the levels reported in protoplasts, reaching 13%, 16%, and 10% with 0, 2, 4x linker PVCs, respectively ( Fig. 5D ). The 2xlinker PVC variant outperformed the rest of the library in the leaf tissue. Given this, the 2xlinker PVC variant was used in all subsequent leaf tissue assays. These experiments are the first demonstrations of efficient PVC-mediated protein delivery to plant cells. The impact of FLS2 receptor density on PVC protein delivery efficiency One notable characteristic of the PVC eCIS binding and delivery is the requirement for sufficiently dense target receptors on the host cell membrane 39 . The current FLS2-targeting PVCs can effectively deliver protein cargoes to plant cells expressing basal levels of the FLS2 receptor, making them powerful tools for applications to wild type organisms. However, receptor over-expression lines might increase the tool’s efficiency when targeting a genetically modified plant by offering heightened receptor levels for PVC binding ( Fig. 6A ). To this end, we developed FLS2 receptor over-expression constructs for transient expression of a GFP-tagged FLS2 receptor in N. benthamiana leaves under either the native FLS2 promoter and terminator or driven by the strong 35S promoter ( Fig. 6B ). While both of these constructs expressed the receptor, visualized as GFP fluorescence under confocal microscopy, when FLS2 expression was driven by its native promoter and terminator, the receptors were better localized at the cell membrane ( Fig. 6B ). After transiently over-expressing the FLS2 receptor in N. benthamiana leaves via Agro-infiltration, we applied the same Cre-loaded PVC delivery workflow as in Fig. 5A using the 2xlinker PVC variant. Notably, in the cells over-expressing the FLS2 receptor with proper membrane localization, Cre-driven YFP expression is significantly more frequent and produces higher intensity signals ( Fig. 6C ). When driven by the native FLS2 promoter, FLS2 over-expression showed increased PVC activity within these cells up to 38% efficiency, a greater than 2.5-fold increase in activity compared to cells with natural FLS2 expression levels ( Fig. 6D ). FLS2 over-expression driven by the strong p35S promoter did not produce an increase in PVC activity. This is potentially due to the fact that, while the receptor is over-expressed using this construct, failure to localize at the cell membrane prevents over-expression from improving the PVC binding and subsequent delivery. Together, these results confirm that increased receptor surface densities can improve PVC delivery capabilities in plants, with the potential to reach efficiencies reliable for translation to field applications. Discussion Plant biomolecule delivery remains a limiting step in the implementation of new and powerful genetic engineering technologies in agriculture. In particular, the direct delivery of functional proteins to plant cells is a big challenge. While many methods are available for transferring protein cargoes to mammalian cells, these methods are either inefficient when applied to plants or entirely unexplored. Recent studies have identified cell-penetrating peptides (CPPs) as a useful tool for translocating proteins into walled plant cells and have discovered novel, plant-specific CPPs that are more effective for plant delivery than their known mammalian counterparts 14 . Even though these are promising developments, CPPs typically achieve low delivery efficiencies and lack any cell-specific targeting capabilities, a hallmark of modern delivery vehicles in mammalian biology. Here, we demonstrated the engineering of the PVC eCIS to target native or overexpressed plant cell surface receptors, serving as a tool for efficient functional protein delivery to plant cells. We specifically engineered PVCs to target the FLS2 receptor, which is widely ubiquitous across both plant species and plant cell types. Notably, we have shown that these engineered PVCs can deliver protein cargoes to both leaf protoplasts and walled leaf cells with efficiencies up to 40%, and the ubiquitous expression of the FLS2 receptor should allow for efficient PVC activity in a multitude of plant species and tissue types. In the engineering of our PVC nanoparticles, we sought to compromise between rigid binding interactions and accessibility to receptor binding via incorporation of flexible (GGSGG) n=0,2,4 linkers flanking the tail fiber flg22 binding domains. Interestingly, the PVC variant with no linker addition was the most efficient in protoplast systems, whereas PVCs containing (GGSGG) 2 linkers were the most efficient in leaf cells with intact walls. This observation is potentially due to the need to traverse the cell wall for effective FLS2 receptor binding in intact cell systems, which is aided by the incorporation of a flexible linker. The potential for cell-type specific delivery in plants using PVCs is as of yet unexplored. PVCs targeted to mammalian receptors have proven highly specific in their recognition of cognate receptors, with minimal off-target activity 32 . Importantly, cell-type specific targeting can provide a source of biocontainment for delivery tool application in field environments. Tightening PVC binding specificity by targeting native plant-specific or cell-type specific receptors could decrease unintended activity while maintaining unparalleled cell-type specific protein delivery efficiencies. Furthermore, by introducing recombinant receptors as PVC targets orthogonal to natural plant receptors, efficiencies and application types can be expanded. Numerous studies have dissected the important role that microbe-associated molecular pattern receptors play in regulating plant immunity in response to pathogens 40 , and the FLS2 receptor is likely the most well-studied. While FLS2 provides a naturally high receptor surface density, making it an ideal candidate for PVC targeting, binding of the flg22 epitope is known to elicit a downstream immune response and induce receptor recycling 36 . However, the consequences of nanoparticle targeting of the FLS2 receptor via flg22 binding is not yet studied. Therefore, further investigation of this interaction, particularly regarding its impact on PVC retargeting, and its effect on plant health is critical and will be part of future studies. Regardless, numerous flg22 epitope variants have been identified that can bind the FLS2 active site without activating the immune response 41 , suggesting possible solutions to circumvent unintended immune response elicitation. In summary, here, we demonstrated the modularity of the PVC delivery system by loading and delivering a novel Cre recombinase cargo. It is known, however, that PVCs are capable of loading and delivering various other biomolecules, seemingly independent of their size 42 . In fact, PVCs retargeted to mammalian cells have been demonstrated to deliver Cas9 ribonucleoprotein complexes with editing efficiencies of ~13% 32 . Such an expansion of the plant-targeting PVC cargo inventory to include gene editing machinery like Cas9-mediated systems could help realize efficient DNA- or transgene-free gene editing in plants. Together, our results identify the PVC eCIS as a novel tool for the efficient delivery of protein cargoes to plant cells, and with further development, this tool may have applications in plant biotechnology and agricultural engineering. Methods Plant growth conditions Nicotiana benthamiana and Arabidopsis thaliana (Col-0) plants were grown in a controlled-environment growth chamber (Conviron) set to 22°C during the day period and 20°C during the night period (16 h:8 h light/dark photoperiod) with 55% relative humidity. Plasmid construction The PVCpnf gene locus had previously been synthesized and domesticated to produce the pStructural (Addgene pAWP78-PVCpnf1-16) and pCargo (Addgene pBR322-PVCpnf17-22). To generate a version of pCargo compatible with Golden Gate Assembly with BsaI, a single BsaI site was removed from the original pCargo construct using the Q5 Site-Directed Mutagenesis Kit (NEB E0554S). All further manipulations of either construct involved standard PCR amplification with Phusion High-Fidelity PCR Master Mix with HF Buffer (ThermoFisher F531) followed by Golden Gate Assembly with BsaI HFv2 (NEB R3733) and T4 DNA Ligase (ThermoFisher EL0014). Assembled constructs were transformed into chemically competent NEB Turbo cell (NEB C2984). For PVC production, final variants of pStructural and pCargo plasmids were co-transformed into chemically competent EPI300 cells (Fisher Scientific NC1583291). PVC purification To generate a given PVC condition, one pStructural variant and one pCargo variant were co-transformed into chemically competent EPI300 cells. The resulting transformants were then cultured and PVC particles harvested using a modified version of a previously developed method 35 . In brief, colonies were grown overnight in 2xYT (ThermoFisher 22712020) media and inoculated (at 1:1,000) into 1 L Terrific Broth (Fisher Scientific BP246850) and shaken at 24°C for 48 h. Cultures were centrifuged at 4,000 g for 20 min at 4°C and the resulting pellet was gently resuspended in 60 mL Buffer P (25 mM Tris-HCl pH 7.5 (ThermoFisher 15567027), 140 mM NaCl (Sigma-Aldrich S5886), 3 mM KCl (Fisher Scientific P217), 5 mM MgCl 2 (Sigma-Aldrich M2393), 200 mg mL -1 lysozyme (ThermoFisher 89833), 50 mg mL -1 DNase I (Sigma-Aldrich DN25), 0.5% Triton X-100 (Sigma-Aldrich X100), and 1 Protease Inhibitor Cocktail (Sigma-Aldrich 11836153001)) using a serological pipette and subsequently shaken at 250 rpm for 30 min at 37°C. Lysates were pelleted at 4,000 g for 30 min at 4°C to remove cell lysate and the supernatant extracted and ultracentrifuged at 120,000 g for 1 h at 4°C to pellet PVCs. The supernatant was discarded and the ultracentrifuge tube swabbed using a Kimwipe. PVC pellets were then washed with 1x PBS (ThermoFisher 70011044), and the tubes were swabbed once more. Pellets were allowed to rehydrate overnight at 4°C in 2 mL PBS before being resuspended via pipetting. Suspensions were agitated for 30 minutes to allow complete rehydration and then centrifuged at 16,000 g for 20 min at 4°C to clarify the solution. Supernatants were then diluted in 60 mL PBS and ultracentrifuged at 120,000 g for 1 h at 4°C once more. Tubes were swabbed after decanting the supernatant and PVC pellets were resuspended via pipetting in 50 mL working solution following a 4 h incubation period. Once again, suspensions were agitated for 30 minutes and then centrifuged at 16,000 g for 20 min at 4°C to clarify the solution. The supernatant was collected as the final PVC product and protein concentration was measured using a Qubit instrument (ThermoFisher Q33211). In silico protein structure prediction All protein structures and self-assemblies were predicted using ColabFold (v1.5.5), an AlphaFold2 implementation based in Google-Colab that generates sequence alignments using MMseqs2. For general protein structure prediction, sequences were queried with default model (AlphaFold2-ptm) and MSA settings. For Pvc13 tail fiber complex predictions, sequences were queried as heterotrimers with the same default model (AlphaFold2-multimer-v3) and MSA settings. The resulting structures were rendered and customized with PyMOL Molecular Graphics System (v3.1.3). Electron microscopy 300-mesh copper grids with Formvar/carbon support (PELCO®, Ted Pella, Inc., 01753-F) were glow-discharged in air for 1 min at 15 mA using negative polarity mode. Purified PVC product suspended in PBS was diluted to 200 ng mL -1 in Milli-Q ultrapure water, and 5 µL was applied to the glow-discharged carbon film side of each grid for 1 min before side-blotting with filter paper. Grids were then washed twice by depositing 5 µL of Milli-Q water followed by immediate blotting. Grids were stained with 5 µL of 1% (w/v) uranyl acetate in Milli-Q water for 30 s and blotted to remove excess stain. Prepared grids were examined on an FEI Tecnai T12 transmission electron microscope (120 kV, LaB₆ filament) equipped with a Gatan Ultrascan 2k × 2k CCD camera (Caltech Biological and Cryo-EM Facility). Images were acquired at nominal magnifications of 6,500×, 15,000×, and 30,000×. SDS-PAGE and denatured Western blotting 20 mg of purified PVCs was prepared for SDS-PAGE by combining with NuPAGE LDS Sample Buffer (ThermoFisher NP0007) and NuPAGE Sample Reducing Reagent (ThermoFisher NP0009) at appropriate dilutions. The mixture was then incubated for 10 min at 70°C in a thermocycler. The denatured PVC preparations were then loaded into NuPAGE Bis-Tris Mini Protein Gels, 4-12% (ThermoFisher NP0321) and run for 50 min at 200 V in 1x NuPAGE MOPS SDS Running Buffer (ThermoFisher NP0001) supplemented with NuPAGE Antioxidant (ThermoFisher NP0005) (at 1:400). For Coomassie staining, gels were rinsed of Running Buffer using Milli-Q ultrapure water prior to incubation in SimplyBlue SafeStain (ThermoFisher LC6065) for 90 min under gentle agitation. Gels were subsequently destained overnight in Milli-Q water and imaged in a Gel Imager (Azure Biosystems). For western blot analysis of loaded PVC payloads, identical sample preparation and electrophoresis conditions were applied. In this case, PVCs were generated with loaded cargoes fused at the C-terminus with a HiBiT peptide tag, and unloaded cargoes were removed from the sample using standard ultracentrifugal separation. Variable quantities of PVC sample were applied to the above SDS-PAGE protocol depending on cargo loading capacity. Following electrophoresis, gels were blotted onto PVDF membranes (BIO-RAD 1704156) using a Trans-Blot Turbo Transfer System (BIO-RAD) set to default settings for turbo transfer of a mini gel (7-minute protocol). Finally, HiBiT-tagged cargo proteins were visualized using the Nano-Glo HiBiT Blotting System (Promega N2410) consisting of a 4 h TBST incubation period and a 2 h LgBiT incubation period, both at room temperature. Chemiluminescent images were captured with an Azure 200 Gel Imager. Band intensity analysis was performed using Fiji, a distribution of ImageJ. Protoplast isolation Protoplasts were isolated from 4-week-old Arabidopsis thaliana leaves as described previously 43 with some modifications. In brief, 7-10 fully developed leaves were gently compressed between Time tape (adhered to the upper epidermis) and 3 M Magic tape (adhered to the lower epidermis). The lower epidermal layer of leaves was removed and discarded along with removal of the Magic tape, thus exposing the leaf mesophyll. The exposed mesophyll was then placed in contact with a cell wall-degrading enzyme solution (20 mM MES pH 5.7, 0.4 M mannitol, 20 mM KCl, 1.5% w/v cellulase R10 Yakult, and 0.4% w/v macerozyme R10 Yakult) in the dark and incubated for 3 h. Protoplasts released into the enzyme solution following incubation were diluted in ice cold W5 solution (2 mM MES pH 5.7, 154 mM NaCl, 125 mM CaCl 2 , and 5 mM KCl) and gently pelleted at 100 g for 5 min with minimal ramp rates. Pellets were suspended in W5 solution and run on a 21% w/v water-based sucrose cushion at 90 g for 10 min. The suspended layer of intact protoplasts was once again diluted in W5 solution and gently pelleted at 100 g for 5 min with minimal ramp rates. The final pellet was resuspended to 5x10 5 cells mL -1 in working solution (see specific experimental method). Plasmid transfection for protoplast assays For assays requiring transfection of a FLEX reporter plasmid and/or a Cre expression plasmid, DNA was transfected into protoplasts using a PEG-mediated transfection procedure. Briefly, 100 mL of protoplasts at 5x10 5 cells mL -1 in an MMG working solution (4 mM MES pH 5.7, 0.4 M mannitol, and 15 mM MgCl 2 ) were gently mixed with 40 mg total plasmid DNA (20 mg:20 mg co-transfections) and 100 mL PEG transfection solution (40% w/v PEG4000, 0.2 M mannitol, 0.1 M CaCl 2 ) in a round-bottom tube. After 15 min of incubation, the protoplasts were washed with 1 mL W5 solution and pelleted at 200 g for 2 min. This wash step was repeated for a total of three times, and the final protoplast pellet was resuspended in WI solution (4 mM MES pH 5.7, 0.5 M mannitol, and 20 mM KCl) at a density of 10 5 cells mL -1 . PVC delivery to protoplasts All protoplast delivery experiments were performed using fresh Arabidopsis thaliana mesophyll protoplasts isolated using the aforementioned procedure. Protoplasts in a working solution of WI media were seeded into clear-bottom 96-well plates (Fisher Scientific 07-000-167) and allowed 30 minutes to settle prior to manipulation. For all experiments, 2x10 4 cells were seeded into a given well, and excess WI solution was aspirated to a final volume of 50 μL. PVCs were then added to a final concentration of 500 ng μL -1 . For fluorescent protein delivery assays, cells were incubated for 18 h and imaged under a Leica STELLARIS 8 FALCON confocal microscope. mScarlet fluorescence was captured using a 569 nm laser excitation from a white light laser. Images were rendered and analyzed using Fiji software. For Cre protein delivery assays, cells were incubated for 48 h and imaged with a Leica STELLARIS 8 FALCON confocal microscope with 434 nm, 517 nm, and 587 nm laser excitations from a white light laser to capture mTurqouis2, YPET, and mCherry expression, respectively. All images were obtained at 20x magnification with water as an immersion media. Quantification of PVC activity in protoplasts For each PVC condition, 3 biological replicates (3 separate wells individually challenged with PVCs) were performed; and for each biological replicate, 10 technical replicates (10 non-overlapping confocal fields of view) were collected. Images were taken around the perimeter of wells due to the tendency for protoplasts to sequester along the well edges. Each field of view was analyzed with Fiji, a distribution of ImageJ, to quantify the total number of YFP expressing nuclei and the total number of mCherry expressing cells for that field of view. The PVC activity for a biological replicate was then calculated by combining YFP and mCherry counts for all technical replicates and calculating PVC activity = (# YFP+ cells)/(# mCherry+ cells). PVC delivery to leaf cells Fully developed leaves (the 3 rd of 4 th true leaf) from Nicotiana benthamiana (3-4 weeks old) plants were selected for all experiments. Biological replicates were sampled from leaves on separate plants generated from the same seed batch. Agrobacterium tumefaciens (GV3101) harboring either of the FLEX switches were grown in 2xYT media (ThermoFisher 22712020) supplemented with 10 μg mL -1 rifampicin (Sigma-Aldrich R3501), 20 μg mL -1 gentamycin (Sigma-Aldrich G1264), 50 μg mL -1 tetracycline (Sigma-Aldrich T7660), and 50 μg mL -1 kanamycin (Sigma-Aldrich K1637) at 30°C and 200 rpm for 24 h. Overnight cultures were then centrifuged at 4,000 g for 20 min, and the pellets were resuspended to an OD600 of 0.4 in infiltration buffer (10 mM MES, pH 5.7 (Sigma-Aldrich M2933), 10 mM MgCl 2 (Sigma-Aldrich M2393), 200 μM acetosyringone (PlantMedia 40100297)). The resuspended cultures were incubated at room temperature and shaken at 120 rpm for 4 h. Following incubation, 100-200 μL of the Agrobacterium mixture was infiltrated against the abaxial surface of a leaf with a 1 ml needleless syringe by applying gentle pressure. Leaves were incubated for 72 h prior to analysis. For PVC delivery experiments, a small puncture was introduced on the abaxial surface of the leaf using a pipette tip, and 50-100 μl of the PVC sample (or of any control solution) was infiltrated against the puncture with a 1 ml needleless syringe by applying gentle pressure. For leaf infiltration experiments, all PVC samples were prepared at 1 mg mL -1 and resuspended in 10 mM HEPES Buffer, pH 7.5 (Sigma-Aldrich H3375). Quantification of PVC activity in leaves PVC-infiltrated Nicotiana benthamiana leaves were prepared for confocal imaging 72 h post-infiltration. A leaf disk puncher (Fisher Scientific NC0769832) was used to extract a 0.25 in leaf section adjacent to the location of PVC infiltration. Leaf disks were then mounted between a glass slide and coverslip of #1 thickness using water as the mounting medium. A Leica STELLARIS 8 FALCON confocal microscope was used to image the plant tissue with 434 nm, 517 nm, and 587 nm laser excitations from a white light laser to capture mTurqouis2, YPET, and mCherry expression, respectively. All images were obtained at 20x magnification with water as an immersion media. For each PVC condition, 3-4 biological replicates (3-4 infiltrations into leaves on 3-4 different plants) were performed; and for each biological replicate, more than 8 technical replicates (8 non-overlapping confocal fields of view) were collected. Each field of view was analyzed with Fiji, a distribution of ImageJ, to quantify the total number of YFP expressing nuclei and the total number of mCherry expressing cells for that field of view. The PVC activity for a biological replicate was then calculated by combining YFP and mCherry counts for all technical replicates and calculating PVC activity = (# YFP+ cells)/(# mCherry+ cells). Statistics and reproducibility Statistical analyses were performed using Prism (10.1.0). Quantitative data are presented as mean ± SEM with n = 3–4 biological replicates per condition; figure legends provide further specification as necessary. Unless otherwise stated, biological replicates represent independent treatments in separate wells ( in vitro assays) or on leaves from separate plants. All micrographs, gels, and blots are representative images from at least 2 independent repeated experiments. Statistical significance was computed using one-way or two-way ( Supplementary Fig. 4E ) ANOVA with Tukey post hoc tests (multiple comparisons correction), as indicated in figure legends. P < 0.05 were considered statistically significant. Declarations Data Availability Statement All data are included either in the manuscript or supplementary files. Conflict of Interest Statement Authors declare no conflict of interest. Author Contributions Conceptualization: MGL, GSD Methodology: MGL, CAH, GSD Data Acquisition: MGL, CAH Supervision: GSD Writing: MGL, GSD Funding Acquisition: GSD Acknowledgements Fluorescence imaging was performed in the Biological Imaging Facility, with the support of the Caltech Beckman Institute and the Arnold and Mabel Beckman Foundation. Electron microscopy imaging was performed in the Caltech Biological and Cryo-EM Facility. Schematics were created with BioRender.com. Funding This work was supported by the Caltech startup funds, Caltech Space-Health Innovation Fund, Henry Luce Foundation, and Shurl and Kay Curci Foundation. MGL is supported through the NSF GRFP program. References Yong, J., Wu, M., Carroll, B. J., Xu, Z. P. & Zhang, R. Enhancing plant biotechnology by nanoparticle delivery of nucleic acids. Trends in Genetics 40 , 352–363 (2024). Demirer, G. S. et al. 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Molecular anatomy of the receptor binding module of a bacteriophage long tail fiber. PLoS Pathog 15 , (2019). Jiang, F. et al. N-terminal signal peptides facilitate the engineering of PVC complex as a potent protein delivery system. Sci. Adv. 8 , (2022). Danov, A. et al. Identification of novel toxins associated with the extracellular contractile injection system using machine learning. Mol Syst Biol 20 , 859–879 (2024). Geller, A. M. et al. The extracellular contractile injection system is enriched in environmental microbes and associates with numerous toxins. Nat Commun 12 , (2021). Ritchie, J. M. et al. An Escherichia coli O157-Specific Engineered Pyocin Prevents and Ameliorates Infection by E. coli O157:H7 in an Animal Model of Diarrheal Disease. Antimicrob Agents Chemother 55 , 5469–5474 (2011). Gebhart, D. et al. A Modified R-Type Bacteriocin Specifically Targeting Clostridium difficile Prevents Colonization of Mice without Affecting Gut Microbiota Diversity. mBio 6 , (2015). Kreitz, J. et al. Programmable protein delivery with a bacterial contractile injection system. Nature 616 , 357–364 (2023). Chen, L. et al. Genome-wide Identification and Characterization of a Superfamily of Bacterial Extracellular Contractile Injection Systems. Cell Reports 29 , 511-521 (2019). Wang, Y. et al. Purification of Photorhabdus Virulence Cassette (PVC) Protein Complexes from Escherichia coli for Artificial Translocation of Heterologous Cargo Proteins. BIO-PROTOCOL 14 , (2024). Jiang, F. et al. Cryo-EM Structure and Assembly of an Extracellular Contractile Injection System. Cell 177 , 370-383 (2019). Chinchilla, D., Bauer, Z., Regenass, M., Boller, T. & Felix, G. The Arabidopsis Receptor Kinase FLS2 Binds flg22 and Determines the Specificity of Flagellin Perception. The Plant Cell 18 , 465–476 (2006). Sun, W. et al. Probing the Arabidopsis Flagellin Receptor: FLS2-FLS2 Association and the Contributions of Specific Domains to Signaling Function. Plant Cell 24 , 1096–1113 (2012). Schnütgen, F. et al. A directional strategy for monitoring Cre-mediated recombination at the cellular level in the mouse. Nat Biotechnol 21 , 562–565 (2003). Storms, Z. J. & Sauvageau, D. Modeling tailed bacteriophage adsorption: Insight into mechanisms. Virology 485 , 355–362 (2015). Ngou, B. P. M., Wyler, M., Schmid, M. W., Kadota, Y. & Shirasu, K. Evolutionary trajectory of pattern recognition receptors in plants. Nat Commun 15 , 308 (2024). Colaianni, N. R. et al. A complex immune response to flagellin epitope variation in commensal communities. Cell Host & Microbe 29 , 635-649 (2021). Kreitz, J. et al. Targeted delivery of diverse biomolecules with engineered bacterial nanosyringes. Nat Biotechnol (2025). Wu, F.-H. et al. Tape-Arabidopsis Sandwich - a simpler Arabidopsis protoplast isolation method. Plant Methods 5 , (2009). Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryTable1.xlsx Supplementary Data Set 1 eCISsupplementfile.docx SUPPLEMENTARY FIGURES Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7412028","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":503550308,"identity":"23117051-76ef-49f6-a7a0-df4e30685add","order_by":0,"name":"Gozde Demirer","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIie2OIQvCQBSAn8WVA+uZ9heeCENBVv0bGwdLQwVBDIaBMKPVn6FlyfDGUMv9gNm0LBlMYlJv2gznouE+rtzjfXwPwGD4S7z3gwZAjT5fwGpKMwIgqqyUIFVV7L7Yrc/b3rB9yE7ZdeoOwJonXKe0ZBHkXhF0ExkgkRRjYPuJXlmFTu5Rhk6ubktj8iMeOj+U4U0pT2yvrCulD6XYF71i87CuFELkTFWissL0CrKiPEwgl+GI5F74MQvGHW1lIYrjnVxsLA6b03Tm+ksr2+TaCn1P6rr1dyX6tWEwGAyGFxSrVuV9HHNdAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-3007-1489","institution":"California Institute of Technology","correspondingAuthor":true,"prefix":"","firstName":"Gozde","middleName":"","lastName":"Demirer","suffix":""},{"id":503550309,"identity":"0838f7fd-92d0-4d6a-9b03-6834bd993295","order_by":1,"name":"Mark Legendre","email":"","orcid":"","institution":"California Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Mark","middleName":"","lastName":"Legendre","suffix":""},{"id":503550310,"identity":"de41edbc-ee39-487b-9aba-97e2837c19dc","order_by":2,"name":"Carlos Heredia","email":"","orcid":"","institution":"California Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Carlos","middleName":"","lastName":"Heredia","suffix":""},{"id":503550311,"identity":"0d1249d0-4d96-471b-90f0-60b20d4c9bd8","order_by":3,"name":"Clair Colee","email":"","orcid":"","institution":"California Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Clair","middleName":"","lastName":"Colee","suffix":""}],"badges":[],"createdAt":"2025-08-19 23:10:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7412028/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7412028/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89882020,"identity":"0aed1e7a-0f66-46be-b96c-23a40bf03898","added_by":"auto","created_at":"2025-08-26 05:58:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":725433,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRational design of PVCs to target the plant cell membrane receptor FLS2. (A) \u003c/strong\u003eSchematic of the gene constructs used to express PVCs in \u003cem\u003eE. coli\u003c/em\u003e: a structural plasmid containing all 16 structural genes (in teal) necessary for particle formation and a cargo plasmid contains 4 regulatory genes in addition to a single cargo-coding DNA sequence (in salmon). The structural gene \u003cem\u003epvc13\u003c/em\u003e and the cargo protein \u003cem\u003ePnf\u003c/em\u003e are marked for their importance in downstream nanoparticle engineering. \u003cstrong\u003e(B)\u003c/strong\u003e AlphaFold predicted structures of a trimeric \u003cem\u003epvc13\u003c/em\u003e tail fiber library designed to target the plant FLS2 receptor. Only the terminal region of the tail fiber heterotrimer is visualized\u003cstrong\u003e, \u003c/strong\u003econtaining part of the tail fiber body (in teal), an engineered flexible linker (in yellow), and the flg22 epitope (in salmon). \u003cstrong\u003e(C)\u003c/strong\u003e ChimeraX alignment of the flg22 epitope of the 4x linker PVC variant’s AlphaFold structure to the flg22 peptide of the crystal structure of flg22 bound to its receptor FLS2 (pdb: 4MN8, in gray). \u003cstrong\u003e(D)\u003c/strong\u003e PVC library preparations were imaged using negative-stain TEM to visualize PVCs in their elongated (non-ejected) states. Scale bars, 100 nm. \u003cstrong\u003e(E)\u003c/strong\u003e SDS-PAGE analysis of PVC libraries visualizing the most abundant structural proteins found within the eCIS particle, including the baseplate proteins (Pvc11 and 12), sheath protein (Pvc2), and our engineered tail fibers (Pvc13).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7412028/v1/932a0a61828251bf45ebfc2a.png"},{"id":89883270,"identity":"732bc89a-e4e3-4768-a63f-130fab82fe35","added_by":"auto","created_at":"2025-08-26 06:06:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":358250,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePVC-mediated delivery of the fluorescent protein mScarlet to mesophyll protoplasts.\u003c/strong\u003e (\u003cstrong\u003eA)\u003c/strong\u003e A general engineering strategy to load novel cargoes into PVCs involves tagging the cargo protein’s N-terminus with the native \u003cem\u003ePnf\u003c/em\u003e packaging domain and its C-terminus with a HiBiT tag. (\u003cstrong\u003eB)\u003c/strong\u003e Denaturing western blot targeting the HiBiT tag on an engineered mScarlet cargo loaded in PVC particles. (\u003cstrong\u003eC)\u003c/strong\u003e Schematic of the PVC-mediated fluorescent protein delivery workflow in mesophyll protoplasts. Following protoplast challenge with mScarlet-loaded PVCs, a short incubation period results in fluorescent protein accumulation within cellular nuclei. (\u003cstrong\u003eD\u003c/strong\u003e) The 0xlinker mScarlet-loaded PVC variant delivery to \u003cem\u003eArabidopsis thaliana\u003c/em\u003emesophyll protoplasts. Scale bar, 50 µm.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7412028/v1/8203a06ee061e83a4466746a.png"},{"id":89883271,"identity":"2d6f1f3c-28c4-41e1-b77c-a5bd24cf83f4","added_by":"auto","created_at":"2025-08-26 06:06:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":679232,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDeveloping an \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein planta\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e Cre recombinase reporter assay\u003c/strong\u003e. \u003cstrong\u003e(A)\u003c/strong\u003e Engineering the native Cre recombinase enzyme with N-terminus PVC packaging domain and plant-optimized NLS, and a C-terminus HiBiT tag allows for loading of the enzyme into PVCs and chemiluminescent detection of loaded Cre. \u003cstrong\u003e(B)\u003c/strong\u003e Denaturing western blot against HiBiT tag visualizes the presence of non-native Cre only within those preparations containing the cargo. \u003cstrong\u003e(C)\u003c/strong\u003e FLEX schematic demonstrating two pairs of orthogonal \u003cem\u003eLoxP\u003c/em\u003e sites interacting with Cre to achieve irreversible inversion of a reporter sequence. The reporter design produces yellow fluorescent signal in response to Cre activation, allowing for visualization of signal using confocal microscopy. \u003cstrong\u003e(D) \u003c/strong\u003eSchematics of different FLEX designs. Flanking the FLEX switch with geminiviral components (FLEX-GV) increases reporter copy numbers. A constitutive Cre expression plasmid allows for validation of the FLEX switch designs as a positive control. \u003cstrong\u003e(E)\u003c/strong\u003e Representative confocal microscopy images of co-transfection of \u003cem\u003eArabidopsis thaliana\u003c/em\u003eprotoplasts with FLEX and Cre plasmids. Scale bar, 100 µm. \u003cstrong\u003e(F) \u003c/strong\u003eRepresentative confocal microscopy images of the \u003cem\u003eN. benthamiana\u003c/em\u003e leaves co-transformed with FLEX and Cre plasmids. Scale bar, 100 µm.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7412028/v1/d6a5296c045b829aa95bdc43.png"},{"id":89882018,"identity":"9e19a3ff-5a34-4439-aec6-f0d460f1513f","added_by":"auto","created_at":"2025-08-26 05:58:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":384476,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReprogrammed PVCs deliver Cre protein cargoes to plant protoplasts.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Schematic of the PVC delivery workflow in plant mesophyll protoplasts. \u003cstrong\u003e(B)\u003c/strong\u003e Representative confocal fluorescence microscopy images of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e protoplasts transfected with the FLEX vectors and treated with Cre-loaded PVC libraries. Scale bar, 100 µm. Inset, 40 µm. \u003cstrong\u003e(C)\u003c/strong\u003eQuantification of percentage of YFP+ cells from confocal images for HEPES buffer control, 0x, 2x, and 4x linker Cre-loaded PVCs and regular FLEX reporter. \u003cstrong\u003e(D) \u003c/strong\u003eQuantification of percentage of YFP+ cells from confocal images for HEPES buffer control, 0x, 2x, and 4x linker Cre-loaded PVCs and regular FLEX-GV reporter. Data in \u003cstrong\u003eC\u003c/strong\u003e, \u003cstrong\u003eD\u003c/strong\u003e are mean ± SEM with n = 3 biological replicates; one-way ANOVA with Tukey post hoc test. *P \u0026lt; 0.05, **P \u0026lt; 0.01, and ****P \u0026lt; 0.0001. ns, not significant.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7412028/v1/efefe55fc6aa15cc1b8313ed.png"},{"id":89882021,"identity":"ba9b0821-ad5d-48bf-aebf-7600e5a68dcf","added_by":"auto","created_at":"2025-08-26 05:58:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":351147,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReprogrammed PVCs deliver Cre protein cargoes to \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eN. benthamiana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e leaves.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003eSchematic of the PVC delivery workflow to \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. \u003cstrong\u003e(B)\u003c/strong\u003eRepresentative confocal fluorescence microscopy images transfected with the FLEX vectors and treated with Cre-loaded PVC libraries. Scale bar, 100 µm. Inset, 40 µm. \u003cstrong\u003e(C)\u003c/strong\u003e Quantification of percentage of YFP+ cells from confocal images for HEPES buffer control, 0x, 2x, and 4x linker Cre-loaded PVCs and regular FLEX reporter. \u003cstrong\u003e(D) \u003c/strong\u003eQuantification of percentage of YFP+ cells from confocal images for HEPES buffer control, 0x, 2x, and 4x linker Cre-loaded PVCs and regular FLEX-GV reporter. Data in \u003cstrong\u003eC\u003c/strong\u003e, \u003cstrong\u003eD\u003c/strong\u003e are mean ± SEM with n = 4 biological replicates; one-way ANOVA with Tukey post hoc test. *P \u0026lt; 0.05, and ****P \u0026lt; 0.0001. ns, not significant.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7412028/v1/b03ecb09304a0f940e392370.png"},{"id":89882022,"identity":"9044692d-8ecb-4a07-8808-d59276f97de8","added_by":"auto","created_at":"2025-08-26 05:58:08","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":492853,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFLS2 over-expression improves PVC-mediated protein delivery. (A)\u003c/strong\u003eSchematic of an activity assay for engineered PVCs based on receptor cell surface density. Cells transiently express the endogenous FLS2 receptor to varying degrees, resulting in a panel of cells with varying FLS2 surface densities. \u003cstrong\u003e(B)\u003c/strong\u003e FLS2 over-expression (OE) can be monitored by a GFP tag. Representative confocal microscopy images of FLS2-GFP overexpression in \u003cem\u003eN. benthamiana\u003c/em\u003eleaves using either the native FLS2 promoter or p35S. Scale bar, 100 µm. \u003cstrong\u003e(C)\u003c/strong\u003eRepresentative confocal microscopy images of FLS2-overexpressing \u003cem\u003eN. benthamiana\u003c/em\u003e leaves when infiltrated with Cre-loaded 2x linker PVC particles. No PVC and no over-expression are tested as controls to the samples overexpressing FLS-GFP either using the native FLS2 promoter or p35S. Scale bar, 100 µm. \u003cstrong\u003e(D)\u003c/strong\u003e Quantification of the percentage of YFP+ cells from confocal images overexpressing FLS2 treated with Cre-loaded 2x linker PVC particles. Data in \u003cstrong\u003eD\u003c/strong\u003e are mean ± SEM with n = 3 biological replicates; one-way ANOVA with Tukey post hoc test. **P \u0026lt; 0.01, and ***P \u0026lt; 0.001. ns, not significant.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7412028/v1/e2cad0a2f10f507eec52f0cf.png"},{"id":89884312,"identity":"5b87ed0e-8c23-406e-b5e0-6872c5fa1f56","added_by":"auto","created_at":"2025-08-26 06:14:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4257380,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7412028/v1/77bb06ab-fc11-4005-9a09-0936f2a2ff02.pdf"},{"id":89882016,"identity":"6d604799-5998-4608-be5b-85b040e81cf9","added_by":"auto","created_at":"2025-08-26 05:58:08","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12506,"visible":true,"origin":"","legend":"Supplementary Data Set 1","description":"","filename":"SupplementaryTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7412028/v1/dc18f3d287b62a0899f371c8.xlsx"},{"id":89882024,"identity":"596e2d76-ca49-4f4b-9e5d-4c0b6dfffd3d","added_by":"auto","created_at":"2025-08-26 05:58:08","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2681960,"visible":true,"origin":"","legend":"SUPPLEMENTARY FIGURES","description":"","filename":"eCISsupplementfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-7412028/v1/793571d0260f799ace804f3d.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Extracellular contractile injection systems for high efficiency protein delivery to plants","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAdvancements in plant biotechnology continue to highlight genetic engineering strategies as a sustainable path to improved crop traits. However, inefficient biomolecule delivery across the rigid cell wall of many species remains a major barrier to plant genetic engineering. Nanotechnology has emerged as a more ubiquitous, higher efficiency, and less destructive alternative to the traditional delivery approaches of \u003cem\u003eAgrobacterium\u003c/em\u003e and biolistics, enabling delivery of diverse biomolecular cargoes to various plant species\u003csup\u003e1\u003c/sup\u003e. While nanomaterials have been developed for efficient nucleic acid delivery to plants\u003csup\u003e2–5\u003c/sup\u003e, nanoparticle-mediated protein delivery remains largely unexplored in plants\u003csup\u003e6\u003c/sup\u003e. The most developed nanomolecular tools for protein delivery to plants to date rely on peptide-based ionic complexes, such as cell-penetrating peptides, to translocate protein cargoes across cell membranes, but these tools remain low in efficiency and are untargeted\u003csup\u003e7–14\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eProtein nanoparticles emerge as an alternative platform with great potential for protein delivery applications\u003csup\u003e15,16\u003c/sup\u003e. In nature, many bacteria have evolved specialized protein nanomachines to translocate effector proteins into eukaryotic host cells. Among these, contractile injection systems (CISs) are phage tail-like protein nanosyringes consisting of a rigid inner tube terminated by a sharp spike protein, encased in a contractile sheath anchored to a baseplate\u003csup\u003e17–20\u003c/sup\u003e. Upon recognition of specific host receptors, sheath contraction drives the inner tube through the target membrane, delivering loaded protein payloads directly into the host cytosol\u003csup\u003e21,22\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eContractile injection systems can be anchored to bacterial membranes for delivery requiring direct cell-to-cell contact\u003csup\u003e23\u003c/sup\u003e, or they can be released as extracellular complexes following suicidal lysis of the producing bacteria. These extracellular contractile injection systems (eCISs) function independently of their bacterial hosts, freely interacting with target cells across all kingdoms of life to deliver protein cargoes for a wide range of purposes\u003csup\u003e24–26\u003c/sup\u003e. Structural and biochemical studies have revealed conserved mechanisms for selective cargo loading\u003csup\u003e27–29\u003c/sup\u003e and demonstrated tail fiber reprogramming to alter host specificity\u003csup\u003e30,31\u003c/sup\u003e. Recent engineering advances have enabled eCIS-mediated delivery of diverse functional proteins, including genome editing machinery, into non-native mammalian hosts for therapeutic purposes\u003csup\u003e27,32\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003ePhotorhabdus\u003c/em\u003e virulence cassette (PVC) is one of the most well-characterized eCIS subfamilies\u003csup\u003e29,33\u003c/sup\u003e. Naturally, PVCs deliver protein toxins into insect host cells, but recent work has retargeted them to deliver heterologous proteins into mammalian cells\u003csup\u003e32,34\u003c/sup\u003e. However, PVCs or any other protein nanoparticles have not been applied to plant systems yet, where targeted protein delivery could address longstanding challenges in plant genetic transformation and enable precise delivery of genome editing machinery into desired cell types, while avoiding the introduction of foreign DNA and unwanted transgenes.\u003c/p\u003e\n\u003cp\u003eHere, we harnessed PVC extracellular contractile injection systems for targeted protein delivery to plants by reprogramming tail fibers to recognize natural plant membrane receptors. We demonstrated efficient loading and delivery of diverse functional protein cargoes, including a fluorescent reporter protein and a recombinase, into both plant cells in culture and intact leaf cells with combined delivery and activity efficiencies up to 40%. This work establishes PVC eCISs as a versatile protein delivery platform in plants, offering a technological breakthrough for targeted protein delivery in agriculture.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eRetargeting PVC eCISs to the plant cell membrane\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOriginating as eCISs produced by the endosymbiotic bacterium \u003cem\u003ePhotorhabdus asymbiotica\u003c/em\u003e, the linear \u003cem\u003ePhotorhabdus\u003c/em\u003e virulence cassette (PVC) gene cluster consists of 16 structural genes (\u003cem\u003epvc1-16\u003c/em\u003e) responsible for the successful expression and self-assembly of the functional nanoparticle\u003csup\u003e35\u003c/sup\u003e. Immediately downstream of these structural genes are two payload constructs (\u003cem\u003ePnf\u0026nbsp;\u003c/em\u003eand \u003cem\u003ePdp1\u003c/em\u003e) that encode its native toxin effector protein cargoes. In addition to these two payload constructs are four accessory genes necessary for cargo loading. Combined, these structural and payload genes constitute the PVCpnf gene locus (\u003cstrong\u003eFig. 1A\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e has previously been engineered to recombinantly express the PVCpnf gene locus from \u003cem\u003eP. asymbiotica ATCC 43949\u003c/em\u003e\u003csup\u003e32\u003c/sup\u003e. In our studies, we used an expression system that reconstitutes the PVCpnf locus into two independent expression vectors for modularity to create diverse libraries, harboring either the structural components of the locus (pStructural) or its cargo components (pCargo). PVC generation and isolation was achieved using previously described methods\u003csup\u003e35\u003c/sup\u003e, where co-transformation of a pStructural and pCargo variants resulted in the production of unique PVC variants in\u003cem\u003e\u0026nbsp;E. coli\u003c/em\u003e (\u003cstrong\u003eFig. 1A\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eTo retarget PVC nanoparticles to the plant cell membrane, we focused on the engineering of the tail fiber binding domain. PVC\u0026rsquo;s homologous tail fiber binding domain has been identified and engineered to retarget PVC tropism to mammalian hosts\u003csup\u003e32\u003c/sup\u003e. We tested whether a similar strategy could be employed to produce functional PVCs that target plant cell surface protein receptors, thus enabling targeted delivery of protein cargoes to plant cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe used AlphaFold to predict the structure of the distal tip of the tail fiber produced by the \u003cem\u003epvc13\u003c/em\u003e gene of the PVCpnf locus (\u003cstrong\u003eFig 1B\u003c/strong\u003e). The tail fiber exists as a Pvc13 homotrimer, and the structural prediction of this supramolecular complex revealed a helical tail fiber body region\u0026mdash;structurally similar to the short tail fibers of bacteriophages\u0026mdash;and a terminal globular region previously identified as the Pvc13 binding domain\u003csup\u003e32\u003c/sup\u003e. We produced a small library of engineered PVCs with binding regions that target a ubiquitous and abundant plant cell surface receptor, FLAGELLIN SENSITIVE2 (FLS2). The FLS2 receptor is a Microbe-Associated Molecular Pattern recognition receptor that binds and detects the conserved 22-amino acid fragment of bacterial flagellin known as flg22\u003csup\u003e36\u003c/sup\u003e. We chose to target PVCs to the FLS2 receptor due to its naturally high expression levels in the leaf tissue of most plant species\u003csup\u003e37\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe PVC library was designed to present the flg22 epitope at various extensions from the tail fiber body using 0x, 2x, and 4x (GGSGG)\u003csub\u003en=0,2,4\u003c/sub\u003e flexible linkers, accounting for any potential steric obstacles to receptor binding (\u003cstrong\u003eFig. 1B\u003c/strong\u003e). AlphaFold simulations confirmed that all members of the PVC library maintained the expected helical structure of the tail fiber body without disturbing the disordered nature of the flg22 epitope. We also used ChimeraX to align the predicted AlphaFold tail fiber structures of the PVC library to the binding region of the FLS2 receptor (\u003cstrong\u003eFig. 1C and Supplementary Fig. 1A, B\u003c/strong\u003e). The resulting alignment verified proper binding of the flg22 epitope to the receptor despite fusion to the larger tail fiber body structure, suggesting the potential functionality of the nanoparticles.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfter expression and purification in \u003cem\u003eE. Coli\u003c/em\u003e, negative-stain transmission electron microscopy (TEM) confirmed proper self-assembly of all PVC library members and demonstrated limited particle instability that would result in spontaneous sheath contraction in various buffers (\u003cstrong\u003eFig. 1D and Supplementary Fig. 2A\u003c/strong\u003e). Generated PVCs showed similar morphology and length distributions independent of their altered tail fiber domains, suggesting that tail fiber modification did not impact sheath structure or loading mechanisms (\u003cstrong\u003eSupplementary Fig. 1C\u003c/strong\u003e). Finally, denaturing gel electrophoresis revealed successful incorporation of the expected PVC structural proteins into each member of the library (\u003cstrong\u003eFig. 1E\u003c/strong\u003e). Together, these results confirm the successful design, generation, and isolation of recombinant PVC nanoparticles.\u003c/p\u003e\n\u003cp\u003eTo test whether FLS2 receptor can be targeted with flg22-displaying PVC particles for protein delivery into plants, we generated the 0xlinker PVC variant carrying the native insect toxin proteins Pnf and Pdp1 and delivered them to \u003cem\u003eArabidopsis thaliana\u003c/em\u003e protoplasts (\u003cstrong\u003eSupplementary Fig. 3\u003c/strong\u003e). Compared to the native insect-targeting tail fiber domain, flg22-displaying PVC particles caused ~4 times more cell lethality, indicating successful targeting and toxin protein delivery into plant cells (\u003cstrong\u003eSupplementary Fig. 3\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eValidating PVC-mediated delivery of novel protein cargoes to plant cells\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhile the exact molecular mechanism of PVC cargo loading is currently unknown, N-terminal protein sequences naturally present on the PnfandPdp1proteins shuttle the effector cargoes to the lumen of the PVC sheath\u003csup\u003e27,28\u003c/sup\u003e. We designed a cargo protein engineering strategy that fuses the Pnf packaging domain to the N-terminus of a novel cargo protein and a HiBiT tag to the protein\u0026rsquo;s C-terminus (\u003cstrong\u003eFig. 2A\u003c/strong\u003e). This dual-tagging strategy allows for loading of novel cargoes as well as their subsequent chemiluminescent detection via HiBiT luminescence complementation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe demonstrated the feasibility of this engineering strategy using an mScarlet fluorescent protein as a non-native cargo for loading into PVCs, generating a 39.8 kDa functional cargo protein. Denaturing western blot analysis against the HiBiT tag on the engineered mScarlet revealed successful loading of the protein into PVCs with a total cargo loading capacity of approximately 4.5 ng mScarlet per 1\u0026nbsp;mg of PVC particle (\u003cstrong\u003eFig. 2B and Supplementary Fig. 2B\u0026nbsp;\u003c/strong\u003efor standard curves), confirming that non-native cargoes can be encoded for shuttling into the FLS2-targeting PVC library for downstream studies.\u003c/p\u003e\n\u003cp\u003eUsing these mScarlet-loaded particles, we tested whether FLS2-targeting PVCs were capable of delivering the fluorescent protein to plant cells. For this, \u003cem\u003eA. thaliana\u003c/em\u003e mesophyll protoplasts were incubated overnight following challenge with 1 mg/mL mScarlet-loaded PVCs (\u003cstrong\u003eFig. 2C\u003c/strong\u003e), where successful PVC binding and delivery were expected to yield nuclear-localized mScarlet signal. When the 0xlinker FLS2-binding PVC variant was loaded with the mScarlet protein cargo and applied, clear nuclear mScarlet signal could be detected in protoplasts in response to PVC activity after 18 h of incubation (\u003cstrong\u003eFig. 2D\u003c/strong\u003e). These initial mScarlet delivery results validated the flg22-binding epitope for PVC retargeting, and ability to deliver non-native cargo into plants, which encouraged further probing to increase delivery efficiency and cargo range.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eInterrogating PVC activity using a Cre-based reporter system\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter confirming fluorescent protein delivery with PVCs, we next investigated the capacity for our engineered particles to load and deliver genome-editing protein cargoes. The Cre recombinase is a well-studied nuclease effector with a molecular weight below that of the native PVC Pnfcargo, making it an ideal PVC cargo candidate. A similar cargo engineering strategy as in \u003cstrong\u003eFig. 2A\u003c/strong\u003e was used to modify the Cre recombinase for PVC loading and cargo detection. Fusion of the PVC packaging domain to the N-terminus of Cre was expected to shuttle the enzyme into the PVC lumen. Addition of a plant-optimized NLS would further improve Cre activity in plant cells and a C-terminal HiBiT tag allowed for chemiluminescent detection of loaded Cre following PVC preparation (\u003cstrong\u003eFig. 3A\u003c/strong\u003e). Denaturing western blot analysis confirmed loading of the Cre cargo into the PVC sheath lumen at an efficiency of approximately 10 ng Cre cargo for every 1\u0026nbsp;mg of PVC nanoparticle (\u003cstrong\u003eFig. 3B and Supplementary Fig. 2C\u0026nbsp;\u003c/strong\u003efor standard curves).\u003c/p\u003e\n\u003cp\u003eUpon producing Cre-loaded nanoparticles for our entire library of FLS2-binding PVC variants, we next developed an \u003cem\u003ein-planta\u003c/em\u003e assay for detecting intracellular Cre activity. The molecular reporter for this assay was a Cre flip-excision (FLEX) reporter\u003csup\u003e38\u003c/sup\u003e: an anti-parallel YFP coding DNA sequence flanked on either side by two orthogonal pairs of \u003cem\u003eLoxP\u003c/em\u003e sites (\u003cstrong\u003eFig. 3C\u003c/strong\u003e). In response to intracellular Cre activity, the reporter irreversibly inverts to produce nuclear-localized YFP signal. Two versions of the FLEX reporter were produced: a traditional FLEX reporter and a FLEX-GV reporter flanked by geminiviral components (\u003cstrong\u003eFig. 3D\u003c/strong\u003e) that is expected to produce high copy numbers within the cell, thus increasing reporter sensitivity and signal output.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFLEX reporter was expressed in plant cells along with a constitutive Cre expression plasmid, where intracellular Cre production should produce positive reporter signal. FLEX reporters were validated in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e protoplasts via PEG transfection (\u003cstrong\u003eFig. 3E\u003c/strong\u003e) and \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaf tissue via \u003cem\u003eAgrobacteria\u003c/em\u003e infiltration (\u003cstrong\u003eFig. 3F\u003c/strong\u003e). Both constructs showed minimal leakage in negative controls and full coverage when co-delivered with a Cre-expression vector. We observed increased signal output intensities under the high copy FLEX-GV reporter compared to the FLEX switch, as indicated by brighter and larger nuclear YFP signals (\u003cstrong\u003eFig. 3E, F\u003c/strong\u003e). Together, these results confirm the design and implementation of two Cre-responsive FLEX reporters for use \u003cem\u003ein planta\u003c/em\u003e. These reporters provide a powerful tool for assessing intracellular activity of an exogenously applied Cre recombinase cargo.\u003c/p\u003e\n\u003cp\u003eAfter confirming Cre loading into PVCs and proper function of FLEX constructs as a tool to report the delivery of Cre protein cargoes, we next attempted to deliver Cre-loaded PVCs into FLEX reporter-expressing plant cells. Initially screening for PVC activity in \u003cem\u003eA. thaliana\u003c/em\u003e mesophyll protoplasts, cells were PEG-transfected with either of the FLEX switches prior to Cre-loaded PVC challenge and subsequent quantification using confocal microscopy (\u003cstrong\u003eFig. 4A\u003c/strong\u003e). PVC activity saturated at relatively low concentrations when applied to mammalian cells\u003csup\u003e32\u003c/sup\u003e. This trend was confirmed in our protoplast assays, where PVC activity saturated at concentrations as low as 100 ng/\u0026micro;L PVC particles (\u003cstrong\u003eSupplementary Fig. 4A-C\u003c/strong\u003e). Additionally, while Cre delivery events saturated beyond 24 hours of incubation, maximal reporter signal intensity occurred at 48 hours during peak FLEX reporter expression (\u003cstrong\u003eSupplementary Fig. 4D, E\u003c/strong\u003e). Together, these results informed optimal assay conditions for screening the FLS2-binding PVC library.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGiven that the cell wall is enzymatically removed from protoplast cells, steric hindrance at receptor binding sites was assumed to be negligible, providing all members of the FLS2-binding PVC library comparable access to membrane receptors. This was confirmed after screening PVC activity in protoplasts using the FLEX reporter, where similar levels of reporter activity, hence Cre delivery, were registered across the entire PVC library of 0, 2, and 4x linker variants (\u003cstrong\u003eFig. 4B\u003c/strong\u003e). The average percentage of cells expressing YFP was ~5%, reflecting both Cre delivery and PVC activity efficiency (\u003cstrong\u003eFig. 4B, C\u003c/strong\u003e). While PVC activity did not depend on the linker length, we confirmed that the flg22 binding epitope was, in fact, required for PVC activity in plant cells, where native insect receptor-binding tail fiber domain did not result in PVC activity in negative control samples (\u003cstrong\u003eSupplementary Fig. 5\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe FLEX-GV switch outperformed the FLEX reporter, registering overall efficiencies of 16%, 10%, and 15% with 0, 2, 4x linker PVCs, respectively (\u003cstrong\u003eFig. 4B, D\u003c/strong\u003e). The lower-copy FLEX switch was not sufficiently sensitive to detect differences in PVC activity across the nanoparticle library, but the higher-copy FLEX-GV reporter identified the 0xlinker variant as the highest performing PVC variant with activities as high as 16% in protoplasts.\u003c/p\u003e\n\u003cp\u003eA similar workflow was employed in \u003cem\u003eN. benthamiana\u0026nbsp;\u003c/em\u003eleaves, where leaves were Agro-transformed with one of the FLEX switches prior to Cre-loaded PVC challenge (\u003cstrong\u003eFig. 5A\u003c/strong\u003e). Cre-driven YFP expression was low and inconsistent across biological replicates when probed using the low copy number FLEX switch (\u003cstrong\u003eFig. 5B, C\u003c/strong\u003e), likely due to insufficient reporter sensitivity. However, when the FLEX-GV reporter was deployed, YFP expression recovered to the levels reported in protoplasts, reaching 13%, 16%, and 10% with 0, 2, 4x linker PVCs, respectively (\u003cstrong\u003eFig. 5D\u003c/strong\u003e). The 2xlinker PVC variant outperformed the rest of the library in the leaf tissue. Given this, the 2xlinker PVC variant was used in all subsequent leaf tissue assays. These experiments are the first demonstrations of efficient PVC-mediated protein delivery to plant cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eThe impact of FLS2 receptor density on PVC protein delivery efficiency\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOne notable characteristic of the PVC eCIS binding and delivery is the requirement for sufficiently dense target receptors on the host cell membrane\u003csup\u003e39\u003c/sup\u003e. The current FLS2-targeting PVCs can effectively deliver protein cargoes to plant cells expressing basal levels of the FLS2 receptor, making them powerful tools for applications to wild type organisms. However, receptor over-expression lines might increase the tool\u0026rsquo;s efficiency when targeting a genetically modified plant by offering heightened receptor levels for PVC binding (\u003cstrong\u003eFig. 6A\u003c/strong\u003e). To this end, we developed FLS2 receptor over-expression constructs for transient expression of a GFP-tagged FLS2 receptor in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves under either the native FLS2 promoter and terminator or driven by the strong 35S promoter (\u003cstrong\u003eFig. 6B\u003c/strong\u003e). While both of these constructs expressed the receptor, visualized as GFP fluorescence under confocal microscopy, when FLS2 expression was driven by its native promoter and terminator, the receptors were better localized at the cell membrane (\u003cstrong\u003eFig. 6B\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eAfter transiently over-expressing the FLS2 receptor in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves via Agro-infiltration, we applied the same Cre-loaded PVC delivery workflow as in \u003cstrong\u003eFig. 5A\u003c/strong\u003e using the 2xlinker PVC variant. Notably, in the cells over-expressing the FLS2 receptor with proper membrane localization, Cre-driven YFP expression is significantly more frequent and produces higher intensity signals (\u003cstrong\u003eFig. 6C\u003c/strong\u003e). When driven by the native FLS2 promoter, FLS2 over-expression showed increased PVC activity within these cells up to 38% efficiency, a greater than 2.5-fold increase in activity compared to cells with natural FLS2 expression levels (\u003cstrong\u003eFig. 6D\u003c/strong\u003e). FLS2 over-expression driven by the strong p35S promoter did not produce an increase in PVC activity. This is potentially due to the fact that, while the receptor is over-expressed using this construct, failure to localize at the cell membrane prevents over-expression from improving the PVC binding and subsequent delivery.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTogether, these results confirm that increased receptor surface densities can improve PVC delivery capabilities in plants, with the potential to reach efficiencies reliable for translation to field applications.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePlant biomolecule delivery remains a limiting step in the implementation of new and powerful genetic engineering technologies in agriculture. In particular, the direct delivery of functional proteins to plant cells is a big challenge. While many methods are available for transferring protein cargoes to mammalian cells, these methods are either inefficient when applied to plants or entirely unexplored. Recent studies have identified cell-penetrating peptides (CPPs) as a useful tool for translocating proteins into walled plant cells and have discovered novel, plant-specific CPPs that are more effective for plant delivery than their known mammalian counterparts\u003csup\u003e14\u003c/sup\u003e. Even though these are promising developments, CPPs typically achieve low delivery efficiencies and lack any cell-specific targeting capabilities, a hallmark of modern delivery vehicles in mammalian biology.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHere, we demonstrated the engineering of the PVC eCIS to target native or overexpressed plant cell surface receptors, serving as a tool for efficient functional protein delivery to plant cells. We specifically engineered PVCs to target the FLS2 receptor, which is widely ubiquitous across both plant species and plant cell types. Notably, we have shown that these engineered PVCs can deliver protein cargoes to both leaf protoplasts and walled leaf cells with efficiencies up to 40%, and the ubiquitous expression of the FLS2 receptor should allow for efficient PVC activity in a multitude of plant species and tissue types. In the engineering of our PVC nanoparticles, we sought to compromise between rigid binding interactions and accessibility to receptor binding via incorporation of flexible (GGSGG)\u003csub\u003en=0,2,4\u003c/sub\u003e linkers flanking the tail fiber flg22 binding domains. Interestingly, the PVC variant with no linker addition was the most efficient in protoplast systems, whereas PVCs containing (GGSGG)\u003csub\u003e2\u003c/sub\u003e linkers were the most efficient in leaf cells with intact walls. This observation is potentially due to the need to traverse the cell wall for effective FLS2 receptor binding in intact cell systems, which is aided by the incorporation of a flexible linker.\u003c/p\u003e\n\u003cp\u003eThe potential for cell-type specific delivery in plants using PVCs is as of yet unexplored. PVCs targeted to mammalian receptors have proven highly specific in their recognition of cognate receptors, with minimal off-target activity\u003csup\u003e32\u003c/sup\u003e. Importantly, cell-type specific targeting can provide a source of biocontainment for delivery tool application in field environments. Tightening PVC binding specificity by targeting native plant-specific or cell-type specific receptors could decrease unintended activity while maintaining unparalleled cell-type specific protein delivery efficiencies. Furthermore, by introducing recombinant receptors as PVC targets orthogonal to natural plant receptors, efficiencies and application types can be expanded.\u003c/p\u003e\n\u003cp\u003eNumerous studies have dissected the important role that microbe-associated molecular pattern receptors play in regulating plant immunity in response to pathogens\u003csup\u003e40\u003c/sup\u003e, and the FLS2 receptor is likely the most well-studied. While FLS2 provides a naturally high receptor surface density, making it an ideal candidate for PVC targeting, binding of the flg22 epitope is known to elicit a downstream immune response and induce receptor recycling\u003csup\u003e36\u003c/sup\u003e. However, the consequences of nanoparticle targeting of the FLS2 receptor via flg22 binding is not yet studied. Therefore, further investigation of this interaction, particularly regarding its impact on PVC retargeting, and its effect on plant health is critical and will be part of future studies. Regardless, numerous flg22 epitope variants have been identified that can bind the FLS2 active site without activating the immune response\u003csup\u003e41\u003c/sup\u003e, suggesting possible solutions to circumvent unintended immune response elicitation.\u003c/p\u003e\n\u003cp\u003eIn summary, here, we demonstrated the modularity of the PVC delivery system by loading and delivering a novel Cre recombinase cargo. It is known, however, that PVCs are capable of loading and delivering various other biomolecules, seemingly independent of their size\u003csup\u003e42\u003c/sup\u003e. In fact, PVCs retargeted to mammalian cells have been demonstrated to deliver Cas9 ribonucleoprotein complexes with editing efficiencies of ~13%\u003csup\u003e32\u003c/sup\u003e. Such an expansion of the plant-targeting PVC cargo inventory to include gene editing machinery like Cas9-mediated systems could help realize efficient DNA- or transgene-free gene editing in plants. Together, our results identify the PVC eCIS as a novel tool for the efficient delivery of protein cargoes to plant cells, and with further development, this tool may have applications in plant biotechnology and agricultural engineering.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePlant growth conditions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNicotiana benthamiana\u0026nbsp;\u003c/em\u003eand \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (Col-0) plants were grown in a controlled-environment growth chamber (Conviron) set to 22°C during the day period and 20°C during the night period (16 h:8 h light/dark photoperiod) with 55% relative humidity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePlasmid construction\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe PVCpnf gene locus had previously been synthesized and domesticated to produce the pStructural (Addgene pAWP78-PVCpnf1-16) and pCargo (Addgene pBR322-PVCpnf17-22). To generate a version of pCargo compatible with Golden Gate Assembly with BsaI, a single BsaI site was removed from the original pCargo construct using the Q5 Site-Directed Mutagenesis Kit (NEB E0554S). All further manipulations of either construct involved standard PCR amplification with Phusion High-Fidelity PCR Master Mix with HF Buffer (ThermoFisher F531) followed by Golden Gate Assembly with BsaI HFv2 (NEB R3733) and T4 DNA Ligase (ThermoFisher EL0014). Assembled constructs were transformed into chemically competent NEB Turbo cell (NEB C2984). For PVC production, final variants of pStructural and pCargo plasmids were co-transformed into chemically competent EPI300 cells (Fisher Scientific NC1583291).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePVC purification\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo generate a given PVC condition, one pStructural variant and one pCargo variant were co-transformed into chemically competent EPI300 cells. The resulting transformants were then cultured and PVC particles harvested using a modified version of a previously developed method\u003csup\u003e35\u003c/sup\u003e. In brief, colonies were grown overnight in 2xYT (ThermoFisher 22712020) media and inoculated (at 1:1,000) into 1 L Terrific Broth (Fisher Scientific BP246850) and shaken at 24°C for 48 h. Cultures were centrifuged at 4,000\u003cem\u003eg\u003c/em\u003e for 20 min at 4°C and the resulting pellet was gently resuspended in 60 mL Buffer P (25 mM Tris-HCl pH 7.5 (ThermoFisher 15567027), 140 mM NaCl (Sigma-Aldrich S5886), 3 mM KCl (Fisher Scientific P217), 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e (Sigma-Aldrich M2393), 200\u0026nbsp;mg mL\u003csup\u003e-1\u003c/sup\u003e lysozyme (ThermoFisher 89833), 50\u0026nbsp;mg mL\u003csup\u003e-1\u003c/sup\u003e DNase I (Sigma-Aldrich DN25), 0.5% Triton X-100 (Sigma-Aldrich X100), and 1 Protease Inhibitor Cocktail (Sigma-Aldrich 11836153001)) using a serological pipette and subsequently shaken at 250 rpm for 30 min at 37°C. Lysates were pelleted at 4,000\u003cem\u003eg\u003c/em\u003e for 30 min at 4°C to remove cell lysate and the supernatant extracted and ultracentrifuged at 120,000\u003cem\u003eg\u003c/em\u003e for 1 h at 4°C to pellet PVCs. The supernatant was discarded and the ultracentrifuge tube swabbed using a Kimwipe. PVC pellets were then washed with 1x PBS (ThermoFisher 70011044), and the tubes were swabbed once more. Pellets were allowed to rehydrate overnight at 4°C in 2 mL PBS before being resuspended via pipetting. Suspensions were agitated for 30 minutes to allow complete rehydration and then centrifuged at 16,000\u003cem\u003eg\u003c/em\u003e for 20 min at 4°C to clarify the solution. Supernatants were then diluted in 60 mL PBS and ultracentrifuged at 120,000\u003cem\u003eg\u003c/em\u003e for 1 h at 4°C once more. Tubes were swabbed after decanting the supernatant and PVC pellets were resuspended via pipetting in 50\u0026nbsp;mL working solution following a 4 h incubation period. Once again, suspensions were agitated for 30 minutes and then centrifuged at 16,000\u003cem\u003eg\u003c/em\u003e for 20 min at 4°C to clarify the solution. The supernatant was collected as the final PVC product and protein concentration was measured using a Qubit instrument (ThermoFisher Q33211).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn silico protein structure prediction\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll protein structures and self-assemblies were predicted using ColabFold (v1.5.5), an AlphaFold2 implementation based in Google-Colab that generates sequence alignments using MMseqs2. For general protein structure prediction, sequences were queried with default model (AlphaFold2-ptm) and MSA settings. For Pvc13 tail fiber complex predictions, sequences were queried as heterotrimers with the same default model (AlphaFold2-multimer-v3) and MSA settings. The resulting structures were rendered and customized with PyMOL Molecular Graphics System (v3.1.3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eElectron microscopy\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e300-mesh copper grids with Formvar/carbon support (PELCO®, Ted Pella, Inc., 01753-F) were glow-discharged in air for 1 min at 15 mA using negative polarity mode. Purified PVC product suspended in PBS was diluted to 200 ng\u0026nbsp;mL\u003csup\u003e-1\u003c/sup\u003e in Milli-Q ultrapure water, and 5 µL was applied to the glow-discharged carbon film side of each grid for 1 min before side-blotting with filter paper. Grids were then washed twice by depositing 5 µL of Milli-Q water followed by immediate blotting. Grids were stained with 5 µL of 1% (w/v) uranyl acetate in Milli-Q water for 30 s and blotted to remove excess stain. Prepared grids were examined on an FEI Tecnai T12 transmission electron microscope (120 kV, LaB₆ filament) equipped with a Gatan Ultrascan 2k × 2k CCD camera (Caltech Biological and Cryo-EM Facility). Images were acquired at nominal magnifications of 6,500×, 15,000×, and 30,000×.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSDS-PAGE and denatured Western blotting\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e20\u0026nbsp;mg of purified PVCs was prepared for SDS-PAGE by combining with NuPAGE LDS Sample Buffer (ThermoFisher NP0007) and NuPAGE Sample Reducing Reagent (ThermoFisher NP0009) at appropriate dilutions. The mixture was then incubated for 10 min at 70°C in a thermocycler. The denatured PVC preparations were then loaded into NuPAGE Bis-Tris Mini Protein Gels, 4-12% (ThermoFisher NP0321) and run for 50 min at 200 V in 1x NuPAGE MOPS SDS Running Buffer (ThermoFisher NP0001) supplemented with NuPAGE Antioxidant (ThermoFisher NP0005) (at 1:400). For Coomassie staining, gels were rinsed of Running Buffer using Milli-Q ultrapure water prior to incubation in SimplyBlue SafeStain (ThermoFisher LC6065) for 90 min under gentle agitation. Gels were subsequently destained overnight in Milli-Q water and imaged in a Gel Imager (Azure Biosystems).\u003c/p\u003e\n\u003cp\u003eFor western blot analysis of loaded PVC payloads, identical sample preparation and electrophoresis conditions were applied. In this case, PVCs were generated with loaded cargoes fused at the C-terminus with a HiBiT peptide tag, and unloaded cargoes were removed from the sample using standard ultracentrifugal separation. Variable quantities of PVC sample were applied to the above SDS-PAGE protocol depending on cargo loading capacity. Following electrophoresis, gels were blotted onto PVDF membranes (BIO-RAD 1704156) using a Trans-Blot Turbo Transfer System (BIO-RAD) set to default settings for turbo transfer of a mini gel (7-minute protocol). Finally, HiBiT-tagged cargo proteins were visualized using the Nano-Glo HiBiT Blotting System (Promega N2410) consisting of a 4 h TBST incubation period and a 2 h LgBiT incubation period, both at room temperature. Chemiluminescent images were captured with an Azure 200 Gel Imager. Band intensity analysis was performed using Fiji, a distribution of ImageJ.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eProtoplast isolation\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtoplasts were isolated from 4-week-old \u003cem\u003eArabidopsis thaliana\u003c/em\u003e leaves as described previously\u003csup\u003e43\u003c/sup\u003e with some modifications. In brief, 7-10 fully developed leaves were gently compressed between Time tape (adhered to the upper epidermis) and 3 M Magic tape (adhered to the lower epidermis). The lower epidermal layer of leaves was removed and discarded along with removal of the Magic tape, thus exposing the leaf mesophyll. The exposed mesophyll was then placed in contact with a cell wall-degrading enzyme solution (20 mM MES pH 5.7, 0.4 M mannitol, 20 mM KCl, 1.5% w/v cellulase R10 Yakult, and 0.4% w/v macerozyme R10 Yakult) in the dark and incubated for 3 h. Protoplasts released into the enzyme solution following incubation were diluted in ice cold W5 solution (2 mM MES pH 5.7, 154 mM NaCl, 125 mM CaCl\u003csub\u003e2\u003c/sub\u003e, and 5 mM KCl) and gently pelleted at 100\u003cem\u003eg\u003c/em\u003e for 5 min with minimal ramp rates. Pellets were suspended in W5 solution and run on a 21% w/v water-based sucrose cushion at 90\u003cem\u003eg\u003c/em\u003e for 10 min. The suspended layer of intact protoplasts was once again diluted in W5 solution and gently pelleted at 100\u003cem\u003eg\u003c/em\u003e for 5 min with minimal ramp rates. The final pellet was resuspended to 5x10\u003csup\u003e5\u003c/sup\u003e cells mL\u003csup\u003e-1\u003c/sup\u003e in working solution (see specific experimental method).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePlasmid transfection for protoplast assays\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor assays requiring transfection of a FLEX reporter plasmid and/or a Cre expression plasmid, DNA was transfected into protoplasts using a PEG-mediated transfection procedure. Briefly, 100\u0026nbsp;mL of protoplasts at 5x10\u003csup\u003e5\u003c/sup\u003e cells mL\u003csup\u003e-1\u003c/sup\u003e in an MMG working solution (4 mM MES pH 5.7, 0.4 M mannitol, and 15 mM MgCl\u003csub\u003e2\u003c/sub\u003e) were gently mixed with 40\u0026nbsp;mg total plasmid DNA (20\u0026nbsp;mg:20\u0026nbsp;mg co-transfections) and 100\u0026nbsp;mL PEG transfection solution (40% w/v PEG4000, 0.2 M mannitol, 0.1 M CaCl\u003csub\u003e2\u003c/sub\u003e) in a round-bottom tube. After 15 min of incubation, the protoplasts were washed with 1 mL W5 solution and pelleted at 200\u003cem\u003eg\u003c/em\u003e for 2 min. This wash step was repeated for a total of three times, and the final protoplast pellet was resuspended in WI solution (4 mM MES pH 5.7, 0.5 M mannitol, and 20 mM KCl) at a density of 10\u003csup\u003e5\u003c/sup\u003e cells mL\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePVC delivery to protoplasts\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll protoplast delivery experiments were performed using fresh \u003cem\u003eArabidopsis thaliana\u003c/em\u003e mesophyll protoplasts isolated using the aforementioned procedure. Protoplasts in a working solution of WI media were seeded into clear-bottom 96-well plates (Fisher Scientific 07-000-167) and allowed 30 minutes to settle prior to manipulation. For all experiments, 2x10\u003csup\u003e4\u0026nbsp;\u003c/sup\u003ecells were seeded into a given well, and excess WI solution was aspirated to a final volume of 50 μL. PVCs were then added to a final concentration of 500 ng μL\u003csup\u003e-1\u003c/sup\u003e. For fluorescent protein delivery assays, cells were incubated for 18 h and imaged under a Leica STELLARIS 8 FALCON confocal microscope. mScarlet fluorescence was captured using a 569 nm laser excitation from a white light laser. Images were rendered and analyzed using Fiji software. For Cre protein delivery assays, cells were incubated for 48 h and imaged with a Leica STELLARIS 8 FALCON confocal microscope with 434 nm, 517 nm, and 587 nm laser excitations from a white light laser to capture mTurqouis2, YPET, and mCherry expression, respectively. All images were obtained at 20x magnification with water as an immersion media.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eQuantification of PVC activity in protoplasts\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor each PVC condition, 3 biological replicates (3 separate wells individually challenged with PVCs) were performed; and for each biological replicate, 10 technical replicates (10 non-overlapping confocal fields of view) were collected. Images were taken around the perimeter of wells due to the tendency for protoplasts to sequester along the well edges. Each field of view was analyzed with Fiji, a distribution of ImageJ, to quantify the total number of YFP expressing nuclei and the total number of mCherry expressing cells for that field of view. The PVC activity for a biological replicate was then calculated by combining YFP and mCherry counts for all technical replicates and calculating PVC activity = (# YFP+ cells)/(# mCherry+ cells).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePVC delivery to leaf cells\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFully developed leaves (the 3\u003csup\u003erd\u003c/sup\u003e of 4\u003csup\u003eth\u003c/sup\u003e true leaf) from \u003cem\u003eNicotiana benthamiana\u003c/em\u003e (3-4 weeks old) plants were selected for all experiments. Biological replicates were sampled from leaves on separate plants generated from the same seed batch. \u003cem\u003eAgrobacterium tumefaciens\u0026nbsp;\u003c/em\u003e(GV3101) harboring either of the FLEX switches were grown in 2xYT media (ThermoFisher 22712020) supplemented with 10 μg mL\u003csup\u003e-1\u003c/sup\u003e rifampicin (Sigma-Aldrich R3501), 20 μg mL\u003csup\u003e-1\u003c/sup\u003e gentamycin (Sigma-Aldrich G1264), 50 μg mL\u003csup\u003e-1\u003c/sup\u003e tetracycline (Sigma-Aldrich T7660), and 50 μg mL\u003csup\u003e-1\u003c/sup\u003e kanamycin (Sigma-Aldrich K1637) at 30°C and 200 rpm for 24 h. Overnight cultures were then centrifuged at 4,000\u003cem\u003eg\u003c/em\u003e for 20 min, and the pellets were resuspended to an OD600 of 0.4 in infiltration buffer (10 mM MES, pH 5.7 (Sigma-Aldrich M2933), 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e (Sigma-Aldrich M2393), 200 μM acetosyringone (PlantMedia 40100297)). The resuspended cultures were incubated at room temperature and shaken at 120 rpm for 4 h. Following incubation, 100-200 μL of the \u003cem\u003eAgrobacterium\u0026nbsp;\u003c/em\u003emixture was infiltrated against the abaxial surface of a leaf with a 1 ml needleless syringe by applying gentle pressure. Leaves were incubated for 72 h prior to analysis.\u003c/p\u003e\n\u003cp\u003eFor PVC delivery experiments, a small puncture was introduced on the abaxial surface of the leaf using a pipette tip, and 50-100 μl of the PVC sample (or of any control solution) was infiltrated against the puncture with a 1 ml needleless syringe by applying gentle pressure. For leaf infiltration experiments, all PVC samples were prepared at 1 mg mL\u003csup\u003e-1\u003c/sup\u003e and resuspended in 10 mM HEPES Buffer, pH 7.5 (Sigma-Aldrich H3375).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eQuantification of PVC activity in leaves\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePVC-infiltrated \u003cem\u003eNicotiana benthamiana\u0026nbsp;\u003c/em\u003eleaves were prepared for confocal imaging 72 h post-infiltration. A leaf disk puncher (Fisher Scientific NC0769832) was used to extract a 0.25 in leaf section adjacent to the location of PVC infiltration. Leaf disks were then mounted between a glass slide and coverslip of #1 thickness using water as the mounting medium. A Leica STELLARIS 8 FALCON confocal microscope was used to image the plant tissue with 434 nm, 517 nm, and 587 nm laser excitations from a white light laser to capture mTurqouis2, YPET, and mCherry expression, respectively. All images were obtained at 20x magnification with water as an immersion media. For each PVC condition, 3-4 biological replicates (3-4 infiltrations into leaves on 3-4 different plants) were performed; and for each biological replicate, more than 8 technical replicates (8 non-overlapping confocal fields of view) were collected. Each field of view was analyzed with Fiji, a distribution of ImageJ, to quantify the total number of YFP expressing nuclei and the total number of mCherry expressing cells for that field of view. The PVC activity for a biological replicate was then calculated by combining YFP and mCherry counts for all technical replicates and calculating PVC activity = (# YFP+ cells)/(# mCherry+ cells).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStatistics and reproducibility\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were performed using Prism (10.1.0). Quantitative data are presented as mean ± SEM with n = 3–4 biological replicates per condition; figure legends provide further specification as necessary. Unless otherwise stated, biological replicates represent independent treatments in separate wells (\u003cem\u003ein vitro\u003c/em\u003e assays) or on leaves from separate plants. All micrographs, gels, and blots are representative images from at least 2 independent repeated experiments. Statistical significance was computed using one-way or two-way (\u003cstrong\u003eSupplementary Fig. 4E\u003c/strong\u003e) ANOVA with Tukey post hoc tests (multiple comparisons correction), as indicated in figure legends. P \u0026lt; 0.05 were considered statistically significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are included either in the manuscript or supplementary files.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors declare no conflict of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: MGL, GSD\u003c/p\u003e\n\u003cp\u003eMethodology: MGL, CAH, GSD\u003c/p\u003e\n\u003cp\u003eData Acquisition: MGL, CAH\u003c/p\u003e\n\u003cp\u003eSupervision: \u0026nbsp;GSD\u003c/p\u003e\n\u003cp\u003eWriting: MGL, GSD\u003c/p\u003e\n\u003cp\u003eFunding Acquisition: GSD\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFluorescence imaging was performed in the Biological Imaging Facility, with the support of the Caltech Beckman Institute and the Arnold and Mabel Beckman Foundation. Electron microscopy imaging was performed in the Caltech Biological and Cryo-EM Facility. Schematics were created with BioRender.com.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Caltech startup funds, Caltech Space-Health Innovation Fund, Henry Luce Foundation, and Shurl and Kay Curci Foundation. MGL is supported through the NSF GRFP program.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eYong, J., Wu, M., Carroll, B. J., Xu, Z. P. \u0026amp; Zhang, R. Enhancing plant biotechnology by nanoparticle delivery of nucleic acids. \u003cem\u003eTrends in Genetics\u003c/em\u003e \u003cstrong\u003e40\u003c/strong\u003e, 352\u0026ndash;363 (2024).\u003c/li\u003e\n\u003cli\u003eDemirer, G. S. \u003cem\u003eet al.\u003c/em\u003e High aspect ratio nanomaterials enable delivery of functional genetic material without DNA integration in mature plants. \u003cem\u003eNat. 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Extracellular contractile injection systems (eCISs) are phage tail-like nanomachines evolved by bacteria to interface with eukaryotic host cells and deliver protein effectors. The Photorhabdus virulence cassette (PVC), a well-characterized eCIS, naturally targets insect hosts but can be reprogrammed for protein cargo delivery in mammalian systems. Here, we adapted PVCs for targeted delivery to plants by engineering their tail fibers to recognize a natural plant immune receptor, FLAGELLIN SENSITIVE2 (FLS2). We designed a library of FLS2-binding PVC variants and demonstrated efficient loading and delivery of non-native cargoes, including a fluorescent reporter protein and the Cre recombinase. We showed that engineered PVCs can deliver these proteins to Arabidopsis thaliana protoplasts and Nicotiana benthamiana leaf cells with efficiencies up to 40%. We elucidated that the delivery efficiency is correlated with receptor surface density, demonstrating that receptor selection and expression level are key parameters for optimization. This work establishes PVCs as novel, programmable protein delivery nanoparticles for plants, capable of targeting plant membrane receptors and effectively delivering diverse functional proteins. By enabling precise, DNA-free delivery of gene editing proteins, plant-targeted PVCs provide the framework for next-generation genome engineering strategies with broad potential in agricultural nanobiotechnology.","manuscriptTitle":"Extracellular contractile injection systems for high efficiency protein delivery to plants","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-26 05:58:04","doi":"10.21203/rs.3.rs-7412028/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-nanotechnology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"nnano","sideBox":"Learn more about [Nature Nanotechnology](http://www.nature.com/nnano/)","snPcode":"","submissionUrl":"","title":"Nature Nanotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"90c6f737-6911-4271-8685-5646244e563e","owner":[],"postedDate":"August 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":53496051,"name":"Biological sciences/Biotechnology/Nanobiotechnology/Nanoparticles"},{"id":53496052,"name":"Biological sciences/Biotechnology/Biomaterials/Drug delivery"}],"tags":[],"updatedAt":"2025-11-10T10:29:15+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-26 05:58:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7412028","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7412028","identity":"rs-7412028","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}