The authors would like to thank Mant Acon for the plants used in this study. The funding was provided by the U.S. Department of Agriculture (USDA) CRIS project 8062-22410-007-000-D and USDA NIFA grant 2020-70029-33176.

1. Production of citrus plants for experimental compound injection
<ol>
	<li>Propagate small potted citrus trees from either rooted vegetative cuttings or seeds. Grow the potted citrus lines under a 16 h/8 h light/dark day length and at 28 &#176;C.</li>
	<li>Select citrus plants of the appropriate size for the experiment. The described applications required the plants to be between 12 cm and 100 cm in height with trunk diameters of 4-14 mm. If new flush growth is required, cut back the plants prior to the experiment.</li>
</ol>
2. Three-dimensional printing of the DPI device and mold components
<ol>
	<li>Apply a thin layer of polyvinyl acetate-based glue to the print bed.</li>
	<li>Load nylon filament into the 3D printer, and prepare the printer according to the manufacturer&#39;s instructions. Import the desired .STL file (Supplementary File 1, Supplementary File 2, Supplementary File 3, Supplementary File 4, Supplementary File 5, Supplementary File 6, Supplementary File 7, Supplementary File 8, Supplementary File 9, Supplementary File 10, Supplementary File 11, Supplementary File 12, Supplementary File 13, and Supplementary File 14), set up, and start printing.</li>
	<li>Remove the piece from the print bed. Rinse the polyvinyl acetate-based glue from the base of the printed component with water, and remove any support structures.</li>
	<li>Spray the DPI component with two coats of clear gloss spray paint.</li>
</ol>
3. Fabrication of the plastisol ring mold
<ol>
	<li>Assemble a mold form large enough to hold the pattern pieces by building a rectangular enclosure using snap-together plastic blocks on a base (Supplementary Figure S1A).</li>
	<li>Mix the RTV silicone monomer and the catalyst together at a 10:1 ratio, and stir without introducing bubbles for 1 min. Color by adding food coloring (3 drops/25 mL of silicone mixture) and hand soap (1 mL of soap/25 mL of silicone mixture) (Supplementary Figure S1B).</li>
	<li>Pour a thin layer of silicone into the mold form that is sufficient to completely cover the bottom. Tamp to flatten the surface, and wait 24 h for the silicone to set (Supplementary Figure S1C).</li>
	<li>Pour a second layer of silicone, as described above, that is deep enough to cover the center hole core print on the pattern (visible at the top of the pattern in Supplementary Figure S1D). Insert the pattern into the liquid silicone with the center hole core print facing down. Ensure that no bubbles are trapped and that the patterns are well separated and not touching (Supplementary Figure S1E).</li>
	<li>Secure the patterns with either a heavy object and/or tape to prevent them from floating out of the silicone while it sets. Wait 24 h for the newly poured layer to set (Supplementary Figure S1F).</li>
	<li>Pour additional layers of silicone until the level is flush with the top of the pattern. Wait 24 h to cure (Supplementary Figure S1G).</li>
	<li>Disassemble the snap-together plastic blocks to release the mold. Remove the patterns (Supplementary Figure S1H). Inspect the mold for tears or deformities (Supplementary Figure S1I).</li>
</ol>
4. Casting of the plastisol rings
<ol>
	<li>Coat the interior of the mold, all the core components, and the O-rings with cooking oil (Supplementary Figure S2A, B). Place an O-ring around the notch in the middle of the center post core, and place the core in the center hole of the plastisol ring mold (Supplementary Figure S2C).</li>
	<li>Insert the delivery channel core into the hole on the side of the plastisol ring mold. Orient the V-shaped tip at the end of the delivery channel core to align with the O-ring on the center post core (Supplementary Figure S2D,E).</li>
	<li>Heat plastisol in the microwave for short 10 s bursts (Supplementary Figure S2F, G). Stir plastisol gently to avoid bubbles (Supplementary Figure S2H). Repeat the heating and stirring until the solution reaches a temperature between 160 &#176;C and 170 &#176;C (Supplementary Figure S2I). 
	CAUTION: Overheating of the plastisol can lead to the release of toxic fumes. Perform the procedure in a well-ventilated area or fume hood. Do not heat the plastisol over 170 &#176;C.</li>
	<li>Pour the molten plastisol into the plastisol ring mold near the outside edge of the mold without introducing bubbles (Supplementary Figure S2J). Wait 1 h for the plastisol rings to cool (Supplementary Figure S2K). Remove the rings from the mold. Ensure proper fit with the DPI device before use in experimental assays (Supplementary Figure S2L).</li>
</ol>
5. Attachment of the DPI device to the plant
<ol>
	<li>Find a smooth, healthy site on the trunk for the device attachment. Avoid any bumps or knots, as these can contribute to device leakage.</li>
	<li>Use a 2 mm diameter drill bit to drill a hole horizontally through the center of the stem at a 90&#176; angle with the trunk surface (Supplementary Figure S3A). Run the drill bit through the hole several times to clean and smooth it to create an unobstructed hole (Supplementary Figure S3B).</li>
	<li>Create a vertical slice in the plastisol ring on the opposite side of the compound delivery channel (Supplementary Figure S3C). Fit the ring around the plant, and line the delivery channel up with the previously drilled hole (Supplementary Figure S3D). 
	NOTE: The ring must fit snugly around the trunk to avoid leakages; core assemblies with various diameters can be used to make plastisol rings for different-sized plant stems.</li>
	<li>Add the DPI device to the plastisol ring, ensuring they fit snugly together. Align the DPI device spout with the plastisol ring delivery channel and drilled hole (Supplementary Figure S3E). 
	NOTE: Using a drill bit to line up the hole and the DPI device spout can be helpful.</li>
	<li>Wrap tightly with silicone tape to hold the apparatus in place (Supplementary Figure S3F). Inspect the fully assembled and attached device to ensure proper alignment and orientation on the plant.</li>
</ol>
6. Applying the compound of interest using the DPI device
<ol>
	<li>Use a syringe or pipette to fill the DPI device with the solution of interest (Supplementary Figure S3G). Use a syringe to penetrate the silicone tape and plastisol ring on the opposite side of the DPI device to pull air out of the interior channel and avoid the introduction of air bubbles into the plant vasculature (Supplementary Figure S3H). Replace any extracted compound back into the DPI device.</li>
	<li>Add an additional small patch of silicone tape over the hole created by the syringe to reinforce the area and prevent tears. Inspect the apparatus for visible leakages at the attachment point, and inspect the device reservoir for a stable liquid level. 
	NOTE: Water can be used initially to test for leaks to avoid waste of test solution.</li>
	<li>Cover the open end of the DPI device with wax sealing film, and pull down to form a seal and reduce the evaporation of the experimental compound. Poke a single hole in the wax film with a syringe tip to prevent the development of a vacuum, and subsequently refill the device (Supplementary Figure S3I).</li>
	<li>Check the apparatus daily to ensure proper alignment of the components (Supplementary Figure S3J), and top off the liquid using a syringe to avoid the reservoir running dry. Repeat this process until the desired amount of experimental solution has been introduced. 
	NOTE: Compounds can be successfully introduced from a given device for up to 1 month. Solution uptake durations that exceed this time frame should be tested empirically before the experimental setup.</li>
</ol>
7. Using CFDA to observe vascular movement with citrus plants
<ol>
	<li>Attach the DPI device to 25 cm tall citron (Citrus medica L.) plants, apply a single 2.0 mL treatment of either 20% DMSO in H2O control or CFDA to the DPI device, and allow it to uptake for 24 h. To follow this protocol, prepare the working CFDA solution by adding 1.6 mL of H2O to 400 &#181;L of CFDA stock solution (the stock solution contains 10 mM CFDA dissolved in 100% dimethyl sulfoxide [DMSO]) to generate a final CFDA concentration of 2 mM and 20% DMSO.</li>
	<li>Make cross-sections of various tissues of the plant, and use a stereoscope or compound microscope with a fluorescent filter that includes the 498 nm wavelength to visualize the CFDA in the plant tissues. Compare the images to the 20% DMSO in H2O controls to account for autofluorescence in the tissue.</li>
</ol>
8. Measuring changes in the CLas titer in leaf samples following streptomycin treatment
<ol>
	<li>Using 0.75 m tall greenhouse-grown CLas-infected Valencia (Citrus sinensis (L.) Osbeck &#34;Valencia&#34;) plants, collect the petiole and midrib of two HLB symptomatic leaves from each plant, chop them into small sections, pool each tissue type, and store them separately in 2 mL impact-resistant sample tubes containing a steel ball of 6.35 mm in diameter.</li>
	<li>Grind the sample using a homogenizer programmed for two 15 s cycles at 3.4 m/s. Extract the total nucleic acid as described previously19.</li>
	<li>Perform qPCR on the CLas-infected leaf samples using the qPCR mix defined in Table 1 and the reaction conditions defined in Table 2. Use plants showing robust CLas infections (robust infection is generally defined as qPCR-based Ct values &#60; 30 for the CLas 16S LasLong (LL) primer set) for further analysis. 
	NOTE: The bacterial titer is estimated using CLas 16S Las Long (LL) and citrus dehydrin DNA primers to estimate the relative genome equivalents, as described previously20.</li>
	<li>Outfit the citrus plants that meet the minimum infection titer outlined above with DPI devices, and subject them to a one-time treatment of 2.0 mL of either H2O solution or streptomycin solution at the desired concentration. To follow this study, use a single 2.0 mL treatment of 9.5 mg/mL (19 mg in total) streptomycin dissolved in H2O.</li>
	<li>Collect leaf samples at 7 days, 14 days, and 28 days post treatment. Assay the sampled leaves for the CLas titer using the same protocol as described above.</li>
	<li>At 60 days post treatment, obtain photographs of the plants treated with H2O or streptomycin. Use visual observations of the CLas infection to score the infection severity. Look for &#60;50% of leaves showing interveinal chlorosis and vein corking along with leaf flush growth as signs of mild infection and greater than 50% of leaves with interveinal chlorosis and vein corking as well as leaf abscission and stunting of the plant growth as signs of more severe infections.</li>
</ol>
9. Measuring the mortality of&#160; D. citri following imidacloprid treatment
<ol>
	<li>Prune 0.5 m tall citron (Citrus medica L.) plants to a height of 12 cm to encourage the growth of new flush. 
	NOTE: The speed of flush growth can be dependent upon the plant health and time of year; so, take this into account during experimental design.</li>
	<li>After &#62;6 flush, 1-2 cm in length, have developed, introduce the plants into cages containing adult D. citri. Leave the plants in the cages for 24 h to allow for the deposition of eggs.</li>
	<li>Conduct representative egg counts on three flush shoots 1-2 days after laying, and subsequently apply the DPI devices. Fill these devices with 2.0 mL of either a water control or the desired concentration of imidacloprid (21.8% active ingredient). To follow this protocol, use 528 &#181;L/L, 52.8 &#181;L/L, and 5.28 &#181;L/L of imidacloprid. 
	NOTE: Egg counts are a destructive measure, so the counts at this stage are used to estimate the number of eggs on the uncounted flush of the same plant and help provide a baseline for the subsequent nymph emergence counts that are conducted at the end of the experiment.</li>
	<li>Collect the D. citri emergence rate on at least three of the remaining flush shoots. Acquire representative plant photographs 7 days after compound introduction.</li>
</ol>

Molecule Delivery in Plants Using a Direct Infusion Device

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The authors have no conflicts of interest. The use of trade, firm, or corporation names in this publication is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the United States Department of Agriculture or the Agricultural Research Service of any product or service to the exclusion of others that may be suitable. The U.S. Department of Agriculture (USDA) prohibits discrimination in all its programs and activities on the basis of race, color, national origin, age, disability, and where applicable, sex, marital status, familial status, parental status, religion, sexual orientation, genetic information, political beliefs, reprisal, or because all or part of an individual's income is derived from any public assistance program. Not all prohibited bases apply to all programs. Persons with disabilities who require alternative means for communication of program information (Braille, large print, audiotape, etc.) should contact USDA's TARGET Center at (202) 720-2600 (voice and TDD). To file a complaint of discrimination, write to USDA, Director, Office of Civil Rights, 1400 Independence Avenue, S.W., Washington, D.C. 20250-9410, or call (800) 795-3272 (voice) or (202) 720-6382 (TDD). USDA is an equal opportunity provider and employer.

For the DPI device to be considered a viable method for exogenous compound delivery into plants, it must contribute to robust and consistent compound uptake into a variety of tissue types. The experiment utilizing CFDA clearly showed both acropetal and basipetal compound movement, as well as in both the vascular system and mesophyll cells of the leaf. Additionally, and presumably because the bored hole used in this DPI device provides a large amount of surface area for compound uptake, the CFDA was present in relatively equal amounts in all sections of the stem, not just in a small subset of the vasculature adjacent to the device, as has been seen in previous dye uptake studies in plants using trunk injection6. Additionally, the delivery of green fluorescent protein and floral dye was tested using the DPI device, and a distribution of these compounds similar to CFDA was observed (data not shown). These data suggest that the device can be used to systemically deliver a variety of compounds that vary in size and molecular structure. However, it is worth noting that there were differences in the compound uptake based on the leaf development stage, with younger developing leaves taking up more compound than older established leaves. This may be due to the changes in the vasculature properties present in sink versus source tissue and should be optimized for a given experiment. 
The DPI device showed sufficient compound uptake for the visualization of CFDA, GFP, and floral dye, and it also took up enough to show the antibacterial and insecticidal effects of streptomycin and imidacloprid, respectively. Both these compounds resulted in changes in the target organism viability 1 week after a single 2.0 mL treatment. These data suggest that the DPI device could be used in whole-plant assays to test the viability of a wide variety of compounds for the control of microbial and insect pests. Moreover, due to its direct contact with the vascular system, this device may even provide an opportunity to test compounds that are not efficiently taken up by the roots or epidermal cells. Of particular interest would be RNA interference (RNAi), as this could be used to modulate the gene expression within the host plant, pathogen, or pathogen vector. Previous research that introduced hairpin RNA through a drilled hole in the trunk of apple and grape plants showed that the RNA molecules were restricted to the xylem tissue, suggesting that these molecules may only be effective against chewing and xylem sap-feeding organisms22. Given that the DPI device uses a similar drilled-hole delivery system, it stands to reason that hairpin RNA delivered with this device may also be restricted to the xylem tissue. However, the observed decrease in the titer of the phloem-limited CLas after streptomycin treatment from the DPI device strongly suggests that this antibiotic was present in the phloem. Therefore, it is likely that the vascular distribution of the compounds delivered using the DPI device is dependent on their size and chemistry, and each molecule should be evaluated on an individual basis.
Although there are a number of commercially available DPI devices available on the market, the device described here can be manufactured in-house and is modifiable. In this way, improvements and changes in size may be made based on the plant species and experimental design being used, and it is not reliant on commercial products. In addition, the device is semi-permanently attached to the plant, meaning that multiple treatments of a given compound can be performed concurrently without having to reinjure the plant with multiple compound injections. On a cautionary note, the device can leak if not installed properly. As a result, the compound is lost to the environment instead of being delivered to the plant. Therefore, care should be taken to inspect the device for any signs of leakage during setup and the first few days afterward. Although drilling a hole in the tree is potentially harmful, this method was chosen to ensure robust and consistent compound uptake. Additionally, no adverse effects on plant health were seen from the attachment of the DPI device in these experiments. However, extra plants should be included in the experimental design to replace those that may lose vigor throughout the course of a given experiment. Lastly, since this device uses passive flow to introduce compounds, it can be difficult to predict the rate of uptake across different plant species or developmental stages of the same species. This may complicate experiments if the speed of compound uptake is a limiting factor. For the best results, experiments should be planned so that sufficient time is provided for the plant to fully absorb the 2.5 mL of compound, which can take up to 1 week. In conclusion, this DPI device is an effective tool for the rapid evaluation of the in planta activity of antimicrobial or insecticidal compounds against CLas and its vector, D. citri, thus providing more information on the systemic effectiveness and influence on plant performance than the previously presented detached leaf assay23. Undoubtedly, the variety of applications for this system reaches well beyond the specific uses described in this study.

The management of plants in commercial and landscape settings often requires the use of chemical compounds to optimize the plant growth and health. How these molecules are delivered depends on the type of molecule, the function of the molecule, the type of plant, and the management system in place. Foliar and soil applications are the easiest delivery strategies, but limitations in the uptake of some molecules necessitate direct delivery. An example of these molecules is therapeutic molecules that function best when they move systemically within the plant but cannot be effectively delivered by simple topical applications1. This is the case with Huanglongbing (HLB), also called citrus greening disease. HLB is a disease associated with a phloem-limited bacterium, Candidatus Liberibacter asiaticus (CLas), which cannot be cultured outside of the plant, or its insect vector, Diaphorina citri Kuwayama (D. citri)2.
If the putative therapeutic molecules are gene products, they can be tested by creating transgenic plants expressing these compounds. However, transgenic plant production can be time- and resource-intensive, is highly genotype-dependent, and can be inhibited by gene silencing3. In addition, even if these transgenics show promising results, regulatory and public perception constraints reduce the likelihood of their commercial acceptance4,5. The exogenous application of compounds, however, simplifies the testing of biological and synthetic molecules because it does not require the production of stable or transiently expressing transgenic plants, which reduces the time and resources for testing a molecule&#39;s effects. A method for the effective and efficient systemic plant delivery of exogenous compounds could be used for a wide variety of research and screening purposes.
One of these applications is the analysis of systemic molecule movement within a plant&#39;s vascular system, which can be done using trackable markers, whether they are fluorescent, visible, or unique chemical isotopes6,7,8,9. One commonly used fluorescent marker is 5,6-carboxyfluorescein-diacetate (CFDA), which is a membrane-permeable dye that is degraded by intracellular esterases into 5,6-carboxyfluorescein (CF) and, subsequently, becomes fluorescent and membrane-impermeable10. CFDA has been extensively used to monitor phloem transport, sink and source relationships, and vasculature patterning in plant tissue11,12.
In addition to these markers, certain compounds may directly alter the plant&#39;s physiology to increase the productivity or kill the plant in the case of herbicides. Both insecticides and antimicrobial compounds are a means of increasing plant productivity, especially in the presence of HLB. An example of an antimicrobial molecule that is used to control CLas is streptomycin. Streptomycin is an aminoglycoside antibiotic that was originally isolated from Streptomyces griseus and has been shown to inhibit bacterial growth through the inhibition of protein biosynthesis13. In terms of insecticides, the main target for HLB research is D. citri, which transmits CLas from tree to tree14. For this purpose, neonicotinoids, such as imidacloprid, are commonly utilized, as they are the gold standard for controlling insect pests15. All these varied uses are important aspects of current plant management strategies, and the development of novel products is dependent on efficient screening assays.
One method that is used for the introduction of compounds into woody plants is direct injection into the trunk. A variety of systems have been designed that vary in their needs for predrilled injection sites, and these systems utilize either pressure-based injection or passive flow16. Although pressure-based systems allow for the quick introduction of a given compound, the potential physical damage caused by forcing liquid through blocked or embolized vasculature needs to be considered17. Although the foliar or drench application of compounds is less time-intensive to implement, direct plant injection reduces waste of the compound of interest due to losses to the air or soil and can also lengthen the time that compounds are in an active state by reducing the exposure to the outside environment18. Both these aspects are important for preserving expensive reagents and ensuring consistency among replicates in research settings.
This study describes the design, construction, and use of an innovative direct plant infusion (DPI) device, which can be used to assess how compounds of interest affect a host plant. A standard 3D printer was used to manufacture both the device itself and several components associated with its construction. This in-house construction method allows researchers to modify the device and device components based on their specific experimental needs and reduces the reliance on commercially available plant injection devices. The device setup is simple and efficient, and all the auxiliary components are readily available and inexpensive. Although the system was designed for use with a variety of plant species, the examples presented here pertain to potted citrus plants. Additionally, this study demonstrates that this device is capable of efficiently delivering multiple types of compounds systemically to young citrus plants without causing lethality. The compounds tested included CFDA, which was used to assess the compound distribution in the plant, and streptomycin and imidacloprid, which were used to verify that the antimicrobial and insecticidal effects of these compounds were observed when delivered via DPI.

<table><tbody><tr><td>0.5 cm Diameter Steel Balls</td><td>Ballistic Products Inc.</td><td>#SHT #T</td><td></td></tr><tr><td>10 mL Luer-Lok Syringe</td><td>Becton Dickinson</td><td>382903029952</td><td></td></tr><tr><td>20 G 1 Syringe Needle</td><td>Becton Dickinson</td><td>305175</td><td></td></tr><tr><td>2 mL Screw Cap Tubes</td><td>USA Scientific</td><td>1420-9710</td><td></td></tr><tr><td>3/32nd Inch Black Oxide Drill Bit</td><td>Sears</td><td>964077</td><td></td></tr><tr><td>3D Printer</td><td>Markforged</td><td>F-PR-2027</td><td></td></tr><tr><td>3D Printing Software</td><td>Markforged</td><td>F-SW-FDVX</td><td></td></tr><tr><td>3D Printing Software</td><td>Markforged</td><td>S-FW-OEVX</td><td></td></tr><tr><td>5(6)-CFDA (5-(and-6)-Carboxyfluorescein Diacetate)</td><td>Invitrogen</td><td>C195</td><td></td></tr><tr><td>5/64th Inch Black Oxide Drill Bit</td><td>Sears</td><td>964502</td><td></td></tr><tr><td>96 Well qPCR Machine</td><td>Roche</td><td>5815916001</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>22621408</td><td></td></tr><tr><td>Fluorescent Microscope</td><td>Olympus</td><td>SP-BX43-BI</td><td></td></tr><tr><td>Fluorescent Microscope Filter</td><td>Chroma</td><td>69401-ET</td><td></td></tr><tr><td>Gloss Clear Spray Paint</td><td>Rustoleum</td><td>249117</td><td></td></tr><tr><td>Grey Lego Baseplate</td><td>Lego</td><td>11024</td><td></td></tr><tr><td>Handheld Cordless Drill</td><td>Makita</td><td>6349D</td><td></td></tr><tr><td>Homogenizer</td><td>Fisher Scientific</td><td>15-340-163</td><td></td></tr><tr><td>Imidacloprid 2F</td><td>Quali-Pro</td><td>83080133</td><td></td></tr><tr><td>Liquid Plastisol Medium Hardness</td><td>Fusion X Fishing Lures</td><td>XSOL-505</td><td></td></tr><tr><td>Red Silicone 70 Shore A O-Ring</td><td>Grainger</td><td>Varies by Size</td><td></td></tr><tr><td>Non-Stick Cooking Spray</td><td>PAM</td><td>64144030217</td><td></td></tr><tr><td>NucleoSpin Plant II</td><td>Macherey-Nagel</td><td>740770.5</td><td></td></tr><tr><td>Parafilm</td><td>Bemis</td><td>HS234526A</td><td></td></tr><tr><td>Poly Viyl Acetate Based Glue</td><td>Elmers</td><td>E301</td><td></td></tr><tr><td>qPCR Master Mix</td><td>Promega</td><td>A6001</td><td></td></tr><tr><td>qPCR Primers</td><td>Integrated DNA Technologies</td><td>Varies by DNA sequence</td><td></td></tr><tr><td>Reverse Transcriptase</td><td>Promega</td><td>A5003</td><td></td></tr><tr><td>Single Edge Razor Blade</td><td>Garvey</td><td>40475</td><td></td></tr><tr><td>Translucent Silicone RTV Rubber</td><td>Aero Marine Products</td><td>AM 115T</td><td></td></tr><tr><td>Transparent Silicone Tape</td><td>Maxwell</td><td>KE30S</td><td></td></tr><tr><td>Truncated Oncocin 112</td><td>Genscript</td><td>Varies by peptide sequence</td><td></td></tr><tr><td>White 1 x 6 Lego Piece</td><td>Lego</td><td>300901</td><td></td></tr><tr><td>White Nylon</td><td>Markforged</td><td>F-MF-0003</td><td></td></tr></tbody></table>

direct infusion device for molecule delivery in plants

Testing the function of therapeutic compounds in plants is an important component of agricultural research. Foliar and soil-drench methods are routine but have drawbacks, including variable uptake and the environmental breakdown of tested molecules. Trunk injection of trees is well-established, but most methods for this require expensive, proprietary equipment. To screen various treatments for Huanglongbing, a simple, low-cost method to deliver these compounds to the vascular tissue of small greenhouse-grown citrus trees infected with the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas) or infested with the phloem-feeding CLas insect vector Diaphorina citri Kuwayama (D. citri) is needed.
To meet these screening requirements, a direct plant infusion (DPI) device was designed that connects to the plant&#39;s trunk. The device is made using a nylon-based 3D-printing system and easily obtainable auxiliary components. The compound uptake efficacy of this device was tested in citrus plants using the fluorescent marker 5,6-carboxyfluorescein-diacetate. Uniform compound distribution of the marker throughout the plants was routinely observed.
Furthermore, this device was used to deliver antimicrobial and insecticidal molecules to determine their effects on CLas and D. citri respectively. The aminoglycoside antibiotic streptomycin was delivered into CLas-infected citrus plants using the device, which resulted in a reduction in the CLas titer from 2 weeks to 4 weeks post treatment. Delivering the neonicotinoid insecticide imidacloprid into D. citri-infested citrus plants resulted in a significant increase in psyllid mortality after 7 days. These results suggest that this DPI device represents a useful system for delivering molecules into plants for&#160;testing and facilitate research and screening purposes.

Watch this Scientific Journal Video about Direct Infusion Device for Molecule Delivery in Plants at JoVE.com

Author Spotlight: Direct Infusion Device for Molecule Delivery in Plants  

This manuscript describes a novel direct plant infusion device for screening the effectiveness of molecules against the bacteria (Candidatus Liberibacter asiaticus) or its insect vector (Diaphorina citri, Kuwayama) that, in combination, are associated with Huanglongbing citrus disease.

Direct Infusion Device for Molecule Delivery in Plants

Testing the function of therapeutic compounds in plants is an important component of agricultural research. Foliar and soil-drench methods are routine but ...

direct-infusion-device-for-molecule-delivery-in-plants

Research

JoVE Journal

Biology

2.2K Views.  United States Department of Agriculture Agricultural Research Service U.S. Horticultural Research Laboratory. Het afleveren van een therapeutisch molecuul aan waar een pathogeen zich in een plant bevindt, kan een uitdaging zijn. De behandeling van Candidatus Liberibacter asiaticus, of CLas, een bacterie die beperkt is tot het floëem, vormt een van de grootste uitdagingen bij het leveren van moleculen ertegen, vooral bij het gebruik van conventionele methoden zoals spraytoepassingen. Het gemak van het printen van het directe installatie-infuusapparaat en de eenvoudige implementatie ervan maakt het vo...

Video: Auteur in de schijnwerpers: Direct infuusapparaat voor molecuulafgifte in planten  

Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as well as new applications in the field of plant biotechnology. Viable methods that currently exist for protein or gene transfer into plant cells, namely Agrobacterium and microprojectile bombardment, have disadvantages of low transformation frequency, limited host range, or a high cost of equipment and microcarriers. The following protocol outlines a simple and versatile method, which employs rationally-designed peptides as delivery agents for a variety of nucleic acid- and protein-based cargoes into plants. Peptides are selected as tools for development of the system due to their biodegradability, reduced size, diverse and tunable properties as well as the ability to gain intracellular/organellar access. The preparation, characterization and application of optimized formulations for each type of the wide range of delivered cargoes (plasmid DNA, double-stranded DNA or RNA, and protein) are described. Critical steps within the protocol, possible modifications and existing limitations of the method are also discussed.

Existing methods for the modification of plants have limited applicability. The novel peptide-derived technology proposed here promises both simplicity and efficiency in the introduction of exogenous protein or genes into the desired intracellular compartments of intact plants.

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as ...

Genetics

Construction and Implantation of a Microinfusion System for Sustained Delivery of Neuroactive Agents.

Sustained delivery of neuroactive agents is widely used in neuroscience, but poses many technical challenges. It is necessary to deliver the agent with high precision while minimizing localized trauma and inflammation. Also, the ability to customize the system to accommodate animals of different species and sizes is desirable. This video presentation demonstrates the construction of an infusion system that can be fitted to any particular research animal. The delivery microcannula diameter is approximately 10-fold smaller than most infusion cannulas presently used. This translates into enhanced accuracy and reduced trauma to the brain region under study. The delivery cannula can also be sculpted to fit the contour of the surface of the animal's skull, thereby allowing closure of the scalp incision neatly over the infusion system, precluding the need for a skull-mounted pedestal, reducing risk of infection, and ensuring a greater level of comfort to the animal. The system is assembled in an air-free environment and requires the researcher to fashion glass micropipettes with a heat source. These construction methods require special skills that are best acquired, if not in person, using video instruction.  (This article is based on work first reported in J Neurosci Methods. 2008 Jan 30;167(2):213-20. Epub 2007 Aug 28.).

As neuroscience inquiry becomes more sophisticated, investigation of brain structures and circuitry requires improved levels of accuracy and higher resolution. We have developed a method for the preparation and implantation of a chronic infusion system within the brain utilizing a borosilicate microcannula with a tip diameter of 50 microns.

Sustained delivery of neuroactive agents is widely used in neuroscience, but poses many technical challenges. It is necessary to deliver the agent with ...

Double-stranded RNA Oral Delivery Methods to Induce RNA Interference in Phloem and Plant-sap-feeding Hemipteran Insects

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran insects account for a number of economically substantial pests of plants that cause damage to crops by feeding on phloem sap. The brown marmorated stink bug (BMSB), Halyomorpha halys (Heteroptera: Pentatomidae) and the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae) are hemipteran insect pests introduced in North America, where they are an invasive agricultural pest of high-value specialty, row, and staple crops and citrus fruits, as well as a nuisance pest when they aggregate indoors. Insecticide resistance in many species has led to the development of alternate methods of pest management strategies. Double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) is a gene silencing mechanism for functional genomic studies that has potential applications as a tool for the management of insect pests. Exogenously synthesized dsRNA or small interfering RNA (siRNA) can trigger highly efficient gene silencing through the degradation of endogenous RNA, which is homologous to that presented. Effective and environmental use of RNAi as molecular biopesticides for biocontrol of hemipteran insects requires the in vivo delivery of dsRNAs through feeding. Here we demonstrate methods for delivery of dsRNA to insects: loading of dsRNA into green beans by immersion, and absorbing of gene-specific dsRNA with oral delivery through ingestion. We have also outlined non-transgenic plant delivery approaches using foliar sprays, root drench, trunk injections as well as clay granules, all of which may be essential for sustained release of dsRNA. Efficient delivery by orally ingested dsRNA was confirmed as an effective dosage to induce a significant decrease in expression of targeted genes, such as juvenile hormone acid O-methyltransferase (JHAMT) and vitellogenin (Vg). These innovative methods represent strategies for delivery of dsRNA to use in crop protection and overcome environmental challenges for pest management.

This article demonstrates novel techniques developed for oral delivery of double-stranded RNA (dsRNA) through the vascular tissues of plants for RNA interference (RNAi) in phloem sap feeding insects.

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran ...

Environment

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to possess medicinally relevant biological activity. However, thus far this potential has not translated into a plethora of triterpene-derived drugs in the clinic. This is arguably (at least partially) a consequence of limited practical synthetic access to this class of compound, a problem that can stifle the exploration of structure-activity relationships and development of lead candidates by traditional medicinal chemistry workflows. Despite their immense diversity, triterpenes are all derived from a single linear precursor, 2,3-oxidosqualene. Transient heterologous expression of biosynthetic enzymes in N. benthamiana can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products that are not naturally produced by this host. Agro-infiltration is an efficient and simple means of achieving transient expression in N. benthamiana. The process involves infiltration of plant leaves with a suspension of Agrobacterium tumefaciens carrying the expression construct(s) of interest. Co-infiltration of an additional A. tumefaciens strain carrying an expression construct encoding an enzyme that boosts precursor supply significantly increases yields. After a period of five days, the infiltrated leaf material can be harvested and processed to extract and isolate the resulting triterpene product(s). This is a process that is linearly and reliably scalable, simply by increasing the number of plants used in the experiment. Herein is described a protocol for rapid preparative-scale production of triterpenes utilizing this plant-based platform. The protocol utilizes an easily replicable vacuum infiltration apparatus, which allows the simultaneous infiltration of up to four plants, enabling batch-wise infiltration of hundreds of plants in a short period of time.

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

Transient heterologous expression of biosynthetic enzymes in Nicotiana benthamiana leaves can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products. Herein is described a detailed protocol for rapid (5 days) preparative-scale production of triterpenes and analogs utilizing this powerful plant-based platform.

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to ...

Biochemistry

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Bioengineering

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a fluorescently tagged-protein of interest is transiently expressed in plant tissue following biolistic delivery of its encoding DNA construct. The intra- and intercellular distribution of the tagged protein is then analyzed by confocal microscopy. We describe this technology in detail, providing step-by-step protocols to assay and evaluate the extent of symplastic protein transport in three plant species, Arabidopsis thaliana, Nicotiana benthamiana and N. tabacum (tobacco).

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Macromolecular trafficking between plant cells can be assessed by transiently expressing a fluorescently-tagged protein of interest and analyzing its intra- and intercellular distribution by confocal microscopy.

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Using Carbon Nanofiber Arrays for Efficient Biomolecule and Dye Delivery to Plants

Using Vertically Aligned Carbon Nanofiber Arrays on Rigid or Flexible Substrates for Delivery of Biomolecules and Dyes to Plants

The delivery of biomolecules and impermeable dyes to intact plants is a major challenge. Nanomaterials are up-and-coming tools for the delivery of DNA to plants. As exciting as these new tools are, they have yet to be widely applied. Nanomaterials fabricated on rigid substrate (backing) are particularly difficult to successfully apply to curved plant structures. This study describes the process for microfabricating vertically aligned carbon nanofiber arrays and transferring them from a rigid to a flexible substrate. We detail and demonstrate how these fibers (on either rigid or flexible substrates) can be used for transient transformation or dye (e.g., fluorescein) delivery to plants. We show how VACNFs can be transferred from rigid silicon substrate to a flexible SU-8 epoxy substrate to form flexible VACNF arrays. To overcome the hydrophobic nature of SU-8, fibers in the flexible film were coated with a thin silicon oxide layer (2-3 nm). To use these fibers for delivery to curved plant organs, we deposit a 1 &#181;L droplet of dye or DNA solution on the fiber side of VACNF films, wait 10 min, place the films on the plant organ and employ a swab with a rolling motion to drive fibers into plant cells. With this method, we have achieved dye and DNA delivery in plant organs with curved surfaces.

Author Spotlight: Unlocking Plant Transformation by Innovating with Carbon Nanofiber Arrays 

Here we describe methods for microfabricating vertically aligned carbon nanofibers (VACNFs), transferring VACNFs to flexible substrates, and applying VACNFs on both rigid and flexible substrates to plants for biomolecule and dye delivery.

The delivery of biomolecules and impermeable dyes to intact plants is a major challenge. Nanomaterials are up-and-coming tools for the delivery of DNA to ...

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

AirID-Based Proximity Labeling for Protein-Protein Interaction in Plants

In mammalian cells and plants, proximity labeling (PL) approaches using modified ascorbate peroxidase (APEX) or the Escherichia coli biotin ligase BirA (known as BioID) have proven successful in identifying protein-protein interactions (PPIs). APEX, BioID, and TurboID, a revised version of BioID have some restrictions in addition to being valuable technologies. The recently developed AirID, a novel version of BioID for proximity identification in protein-protein interactions, overcame these restrictions. Previously, AirID has been used in animal models, while the current study demonstrates the use of AirID in plants, and the results confirmed that AirID performs better in plant systems as compared to other PL enzymes such as BioID and TurboID for protein labeling that are proximal to the target proteins. Here is a step-by-step protocol for identifying protein interaction partners using AT4G18020 (APRR2) protein as a model. The methods describe the construction of vector, the transformation of construct through agroinfiltration, biotin transformation, extraction of proteins, and enrichment of biotin-labeled proteins through affinity purification technique. The results conclude that AirID is a novel and ideal enzyme for analyzing PPIs in plants. The method can be applied to study other proteins in plants.

Here, we present a step-by-step protocol for performing the proximity labeling (PL) experiment in cucumber (Cucumis sativus L.) using AT4G18020 (APRR2)-AirID protein as a model. The method describes the construction of a vector, the transformation of a construct through agroinfiltration, biotin infiltration, protein extraction, and purification of biotin-labeled proteins through affinity purification technique.

In mammalian cells and plants, proximity labeling (PL) approaches using modified ascorbate peroxidase (APEX) or the Escherichia coli biotin ligase BirA ...

Direct Infusion Device for Molecule Delivery in Plants

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