The authors would like to thank Bob Farrar and Alexis Park for providing insects used in this study and for their expertise in larval staging. Additional thanks to Michael Blackburn and Saikat Ghosh for critical review of the manuscript.
Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.
USDA is an Equal Opportunity Provider and Employer.

NOTE: The following protocol is designed for one person to set up, make observations and collect samples. Multiple runs of the same setup may be combined to increase biological replication. Any additional repetitions of the experiment should be set up at the same time of day to eliminate possible diurnal influences on gene expression. The protocol is designed to create 3 &#8216;infested&#8217; leaves for 5 separate harvest time points. Matched control leaves for each time point create a total of 30 samples. The experiment may be performed with a variety of leaf sizes and larval stages, but it is recommended that leaf size, larval stage and infestation time be consistent throughout the procedure.
1. Preparation of the potato plants
NOTE: All steps requiring sterile technique must be performed in a tissue culture hood18,19.
<ol>
	<li>Prepare Kennebec plantlets from explant source.
	<ol>
		<li>Prepare propagation medium.
		<ol>
			<li>Add 4.43 g of Murashige and Skoog (MS) with vitamins powder, 20 g of sucrose and 2 g of agar substitute to 1 L of reverse osmosis (RO)-purified water in a 2 L flask with a spin bar. Transfer flask to a stir plate and mix. Adjust pH to 5.8 using NaOH while continuing to stir. 
			NOTE: Agar will not dissolve until autoclaved.</li>
			<li>Add 1 mL of preservative/biocide and autoclave on liquid cycle for 20 min (121 &#176;C, 101.3 kPa). Remove medium from autoclave immediately after the cycle is finished and cool it to 50 &#176;C. Transfer 100 mL of sterile and cooled propagation medium to sterile culture vessels (e.g., Magenta or Plantcon) using sterile technique in a tissue culture hood.</li>
		</ol>
		</li>
		<li>Prepare explant material.
		<ol>
			<li>Obtain explant source as Kennebec seed potatoes and remove all traces of soil by washing with tap H2O. Remove sprouts and cut into 2 cm pieces with a sterile scalpel.</li>
			<li>Sterilize sprout pieces by soaking for 15 s in 70% ethanol, followed by 13 min in 1:1 bleach (1 part RO-water: 1 part concentrated germicidal/commercial grade bleach). Rinse 5 times in sterile H2O.</li>
			<li>Transfer explant material to sterile tissue culture vessels with propagation medium in a tissue culture hood using sterile technique. Transfer vessels to a plant tissue culture chamber and grow for 2&#8210;3 weeks or until plantlets form at 24 &#176;C, 16 h light (140 &#181;mol&#183;m-2&#183;s-1)/8 h dark photoperiod.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Prepare nodal-propagated tissue culture potato plants.
	<ol>
		<li>Prepare nodal transfer medium. 
		NOTE: This will make 10 tissue culture vessels which may be used the same day or may be made ahead of time and stored at 4 &#176;C until nodal transfer.
		<ol>
			<li>Add 36.43 g of nutrient agar mix to 1 L of RO-purified water in a 2 L flask with a spin bar. Transfer flask to a stir plate and mix. Adjust pH to 5.8 using KOH while continuing to stir. 
			NOTE: Agar will not dissolve until autoclaved.</li>
			<li>Autoclave on liquid cycle for 20 min (121 &#176;C, 101.3 kPa). Remove medium from autoclave immediately after the cycle is finished and cool it to 50 &#176;C. Transfer 100 mL of sterile cooled nodal transfer medium to sterile culture vessels (see the Table of Materials) using sterile technique in a tissue culture hood.</li>
		</ol>
		</li>
		<li>Prepare nodal cuttings.
		<ol>
			<li>Obtain Kennebec plantlets grown from explant material (produced in step 1.1.2). Plantlets should have at least 3 to 4 nodes (branch points). Remove leaves using sterile scissors or scalpel. Cut branches close to the main stem, leaving about 2 mm of branch tissue.</li>
			<li>Remove nodal sections from stem by cutting approximately 2 mm above and below each branch point or node. Arrange nodal cuttings in a sterile tissue culture vessel containing nodal transfer medium (produced in step 1.2.1.2) in the same orientation as in the previous vessel (branch pointing up).</li>
			<li>Transfer vessels with nodal cuttings to a plant tissue culture chamber and grow for 2&#8210;3 weeks at 24 &#176;C, 16 h light (140 &#181;mol&#183;m-2&#183;s-1)/8 h dark photoperiod. 
			NOTE: Each nodal cutting will grow into a new plantlet. The number of cuttings in each vessel determines leaf size. Fewer cuttings transferred per vessel will result in larger leaves. Three plants per vessel will have 15 mm x 20 mm leaves in 2&#8210;3 weeks.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>(Optional) Prepare soil grown Kennebec potato plants. 
	NOTE: To produce larger leaves, nodal propagated potato plantlets can be transferred to soil.
	<ol>
		<li>Transfer potato plantlets grown in nodal transfer medium to soil by gently pulling the plant from the medium until all the root tissue is released from the agar.</li>
		<li>Transfer to soil just above the 1st node from the roots and lightly pack the soil around the transplant. Water gently to ensure soil contact with the root system.</li>
		<li>Place in a growth chamber with 16 h light (140 &#181;mol&#183;m-2&#183;s-1)/8 h dark photoperiod and 25/20 &#176;C day/night temperatures. 
		NOTE: Plants are ready when the top two fully expanded leaves have reached the size appropriate for the assay.</li>
	</ol>
	</li>
	<li>(Optional) Prepare tuber-grown potato plants. 
	NOTE: Potato plants grown from tubers are larger and more robust and can be useful if rearing larvae on plants or if overnight larval feeding is desired.
	<ol>
		<li>Place a potato tuber 6 in deep in a 10 in pot containing soil mix supplemented with 10 mL pelleted slow release fertilizer.</li>
		<li>Place in a growth chamber with 16 h light (140 &#181;mol&#183;m-2&#183;s-1)/8 h dark photoperiod and 25/20 &#176;C day/night temperature. Plants are ready approximately 30&#8210;40 days from tuber planting. 
		NOTE: Do not use plants that have begun to flower.</li>
	</ol>
	</li>
</ol>
2. Preparation of insects for feeding
<ol>
	<li>Obtain desired larval stage of M. sexta. 
	NOTE: Larvae for this study were reared on artificial diet through the 5th instar20 and staged by an experienced individual from the in-house insectary. Larvae may also be reared partially or completely on plant tissue. Larvae do not eat immediately before a molt and are most likely to eat right after molt, so appropriate staging is important21.</li>
	<li>Transfer larvae to an appropriate containment vessel (e.g., 6-, 12-, 24-well tissue culture dish) depending on larval size. There should be one larva per well in the dish. 
	NOTE: Larvae may display territorial or cannibalistic behavior without a food source and may become injured if housed together.</li>
	<li>(Optional) Starve larvae for up to 2 h as this may improve larval feeding. 
	NOTE: Larvae should be in a containment vessel stored in the growth chamber during this time.</li>
</ol>
3. Components setup for the infestation experiment
NOTE: See the schematic summary in Figure 1.
<ol>
	<li>Make placement templates for each harvest time point. 
	NOTE: It is helpful to set up 5 different trays for each harvest time point. This keeps samples organized and allows the dishes to be moved around more efficiently as a set without changing their arrangement. If percentage-of-damage calculations will be performed, this is essential as before- and after-infestation images must be captured at the same focal length.
	<ol>
		<li>Obtain 5 sturdy trays capable of holding a set of six appropriately sized Petri dishes and line with white paper. 
		NOTE: The size of the Petri dish is based on the leaf size chosen for the feeding assay. The leaf should fit easily in the dish without touching the sides. For instance, a 60 mm x 15 mm dish is suitable for leaves up to 50 mm in length or width.</li>
		<li>Trace a set of six circles using the appropriately sized Petri dish on the paper in each tray. Label one set of circles &#8216;control&#8217; A, B and C and the other &#8216;infested&#8217; A, B, and C. Also label each placement template with the appropriate harvest time.</li>
	</ol>
	</li>
	<li>(Optional) Set up a camera for &#8216;before infestation&#8217; and &#8216;after infestation&#8217; image capture.
	<ol>
		<li>Secure a camera on a stand at the appropriate focal length for image capture of all Petri dishes in the placement template.</li>
	</ol>
	</li>
	<li>Label the harvest time point tubes.
	<ol>
		<li>Label a set of 30, 1.7 mL microcentrifuge tubes corresponding to each circle in the placement template. Label the tubes to appropriately identify the perturbation (control/infested), the plant replication letter (A, B or C) and the harvest time point (number of minutes post infestation period).</li>
	</ol>
	</li>
	<li>Prepare Petri dishes chosen in step 3.1.1.
	<ol>
		<li>Place a sterile filter paper disc in each of the 30 Petri dishes from step 3.1.1. Add sterile water to moisten the discs; do not allow excess water to pool in the dish. Place each dish in each of the six circles in each placement template.</li>
		<li>Position three potato plants next to each placement template. Ensure that plants are all the same age and relative size.</li>
	</ol>
	</li>
</ol>
4. Performing infestation
NOTE: One harvest time point/placement template is set up at a time.
<ol>
	<li>Remove the top two size-matched leaves from each plant with sterile scissors and place one leaf in the control Petri dish and one in the infested Petri dish for each plant (A, B and C). Carry out this process as quickly as possible.</li>
	<li>(Optional) Transfer the placement template to the camera stand to capture a &#8216;before infestation&#8217; image.</li>
	<li>Transfer larvae to each infested dish using soft touch forceps as quickly as possible. Set the timer for desired &#8216;infestation&#8217; time. 
	NOTE: The period of time when larvae are consuming the leaf tissue is the &#8216;infestation time&#8217;. This is something that can be determined empirically using a few test leaves/larvae before the start of the actual infestation. The chosen &#8216;infestation time&#8217; should be consistent for all infested leaves. 
	NOTE: Larvae should be handled with care by soft touch forceps. 2nd through 4th instar may be grasped gently by their horn or midsection.</li>
	<li>Observe feeding to make sure all larvae are eating. Larvae should be added/removed based on feeding behavior. Keep lids on the Petri dishes as much as possible. 
	NOTE: Multiple larvae may be used per leaf.</li>
	<li>Remove larvae from leaves at the end of the infestation time. Start the timer for the harvest time.</li>
	<li>(Optional) Transfer the placement template to the camera stand to capture the &#8216;after infestation&#8217; image. 
	NOTE: All six leaves in the placement template are harvested at the specific harvest time.</li>
</ol>
5. Harvesting leaves
<ol>
	<li>Transfer each leaf to the corresponding labeled tube at the end of each harvest time point and immediately freeze by dropping the tube into liquid N2. Store the harvested plant tissue at -80 &#176;C until the isolation of RNA.</li>
	<li>Repeat steps 4&#8210;5.1 for each harvest time point.</li>
</ol>
6. Processing leaf tissue for gene expression analysis
<ol>
	<li>Grind the frozen leaf tissue to a powder with a micropestle.</li>
	<li>Isolate total RNA and process for gene expression as previously described22.</li>
</ol>
7. (Optional) Estimating leaf damage
<ol>
	<li>Visually estimate percent of leaf damage or calculate leaf area in the before- and after-infestation images.</li>
	<li>Measure leaf area with software tools (e.g., Phenophyte)23.</li>
	<li>From leaf area measurements, calculate percentage of damage as 
	% damage = [(leaf area before &#8211; leaf area after)/leaf area before] x 100.</li>
</ol>

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U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appraisal+of+artificial+screening+techniques+of+tomato+to+accurately+reflect+field+performance+of+the+Late+Blight+resistance.">Appraisal of artificial screening techniques of tomato to accurately reflect field performance of the Late Blight resistance.</a> Plos One. 9, e109328 (2014).</li><li>Arimura, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herbivory-induced+volatiles+elicit+defence+genes+in+lima+bean+leaves.">Herbivory-induced volatiles elicit defence genes in lima bean leaves.</a> Nature. 406, 512-515 (2000).</li><li>Erb, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volatiles+as+inducers+and+suppressors+of+plant+defense+and+immunity-origins,+specificity,+perception+and+signaling.">Volatiles as inducers and suppressors of plant defense and immunity-origins, specificity, perception and signaling.</a> Current Opinion in Plant Biology. 44, 117-121 (2018).</li><li>Hasegawa, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+analysis+of+wounding-induced+root-to-shoot+communication+in+Arabidopsis+thaliana.">Gene expression analysis of wounding-induced root-to-shoot communication in Arabidopsis thaliana.</a> Plant, Cell and Environment. 34, 705-716 (2011).</li><li>Ryan, C. A., Moura, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+wound+signaling+in+plants:+A+new+perception.">Systemic wound signaling in plants: A new perception.</a> Proceedings of the National Academy of Sciences, USA. 99, 6519-6520 (2002).</li><li>Hilleary, R., Gilroy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+signaling+in+response+to+wounding+and+pathogens.">Systemic signaling in response to wounding and pathogens.</a> Current Opinion in Plant Biology. 43, 57-62 (2018).</li><li>. Hornworms Available from: <a target="_blank" href="https://www.carolina.com/hornworm/hornworms/FAM_143880.pr">https://www.carolina.com/hornworm/hornworms/FAM_143880.pr</a> (2018)</li><li>. Products Available from: <a target="_blank" href="https://www.greatlakeshornworm.com/products/">https://www.greatlakeshornworm.com/products/</a> (2018)</li><li>. Raising Manduca sexta Available from: <a target="_blank" href="https://acad.carleton.edu/curricular/Biol/resources/rlink/description2.html">https://acad.carleton.edu/curricular/Biol/resources/rlink/description2.html</a> (2018)</li><li>. Teach life cycles with the tobacco hornworm Available from: <a target="_blank" href="https://www.carolina.com/teacher-resources/Interactive/teach-life-cycles-with-the-tobacco-hornworm/tr30179.tr">https://www.carolina.com/teacher-resources/Interactive/teach-life-cycles-with-the-tobacco-hornworm/tr30179.tr</a> (2018)</li><li>Chung, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+plant+species+determines+symbiotic+bacterial+community+mediating+suppression+of+plant+defenses.">Host plant species determines symbiotic bacterial community mediating suppression of plant defenses.</a> Scientific Reports. 7, 1-13 (2017).</li></ol>

The authors have nothing to disclose.

The use of existing whole plant herbivory methodologies is unnecessary to achieve the goal of this particular study (i.e., screen a set of candidate genes for their response to infestation). The obvious benefit of the detached leaf refinement is shortening the time it takes to perform herbivory assays. The unwieldy nature of whole plants with clip cages is eliminated and assays are performed sooner, since plants as young as 2 weeks can be used to harvest leaves. It also requires a much smaller footprint during feeding an...

Herbivory sets in motion a series of molecular events during which a plant can both identify the attack and mount an appropriate response for its survival. A plant receives two basic cues from chewing insects; one from the physical damage to the tissue and the other from insect-specific substances. Damage-associated molecular patterns (DAMPs) are released in response to damage created by larval mouthparts and trigger a well-defined wound response that results in an increase in the hormone jasmonic acid and the transcription of defense genes1. One of the best-known DAMPs is systemin, a polypeptide that is formed by the cleavage of the larger prosystemin protein after a leaf is wounded2,3. The jasmonic acid wound response is further modulated by herbivore-associated molecular patterns (HAMPs), which can be derived from caterpillar saliva, gut contents (regurgitant) and feces (frass)4. Insects use these substances to either boost or evade the defense response5. Transcription factors then relay the message from hormone signals in the defense response via regulation of downstream defense genes6,7,8.
Some plant-insect interaction studies used in laboratory settings are of the simulated type, with a goal of approximating the natural method of feeding by the insect. Simulated herbivory is usually accomplished by creating artificial damage to plant tissues with various tools that mimic the specific mechanism of insect mouthparts sufficient to cause the release of DAMPs and trigger the production of defense genes. Other insect-specific components such as oral secretions or regurgitant are often added to replicate the contribution from HAMPs9,10,11. The creation of a specific size and type of wound and the application of precise amounts of HAMPs is one advantage to these types of studies and can offer more reproducible results. Natural herbivory studies, where damage to plant tissue is accomplished by the application of field-acquired or laboratory-reared insects, are often more challenging because wound-size and HAMP amounts are governed by insect behavior and add variability to the data. The natural versus simulated methods and their advantages and disadvantages are well debated in the literature12,13,14.
To study early signaling events such as transcription factors, a certain percentage of the leaf must be consumed in a relatively short amount of time, so larvae must begin to chew immediately and maintain consumption until the leaf is frozen for analysis. M. sexta is a voracious feeder on multiple solanaceous plants during many of its larval stages, making it ideal for imparting maximum damage in a relatively short amount of time15. This is convenient when studying early signaling events, as the plant response occurs almost immediately after an insect contacts the leaf surface16,17. The commonly used clip cage method of containment proves clumsy, as multiple cages would require continual adjustments throughout the experiment to allow for the removal or addition of larvae. The leaves must also be large enough and strong enough to support multiple insects feeding at the same time. These types of potato plants require a large amount of space to observe feeding. Larvae will often relocate to the underside of the leaf surface which also makes feeding observations quite difficult. Using whole plants to perform these experiments is clearly cumbersome.
The current study uses detached leaves isolated in Petri dishes rather than whole plants to streamline and simplify the whole plant approach to studying herbivory. The application of the protocol in this study is limited to the observation of a group of C2H2 transcription factors induced early in potato leaves after herbivorous damage by M. sexta larvae.

<table border="0" cellpadding="0" cellspacing="0" style="width:1140px;" width="1139">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">agar substitute</td>
			<td style="width:217px;">PhytoTechnology Laboratories</td>
			<td style="width:173px;">G3251</td>
			<td style="width:471px;">product is Gelzan</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">containment vessel (6,12 or 24 well dish)</td>
			<td style="width:217px;">Fisher Scientific&#160;</td>
			<td style="width:173px;">08-772-49, 08-772-50, 08-72-51</td>
			<td>many other companies sell these products</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">manduca eggs&#160;</td>
			<td style="width:217px;">Carolina Biological Supply Company</td>
			<td style="width:173px;">143880</td>
			<td>30-50 eggs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">manduca eggs&#160;</td>
			<td style="width:217px;">Great Lakes Hornworm</td>
			<td style="width:173px;">NA</td>
			<td>50, 100, 250 or 500 eggs</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">manduca larvae</td>
			<td style="width:217px;">Carolina Biological Supply Company</td>
			<td style="width:173px;">call for specific larval instar requests</td>
			<td>any instar</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">manduca larvae</td>
			<td style="width:217px;">Great Lakes Hornworm</td>
			<td style="width:173px;">call for specific larval instar requests</td>
			<td>any instar</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">microcentrifuge tubes, 1.7 ml&#160;</td>
			<td style="width:217px;">Thomas Scientific</td>
			<td style="width:173px;">1158R22</td>
			<td>these have been tested in liquid N2 and will not explode</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Murashige &#38; Skoog (MS) Basal Medium w/Vitamins</td>
			<td style="width:217px;">PhytoTechnology Laboratories</td>
			<td style="width:173px;">M519</td>
			<td>used to make propagation medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">nutrient agar mix</td>
			<td style="width:217px;">PhytoTechnology Laboratories</td>
			<td style="width:173px;">M5825</td>
			<td>product is Murashige &#38; Skoog Basal Medium with vitamins, sucrose, and Gelzan</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">paper filter discs</td>
			<td style="width:217px;">Fisher Scientific&#160;</td>
			<td style="width:173px;">09-805A</td>
			<td>Whatman circles-purchase to fit in petri dish</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">petri dish, 60X15 mm or 100X15 mm</td>
			<td style="width:217px;">Fisher Scientific&#160;</td>
			<td style="width:173px;">FB0875713A or FB0875712</td>
			<td>purchase size appropriate for leaf size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">potato tubers&#160;</td>
			<td style="width:217px;">any</td>
			<td style="width:173px;">B size (not organic)</td>
			<td style="width:471px;">suggest Maine Farmer&#8217;s Exchange</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">pots, 10&#34;&#160;</td>
			<td style="width:217px;">Griffin Greenhouse Supplies, Inc.</td>
			<td style="width:173px;">41PT1000CN2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">preservative/biocide</td>
			<td style="width:217px;">Plant Cell Technology</td>
			<td style="width:173px;">NA</td>
			<td>product is PPM (Plant Preservative Mixture)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">seed potatoes for explant source</td>
			<td style="width:217px;">any</td>
			<td style="width:173px;">B size (not organic)</td>
			<td style="width:471px;">suggest Maine Farmer&#8217;s Exchange</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">slow release fertilizer (14-14-14 )</td>
			<td style="width:217px;">any</td>
			<td style="width:173px;">NA</td>
			<td>Osmocote is a popular brand name</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">soft touch forceps</td>
			<td style="width:217px;">BioQuip</td>
			<td style="width:173px;">4750</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">soil mix</td>
			<td style="width:217px;">Griffin Greenhouse Supplies, Inc.</td>
			<td style="width:173px;">65-51121</td>
			<td>product is Sunshine LC1 mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">sterile culture vessel&#160;</td>
			<td style="width:217px;">PhytoTechnology Laboratories</td>
			<td style="width:173px;">C2100</td>
			<td>Magenta-type vessel, PTL-100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">sterile culture vessel&#160;</td>
			<td style="width:217px;">Fisher Scientific&#160;</td>
			<td style="width:173px;">ICN2672206</td>
			<td>product is MP Biomedicals Plantcon</td>
		</tr>
	</tbody>
</table>

detached leaf assays to simplify gene expression studies in potato during infestation by chewing insect manduca sexta

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more streamlined herbivory protocols. Perturbations of chewing insects are usually studied in whole plant systems. While this whole organism strategy is popular, it is not necessary if similar observations can be replicated in a single detached leaf. The assumption is that basic elements required for signal transduction are present within the leaf itself. In the case of early events in signal transduction, cells need only to receive the signal from the perturbation and transmit that signal to neighboring cells which are assayed for gene expression.
The proposed method simply changes the timing of the detachment. In whole plant experiments, larvae are confined to a single leaf which is eventually detached from the plant and assayed for gene expression. If the order of excision is reversed, from last in whole plant studies, to first in the detached study, the feeding experiment is simplified.
Solanum tuberosum var. Kennebec is propagated by nodal transfer in a simple tissue culture medium and transferred to soil for further growth if desired. Leaves are excised from the parent plant and relocated to Petri dishes where the feeding assay is conducted with the larval stages of M. sexta. Damaged leaf tissue is assayed for the expression of relatively early events in signal transduction. Gene expression analysis identified infestation-specific Cys2-His2 (C2H2) transcription factors, confirming the success of using detached leaves in early response studies. The method is easier to perform than whole plant infestations and uses less space.

Leaf consumption defines success of the protocol. Healthy, accurately staged larvae should begin feeding immediately after placement on the leaf surface and feeding should continue in a fairly consistent manner throughout the infestation time. In Video 1, the larva at the top begins to chew immediately after placement and maintains a consistent rate while feeding. This is especially important if assaying early gene expression events after infestation. The larva at the bot...

Watch this Scientific Journal Video about Detached Leaf Assays to Simplify Gene Expression Studies in Potato During Infestation by Chewing Insect Manduca sexta at JoVE.com

Detached Leaf Assays to Simplify Gene Expression Studies in Potato During Infestation by Chewing Insect Manduca sexta

The presented method creates natural herbivore damaged plant tissue through the application of Manduca sexta larvae to detached leaves of potato. The plant tissue is assayed for expression of six transcription factor homologs involved in early responses to insect herbivory.

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more ...

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6.6K Views.  United States Department of Agriculture (USDA), Agricultural Research Service (ARS). The presented method creates natural herbivore damaged plant tissue through the application of Manduca sexta larvae to detached leaves of potato. The plant tissue is assayed for expression of six transcription factor homologs involved in early responses to insect herbivory.

Video: Detached Leaf Assays to Simplify Gene Expression Studies in Potato During Infestation by Chewing Insect Manduca sexta

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5&#8217; mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

A Technique to Screen American Beech for Resistance to the Beech Scale Insect (Cryptococcus fagisuga Lind.)

Beech bark disease (BBD) results in high levels of initial mortality, leaving behind survivor trees that are greatly weakened and deformed.&#160;The disease is initiated by feeding activities of the invasive beech scale insect, Cryptococcus fagisuga, which creates entry points for infection by one of the Neonectria species of fungus.&#160;Without scale infestation, there is little opportunity for fungal infection. Using scale eggs to artificially infest healthy trees in heavily BBD impacted stands demonstrated that these trees were resistant to the scale insect portion of the disease complex1.&#160;Here we present a protocol that we have developed, based on the artificial infestation technique by Houston2, which can be used to screen for scale-resistant trees in the field and in smaller potted seedlings and grafts.&#160;The identification of scale-resistant trees is an important component of management of BBD through tree improvement programs and silvicultural manipulation.

A Technique to Screen American Beech for Resistance to the Beech Scale Insect Cryptococcus fagisuga Lind.

Beech bark disease is initiated by feeding activities of the beech scale insect that create fungal entry points in the bark.&#160;Trees that are resistant to the scale insect are also disease resistant.&#160;Here we present the protocol we have developed to screen individual beech trees for beech scale resistance.

Beech bark disease (BBD) results in high levels of initial mortality, leaving behind survivor trees that are greatly weakened and deformed.&#160;The ...

Measuring Rates of Herbicide Metabolism in Dicot Weeds with an Excised Leaf Assay

In order to isolate and accurately determine rates of herbicide metabolism in an obligate-outcrossing dicot weed, waterhemp (Amaranthus tuberculatus), we developed an excised leaf assay combined with a vegetative cloning strategy to normalize herbicide uptake and remove translocation as contributing factors in herbicide-resistant (R) and &#8211;sensitive (S) waterhemp populations. Biokinetic analyses of organic pesticides in plants typically include the determination of uptake, translocation (delivery to the target site), metabolic fate, and interactions with the target site. Herbicide metabolism is an important parameter to measure in herbicide-resistant weeds and herbicide-tolerant crops, and is typically accomplished with whole-plant tests using radiolabeled herbicides. However, one difficulty with interpreting biokinetic parameters derived from whole-plant methods is that translocation is often affected by rates of herbicide metabolism, since polar metabolites are usually not mobile within the plant following herbicide detoxification reactions. Advantages of the protocol described in this manuscript include reproducible, accurate, and rapid determination of herbicide degradation rates in R and S populations, a substantial decrease in the amount of radiolabeled herbicide consumed, a large reduction in radiolabeled plant materials requiring further handling and disposal, and the ability to perform radiolabeled herbicide experiments in the lab or growth chamber instead of a greenhouse. As herbicide resistance continues to develop and spread in dicot weed populations worldwide, the excised leaf assay method developed and described herein will provide an invaluable technique for investigating non-target site-based resistance due to enhanced rates of herbicide metabolism and detoxification.

This manuscript describes how herbicide metabolism rates can be effectively quantified with excised leaves from a dicot weed, thereby reducing variability and removing any possible confounding effects of herbicide uptake or translocation typically observed in whole-plant assays.

In order to isolate and accurately determine rates of herbicide metabolism in an obligate-outcrossing dicot weed, waterhemp (Amaranthus tuberculatus), we ...

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 ...

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly relevant as a model for the studies of osmoregulatory mechanisms because of its extreme tolerance to low salinity. It is also widely used as a model of settling biology, in particular in relation to antifouling research. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae for studies. A protocol for the all-year-round culturing of B. improvisus has been developed and a detailed description of all steps in the production line is outlined (i.e., the establishment of adult cultures on panels, the collection and rearing of barnacle larvae, and the administration of feed for adults and larvae). The description also provides guidance on troubleshooting and discusses critical parameters (e.g., the removal of contamination, the production of high-quality feed, the manpower needed, and the importance of high-quality seawater). Each batch from the culturing system maximally yields roughly 12,000 nauplii and can deliver four batches in a week, so up to almost 50,000 larvae per week can be produced. The method used to culture B. improvisus is, probably, to a large extent also applicable to other marine invertebrates with free-swimminglarvae. Protocols are presented for the dissection of various tissues from adults as well as the production of high-quality RNA for studies on gene expression. It is also described how cultured adults and reared cyprids can be utilized in a wide array of experimental designs for examining gene expression in relation to external factors. The use of cultured barnacles in gene expression is illustrated with studies of possible osmoregulatory roles of Na+/K+ ATPase and aquaporins.

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

The barnacle Balanus (Amphibalanus) improvisus is a model for studying osmoregulation and antifouling. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae. Here, a protocol for the all-year-round culturing of B. improvisus is described, including the production of larvae. The use of cultured barnacles in gene expression studies is illustrated.

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly ...

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and toxicological studies. Using automated digital tracking platforms, recent efforts in neuropharmacology are leveraging larval fish locomotor behaviors to identify potential therapeutic targets for novel small molecules. Similar to these efforts, research in the environmental sciences and comparative pharmacology and toxicology is examining various behaviors of fish models as diagnostic tools in tiered evaluation of contaminants and real-time monitoring of surface waters for contaminant threats. Whereas the zebrafish is a popular larval fish model in the biomedical sciences, the fathead minnow is a common larval fish model in ecotoxicology. Unfortunately, fathead minnow larvae have received considerably less attention in behavioral studies. Here, we develop and demonstrate a behavioral profile protocol using caffeine as a model neurostimulant. Though photomotor responses of fathead minnows were occasionally affected by caffeine, zebrafish&#160;were markedly more sensitive for photomotor and locomotor endpoints, which responded at environmentally relevant levels. Future studies are needed to understand comparative behavioral sensitivity differences among fish with age and time of day, and to determine whether similar behavioral effects would occur in nature and be indicative of adverse outcomes at the individual or population levels of biological organization.

Here, we present a protocol to examine larval zebrafish and fathead minnow locomotor activities and photomotor responses (PMR) using an automated tracking software. When incorporated in common toxicology bioassays, analyses of these behaviors provide a diagnostic tool to examine chemical bioactivity. This protocol is described using caffeine, a model neurostimulant.

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and ...

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into the plant&#39;s genome. Here, we present two transformation systems to genetically modify potato (Solanum tuberosum) plants. In A. tumefaciens transformation, leaves are infected, the transformed cells are selected and a new complete transformed plant is regenerated using phytohormones in 18 weeks. In A. rhizogenes transformation, stems are infected by injecting the bacteria with a needle, the new emerged transformed hairy roots are detected using a red fluorescent marker and the non-transformed roots are removed. In 5-6 weeks, the resulting plant is a composite of a wild type shoot with fully developed transformed hairy roots. To increase the biomass, the transformed hairy roots can be excised and self-propagated. We applied both Agrobacterium-mediated transformation methods to obtain roots expressing the GUS reporter gene driven by a suberin biosynthetic gene promoter. The GUS staining procedure is provided and allows the cell localization of the promoter induction. In both methods, the transformed potato roots showed GUS staining in the suberized endodermis and exodermis, and additionally, in A. rhizogenes transformed roots the GUS activity was also detected in the emergence of lateral roots. These results suggest that A. rhizogenes can be a fast alternative tool to study the genes that are expressed&#160;in roots.

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Here, we present two protocols to transform potato plants. The Agrobacterium tumefaciens transformation leads to a complete transgenic plant while the Agrobacterium rhizogenes produces transgenic hairy roots in a wild type shoot that can be self-propagated. We then detect promoter activity by GUS staining in the transformed roots.

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into ...

Leaf Area Index Estimation Using Three Distinct Methods in Pure Deciduous Stands

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for describing the vegetation structure in the fields of ecology, forestry, and agriculture. Therefore, procedures of three commercially used methods (litter traps, needle technique, and a plant canopy analyzer) for performing LAI estimation were presented step-by-step. Specific methodological approaches were compared, and their current advantages, controversies, challenges, and future perspectives were discussed in this protocol. Litter traps are usually deemed as the reference level. Both the needle technique and the plant canopy analyzer (e.g., LAI-2000) frequently underestimate LAI values in comparison with the reference. The needle technique is easy to use in deciduous stands where the litter completely decomposes each year (e.g., oak and beech stands). However, calibration based on litter traps or direct destructive methods is necessary. The plant canopy analyzer is a commonly used device for performing LAI estimation in ecology, forestry, and agriculture, but is subject to potential error due to foliage clumping and the contribution of woody elements in the field of view (FOV) of the sensor. Eliminating these potential error sources was discussed. The plant canopy analyzer is a very suitable device for performing LAI estimations at the high spatial level, observing a seasonal LAI dynamic, and for long-term monitoring of LAI.

An accurate estimation of leaf area index (LAI) is crucial for many models of material and energy fluxes within plant ecosystems and between an ecosystem and the atmospheric boundary layer. Therefore, three methods (litter traps, needle technique, and PCA) for taking precise LAI measurements were in the presented protocol.

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for ...

Potato Virus X-Based microRNA Silencing (VbMS) In Potato.

Virus-based microRNA silencing (VbMS) is a rapid and efficient tool for functional characterization of microRNAs (miRNAs) in plants. The VbMS system has been developed and applied for various plant species including Nicotiana benthamiana, tomato, Arabidopsis, cotton, and monocot plants such as wheat and maize. Here, we describe a detailed protocol using PVX-based VbMS vectors to silence endogenous miRNAs in potato. To knock down the expression of a specific miRNA, target mimic (TM) molecules of miRNA of interest are designed, integrated into plant virus vectors, and expressed in potato by Agrobacterium infiltration to bind directly to the endogenous miRNA of interest and block its function.

Potato Virus X-Based microRNA Silencing VbMS In Potato.

We present a detailed protocol for potato virus X (PVX)-based microRNA silencing (VbMS) system to functionally characterize endogenous microRNAs (miRNAs) in potato. Target mimic (TM) molecules of miRNA of interest are integrated into the PVX vector and transiently expressed in potato to silence the target miRNA or miRNA family.

Virus-based microRNA silencing (VbMS) is a rapid and efficient tool for functional characterization of microRNAs (miRNAs) in plants. The VbMS system has ...

RNA Interference in Aquatic Beetles as a Powerful Tool for Manipulating Gene Expression at Specific Developmental Time Points

RNA interference (RNAi) remains a powerful technique that allows for the targeted reduction of gene expression through mRNA degradation. This technique is applicable to a wide variety of organisms and is highly efficient in the species-rich order Coleoptera (beetles). Here, we summarize the necessary steps for developing this technique in a novel organism and illustrate its application to the different developmental stages of the aquatic diving beetle Thermonectus marmoratus. Target gene sequences can be obtained cost-effectively through the assembly of transcriptomes against a close relative with known genomics or de novo. Candidate gene cloning utilizes a specific cloning vector (the pCR4-TOPO plasmid), which allows the synthesis of double-stranded RNA (dsRNA) for any gene with the use of a single common primer. The synthesized dsRNA can be injected into either embryos for early developmental processes or larvae for later developmental processes. We then illustrate how RNAi can be injected into aquatic larvae using immobilization in agarose. To demonstrate the technique, we provide several examples of RNAi experiments, generating specific knockdowns with predicted phenotypes. Specifically, RNAi for the tanning gene laccase2 leads to cuticle lightening in both larvae and adults, and RNAi for the eye pigmentation gene white produces a lightening/lack of pigmentation in eye tubes. In addition, the knockdown of a key lens protein leads to larvae with optical deficiencies and a reduced ability to hunt prey. Combined, these results exemplify the power of RNAi as a tool for investigating both morphological patterning and behavioral traits in organisms with only transcriptomic databases.

RNA interference is a widely applicable, powerful technique for manipulating gene expression at specific developmental stages. Here, we describe the necessary steps for implementing this technique in the aquatic diving beetle Thermonectus marmoratus, from the acquisition of gene sequences to the knockdown of genes that affect structure or behavior.

RNA interference (RNAi) remains a powerful technique that allows for the targeted reduction of gene expression through mRNA degradation. This technique is ...

Detached Leaf Assays to Simplify Gene Expression Studies in Potato During Infestation by Chewing Insect Manduca sexta

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Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Leaf Area Index Estimation Using Three Distinct Methods in Pure Deciduous Stands

Potato Virus X-Based microRNA Silencing (VbMS) In Potato.

RNA Interference in Aquatic Beetles as a Powerful Tool for Manipulating Gene Expression at Specific Developmental Time Points