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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<li>Nowakowska, M., Nowicki, M., Kłosińska, U., Maciorowski, R., Kozik, E. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Appraisal+of+artificial+screening+techniques+of+tomato+to+accurately+reflect+field+performance+of+the+Late+Blight+resistance%5BTitle%5D)+AND+%22Plos+One%22%5BJournal%5D)">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/pubmed?term=((Herbivory-induced+volatiles+elicit+defence+genes+in+lima+bean+leaves%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Volatiles+as+inducers+and+suppressors+of+plant+defense+and+immunity-origins%2C+specificity%2C+perception+and+signaling%5BTitle%5D)+AND+%22Current+Opinion+in+Plant+Biology%22%5BJournal%5D)">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/pubmed?term=((Gene+expression+analysis+of+wounding-induced+root-to-shoot+communication+in+Arabidopsis+thaliana%5BTitle%5D)+AND+%22Plant%2C+Cell+and+Environment%22%5BJournal%5D)">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/pubmed?term=((Systemic+wound+signaling+in+plants%3A+A+new+perception%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%2C+USA%22%5BJournal%5D)">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/pubmed?term=((Systemic+signaling+in+response+to+wounding+and+pathogens%5BTitle%5D)+AND+%22Current+Opinion+in+Plant+Biology%22%5BJournal%5D)">Systemic signaling in response to wounding and pathogens.</a> Current Opinion in Plant Biology. 43, 57-62 (2018).</li>
<li>Carolina Biological Supply Company. 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>Great Lakes Hornworm. Products. , Available from:
 <a target="_blank" href="https://www.greatlakeshornworm.com/products/">https://www.greatlakeshornworm.com/products/</a> (2018).</li>
<li>The Board of regents of the University of Wisconsin System. Manduca. , Available from:
 <a target="_blank" href="http://labs.russell.wisc.edu/manduca/lifecycle/">http://labs.russell.wisc.edu/manduca/lifecycle/</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>Berkley, C. Teach life cycles with the tobacco hornworm. , Available from:
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<li>Chung, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Host+plant+species+determines+symbiotic+bacterial+community+mediating+suppression+of+plant+defenses%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">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><tbody><tr><td>agar substitute</td><td>PhytoTechnology Laboratories</td><td>G3251</td><td>product is Gelzan</td></tr><tr><td>containment vessel (6,12 or 24 well dish)</td><td>Fisher Scientific&amp;nbsp;</td><td>08-772-49, 08-772-50, 08-72-51</td><td>many other companies sell these products</td></tr><tr><td>manduca eggs&amp;nbsp;</td><td>Carolina Biological Supply Company</td><td>143880</td><td>30-50 eggs</td></tr><tr><td>manduca eggs&amp;nbsp;</td><td>Great Lakes Hornworm</td><td>NA</td><td>50, 100, 250 or 500 eggs</td></tr><tr><td>manduca larvae</td><td>Carolina Biological Supply Company</td><td>call for specific larval instar requests</td><td>any instar</td></tr><tr><td>manduca larvae</td><td>Great Lakes Hornworm</td><td>call for specific larval instar requests</td><td>any instar</td></tr><tr><td>microcentrifuge tubes, 1.7 ml&amp;nbsp;</td><td>Thomas Scientific</td><td>1158R22</td><td>these have been tested in liquid N2 and will not explode</td></tr><tr><td>Murashige &amp;amp; Skoog (MS) Basal Medium w/Vitamins</td><td>PhytoTechnology Laboratories</td><td>M519</td><td>used to make propagation medium</td></tr><tr><td>nutrient agar mix</td><td>PhytoTechnology Laboratories</td><td>M5825</td><td>product is Murashige &amp;amp; Skoog Basal Medium with vitamins, sucrose, and Gelzan</td></tr><tr><td>paper filter discs</td><td>Fisher Scientific&amp;nbsp;</td><td>09-805A</td><td>Whatman circles-purchase to fit in petri dish</td></tr><tr><td>petri dish, 60X15 mm or 100X15 mm</td><td>Fisher Scientific&amp;nbsp;</td><td>FB0875713A or FB0875712</td><td>purchase size appropriate for leaf size</td></tr><tr><td>potato tubers&amp;nbsp;</td><td>any</td><td>B size (not organic)</td><td>suggest Maine Farmer&amp;rsquo;s Exchange</td></tr><tr><td>pots, 10&quot;&amp;nbsp;</td><td>Griffin Greenhouse Supplies, Inc.</td><td>41PT1000CN2</td><td></td></tr><tr><td>preservative/biocide</td><td>Plant Cell Technology</td><td>NA</td><td>product is PPM (Plant Preservative Mixture)</td></tr><tr><td>seed potatoes for explant source</td><td>any</td><td>B size (not organic)</td><td>suggest Maine Farmer&amp;rsquo;s Exchange</td></tr><tr><td>slow release fertilizer (14-14-14 )</td><td>any</td><td>NA</td><td>Osmocote is a popular brand name</td></tr><tr><td>soft touch forceps</td><td>BioQuip</td><td>4750</td><td></td></tr><tr><td>soil mix</td><td>Griffin Greenhouse Supplies, Inc.</td><td>65-51121</td><td>product is Sunshine LC1 mix</td></tr><tr><td>sterile culture vessel&amp;nbsp;</td><td>PhytoTechnology Laboratories</td><td>C2100</td><td>Magenta-type vessel, PTL-100</td></tr><tr><td>sterile culture vessel&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>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 ...

detached-leaf-assays-to-simplify-gene-expression-studies-potato

Research

JoVE Journal

Environment

6.6K Views.  United States Department of Agriculture (USDA), Agricultural Research Service (ARS). Dit protocol maakt gebruik van een gestroomlijnde aanpak voor het meten van plantengenexpressie in reactie op insectenherbisder. Door het gebruik van losstaande bladeren geïsoleerd in petrischaaltjes, zijn de insecten gemakkelijker toe te passen en te controleren tijdens de relatief korte periode van besmetting die bijdraagt aan reproduceerbare genexpressiegegevens. Dit protocol kan worden aangepast aan vele interacties tussen planten en insecten.als rekeningmetswaarnemingen van hele ...

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

Experimental Design for Laser Microdissection RNA-Seq: Lessons from an Analysis of Maize Leaf Development

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not detected or are not identified as differentially expressed (DE) in transcriptomic analyses of whole plant organs. Laser Microdissection RNA-Seq (LM RNA-Seq) is a powerful tool to identify genes that are DE in specific developmental domains. However, the choice of cellular domains to microdissect and compare, and the accuracy of the microdissections are crucial to the success of the experiments. Here, two examples illustrate design considerations for transcriptomics experiments; a LM RNA-seq analysis to identify genes that are DE along the maize leaf proximal-distal axis, and a second experiment to identify genes that are DE in liguleless1-R (lg1-R) mutants compared to wild-type. Key elements that contributed to the success of these experiments were detailed histological and in situ hybridization analyses of the region to be analyzed, selection of leaf primordia at equivalent developmental stages, the use of morphological landmarks to select regions for microdissection, and microdissection of precisely measured domains. This paper provides a detailed protocol for the analysis of developmental domains by LM RNA-Seq. The data presented here illustrate how the region selected for microdissection will affect the results obtained.

Many developmentally important genes have cell- or tissue-specific expression patterns. This paper describes LM RNA-seq experiments to identify genes that are differentially expressed at the maize leaf blade-sheath boundary and in lg1-R mutants compared to wild-type. The experimental considerations discussed here apply to transcriptomic analyses of other developmental phenomena.

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not ...

Biochemistry

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 ...

A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for assessing plant responses to chemicals and other substances of interest is presented. This system is highly efficient in obtaining homogeneous and healthy seedlings of the C3 and C4 model species Arabidopsis thaliana and Setaria viridis, respectively. The sterile cultivation avoids algae and microorganism contamination, which are known limiting factors for plant normal growth and development in hydroponics. In addition, this system is scalable, enabling the harvest of plant material on a large scale with minor mechanical damage, as well as the harvest of individual parts of a plant if desired. A detailed protocol demonstrating that this system has an easy and low-cost assembly, as it uses pipette racks as the main platform for growing plants, is provided. The feasibility of this system was validated using Arabidopsis seedlings to assess the effect of the drug AZD-8055, a chemical inhibitor of the target of rapamycin (TOR) kinase. TOR inhibition was efficiently detected as early as 30 min after an AZD-8055 treatment in roots and shoots. Furthermore, AZD-8055-treated plants displayed the expected starch-excess phenotype. We proposed this hydroponic system as an ideal method for plant researchers aiming to monitor the action of plant inducers or inhibitors, as well as to assess metabolic fluxes using isotope-labeling compounds which, in general, requires the use of expensive reagents.

A simple, versatile, and low-cost in vitro hydroponic system was successfully optimized, enabling large-scale experiments under sterile conditions. This system facilitates the application of chemicals in a solution and their efficient absorption by roots for molecular, biochemical, and physiological studies.

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for ...

Screening and Identification of RNA Silencing Suppressors from Secreted Effectors of Plant Pathogens

RNA silencing is an evolutionarily conserved, sequence-specific gene regulation mechanism in eukaryotes. Several plant pathogens have evolved proteins with the ability to inhibit the host plant RNA silencing pathway. Unlike virus effector proteins, only several secreted effector proteins have showed the ability to suppress RNA silencing in bacterial, oomycete, and fungal pathogens, and the molecular functions of most effectors remain largely unknown. Here, we describe in detail a slightly modified version of the co-infiltration assay that could serve as a general method for observing RNA silencing and for characterizing effector proteins secreted by plant pathogens. The key steps of the approach are choosing the healthy and fully developed leaves, adjusting the bacteria culture to the appropriate optical density (OD) at 600 nm, and observing green fluorescent protein (GFP) fluorescence at the optimum time on the infiltrated leaves in order to avoid omitting effectors with weak suppression activity. This improved protocol will contribute to rapid, accurate, and extensive screening of RNA silencing suppressors and serve as an excellent starting point for investigating the molecular functions of these proteins.

Here, we present a modified screening method that can be extensively used to quickly screen RNA silencing suppressors in plant pathogens.

RNA silencing is an evolutionarily conserved, sequence-specific gene regulation mechanism in eukaryotes. Several plant pathogens have evolved proteins ...

Genetics

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 ...

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend themselves against pathogen attack. The contribution of a specific Arabidopsis gene to the overall plant immune response can be specifically and quantitatively assessed by assaying the pathogen growth within the infected tissue. For over three decades, the hemibiotrophic bacterium Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326) has been widely applied as the model pathogen to investigate the molecular mechanisms underlying the Arabidopsis immune response. To deliver pathogens into the leaf tissue, multiple inoculation methods have been established, e.g., syringe infiltration, dip inoculation, spray, vacuum infiltration, and flood inoculation. The following protocol describes an optimized syringe infiltration method to deliver virulent Psm ES4326 into leaves of adult soil-grown Arabidopsis plants and accurately screen for enhanced disease susceptibility (EDS) towards this pathogen. In addition, this protocol can be supplemented with multiple pre-treatments to further dissect specific immune defects within different layers of plant defense, including Salicylic Acid (SA)-Triggered Immunity (STI) and MAMP-Triggered Immunity (MTI).

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

Quantification of pathogen growth is a powerful tool to characterize various Arabidopsis thaliana (hereafter: Arabidopsis) immune responses. The method described here presents an optimized syringe infiltration assay to quantify the Pseudomonas syringae pv. maculicola ES4326 growth in adult Arabidopsis leaves.

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend ...

Immunology and Infection

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most commonly used agent for agroinfiltration is Agrobacterium tumefaciens, a pathogen of many dicot plant species. This implies that agroinfiltration can be applied to many plant species. Here, we present our protocols and expected results when applying these methods to the potato (Solanum tuberosum), its related wild tuber-bearing Solanum species (Solanum section Petota) and the model plant Nicotiana benthamiana. In addition to functional analysis of single genes, such as resistance (R) or avirulence (Avr) genes, the agroinfiltration assay is very suitable for recapitulating the R-AVR interactions associated with specific host pathogen interactions by simply delivering R and Avr transgenes into the same cell. However, some plant genotypes can raise nonspecific defense responses to Agrobacterium, as we observed for example for several potato genotypes. Compared to agroinfiltration, detection of AVR activity with PVX agroinfection is more sensitive, more high-throughput in functional screens and less sensitive to nonspecific defense responses to Agrobacterium. However, nonspecific defense to PVX can occur and there is a risk to miss responses due to virus-induced extreme resistance. Despite such limitations, in our experience, agroinfiltration and PVX agroinfection are both suitable and complementary assays that can be used simultaneously to confirm each other&#39;s results.

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are routine functional assays for transient ectopic expression of genes in plants. These methods are efficient assays in effectoromics strategies (rapid resistance and avirulence gene discovery) and crucial to modern research in molecular plant pathology. They meet the demand for robust high-throughput functional analysis in plants.

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most ...

Biology

Plant-Microbe Interaction: Transcriptional Response of Bacillus Mycoides to Potato Root Exudates

Beneficial plant-associated bacteria play an important role in promoting growth and preventing disease in plants. The application of plant growth-promoting rhizobacteria (PGPR) as biofertilizers or biocontrol agents has become an effective alternative to the use of conventional fertilizers and can increase crop productivity at low cost. Plant-microbe interactions depend upon host plant-secreted signals and a reaction hereon by their associated bacteria. However, the molecular mechanisms of how beneficial bacteria respond to their associated plant-derived signals are not fully understood. Assessing the transcriptomic response of bacteria to root exudates is a powerful approach to determine the bacterial gene expression and regulation under rhizospheric conditions. Such knowledge is necessary to understand the underlying mechanisms involved in plant-microbe interactions. This paper describes a detailed protocol to study the transcriptomic response of B. mycoides EC18, a strain isolated from the potato endosphere, to potato root exudates. With the help of recent high-throughput sequencing technology, this protocol can be performed in several weeks and produce massive datasets. First, we collect the root exudates under sterile conditions, after which they are added to B. mycoides cultures. The RNA from these cultures is isolated using a phenol/chloroform method combined with a commercial kit and subjected to quality control by an automated electrophoresis instrument. After sequencing, data analysis is performed with the web-based T-REx pipeline and a group of differentially expressed genes is identified. This method is a useful tool to facilitate new discoveries on the bacterial genes involved in plant-microbe interactions.

Plant-Microbe Interaction: Transcriptional Response of Bacillus Mycoides to Potato Root Exudates

The goal of the protocol presented here is to study the transcriptomic response of endosphere-isolated Bacillus mycoides to potato root exudates. This method facilitates the identification of important bacterial genes involved in plant-microbe interactions and is in principle applicable to other endophytes and plants, with minor adjustments.

Beneficial plant-associated bacteria play an important role in promoting growth and preventing disease in plants. The application of plant ...

Direct Agroinoculation of Maize Seedlings by Injection with Recombinant Foxtail Mosaic Virus and Sugarcane Mosaic Virus Infectious Clones

Agrobacterium-based inoculation approaches are widely used for introducing viral vectors into plant tissues. This study details a protocol for the injection of maize seedlings near meristematic tissue with Agrobacterium carrying a viral vector. Recombinant foxtail mosaic virus (FoMV) clones engineered for gene silencing and gene expression were used to optimize this method, and its use was expanded to include a recombinant sugarcane mosaic virus (SCMV) engineered for gene expression. Gene fragments or coding sequences of interest are inserted into a modified, infectious viral genome that has been cloned into the binary T-DNA plasmid vector pCAMBIA1380. The resulting plasmid constructs are transformed into Agrobacterium tumefaciens strain GV3101. Maize seedlings as young as 4 days old can be injected near the coleoptilar node with bacteria resuspended in MgSO4 solution. During infection with Agrobacterium, the T-DNA carrying the viral genome is transferred to maize cells, allowing for the transcription of the viral RNA genome. As the recombinant virus replicates and systemically spreads throughout the plant, viral symptoms and phenotypic changes resulting from the silencing of the target genes lesion mimic 22 (les22)&#160;or&#160;phytoene desaturase (pds) can be observed on the leaves, or expression of green fluorescent protein (GFP) can be detected upon illumination with UV light or fluorescence microscopy. To detect the virus and assess the integrity of the insert simultaneously, RNA is extracted from the leaves of the injected plant and RT-PCR is conducted using primers flanking the multiple cloning site (MCS) carrying the inserted sequence. This protocol has been used effectively in several maize genotypes and can readily be expanded to other viral vectors, thereby offering an accessible tool for viral vector introduction in maize.

An Agrobacterium-based injection (agroinjection) protocol is presented for the inoculation of foxtail mosaic virus&#160;and sugarcane mosaic virusclones into maize seedlings. Inoculation in this manner leads to viral infection, virus-induced gene silencing of marker genes, and viral overexpression of GFP.

Agrobacterium-based inoculation approaches are widely used for introducing viral vectors into plant tissues. This study details a protocol for the ...

Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue

A plethora of pathogenic microorganisms constantly attack plants. The Pseudomonas syringae species complex encompasses Gram-negative plant-pathogenic bacteria of special relevance for a wide number of hosts. P. syringae enters the plant from the leaf surface and multiplies rapidly within the apoplast, forming microcolonies that occupy the intercellular space. The constitutive expression of fluorescent proteins by the bacteria allows for visualization of the microcolonies and monitoring of the development of the infection at the microscopic level. Recent advances in single-cell analysis have revealed the large complexity reached by clonal isogenic bacterial populations. This complexity, referred to as phenotypic heterogeneity, is the consequence of cell-to-cell differences in gene expression (not linked to genetic differences) among the bacterial community. To analyze the expression of individual loci at the single-cell level, transcriptional fusions to fluorescent proteins have been widely used. Under stress conditions, such as those occurring during colonization of the plant apoplast, P. syringae differentiates into distinct subpopulations based on the heterogeneous expression of key virulence genes (i.e., the Hrp type III secretion system). However, single-cell analysis of any given P. syringae population recovered from plant tissue is challenging due to the cellular debris released during the mechanical disruption intrinsic to the inoculation and bacterial extraction processes. The present report details a method developed to monitor the expression of P. syringae genes of interest at the single-cell level during the colonization of Arabidopsis and bean plants. The preparation of the plants and the bacterial suspensions used for inoculation using a vacuum chamber are described. The recovery of endophytic bacteria from infected leaves by apoplastic fluid extraction is also explained here. Both the bacterial inoculation and bacterial extraction methods are empirically optimized to minimize plant and bacterial cell damage, resulting in bacterial preparations optimal for microscopy and flow cytometry analysis.

Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue

The present protocol describes a method that allows single-cell gene expression analysis on Pseudomonas syringae populations grown within the plant apoplast.

A plethora of pathogenic microorganisms constantly attack plants. The Pseudomonas syringae species complex encompasses Gram-negative plant-pathogenic ...

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

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