Name: detached leaf assays to simplify gene expression studies in potato during infestation by chewing insect manduca sexta
Uploaded: 2019-05-15T12:30:00
Description: 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

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 experimen...

<ol>
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A., 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=A+laboratory+assay+for+Phytophthora+infestans+resistance+in+various+Solanum+species+reflects+the+field+situation.">A laboratory assay for Phytophthora infestans resistance in various Solanum species reflects the field situation.</a> European Journal of Plant Pathology. 105, 241-250 (1999).</li><li>Kamoun, 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=A+gene+encoding+a+protein+elicitor+of+Phytophthora+infestans+is+down-regulated+during+infection+of+potato.">A gene encoding a protein elicitor of Phytophthora infestans is down-regulated during infection of potato.</a> Molecular Plant-Microbe Interactions. 10, 13-20 (1997).</li><li>Nowakowska, M., Nowicki, M., K&#322;osi&#324;ska, U., Maciorowski, R., Kozik, E. 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. 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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. 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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.

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			<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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