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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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 pro....

Protocol

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 ‘infested’ leaves for 5 separate harvest time points. Matched control leaves for each time point create a total of 30 samples. The experimen.......

Representative Results

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.......

Discussion

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.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
agar substitutePhytoTechnology LaboratoriesG3251product is Gelzan
containment vessel (6,12 or 24 well dish)Fisher Scientific 08-772-49, 08-772-50, 08-72-51many other companies sell these products
manduca eggs Carolina Biological Supply Company14388030-50 eggs
manduca eggs Great Lakes HornwormNA50, 100, 250 or 500 eggs
manduca larvaeCarolina Biological Supply Companycall for specific larval instar requestsany instar
manduca larvaeGreat Lakes Hornwormcall for specific larval instar requestsany instar
microcentrifuge tubes, 1.7 ml Thomas Scientific1158R22these have been tested in liquid N2 and will not explode
Murashige & Skoog (MS) Basal Medium w/VitaminsPhytoTechnology LaboratoriesM519used to make propagation medium
nutrient agar mixPhytoTechnology LaboratoriesM5825product is Murashige & Skoog Basal Medium with vitamins, sucrose, and Gelzan
paper filter discsFisher Scientific 09-805AWhatman circles-purchase to fit in petri dish
petri dish, 60X15 mm or 100X15 mmFisher Scientific FB0875713A or FB0875712purchase size appropriate for leaf size
potato tubers anyB size (not organic)suggest Maine Farmer’s Exchange
pots, 10" Griffin Greenhouse Supplies, Inc.41PT1000CN2
preservative/biocidePlant Cell TechnologyNAproduct is PPM (Plant Preservative Mixture)
seed potatoes for explant sourceanyB size (not organic)suggest Maine Farmer’s Exchange
slow release fertilizer (14-14-14 )anyNAOsmocote is a popular brand name
soft touch forcepsBioQuip4750
soil mixGriffin Greenhouse Supplies, Inc.65-51121product is Sunshine LC1 mix
sterile culture vessel PhytoTechnology LaboratoriesC2100Magenta-type vessel, PTL-100
sterile culture vessel Fisher Scientific ICN2672206product is MP Biomedicals Plantcon

References

  1. Choi, H. W., Klessig, D. F. DAMPs, MAMPs, and NAMPS in plant innate immunity. BMC Plant Biology. 16, 1-10 (2016).
  2. Pearce, G., Strydom, D., Johnson, S., Ryan, C. A. A polypeptide from tomato leaves induces wound-induci....

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Detached Leaf AssayGene ExpressionPotatoManduca SextaInsect HerbivoryChewing InsectTissue CultureNodal PropagationLarval StagePlant insect InteractionReproducible DataPlant Genetic Improvement

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