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

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.......

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.......

Materials

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