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 prosystemin protein after a leaf is wounded^2,3. 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 genes^6,7,8.

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 HAMPs^9,10,11. 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 literature^12,13,14.

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 surface^16,17. 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.

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 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 hood^18,19.

1. Prepare Kennebec plantlets from explant source.
   1. Prepare propagation medium.
      1. 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.
      2. Add 1 mL of preservative/biocide and autoclave on liquid cycle for 20 min (121 °C, 101.3 kPa). Remove medium from autoclave immediately after the cycle is finished and cool it to 50 °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.
   2. Prepare explant material.
      1. 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.
      2. 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.
      3. 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 2‒3 weeks or until plantlets form at 24 °C, 16 h light (140 µmol·m^-2·s^-1)/8 h dark photoperiod.
2. Prepare nodal-propagated tissue culture potato plants.
   1. 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 °C until nodal transfer.
      1. 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.
      2. Autoclave on liquid cycle for 20 min (121 °C, 101.3 kPa). Remove medium from autoclave immediately after the cycle is finished and cool it to 50 °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.
   2. Prepare nodal cuttings.
      1. 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.
      2. 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).
      3. Transfer vessels with nodal cuttings to a plant tissue culture chamber and grow for 2‒3 weeks at 24 °C, 16 h light (140 µmol·m^-2·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 2‒3 weeks.
3. (Optional) Prepare soil grown Kennebec potato plants.
   NOTE: To produce larger leaves, nodal propagated potato plantlets can be transferred to soil.
   1. 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.
   2. 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.
   3. Place in a growth chamber with 16 h light (140 µmol·m^-2·s^-1)/8 h dark photoperiod and 25/20 °C day/night temperatures.
      NOTE: Plants are ready when the top two fully expanded leaves have reached the size appropriate for the assay.
4. (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.
   1. Place a potato tuber 6 in deep in a 10 in pot containing soil mix supplemented with 10 mL pelleted slow release fertilizer.
   2. Place in a growth chamber with 16 h light (140 µmol·m^-2·s^-1)/8 h dark photoperiod and 25/20 °C day/night temperature. Plants are ready approximately 30‒40 days from tuber planting.
      NOTE: Do not use plants that have begun to flower.

2. Preparation of insects for feeding

1. 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.
2. 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.
3. (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.

3. Components setup for the infestation experiment

NOTE: See the schematic summary in Figure 1.

1. 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.
   1. 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.
   2. Trace a set of six circles using the appropriately sized Petri dish on the paper in each tray. Label one set of circles ‘control’ A, B and C and the other ‘infested’ A, B, and C. Also label each placement template with the appropriate harvest time.
2. (Optional) Set up a camera for ‘before infestation’ and ‘after infestation’ image capture.
   1. Secure a camera on a stand at the appropriate focal length for image capture of all Petri dishes in the placement template.
3. Label the harvest time point tubes.
   1. 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).
4. Prepare Petri dishes chosen in step 3.1.1.
   1. 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.
   2. Position three potato plants next to each placement template. Ensure that plants are all the same age and relative size.

4. Performing infestation

NOTE: One harvest time point/placement template is set up at a time.

1. 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.
2. (Optional) Transfer the placement template to the camera stand to capture a ‘before infestation’ image.
3. Transfer larvae to each infested dish using soft touch forceps as quickly as possible. Set the timer for desired ‘infestation’ time.
   NOTE: The period of time when larvae are consuming the leaf tissue is the ‘infestation time’. This is something that can be determined empirically using a few test leaves/larvae before the start of the actual infestation. The chosen ‘infestation time’ 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.
4. 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.
5. Remove larvae from leaves at the end of the infestation time. Start the timer for the harvest time.
6. (Optional) Transfer the placement template to the camera stand to capture the ‘after infestation’ image.
   NOTE: All six leaves in the placement template are harvested at the specific harvest time.

5. Harvesting leaves

1. 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 °C until the isolation of RNA.
2. Repeat steps 4‒5.1 for each harvest time point.

6. Processing leaf tissue for gene expression analysis

1. Grind the frozen leaf tissue to a powder with a micropestle.
2. Isolate total RNA and process for gene expression as previously described²².

7. (Optional) Estimating leaf damage

1. Visually estimate percent of leaf damage or calculate leaf area in the before- and after-infestation images.
2. Measure leaf area with software tools (e.g., Phenophyte)²³.
3. From leaf area measurements, calculate percentage of damage as
   % damage = [(leaf area before – leaf area after)/leaf area before] x 100.

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...

Materials

List of materials used in this article

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