51

Alain Goossens and Laurens Pauwels (eds.), Jasmonate Signaling: Methods and Protocols, Methods in Molecular Biology, vol. 1011, DOI 10.1007/978-1-62703-414-2_5, © Springer Science+Business Media, LLC 2013

Chapter 5

Elicitation of Jasmonate-Mediated Defense Responses by Mechanical Wounding and Insect Herbivory

Marco Herde , Abraham J. K. Koo, and Gregg A. Howe

Abstract

Many plant immune responses to biotic stress are mediated by the wound hormone jasmonate (JA). Functional and mechanistic studies of the JA signaling pathway often involve plant manipulations that elicit JA production and subsequent changes in gene expression in local and systemic tissues. Here, we describe a simple mechanical wounding procedure to effectively trigger JA responses in the Arabidopsis thaliana rosette. For comparison, we also present a plant–insect bioassay to elicit defense responses with the chewing insect Trichoplusia ni . This latter procedure can be used to determine the effect of JA-regulated defenses on growth and development of insect herbivores.

Key words Jasmonate , Plant–insect interaction , Mechanical wounding , Wound response , Herbivory , Plant defense , Arabidopsis , Trichoplusia ni

A pioneering study by C.A. Ryan and colleagues demonstrated that leaf damage in fl icted by insect herbivory or mechanical wound-ing results in rapid expression of defensive proteinase inhibitors [ 1 ] . Furthermore, tissue damage had been reported to elicit defense responses not only in wounded leaves of the plant but also in undamaged ones. This work [ 1 ] set the stage for decades of inten-sive research to elucidate the ecophysiological relevance and under-lying molecular mechanism of induced resistance to arthropod herbivores. A major conclusion from a large body of research is that plant defenses induced by wounding and insects are regulated largely by the plant stress hormone jasmonate (JA) [ 2– 4 ] .

JA-regulated plant defense responses are modulated by many environmental inputs [ 5, 6 ] . The use of de fi ned experimental con-ditions to induce this form of plant immunity is therefore para-mount to achieving reproducible results within a study, as well as for comparing results generated in different laboratories. Exogenous JA, mechanical wounding (or simulated herbivory), and herbivory

1 Introduction

52 Marco Herde et al.

are among the most common experimental treatments used to induce resistance. Each of these approaches has its own merits and shortcomings. Although exogenous JA is widely utilized as a potent elicitor of defense responses [ 7 ] , typical compounds (such as MeJA) are now recognized to be inactive per se at the level of the COI1-JAZ receptor and must be metabolized in planta to the bioactive form of the hormone, jasmonoyl-isoleucine (JA-Ile) [ 8– 10 ] . Such treatments may generate nonphysiological concentrations of the hormone and override the cell- and tissue-speci fi c control of the pathway.

Mechanical wounding provides a simple alternative approach to elicit JA/JA-Ile production and associated responses in dam-aged (local) and undamaged (systemic) tissues. Among the imple-ments used for mechanical wounding are hole punchers, razor blades or scalpels to cut the leaf surface, pattern wheels to create multiple small wounds, and hemostatic forceps (hemostats) to crush the leaf lamina. These treatments are typically administered at a single time point to activate the JA pathway in a reproducible manner. Relatively severe wounds in fl icted, for example, by a hemostat have been used extensively to study the timing of wound-induced changes in JA content in local and systemic tissues, the dynamic behavior of JA signaling components [ 11 ] , and genome-wide alterations in transcript pro fi les [ 12, 13 ] . This approach is applicable to monocots and dicots, as well as gymnosperms [ 14 ] . The speci fi c method of mechanical wounding should be tailored to the particular plant species under study.

Herbivore challenge is arguably the best approach for eliciting defense responses in the context of the natural plant–herbivore interaction. Although herbivory and severe mechanical wounding often elicit similar responses [ 1, 2 ] , these two treatments differ in important ways. For example, arthropod herbivores (such as chew-ing insects) use highly specialized mouthparts to continuously remove small pieces of leaf tissue. Specialized robotic devices have been developed to more accurately simulate the spatial and tempo-ral aspects of mechanical damage resulting from insect herbivory [ 15 ] . Moreover, oral secretions of herbivores contain chemical fac-tors that modulate the timing and amplitude of the host defense response [ 2 ] . For these reasons, caution should be exercised when drawing conclusions about the underlying causes of differential plant responses to mechanical wounding versus herbivory.

Below, we describe the use of hemostat wounding to elicit JA responses in the Arabidopsis thaliana rosette as well as a simple protocol to challenge Arabidopsis plants with the lepidopteran her-bivore Trichoplusia ni (cabbage looper). In addition to its utility for studying host plant responses to herbivory, this procedure is also useful for determining the effectiveness of host defenses on the herbivore.

53Elicitation of JA-dependent Wound Responses

1. Commercial bleach 40 % (v/v). 2. Sterile water. 3. Autoclavable bags. 4. Pots (at least 3.5-in. diameter). 5. Scale with 1-mg accuracy. 6. Weigh paper or weigh boats. 7. Prelabeled collection tubes or aluminum foil. 8. Liquid nitrogen in portable container. 9. Hemostats with straight 1.5–2 mm serrated tip and locking

mechanism disabled by wrapping tape around the interlocking teeth (Fig. 1a ).

10. Razor blade or mini-bud trimmer. 11. Tissue storage box (precooled in −80 °C freezer). 12. Timer.

1. Trichoplusia ni (Benzon Research, or obtained from an in-house rearing facility).

2. Featherweight forceps. 3. Paintbrush. 4. Clear plastic cups (9 oz. or 250-mL tumblers). 5. Glue gun.

2 Materials

2.1 Elicitation of JA Responses by Mechanical Wounding

2.2 Plant–Insect Bioassay

2.2.1 Insects

Fig. 1 Eliciting JA-mediated defense responses in Arabidopsis . ( a ) Use of hemostats to mechanically wound Arabidopsis leaves. ( b ) Experimental setup for caging insect larvae on Arabidopsis

54 Marco Herde et al.

1. Pots (3.5 in. in diameter). 2. Standard Arabidopsis soil mix. 3. Arabidopsis thaliana (L.) Heyhn. plants not yet at the repro-

ductive state. 4. Para fi lm.

1. Remove the bottom of an inverted clear plastic cup with a heated metal piece.

2. Cover the opening with Miracloth attached with a hot glue gun. 3. Af fi x to the pot top (Fig. 1b ). 4. Prepare a suf fi cient number of cup cages prior to initiating the

feeding assay.

1. Surface sterilize Arabidopsis seeds with 40 % (v/v) commercial bleach for 5 min.

2. Rinse seeds thoroughly with sterile water. 3. Imbibe in water for 2–3 days at 4 °C in the dark ( see Note 1 ). 4. Sterilize soil prior to sowing ( see Note 2 ) by placing the desired

amount of moist soil in an autoclavable bag. Autoclave for an appropriate amount of time (such as 60-min dry cycle for a 45-L bag).

5. Allow soil to cool after autoclaving, but keep the bag unopened until seeds are sown to prevent recontamination.

6. Add soil to the pots and immediately sow seeds. Taking the seed germination ef fi ciency into account, sow a suf fi cient num-ber of seeds to ensure growth of two to four plants per pot ( see Note 3 ).

7. Grow plants in a growth chamber maintained at 22 °C with a photoperiod of 16-h light (100 μ E/m 2 /s) and 8-h darkness. In our standard wound assay we use 30-day-old plants with fully expanded rosette leaves but that have not bolted yet.

1. Plan the location and timing of your experiment, with particu-lar attention to environmental factors that affect JA-induced responses ( see Notes 4 and 5 ).

2. Select speci fi c plants to be included in the experiment and arrange pots so as to facilitate wounding and collection of sam-ples in a coordinated manner. It is critical to use plants that have similarly sized leaves so that the relative proportion of damaged and undamaged areas on the leaf is consistent between plants ( see Notes 6 and 7 ).

2.2.2 Plants

2.2.3 Plant–Insect Arena

3 Methods

3.1 Preparation of Plants for Elicitation

3.2 Elicitation of JA Responses by Mechanical Wounding

55Elicitation of JA-dependent Wound Responses

3. Administer the wound treatment with the device of choice ( see Note 8 ) and immediately start the timer. If using a hemostat, brie fl y clamp the device fi rmly across the leaf (perpendicular to the midvein; Fig. 1a ) and then release ( see Note 9 ). Serrated wounds should be clearly visible on the leaf.

4. Make one or two such wounds per leaf ( see Note 10 ), damag-ing at least fi ve leaves in less than 1 min. Continue to wound a suf fi cient number of leaves for one biological replicate ( see Note 11 ).

5. With the timer still running, wound additional plants for repli-cates two and three for that particular time point. Typically, we do not pause the wound treatment between replicates or between different plant genotypes.

6. Start wounding independent plants for additional time points ( see Note 12 ).

7. At the appropriate time after wounding, harvest the damaged leaves by cutting the petiole with a razor blade or mini-bud trimmers.

8. Work quickly to measure the fresh weight of the excised leaves. 9. Transfer the tissue to a suitable container (such as aluminum

foil) and immediately freeze in liquid nitrogen ( see Note 13 ). Frozen tissue should be stored at −80 °C until use.

10. Harvest leaf tissue from undamaged control plants at the beginning of the time-course experiment or, alternatively, in parallel with the damaged leaves from a matched set of wounded plants.

Reproducibility in plant–insect bioassays designed to measure plant and insect performance (or fi tness) is of the utmost importance. One critical factor is the ability to grow healthy plants under con-trolled conditions. To achieve meaningful results, the number of plants, insects, and replicates required should also be taken into account. Experimental results are in fl uenced by many factors, including larval mortality, the amount of plant tissue consumed during the feeding assay, and the extent to which larval growth on a particular host genotype varies between insects ( see Note 14 ). If the larval mortality is high, plants can be challenged with multiple larvae. The use of many larvae per plant might result in overcon-sumption of plant tissue without suf fi cient larval weight gain dur-ing the feeding trial. For statistical analyses, we typically use the average of all larval weights from one plant as a single data point.

The plant and insect species used for bioassays will in fl uence the conclusions drawn from the experiment. For example, the per-formance of an insect species (such as Pieris rapae ) that is special-ized for feeding on Arabidopsis may be unaffected by the presence of glucosinolates, which are the major class of chemical defenses in

3.3 Plant–Insect Bioassay

56 Marco Herde et al.

Arabidopsis . In contrast, growth and development of crucifer nonspecialists (such as Spodoptera exigua ) will be negatively affected by glucosinolates. The ecological relevance of the plant–insect interaction (i.e., whether a particular interaction actually occurs in nature) may also be relevant for the experimental design.

Moreover, the defense status of a plant is strongly affected by the plant’s developmental age. When assessing the effect of JA-inducible defenses expressed in vegetative tissue (for instance, leaves), it is desirable to use plants that have not yet entered the reproductive phase of development. Prolonged vegetative growth (i.e., in the absence of bolting) of Arabidopsis can be accomplished by the use of short-day growth conditions.

Finally, the genetic variation between individual insects is a potential source of experimental variation in insect growth on a given diet. This variation could be minimized by the use of an insect colony that has been inbred for many generations. However, excessive inbreeding within the population may result in the loss of traits (such as host range) exhibited by populations in the wild.

The following protocol describes a bioassay with Trichoplusia ni reared on Arabidopsis (accession Col-0). The procedure can readily be adapted for use with other lepidopteran herbivores, such as Spodoptera exigua and Pieris rapae .

1. Grow one plant per pot with soil under short-day conditions to maximize vegetative growth. Our standard growth condi-tions are 22 °C with a photoperiod of 10 h light (100 μ E/m 2 /s) and 14 h darkness. Plants should approximately be 5 weeks old at the time of insect challenge ( see Note 15 ).

2. Place insect eggs (such as commercially obtained T . ni eggs) in a Petri dish together with a small piece of moist fi lter paper to maintain humidity and prevent desiccation of larvae.

3. Seal the dish with para fi lm and incubate eggs at room tempera-ture ( see Note 16 ). Eggs typically begin to hatch within 48 h. Please keep in mind the day/night light cycle under which plants and insects are grown ( see Note 17 ).

4. Water plants well the evening before placing insects on plants. 5. Use a small paintbrush to transfer larvae to leaves near the

center of the rosette ( see Notes 18 and 19 ). Remain consis-tent with the location of the insect placement on the plant ( see Note 20 ).

6. Use cup cages to secure the insect on the plant and tightly seal the junction between the pot and cage with para fi lm.

7. Return the plants to the growth chamber in which the plants had been grown previously.

8. Remove the cup cage at different time points (i.e., various days after infestation) to determine the weight of insects ( see Notes 21 and 22 ).

57Elicitation of JA-dependent Wound Responses

9. Use a scale with appropriate accuracy for the particular larvae measured ( see Note 22 ). A setup for automated transfer of weight measurements from the scale to an appropriate com-puter program (such as Excel) facilitates data recording.

10. Handle heavier larvae carefully with featherweight forceps without damaging or stressing the insects. For this reason, we return individually weighed larvae to their plant of origin prior to processing to the next insect.

11. Thoroughly search the host plant, soil surface, and cage to identify larvae. The number of viable larvae identi fi ed provides a measure of insect mortality.

12. Analyze the host plant as dictated by the needs of the experi-ment, which might include photographing leaves to evaluate the consumed leaf area with an appropriate imaging software (such as ImageJ; http://rsbweb.nih.gov/ij/ ) and harvesting leaf tissue for RNA isolation or chemical analysis.

1. Alternatively, seeds can be sown and the pots placed in a cold room for 3 days. The drawback of this procedure is that suf fi cient space is needed in a cold room and plants may inad-vertently be exposed to fungal spores. Growth of healthy, unstressed plants is a key factor in obtaining reliable results.

2. We use autoclaved soil for growing Arabidopsis because it reduces the risk of unintended interactions with soil-borne fungi.

3. Sowing too many seeds per pot will necessitate the additional manual removal of plants to avoid overcrowding. Extra han-dling of plants at this stage may cause inadvertent stress to the plants.

4. We typically perform short-term (i.e., <12 h) time-course experiments with plants that have been moved from the growth chamber to a work area within the laboratory with similar light intensity and temperature. Care should be taken to minimize unintentional stress to the plants during transport and han-dling. For example, mechanical agitation may activate a basal response in unwounded control plants, or may prime plants for a stronger wound response. As an alternative experimental setup, wound elicitation and tissue harvesting may be per-formed without removing plants from the growth chamber. In this case, all materials should be readily available in the growth chamber.

5. Recent studies indicate that JA-regulated defense responses to herbivore attack are in fl uenced by circadian and/or diurnal inputs [ 16– 18 ] . Therefore, it is important to plan the timing

4 Notes

58 Marco Herde et al.

of the experiments with respect to the light/dark cycle and to be as consistent as possible from experiment to experiment. In our hands, wound treatments administered in the “morning” (i.e., within a few hours of the dark-to-light transition) result in more robust JA-mediated responses in Arabidopsis and Solanum lycopersicum (tomato).

6. Generally, the correlation is positive between the area of dam-aged tissue (as a percentage of the total leaf area) and the accu-mulated levels of JA and JA-responsive transcripts. If a particular mutant under study exhibits a morphological phenotype (such as a semidwarf stature), the damage (relative to the total leaf area) in fl icted to the mutant will be greater than that adminis-tered to the wild type. Accordingly, the responses in the mutant might appear stronger than those in the wild type.

7. To analyze wound-induced systemic responses in the Arabidopsis rosette, the extent of the vascular connectivity between damaged (local response) and undamaged (systemic response) leaves of the same plant should be considered. In Arabidopsis and nonrosette plants as well, parastichous leaves with a strong interconnective vasculature exhibit stronger sys-temic responses than nonparastichous leaves [ 19 ] . In the case of Arabidopsis , we typically use three or four developmentally older leaves at the base of the rosette and younger undamaged (parastichous) leaves on the same rosette to assess the local and systemic responses, respectively [ 11 ] .

8. The amplitude of the wound-induced JA accumulation and JA-dependent gene expression may vary greatly depending on the particular wounding method. In general, the severity of tissue damage correlates with the strength of the response. Crushing-type wounds in fl icted with a hemostat are relatively severe and seemingly activate maximal responses. Accordingly, this simple procedure is especially useful for eliciting rapid and uniform responses to be monitored in time-course studies.

9. Wounds in fl icted either parallel or perpendicular to the mid-vein are effective in activating JA responses. However, for the ease and consistency of the treatment, we typically administer wounds perpendicular to the midvein (Fig. 1a ).

10. Although multiple wounds in fl icted to a single leaf intensify the JA responses, it also increases the time required for the treatment. We found that two wounds per leaf is a good com-promise for most applications. If administering more than one wound per leaf, make the initial wound at the distal end of the leaf and the subsequent wounds at the proximal end.

11. We use three biological replicates (i.e., different plants) for each data point. Statistical variation between replicates may be reduced by pooling leaves from multiple plants. In general, we

59Elicitation of JA-dependent Wound Responses

pool three to fi ve fully expanded rosette leaves (approximately 200–300 mg fresh weight) as a single replicate in hormone measurements and RNA preparations.

12. In short time-course experiments (for instance, tissue harvested 5 min post wounding) or experiments with multiple sampling points in which wounding and harvesting procedures tempo-rally overlap, it is convenient to use multiple timers to keep track of the different samples.

13. Recent studies with Arabidopsis [ 11, 20 ] indicate that it is cru-cial to minimize the time between harvesting (i.e., excising) and freezing of tissue in liquid nitrogen because JA levels increase very rapidly in excised leaves. Therefore, it is impor-tant to perform the various steps of the protocol (leaf excision, weighing and recording, securing tissue in an appropriate con-tainer, and fl ash freezing) as fast as possible without compro-mising the accuracy. Wrapping tissue in prelabeled aluminum foil is useful for this purpose. In general, the total time elapsed between excising and freezing leaves in liquid nitrogen should be shorter than 1 min.

14. As for the wound assays, host responses and insect performance on plant genotypes that differ signi fi cantly in morphology or development should be compared with caution.

15. The physical position of individual plants within a growth chamber (and watering tray, if used) may have a signi fi cant effect on the plant physiology, which, in turn, can in fl uence insect performance. To correct this potential bias, the position of all plants within the growth chamber and watering tray should be randomized at the onset of the experiment.

16. The time required to hatch eggs can be modi fi ed by changing the temperature. Higher temperatures (such as 28 °C) acceler-ate the process, whereas cooler temperatures are useful to slow the hatch rate. Although some insect eggs can be stored tem-porarily at 4 °C to delay hatching, we recommend that eggs should be hatched as soon as possible to maximize hatching viability and uniformity.

17. The circadian regulation defense responses of Arabidopsis are coordinated with the circadian cycle of the T . ni larvae [ 18 ] . Thus, consistency in the timing (relative to the light/dark cycle) of the plant and insect manipulations should be main-tained. In general, we perform these procedures during the second half of the light period, prior to the beginning of the dark period.

18. Neonate (newly hatched) larvae are a suitable choice for feed-ing trials. Typically, an excess number of eggs are hatched and, for plant infestation, neonates are selected that hatch at a simi-lar time. In the event of unacceptably high neonate mortality

60 Marco Herde et al.

on a particular host genotype, neonates may be reared on an arti fi cial diet for a brief period (for instance, 1 day) prior to transfer to the plant. Although this method tends to reduce larval mortality and facilitates more effective damage to the host plant, bypassing the initial neonate–host interaction may be unsuitable for all purposes.

19. As neonate larvae are very delicate, physical damage during the transfer process should be avoided.

20. Because of inter- and intra-leaf variation in the expression of host defenses [ 21 ] , every effort should be made to standardize the position of the larvae on the leaves at the onset of the experiment.

21. T . ni larvae usually empty their gut content prior to molting and pupation with a signi fi cant effect on larval weight as a consequence.

22. Speci fi c time points chosen for the determination of larval weight depend on the objective of the experiment. In general, differences in larval performance on well-defended (such as wild type) versus defense-compromised (such as a JA mutant) host genotypes become more apparent at the later stages of the larval development. A scale with a 0.1-mg accuracy will facili-tate weight measurements of early-instar larvae.

Acknowledgments

This work was supported by the National Institutes of Health (grant R01GM57795) and the Chemical Sciences, Geosciences and Biosciences Division, Of fi ce of Basic Energy Sciences, Of fi ce of Science, US Department of Energy (grant DE–FG02–91ER20021) for partial support of M.H. M.H. was also the recipient of a fellow-ship (HE 5949/1-1) from the German Research Foundation (DFG).

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