A tale of two tissues: Probing gene expression in a complex insect-induced gall.

Plant galls are novel and sometimes dramatic plant organs whose development is initiated and controlled by parasitic microbes, nematodes, insects and mites. For arthropods, galls provide relative safety from enemies and abiotic stresses while providing nutrition. Galls are formed entirely by the plant, whose transcriptional pathways are modified and coopted to produce a structure specific to the galler species; they comprise a classic example of Dawkins' "extended phenotype". Arthropod-elicited galls are unique in that they are often anatomically complex (Figure 1a), with multiple differentiated tissue types (Figure 1b). A growing number of investigators have studied changes in hostplant gene expression to understand arthropod gall development. In this issue of Molecular Ecology, Martinson et al. (2021) report using RNA sequencing to explore tissue-specific gene expression associated with anatomical and functional gall complexity, demonstrating for the first time that gall tissues are as different transcriptionally as they are anatomically.

Impact of chronic stylet-feeder infestation on folivore-induced signaling and defenses in a conifer.

Our understanding of how conifers respond biochemically to multiple simultaneous herbivore attacks is lacking. Eastern hemlock (Tsuga canadensis; 'hemlock') is fed on by hemlock woolly adelgid (Adelges tsugae; 'adelgid') and by later-instar gypsy moth (Lymantria dispar; 'gypsy moth') caterpillars. The adelgid is a stylet-feeding insect that causes a salicylic acid (SA)-linked response in hemlock, and gypsy moth larvae are folivores that presumably cause a jasmonic acid (JA)-linked response. This system presents an opportunity to study how invasive herbivore-herbivore interactions mediated through host biochemical responses. We used a factorial field experiment to challenge chronically adelgid-infested hemlocks with gypsy moth caterpillars. We quantified 17 phytohormones, 26 phenolic and terpene metabolites, and proanthocyanidin, cell wall-bound (CW-bound) phenolic, and lignin contents. Foliage infested with adelgid only accumulated gibberellins and SA; foliage challenged by gypsy moth only accumulated JA phytohormones. Gypsy moth folivory on adelgid-infested foliage reduced the accumulation of JA phytohormones and increased the SA levels. Both herbivores increased CW-bound phenolics and gypsy moth increased lignin content when feeding alone but not when feeding on adelgid-infested foliage. Our study illustrates the importance of understanding the biochemical mechanisms and signaling antagonism underlying tree responses to multiple stresses and of disentangling local and systemic stress signaling in trees.

Caterpillar Chewing Vibrations Cause Changes in Plant Hormones and Volatile Emissions in Arabidopsis thaliana.

Plant perception of insect feeding involves integration of the multiple signals involved: wounding, oral secretions, and substrate borne feeding vibrations. Although plant responses to wounding and oral secretions have been studied, little is known about how signals from the rapidly transmitted vibrations caused by chewing insect feeding are integrated to produce effects on plant defenses. In this study, we examined whether 24 h of insect feeding vibrations caused changes in levels of phytohormones and volatile organic compounds (VOCs) produced by leaves of Arabidopsis thaliana when they were subjected to just feeding vibrations or feeding vibrations and wounding + methyl jasmonate (MeJA), compared to their respective controls of silent sham or wounding + MeJA. We showed that feeding vibrations alone caused a decrease in the concentrations of most phytohormones, compared to those found in control plants receiving no vibrations. When feeding vibrations were combined with wounding and application of MeJA, the results were more complex. For hormones whose levels were induced by wounding and MeJA (jasmonic acid, indole-3-butyric acid), the addition of feeding vibrations caused an even larger response. If the level of hormone was unchanged by wounding and MeJA compared with controls, then the addition of feeding vibrations had little effect. The levels of some VOCs were influenced by the treatments. Feeding vibrations alone caused an increase in ß-ionone and decrease in methyl salicylate, and wounding + MeJA alone caused a decrease in benzaldehyde and methyl salicylate. When feeding vibrations were combined with wounding + MeJA, the effects on ß-ionone and methyl salicylate were similar to those seen with feeding vibrations alone, and levels of benzaldehyde remained low as seen with wounding + MeJA alone. The widespread downregulation of plant hormones observed in this study is also seen in plant responses to cold, suggesting that membrane fluidity changes and/or downstream signaling may be common to both phenomena.

A galling insect activates plant reproductive programs during gall development.

Many insect species have acquired the ability to redirect plant development to form unique organs called galls, which provide these insects with unique, enhanced food and protection from enemies and the elements. Many galls resemble flowers or fruits, suggesting that elements of reproductive development may be involved. We tested this hypothesis using RNA sequencing to quantify the transcriptional responses of wild grapevine (Vitis riparia) leaves to a galling parasite, phylloxera (Daktulosphaira vitifoliae). If development of reproductive structures is part of gall formation, we expected to find significantly elevated expression of genes involved in flower and/or fruit development in developing galls as opposed to ungalled leaves. We found that reproductive gene ontology categories were significantly enriched in developing galls, and that expression of many candidate genes involved in floral development were significantly increased, particularly in later gall stages. The patterns of gene expression found in galls suggest that phylloxera exploits vascular cambium to provide meristematic tissue and redirects leaf development towards formation of carpels. The phylloxera leaf gall appears to be phenotypically and transcriptionally similar to the carpel, due to the parasite hijacking underlying genetic machinery in the host plant.

Heritable Phytohormone Profiles of Poplar Genotypes Vary in Resistance to a Galling Aphid.

Insect galls are highly specialized structures arising from atypical development of plant tissue induced by insects. Galls provide the insect enhanced nutrition and protection against natural enemies and environmental stresses. Galls are essentially plant organs formed by an intimate biochemical interaction between the gall-inducing insect and its host plant. Because galls are plant organs, their development is likely to be governed by phytohormones involved in normal organogenesis. We characterized concentrations of both growth and defensive phytohormones in ungalled control leaves and galls induced by the aphid Pemphigus betae on narrowleaf cottonwood Populus angustifolia that differ genotypically in resistance to this insect. We found that susceptible trees differed from resistant trees in constitutive concentrations of both growth and defense phytohormones. Susceptible trees were characterized by significantly higher constitutive cytokinin concentrations in leaves, significantly greater ability of aphids to elicit cytokinin increases, and significantly lower constitutive defense phytohormone concentrations than observed in resistant trees. Phytohormone concentrations in both constitutive and induced responses in galled leaves exhibited high broad-sense heritability that, respectively, ranged from 0.39 to 0.93 and from 0.28 to 0.66, suggesting that selection can act upon these traits and that they might vary across the landscape. Increased cytokinin concentrations may facilitate forming strong photosynthate sinks in the galls, a requirement for galling insect success. By characterizing for the first time the changes in 15 phytohormones belonging to five different classes, this study offers a better overview of the signaling alteration occurring in galls that has likely been important for their ecology and evolution. Copyright © 2019 The Author(s). This is an open-access article distributed under the CC BY-NC-ND 4.0 International license .

Morphometric analysis of young petiole galls on the narrow-leaf cottonwood, Populus angustifolia, by the sugarbeet root aphid, Pemphigus betae.

An insect-induced gall is a highly specialized structure resulting from atypical development of plant tissue induced by a reaction to the presence and activity of an insect. The insect induces a differentiation of tissues with features and functions of an ectopic organ, providing nutrition and protection to the galling insect from natural enemies and environmental stresses. In this anatomical and cytological study, we characterized how the gall-inducing aphid Pemphigus betae reshapes the leaf morphology of the narrow-leaf cottonwood Populus angustifolia to form a leaf fold gall. Young galls displayed a bend on one side of the midvein toward the center of the leaf and back to create a fold on the abaxial side of the leaf. This fold was formed abaxially by periclinal and anticlinal divisions, effectively eliminating intercellular spaces from the spongy parenchyma. Galls at this stage exhibited both cell hypertrophy and tissue hyperplasia. Cells on the adaxial surface were more numerous and smaller than cells near the abaxial surface were, creating the large fold that surrounds the insect. Mesophyll cells exhibited some features typical of nutritive cells induced by other galling insects, including conspicuous nucleolus, reduced and fragmented vacuole, smaller and degraded chloroplasts, and dense cytoplasm compared to ungalled tissue. Even though aphids feed on the contents of phloem and do not directly consume the gall tissue, they induce changes in the plant vascular system, which lead to nutrient accumulation to support the growing aphid numbers in mature galls.

The Arabidopsis immune regulator SRFR1 dampens defences against herbivory by Spodoptera exigua and parasitism by Heterodera schachtii.

Plants have developed diverse mechanisms to fine tune defence responses to different types of enemy. Cross-regulation between signalling pathways may allow the prioritization of one response over another. Previously, we identified SUPPRESSOR OF rps4-RLD1 (SRFR1) as a negative regulator of ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1)-dependent effector-triggered immunity against the bacterial pathogen Pseudomonas syringae pv. tomato strain DC3000 expressing avrRps4. The use of multiple stresses is a powerful tool to further define gene function. Here, we examined whether SRFR1 also impacts resistance to a herbivorous insect in leaves and to a cyst nematode in roots. Interestingly, srfr1-1 plants showed increased resistance to herbivory by the beet army worm Spodoptera exigua and to parasitism by the cyst nematode Heterodera schachtii compared with the corresponding wild-type Arabidopsis accession RLD. Using quantitative real-time PCR (qRT-PCR) to measure the transcript levels of salicylic acid (SA) and jasmonate/ethylene (JA/ET) pathway genes, we found that enhanced resistance of srfr1-1 plants to S. exigua correlated with specific upregulation of the MYC2 branch of the JA pathway concurrent with suppression of the SA pathway. In contrast, the greater susceptibility of RLD was accompanied by simultaneously increased transcript levels of SA, JA and JA/ET signalling pathway genes. Surprisingly, mutation of either SRFR1 or EDS1 increased resistance to H. schachtii, indicating that the concurrent presence of both wild-type genes promotes susceptibility. This finding suggests a novel form of resistance in Arabidopsis to the biotrophic pathogen H. schachtii or a root-specific regulation of the SA pathway by EDS1, and places SRFR1 at an intersection between multiple defence pathways.

Shared weapons of blood- and plant-feeding insects: Surprising commonalities for manipulating hosts.

Insects that reprogram host plants during colonization remind us that the insect side of plant-insect story is just as interesting as the plant side. Insect effectors secreted by the salivary glands play an important role in plant reprogramming. Recent discoveries point to large numbers of salivary effectors being produced by a single herbivore species. Since genetic and functional characterization of effectors is an arduous task, narrowing the field of candidates is useful. We present ideas about types and functions of effectors from research on blood-feeding parasites and their mammalian hosts. Because of their importance for human health, blood-feeding parasites have more tools from genomics and other - omics than plant-feeding parasites. Four themes have emerged: (1) mechanical damage resulting from attack by blood-feeding parasites triggers "early danger signals" in mammalian hosts, which are mediated by eATP, calcium, and hydrogen peroxide, (2) mammalian hosts need to modulate their immune responses to the three "early danger signals" and use apyrases, calreticulins, and peroxiredoxins, respectively, to achieve this, (3) blood-feeding parasites, like their mammalian hosts, rely on some of the same "early danger signals" and modulate their immune responses using the same proteins, and (4) blood-feeding parasites deploy apyrases, calreticulins, and peroxiredoxins in their saliva to manipulate the "danger signals" of their mammalian hosts. We review emerging evidence that plant-feeding insects also interfere with "early danger signals" of their hosts by deploying apyrases, calreticulins and peroxiredoxins in saliva. Given emerging links between these molecules, and plant growth and defense, we propose that these effectors interfere with phytohormone signaling, and therefore have a special importance for gall-inducing and leaf-mining insects, which manipulate host-plants to create better food and shelter.

Plant vascular architecture determines the pattern of herbivore-induced systemic responses in Arabidopsis thaliana.

The induction of systemic responses in plants is associated with the connectivity between damaged and undamaged leaves, as determined by vascular architecture. Despite the widespread appreciation for studying variation in induced plant defense, few studies have characterized spatial variability of induction in the model species, Arabidopsis thaliana. Here we show that plant architecture generates fine scale spatial variation in the systemic induction of invertase and phenolic compounds. We examined whether the arrangement of leaves along the stem (phyllotaxy) produces predictable spatial patterns of cell-wall bound and soluble invertase activities, and downstream phenolic accumulation following feeding by the dietary specialist herbivore, Pieris rapae and the generalist, Spodoptera exigua. Responses were measured in leaves within and outside of the damaged orthostichy (leaves sharing direct vascular connections), and compared to those from plants where source-sink transport was disrupted by source leaf removal and by an insertional mutation in a sucrose transporter gene (suc2-1). Following herbivore damage to a single, middle-aged leaf, induction of cell-wall and soluble invertase was most pronounced in young and old leaves within the damaged orthostichy. The pattern of accumulation of phenolics was also predicted by these vascular connections and was, in part, dependent on the presence of source leaves and intact sucrose transporter function. Induction also occurred in leaves outside of the damaged orthostichy, suggesting that mechanisms may exist to overcome vascular constraints in this system. Our results demonstrate that systemic responses vary widely according to orthostichy, are often herbivore-specific, and partially rely on transport between source and sink leaves. We also provide evidence that patterns of induction are more integrated in A. thaliana than previously described. This work highlights the importance of plant vascular architecture in determining patterns of systemic induction, which is likely to be ecologically important to insect herbivores and plant pathogens.

Transcriptional and metabolic signatures of Arabidopsis responses to chewing damage by an insect herbivore and bacterial infection and the consequences of their interaction.

Plants use multiple interacting signaling systems to identify and respond to biotic stresses. Although it is often assumed that there is specificity in signaling responses to specific pests, this is rarely examined outside of the gene-for-gene relationships of plant-pathogen interactions. In this study, we first compared early events in gene expression and later events in metabolite profiles of Arabidopsis thaliana following attack by either the caterpillar Spodoptera exigua or avirulent (DC3000 avrRpm1) Pseudomonas syringae pv. tomato at three time points. Transcriptional responses of the plant to caterpillar feeding were rapid, occurring within 1 h of feeding, and then decreased at 6 and 24 h. In contrast, plant response to the pathogen was undetectable at 1 h but grew larger and more significant at 6 and 24 h. There was a surprisingly large amount of overlap in jasmonate and salicylate signaling in responses to the insect and pathogen, including levels of gene expression and individual hormones. The caterpillar and pathogen treatments induced different patterns of expression of glucosinolate biosynthesis genes and levels of glucosinolates. This suggests that when specific responses develop, their regulation is complex and best understood by characterizing expression of many genes and metabolites. We then examined the effect of feeding by the caterpillar Spodoptera exigua on Arabidopsis susceptibility to virulent (DC3000) and avirulent (DC3000 avrRpm1) P. syringae pv. tomato, and found that caterpillar feeding enhanced Arabidopsis resistance to the avirulent pathogen and lowered resistance to the virulent strain. We conclude that efforts to improve plant resistance to bacterial pathogens are likely to influence resistance to insects and vice versa. Studies explicitly comparing plant responses to multiple stresses, including the role of elicitors at early time points, are critical to understanding how plants organize responses in natural settings.

Roles for jasmonate- and ethylene-induced transcription factors in the ability of Arabidopsis to respond differentially to damage caused by two insect herbivores.

Plant responses to insects and wounding involve substantial transcriptional reprogramming that integrates hormonal, metabolic, and physiological events. The ability to respond differentially to various stresses, including wounding, generally involves hormone signaling and trans-acting regulatory factors. Evidence of the importance of transcription factors (TFs) in responses to insects is also accumulating. However, the relationships among hormone signaling, TF activity, and ability to respond specifically to different insects are uncertain. We examined transcriptional and hormonal changes in Arabidopsis thaliana after herbivory by larvae of two lepidopteran species, Spodoptera exigua (Hübner) and Pieris rapae L. over a 24-h time course. Transcriptional responses to the two insects differed and were frequently weaker or absent in response to the specialist P. rapae. Using microarray analysis and qRT-PCR, we found 141 TFs, including many AP2/ERFs (Ethylene Response Factors) and selected defense-related genes, to be differentially regulated in response to the two insect species or wounding. Jasmonic Acid (JA), JA-isoleucine (JA-IL), and ethylene production by Arabidopsis plants increased after attack by both insect species. However, the amounts and timing of ethylene production differed between the two herbivory treatments. Our results support the hypothesis that the different responses to these two insects involve modifications of JA-signaling events and activation of different subsets of ERF TFs, resulting in different degrees of divergence from responses to wounding alone.

Flexible resource allocation during plant defense responses.

Plants are organisms composed of modules connected by xylem and phloem transport streams. Attack by both insects and pathogens elicits sometimes rapid defense responses in the attacked module. We have also known for some time that proteins are often reallocated away from pathogen-infected tissues, while the same infection sites may draw carbohydrates to them. This has been interpreted as a tug of war in which the plant withdraws critical resources to block microbial growth while the microbes attempt to acquire more resources. Sink-source regulated transport among modules of critical resources, particularly carbon and nitrogen, is also altered in response to attack. Insects and jasmonate can increase local sink strength, drawing carbohydrates that support defense production. Shortly after attack, carbohydrates may also be drawn to the root. The rate and direction of movement of photosynthate or signals in phloem in response to attack is subject to constraints that include branching, degree of connection among tissues, distance between sources and sinks, proximity, strength, and number of competing sinks, and phloem loading/unloading regulators. Movement of materials (e.g., amino acids, signals) to or from attack sites in xylem is less well understood but is partly driven by transpiration. The root is an influential sink and may regulate sink-source interactions and transport above and below ground as well as between the plant and the rhizosphere and nearby, connected plants. Research on resource translocation in response to pathogens or herbivores has focused on biochemical mechanisms; whole-plant research is needed to determine which, if any, of these plant behaviors actually influence plant fitness.

Temporal changes in allocation and partitioning of new carbon as (11)C elicited by simulated herbivory suggest that roots shape aboveground responses in Arabidopsis.

Using the short-lived isotope (11)C (t(1/2) = 20.4 min) as (11)CO(2), we captured temporal changes in whole-plant carbon movement and partitioning of recently fixed carbon into primary and secondary metabolites in a time course (2, 6, and 24 h) following simulated herbivory with the well-known defense elicitor methyl jasmonate (MeJA) to young leaves of Arabidopsis (Arabidopsis thaliana). Both (11)CO(2) fixation and (11)C-photosynthate export from the labeled source leaf increased rapidly (2 h) following MeJA treatment relative to controls, with preferential allocation of radiolabeled resources belowground. At the same time, (11)C-photosynthate remaining in the aboveground sink tissues showed preferential allocation to MeJA-treated, young leaves, where it was incorporated into (11)C-cinnamic acid. By 24 h, resource allocation toward roots returned to control levels, while allocation to the young leaves increased. This corresponded to an increase in invertase activity and the accumulation of phenolic compounds, particularly anthocyanins, in young leaves. Induction of phenolics was suppressed in sucrose transporter mutant plants (suc2-1), indicating that this phenomenon may be controlled, in part, by phloem loading at source leaves. However, when plant roots were chilled to 5°C to disrupt carbon flow between above- and belowground tissues, source leaves failed to allocate resources belowground or toward damaged leaves following wounding and MeJA treatment to young leaves, suggesting that roots may play an integral role in controlling how plants respond defensively aboveground.

Is polyphenol induction simply a result of altered carbon and nitrogen accumulation?

Carbon translocation in plants is shaped by phyllotaxis and regulated by source/sink interactions that respond to the demands of growth and defense. We have studied this extensively in poplar saplings, and recently showed that unlike carbon import, nitrogen is not translocated to sink leaves in response to application of jasmonic acid. Here we report that this is also true for young trees in the field. We discuss the importance of transport processes in establishing local C:N ratios, and suggest that the JA-induced flow of C but not N to sink tissues, and their corresponding increases in C-based defenses, may simply reflect a plant adaptation to handle excess reduced carbon and energy.

Novel application of 2-[(18)F]fluoro-2-deoxy-D-glucose to study plant defenses.

INTRODUCTION: Since its first use in humans in 1976, 2-[¹8F]fluoro-2-deoxy-d-glucose (¹8FDG) continues to serve as a tracer to measure tissue glucose metabolism in medical imaging. Here we demonstrate a novel use for this tracer to study glycoside biosynthesis in plants as a measure of plant response to defense induction. METHODS: Coupling autoradiography with radio high-performance liquid chromatography analysis of tissue extracts, we examined the combined effects of leaf wounding and treatment using the potent plant defense hormone, methyl jasmonate (MeJA), to measure tracer distribution and tracer use in secondary defense chemistry in Arabidopsis thaliana. We hypothesized that competing sinks like roots and reproductive tissues, as well as vascular architecture, would impact the induction of phenolic defenses of the plant that make use of glucose in glycoside formation by altering distribution and metabolic utilization of ¹8FDG. RESULTS: Our studies showed that leaf orthostichy defined the major route of ¹8FDG transport in both vegetative and reproductive plants when a single petiole was cut as the entry point for tracer introduction. However, when nonorthostichous leaves were damaged and treated with MeJA, ¹8FDG was transported in its intact form to these leaves 3 h later, where it was incorporated into phenolic glycosides. CONCLUSIONS: Our work demonstrates a new use for ¹8FDG in plant science with insights into carbohydrate allocation that contradict conclusions of previous studies showing transport of resources away from damaged sites.

Effects of jasmonic acid, branching and girdling on carbon and nitrogen transport in poplar.

• Here, we examined the impact of jasmonate (JA) treatment, branching and phloem girdling on ¹³C and ¹5N import, invertase activity and polyphenol accumulation in juvenile tissues of unbranched and branched hybrid poplar saplings (Populus nigra × P. deltoides). • The import of ¹³C to juvenile tissues was positively correlated with invertase activity at the treatment site and enhanced by JA. Both invertase activity and ¹³C import were greater in shorter, younger branches and smaller, younger leaves. By contrast, JA treatments, branching and girdling had little or no impact on ¹5N import. • In poplar saplings with multiple lateral branches, we observed almost no ¹³C movement from subtending source leaves into lateral branches above them, with or without JA treatment. The presence of potentially competing branches, treated with JA or not, girdled or not, had no impact on carbohydrate (CHO) import or polyphenol accumulation in target branches. • We conclude that poplar branches comprise modules that are relatively independent from each other and from the stem below in terms of CHO movement, carbon-based defence production and response to elicitors. By contrast, branches are closely linked modules in terms of nitrogen movement. This should produce trees that are highly heterogeneous in quality for herbivores.

Adaptive two-dimensional microgas chromatography.

We proposed and investigated a novel adaptive two-dimensional (2-D) microgas chromatography system, which consists of one 1st-dimensional column, multiple parallel 2nd-dimensional columns, and a decision-making module. The decision-making module, installed between the 1st- and 2nd-dimensional columns, normally comprises an on-column nondestructive vapor detector, a flow routing system, and a computer that monitors the detection signal from the detector and sends out the trigger signal to the flow routing system. During the operation, effluents from the 1st-dimensional column are first detected by the detector and, then, depending on the signal generated by the detector, routed to one of the 2nd-dimensional columns sequentially for further separation. As compared to conventional 2-D GC systems, the proposed adaptive GC scheme has a number of unique and advantageous features. First and foremost, the multiple parallel columns are independent of each other. Therefore, their length, stationary phase, flow rate, and temperature can be optimized for best separation and maximal versatility. In addition, the adaptive GC significantly lowers the thermal modulator modulation frequency and hence power consumption. Finally, it greatly simplifies the postdata analysis process required to reconstruct the 2-D chromatogram. In this paper, the underlying working principle and data analysis of the adaptive GC was first discussed. Then, separation of a mixture of 20 analytes with various volatilities and polarities was demonstrated using an adaptive GC system with a single 2nd-dimensional column. Finally, an adaptive GC system with dual 2nd-dimensional columns was employed, in conjunction with temperature ramping, in a practical application to separate a mixture of plant emitted volatile organic compounds with significantly shortened analysis time.

Hormaphis hamamelidis fundatrices benefit by manipulating phenolic metabolism of their host.

We investigated the pattern and potential adaptive value of phenolic concentrations in galls induced by the aphid Hormaphis hamamelidis on leaves of Hamamelis virginiana. By the time that founding females began reproduction, galls had higher concentrations of condensed tannins and lower concentrations of hydrolyzable tannins than leaves. Galled and ungalled leaf laminas never differed significantly in any phenolic measure. Condensed tannin concentrations also were positively related to the number of offspring per gall when gall dry weight, another important correlate of fecundity, was accounted for. This could indicate the prior sink strength of the gall. Polyphenols may act as a repository for excess carbon drawn to the gall by increased sink strength, or be an indication of the fundatrix' ability to manipulate host physiology. This study is the first to demonstrate a tangible, quantitative association between phenolic accumulation in galls and gall-former reproductive performance, and illustrates that condensed tannins may play roles other than plant defense.

Measuring 'normalcy' in plant gene expression after herbivore attack.

Plants make drastic changes to their transcriptome to appropriately respond to environmental change, and the regulation of genes that are specific to abiotic and biotic stresses is a key to plant survival. The coordination of defence gene transcription is often coupled with significant adjustments in the levels of expression of primary metabolic and structural genes to relocate resources, repair damage and/or induce senescence. This complicates the process of finding suitable 'housekeeping' or reference genes to use in measurements of gene expression by real-time reverse transcription (RT-PCR) in response to herbivore attack. Several software programs have been developed to identify candidate reference genes, but measurement of their expression may still not yield an appropriate gene or suite of genes for normalization. This is especially true in plant-herbivore interactions where tissue damage is immediate and continuous. Here, we show that 12 traditional reference genes customarily used in RT-PCR analysis are not stably expressed after insect attack. We describe the pitfalls of using traditional reference genes and why insect attack may be affecting whole cell metabolism. We propose a method using RNA quantification in combination with an external spike of commercially available mRNA as normalization factors in studies involving herbivory, multiple stress treatments or species where stable reference genes are unknown.