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Consequences of russet mite-induced tomato defenses for community interactions

Glas, J.J.

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Citation for published version (APA):Glas, J. J. (2014). Consequences of russet mite-induced tomato defenses for community interactions.

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Analysis of transcriptome-wide changesin tomato during defense suppressionby spider mites and russet mites revealsdistinct differences in cell wall modification and hormone metabolism

J.J. Glas, J.M. Alba, R.C. Schuurink, M.W. Sabelis & M.R. Kant

Tomatoes are commonly attacked by different herbivores. Among these are phy-tophagous mites, including the generalist Tetranychus urticae and the specialistsTetranychus evansi and Aculops lycopersici. Tomato defense responses to spidermites have been relatively well characterized: whereas the common genotypes ofT. urticae activate the jasmonic acid (JA)- and salicylic acid (SA)-dependent path-ways, T. evansi was found to suppress both these defense pathways. In contrast,A. lycopersici up-regulates SA-mediated defenses but down-regulates JA-dependent defenses. In order to get a broader overview of these differences, wecompared phytohormone- and transcriptome-wide responses in leaflets afterattack by T. urticae, T. evansi, A. lycopersici or the combination of T. urticae and A.lycopersici using LC-MS and microarray analysis, respectively. Tetranychus urticaeand A. lycopersici altered the expression of a comparable number of genes (ca.15%) compared to uninfested control plants of which approximately 50% was reg-ulated similarly by both mite species. In contrast, T. evansi influenced the expres-sion of only 2% of the plant genes in comparison to uninfested plants. Moreover,the absolute magnitude of up- or down-regulation of tomato genes was muchsmaller in the case of T. evansi. In particular genes annotated as having a role inphotosynthesis, cell wall modification, hormone metabolism and stress responses(e.g., PR-proteins; glutathione S-transferases; cytochrome P450s; peroxidases;WRKY transcription factors) were affected by T. urticae as well as by A. lycopersi-ci. Both defense-inducing and defense-suppressing mites caused a down-regula-tion of photosynthesis-related genes with the largest number of genes beingaffected in leaflets infested with both species simultaneously. In addition, uniquely-regulated genes were identified as well, for instance a xylanase inhibitor up-regu-lated only by A. lycopersici and possibly important in mediating plant defenseresponses. Finally, several hypothesizes are discussed that may explain our obser-vations in the context of recent work on the relation between defenses andresource allocation.

In nature plants are regularly confronted with biotic attackers against which theyhave to defend themselves. Induced defense responses, which, together with con-

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stitutive defenses, determine the capability of a plant to withstand these attackers,depend mainly on the action of the phytohormones jasmonic acid (JA) and salicylicacid (SA). These two phytohormones, together with several others (Erb et al., 2012),regulate anti-herbivore and pathogen defenses, which includes the synthesis of tox-ins and inhibitors of food digestion (Howe & Jander, 2008) and re-allocation ofresources (Gómez et al., 2012).

Tomato (Solanum lycopersicum) has become a well-established model organismto elucidate wound- and herbivore-induced defense mechanisms in plants (e.g.,Ryan, 2000; Li et al., 2004; Scranton et al., 2013). Only a limited number of microar-ray studies have been undertaken to identify tomato transcriptional responses to her-bivory, for instance to the two-spotted spider mite Tetranychus urticae (Ament et al.,2004; Kant et al., 2004; Li et al., 2004), caterpillars of the beet armyworm(Spodoptera exigua) (Rodriguez-Saona et al., 2010) and aphids (Macrosiphumeuphorbiae) (Rodriguez-Saona et al., 2010). Spider mites (Tetranychus spp.) use theirpaired stylets to feed from parenchyma cells leading to chlorotic lesions were cellshave been emptied and it has been estimated that approximately 1 mm2 of leaf areais damaged per day per adult spider mite on tomato (Kant et al., 2004).

The two-spotted spider mite T. urticae, which is highly polyphagous (Dermauw etal., 2012), induces both JA- and SA-regulated genes, among which are JA-depend-ent wound-induced proteinase inhibitors (PINs), SA-responsive pathogenesis-relatedproteins (PR-proteins), and genes depending on both JA and SA signaling, such asGeranylgeranyl Pyrophosphate Synthase1 (GGPS1), which is involved in productionof plant volatiles (Kant et al., 2004; Ament et al., 2006).

Closely related to T. urticae is the red tomato spider mite T. evansi which has spe-cialized to feed on solanaceous plants in Brazil and Africa and has recently made itsentry into Europe (Boubou et al., 2012). Just like some rare T. urticae genotypes (Kantet al., 2008), T. evansi was found to suppress the plant’s defense responses(Sarmento et al., 2011). The mechanism by which this suppression occurs is notknown, but appears to be independent from the JA/SA-antagonism, since bothdefense pathways are suppressed by T. evansi (Alba et al., submitted). Adult femalesof both spider mite species produce, depending on the temperature, around 5-12eggs per day on tomato (Sarmento et al., 2011), which develop in reproducing adultsthemselves within 2 weeks, resulting in exponential population growth.

A third phytophagous mite pest of tomato is the specialist tomato russet miteAculops lycopersici, which, like T. evansi, has specialized to use solanaceous plantsas host. However, since adult russet mites have much shorter stylets (ca. 10 μm)compared to adult spider mites (ca. 150 μm) (Park & Lee, 2002), they do not reachthe mesophyll layer but instead feed only on epidermal cells of leaves and stems oftomato. Russet mites do not only target very different tomato tissues compared to

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spider mites, but also have a different effect on induced responses as they werefound to induce SA-mediated defenses but suppress JA-dependent defenses(CHAPTER 2). Hence, the A. lycopersici-induced response is distinct from T. urticae,which induces both the JA- and SA-pathway, as well as that from T. evansi, whichsuppresses both defense pathways.

To enhance our understanding of the differences between defense suppressionand induction by these three mite species, we compared transcriptional changes inleaflets that had been infested with T. urticae, T. evansi, A. lycopersici or with thecombination of T. urticae and A. lycopersici using a 60-mer oligonucleotide microar-ray (Agilent/Nimblegen) carrying 44195 probes designed on public tomato ESTlibraries. In addition, we measured phytohormone levels in the same tissues used forthe transcriptome analysis to compare transcriptional responses with the accumula-tion of the phytohormones 12-oxo-phytodienoic acid (OPDA), JA, jasmonic acid-isoleucine (JA-Ile), SA and abscisic acid (ABA).

Material and methodsPlants and mitesTomato seeds [S. lycopersicum cv. Castlemart (CM)] were germinated in soil andgrown in a greenhouse compartment at a temperature of 25ºC and a 15/9 h light/darkregime. One week prior to the start of the experiment, plants were transferred to aclimate room at 25ºC and a 16/8 h light/dark regime with 300 μE m-2 s-1 and 60%RH. Plants were infested when they were 21 days old and samples when 28 days old.

The tomato russet mite (A. lycopersici) was obtained from Koppert BiologicalSystems (Berkel en Rodenrijs, The Netherlands) who in turn had collected them in2008 from a tomato greenhouse in the Westland area (The Netherlands). Russetmites were reared in insect cages (BugDorm-44590DH, Bug Dorm Store, MegaViewScience, Taichung, Taiwan) in a climate room (day/night temperature of 27ºC/25ºC, a16/8 h light/dark regime and 60% RH) on tomato plants (cv. CM) that were between3 and 5 weeks old.

The two-spotted spider mite (T. urticae) strain we used was originally obtained in2001 from a single European spindle tree (Euonymus europaeus) in the dunes nearSantpoort (The Netherlands) (GPS coordinates: 52 26.503 N 4 36.315 E), and hasbeen described as T. urticae Santpoort-2 (Alba et al., submitted). In a previous study,where it was referred to as ‘KMB’, T. urticae Santpoort-2 was characterized as an‘inducer’ of JA-defenses and as susceptible to these defenses (Kant et al., 2008).Since its collection from the field, the strain has been propagated on detached bean(Phaseolus vulgaris) leaves that were placed with the abaxial surface on wet cottonwool and maintained in a climate room (temperature of 25ºC, a 16/8 h light/dark and60% RH).

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The tomato red spider mite (T. evansi) was collected in a tomato greenhouse onthe campus of the University of Viçosa (Brazil) (GPS coordinates: 20 45.473 S 4252.163 W) where it was maintained on S. lycopersicum (cv. Santa Clara) as describedpreviously (Sarmento et al., 2011). In 2009, 10 heavily infested leaves from this pop-ulation were transferred to Amsterdam where it was subsequently propagated ondetached leaves of S. lycopersicum (cv. CM) in a climate room (temperature of 25ºC,a 16/8 h light/dark and 60% RH).

The species identity of A. lycopersici was confirmed on the basis of the morphol-ogy of the prodorsal shield (F. Faraji, personal observation). The species identity of T.urticae and T. evansi was confirmed on the basis of the morphology of the malereproductive organ (the aedeagus) and on the basis of a phylogenetic constructionusing the mitochondrial cytochrome oxidase subunit 1 (CO1) sequences (cf. Alba etal., submitted). The CO1-sequences and sequencing primers can be found inGenBank under the following IDs: T. urticae: KF447571 and T. evansi: KF447575.

Infestation and sampling of plantsAt the start of the experiments, 21-day-old tomato plants were infested simultane-ously with T. evansi, T. urticae, A. lycopersici or with T. urticae and A. lycopersicitogether. Spider mite infestations were performed by individually transferring adultfemales, randomly collected from the rearing colony, with a fine brush to each ofthree leaflets per plant. Each leaflet received 15 spider mites so that in total eachplant was infested with 45 spider mites. Russet mite infestations were performed bytransferring mites on small pieces of leaflets (ca. 0.5 cm²) to the leaflets of uninfest-ed plants. These leaflet pieces had been cut from leaves picked from a well-infestedtomato plant and each piece contained ca. 250 mobile stages of RM as determinedwith a stereomicroscope. To prevent mites from dispersing we applied a thin barrierof lanolin (Sigma-Aldrich Chemie, Zwijndrecht, The Netherlands) on the petioles ofleaflets that were chosen for infestation. Uninfested control plants received the lano-lin barrier, but no mites. Within an experiment, each plant was taken as one replicate(i.e., three leaflets per plant were later on pooled together for RNA extraction). In totalsix plants were infested per treatment. Sampled leaflets were flash-frozen in liquidnitrogen and stored at -80°C until total RNA was extracted. Care was taken to alwayspick leaflets with the same position for infestation, i.e., one leaflet of the second com-pound leaf, one from the third compound leaf and the terminal leaflet of the fourthcompound leaf (counted from the bottom to the top of the plant). Samplings wereperformed at the same time of the day on day 7 after infestation. The experiment wasrepeated 4× during four consecutive weeks in March 2012. Hence, in total there were120 samples (6 samples × 5 treatments × 4 replicate trials).


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RNA isolationLeaflets were ground in liquid nitrogen, and total RNA was extracted using a phenol-LiCl-based method as described by Verdonk et al. (2003). The integrity of RNA waschecked on 1% agarose gels. Total RNA was subsequently quantified using aNanoDrop 100 spectrophotometer (Fisher Scientific, Loughborough, UK).

Microarray hybridizationsFor the microarray hybridizations, equal amounts of RNA were pooled per treatment(i.e., the six RNA samples obtained from the same trail week and the same treatmenttype were pooled). Per pooled treatment one array was hybridized so that in total 20arrays were used (5 treatments × 4 replicate trials weeks). The probe sequences (60-mer oligos) were identical to those from the tomato Agilent 44k array (Agilent, SantaClara, CA, USA) and included ~600 custom sequences. The probes were synthe-sized in spots on the 135k Nimblegen array format (Nimblegen, Madison, WI, USA)having each probe represented by three spots on each array. Preparation, labeling,purification and hybridizations were carried out according to protocols provided bythe manufacturer (Nimblegen). Samples were cy3 labeled and these were normalizedacross arrays via a common reference design using a cy5 labeled pool of all the sam-ples as the reference on each array.

Microarray analysisFirst, all signal intensities were normalized within each array using Lowess.Subsequently, all triplicate probe-sets were averaged. These averages were thennormalized across arrays by means of the averaged common reference signals.These relative intensities are equivalent to the relative expression levels of the genesrepresented by each probe.

For data analysis we selected only those probes that gave a good hit on the pre-dicted genome CDSs available at the SGN database (Bombarely et al., 2011), i.e.,those that had a match smaller or equal to 1E-20 (corresponding to maximally threemismatches between the 60-mer probe and the target sequence). Applying this cri-terion resulted in a list of 28077 probes suitable for the analysis (64% of the total of44195 that were spotted on the array). Since many of these probes were duplicatedon the array (i.e., different probes corresponding to the same gene) we subsequent-ly filtered the array to select for the unique genes. This yielded a total number of15504 genes that were represented on the array (~50% of the total number of 31760tomato genes). In the case of duplicated probes the values for the individual probeswere averaged. Significant differences in gene expression between control and treat-ment samples were identified using a nested ANOVA on the log2-transformed rela-tive expression values. P-values were adjusted according to Benjamini & Hochberg’s


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step-up procedure for controlling the False Discovery Rate (FDR) (Benjamini &Hochberg, 1995). The P-values of the arrays on which <5% of all genes were signif-icantly regulated before P-value correction (which was the case for the T. evansi sam-ples) were adjusted via Simes’ step up FDR procedure (Simes, 1986) which behavesa little less stringent at small fractions of regulated genes (Bretz et al., 2005).Differentially expressed genes were identified based on an adjusted P-value <0.05.Differences in gene expression between treatments were visualized using MapMan.In MapMan, large data sets can be projected onto metabolic pathways or assignedto functional categories (called ‘BINs’, ‘subBINs’) thereby allowing for detection oftrends that might be less apparent when looking at individual genes (Thimm et al.,2004). In MapMan, we performed a so-called over/under-representation or enrich-ment analysis (FIGURE 4.2). Here, the experiment is divided into up- and down-regu-lated genes. In both cases, if a category has more genes than expected exceedingthe threshold it is colored in green, if it has less than colored in red. Over- or under-representation was tested by applying Fisher’s test for over representation analysis(ORA) with the threshold set to 1. Since the Benjamini & Hochberg multiple testingcorrection is known as a conservative approach, which can sometimes be too strin-gent (Huang et al., 2009) we performed the same analysis with and without theBenjamini & Hochberg correction. Classes in FIGURE 4.2 that were found to be signif-icantly different also after the Benjamini & Hochberg correction are indicated by anasterisk. For the ORA only those genes where selected where at least one of thetreatments was significantly different from the control (adjusted P-value <0.05).

Phytohormone analysisPhytohormones were extracted and quantified by means of LC-MS as described inCHAPTER 2. Phytohormone data were log-transformed and analyzed using ANOVA,with ‘Treatment’ (with the levels: ‘Control’, ‘T. urticae’, ‘T. evansi’, ‘A. lycopersici’ and‘T. urticae + A. lycopersici’) as fixed factor and ‘Experimental Replicate’ (with levels1-4) included as random factor in the model. The means of each group were com-pared using Fisher’s least significant difference (LSD) post hoc test.

Results and DiscussionPhytohormone analysisSince transcriptional responses are tightly regulated by phytohormones, we firstmeasured phytohormone accumulation in the same samples as used for the tran-scriptome analysis. Tetranychus urticae induced a significant increase in levels of theJA-precursor OPDA, JA and JA-Isoleucine (JA-Ile), the bio-active form of JA, as wellas SA and ABA, when compared to uninfested control plants (FIGURE 4.1). In contrast,T. evansi did not induce the accumulation of OPDA, JA and ABA, whereas levels of


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JA-Ile and SA were slightly higher in T. evansi-infested leaflets in comparison to con-trols, but ca. 6- and 3-fold lower when compared to T. urticae-infested leaflets.Similar to T. urticae, also A. lycopersici induced a significant increase in levels ofOPDA, JA and JA-Ile and SA although the levels were lower in comparison to leafletsinfested with T. urticae. Aculops lycopersici also induced a significant accumulation

FIGURE 4.1 Phytohormone levels in leaflets infested with different mite species. The figure shows the mean(+SEM) amounts (ng/g fresh weight; n=21-24 from four replicate experiments) of endogenous OPDA (A), JA (B),JA-Ile (C), SA (D) and ABA (E) in uninfested and mite-infested wild-type (cv. CM) leaflets after 7 days of infesta-tion. C = Uninfested control plants, U = Tetranychus urticae. E = T. evansi, A = Aculops lycopersici. Different let-ters capping the bars indicate significant differences (ANOVA followed by Fisher’s LSD test: P<0.05). The samesamples were used for the transcriptome analysis.


of ABA, up to levels that were similar to those measured in leaflets infested with T.urticae.

In leaflets co-infested with T. urticae and A. lycopersici simultaneously, levels ofJA-Ile were significantly increased in comparison to leaflets that had been infestedwith T. urticae only or A. lycopersici only, whereas levels of SA and ABA in theseleaflets were similar to those in leaflets infested with T. urticae only (FIGURE 4.1).

Overview of transcriptional changesTetranychus evansi regulated 2% of the total number of the 15504 unique genes onthe array (98 up and 176 down compared to control plants), T. urticae 13% (1004 upand 1010 down compared to control plants), A. lycopersici 14% (1239 up and 980down compared to control plants) and the combination of T. urticae and A. lycoper-sici 16% (1232 up and 1287 down compared to control plants) (TABLE 4.1). One hun-dred eighteen genes were specifically regulated by T. evansi alone (1%) (22 up and96 down), 531 genes specifically by T. urticae alone (3%) (200 up and 331 down), 634genes specifically by A. lycopersici alone (5%) (329 up and 305 down) and 795specifically by the combination of T. urticae and A. lycopersici (6%) (289 up and 506down). Seventy-four genes were similarly regulated in all four treatments (0.5%) (56up and 18 down).

Six hundred sixty-nine genes were up-regulated by T. urticae and A. lycopersici(67% of the total number of T. urticae-induced genes; 54% of the total number of A.lycopersici-induced genes), whereas 443 genes were down-regulated by both mitespecies (44% of the total number of genes down-regulated by T. urticae; 34% of thetotal number of genes down-regulated by A. lycopersici). Three genes, among whichtwo GDSL esterases, were induced by A. lycopersici, yet down-regulated by T.urticae, whereas no genes were found that followed the reverse pattern, i.e., inducedby T. urticae but down-regulated by A. lycopersici. A total of 69 genes were inducedby A. lycopersici and T. evansi (70% of the total number of T. evansi-induced genes;21% of the total number of A. lycopersici-induced genes), whereas 38 genes weredown-regulated by both T. evansi and A. lycopersici (22% of the total number ofgenes down-regulated by T. evansi; 12% of the total number of genes down-regulat-ed by A. lycopersici). Two genes, i.e., a vesicle-associated membrane protein and asmall nucleolar ribonucleoprotein, were up-regulated by A. lycopersici, yet down-regulated by T. evansi. No genes were found that were up-regulated by T. evansi anddown-regulated by A. lycopersici.

Generally, T. evansi had a much weaker effect on the magnitude of tomato’s tran-scriptional up- and down regulation than the other two mite species as indicated bythe fact that not only the number of regulated genes was smaller in the case of T.evansi (TABLE 4.1), but also the fold-regulations were smaller when compared to the

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other two species. For instance, of the genes that were significantly up-regulated, A.lycopersici and T. urticae regulated, respectively, 583 (47% of the total number of A.lycopersici-induced genes) and 494 (49% of the total number of T. urticae-inducedgenes) genes more than 2-fold up, whereas T. evansi regulated only 24 genes (22%of the total number of T. evansi-induced genes) more than 2-fold up. Similarly, of thegenes that were significantly down-regulated, A. lycopersici and T. urticae regulated,respectively, 165 (17% of the total number of genes down-regulated by A. lycopersi-ci) and 96 genes (10% of the total number of genes down-regulated by T. urticae)more than 2-fold, whereas T. evansi did not regulate any gene (0%) more than 2-folddown.

MapMan functional categories enriched in differentially regulated genesIn FIGURE 4.2 the result of a so-called PageMan analysis (Usadel et al., 2006) is shownfor which the experiment is divided in up- and down-regulated genes. The colorsgreen and red indicate whether genes from a particular category are over- or under-represented, respectively, in the total group of genes that are up- or down-regulatedby a particular treatment. The PageMan analysis confirms that the effect of T. evan-si on the tomato transcriptome is much more subtle than the effect of the other twomite species: whereas T. urticae and A. lycopersici had a significant effect on 18functional categories, only six of those [i.e., photosynthesis (‘PS’); ‘TCA/organic acidtransformation’; ‘N-metabolism’; ‘hormone metabolism’; ‘miscellaneous’; ‘protein’]were significantly affected by T. evansi as well.

Genes in the category ‘PS’, including those related to photosystem I and II, weresignificantly overrepresented in the group of down-regulated genes, whereas theywere underrepresented in the group of up-regulated genes and this was the case forall treatments. This suggests that also T. evansi affected photosynthesis, in a similarway as T. urticae and A. lycopersici, although the effect was weaker as indicated bythe fact that only three photosynthesis subcategories were significantly affected by T.evansi, whereas 11 and six categories were affected by A. lycopersici and T. urticae,respectively (FIGURE 4.2). Furthermore, in plants simultaneously infested with T. urticaeand A. lycopersici, 16 photosynthesis subcategories were significantly influencedindicating that the effect on photosynthesis was more extensive in these plants com-pared to plants that had been infested with either of the two mite species alone.

TABLE 4.1 Tomato genes differentially expressed in response to mite feeding.Treatment Up-regulated No. genes down-regulated TotalTetranychus evansi 98 176 274T. urticae 1004 1010 2014Aculops lycopersici 1239 980 2219T. urticae and A. lycopersici 1232 1287 2519


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FIGURE 4.2 Pageman functional categories enriched in the genes that are differentially regulated by mites. Thedegree of enrichment among the up- and down-regulated genes is indicated by the different shades of green andred, respectively (green=overrepresented; red=underrepresented). Differentially regulated genes were identified asthose with adjusted P<0.05. E = Tetranychus evansi, A = Aculops lycopersici, U = T. urticae. Functional categoriesthat tested significantly different also after the Benjamini & Hochberg correction are indicated by an asterisk.



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Genes involved in ‘minor carbohydrate (CHO) metabolism’ (i.e., the hexosemonophosphate pathway and uronic acid pathway) and ‘glycolysis’ were overrepre-sented among the down-regulated genes in T. urticae- and A. lycopersici-infestedleaflets, whereas genes related to the ‘TCA (tricarboxylic acid) cycle’ were generallyoverrepresented among the genes up-regulated by T. urticae and A. lycopersici,although not always (cf., e.g., the ‘carbonic anhydrases’ which were more oftenfound among the genes down-regulated by A. lycopersici).

Down-regulation of CHO metabolism genes has been observed before in plant-pathogen interactions, for instance in hot pepper (Capsicum annuum) plants that hadbeen infected with the pathogen Xanthomonas axonopodis (Lee et al., 2004). As sug-gested by Lee et al. (2004), this could indicate that attacked plants switch their car-bon flow from the primary metabolic pathway to secondary metabolism, for instanceto activate defenses.

Expression levels of cell wall-associated genes were affected by both T. urticaeand A. lycopersici when compared to uninfested control plants. For instance, genesin the subcategory ‘cell-wall proteins’, including the arabinogalactan proteins (AGPs),were overrepresented among the genes down-regulated by A. lycopersici and T.urticae. AGPs have been found on the plasma membrane and in the cell wall ofplants but their function has remained elusive (Ellis et al., 2010). Genes in the cate-gory ‘cell wall modification’ were overrepresented among the up-regulated genes intomatoes infested with either T. urticae or A. lycopersici. However, after applying theBenjamini & Hochberg correction, cell wall modification-related genes were still over-represented among the A. lycopersici-induced genes, but not among those up-reg-ulated by T. urticae, suggesting that the effect of A. lycopersici on cell wall modifica-tion is more pronounced compared to T. urticae.

Genes of the ‘aspartate family pathway’ involved in amino acid metabolism wereenriched in the A. lycopersici down-regulated gene-pool but not in the T. urticaedown-regulated gene pool. The aspartate family pathway is involved in the synthesisof essential amino acids, including lysine, threonine, methionine and isoleucine(Kirma et al., 2012). In contrast, for both T. urticae and A. lycopersici, genes involvedin the biosynthesis of the aromatic amino acids were more often found among theup-regulated genes. Furthermore, also genes of the phenylpropanoid pathway wereoverrepresented among the genes induced by A. lycopersici alone as well as amongthose induced by A. lycopersici and T. urticae together. Notably, genes in the cate-gory ‘terpenoids’ were specifically overrepresented among the T. urticae-inducedgenes. In addition, genes that have a role in lignin biosynthesis were overrepresent-ed among the up-regulated genes in the samples infested with both mite species andgenes of the subcategory ‘flavonoid chalcones’ were specifically overrepresentedamong the genes down-regulated by A. lycopersici (FIGURE 4.2).

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Phytohormone-related genes were affected by all three mite species, although notin the same way. The total pool of T. urticae-induced genes was enriched in JA-metabolism-related genes, whereas this was not the case for A. lycopersici.Remarkably, among the genes up-regulated by T. evansi there was a significantenrichment in transcripts encoding for the JA biosynthesis enzyme allene oxide syn-thase (AOS). Also, the pool of T. evansi-induced genes appeared to be enriched in SA-metabolism related genes. Genes related to auxin, cytokinin and ethylene signalingwere significantly more often found among the up-regulated genes in plants co-infest-ed with T. urticae and A. lycopersici. Strikingly, the group of A. lycopersici-inducedgenes was specifically enriched in gibberellin-related genes, whereas these were alsooverrepresented among the genes down-regulated by T. urticae. Genes involved in‘tetrapyrrole synthesis’ were overrepresented among the genes down-regulated by A.lycopersici, but not by T. urticae, although the reverse was true for the tetrapyrrolesubcategory ‘uroporphyrinogen carboxylase’. Tetrapyrroles, like chlorophyll, playimportant roles in photosynthesis and respiration in plants (Tanaka & Tanaka, 2007).

Then, a number of gene groups annotated as ‘stress proteins’, including PR-pro-teins, glutathione S transferases, peroxidases and WRKY transcription factors wereoverrepresented among the genes up-regulated by T. urticae and A. lycopersici,whereas auxin-related transcription factors were also overrepresented among thegenes down-regulated by T. urticae and by A. lycopersici. Genes involved in the cat-egory ‘signalling’ were significantly more often found in the pool of genes up-regu-lated by A. lycopersici and T. urticae. Genes in the signaling subcategory ‘sugar andnutrient physiology’ were specifically overrepresented in the gene pool induced byboth mite species. Finally, among the A. lycopersici up-regulated genes the ‘leucinerich repeat XI receptor kinases’ were found to be overrepresented and this was thecase only in the A. lycopersici-infested samples, whereas genes in the other two sig-naling subcategories (‘receptor kinases’ and ‘receptor kinases DUF26’) were over-represented in the pool of T. urticae-induced genes as well.

Transcriptional regulation of defense pathways by the different mitespeciesJA and SA are known as the major plant hormones responsible for defenses againstherbivores and pathogens, respectively. Hence, in the following text we discuss theeffect of the mites on these specific defense pathways in more detail.

The octadecanoid pathwayInduced defense responses against herbivores in tomato depend on the signalingpeptide systemin, which itself is derived from a larger protein, prosystemin, the lev-els of which are known to increase in response to wounding in tomato (Ryan, 2000).Systemin is perceived by the membrane-bound receptor SR160 resulting in the acti-

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vation of the octadecanoid pathway, as indicated by the activation of mitogen-acti-vated protein kinases (MAPKs) (MPK1, 2 and 3), synthesis of JA and expression ofdownstream defense genes (Kandoth et al., 2007). Even though prosystemin levelsare known to increase in response to wounding, on our array none of the mite-infes-tations changed the expression of this gene (FIGURE 4.3). JA synthesis takes placepartly in the chloroplast and partly in the peroxisome. First, the fatty acid substratelinolenic acid (LA) is released from membrane lipids by the action of phospholipasesand converted via the action of several enzymes into JA. In tomato it was shown thatwounding results in an increase in the levels of the early second messenger phos-phatidic acid (PA) (Munnik, 2001) as well as lysophospholipids, suggesting increasedactivity of the phospholipases PLA, PLD and/or PLC (Lee et al., 1997). Indeed,Narváez-Vásquez et al. (1999) showed that wounding as well as treatments with sys-temin increased activity of a phospholipase2 (PLA2) and this increased the concen-trations of lysophosphatidylcholine in tomato leaves. It is important to note that,although it is known that phospholipases hydrolyze phospholipids thereby releasinglysophosphatidylcholine and LA, there is much controversy about exactly whichlipases are responsible for the generation of the JA substrate. Candidates that havebeen proposed to be involved in JA biosynthesis in Arabidopsis include the phos-pholipases1 (PLA1) DEFECTIVE IN ANTHER DEHISCENSE 1 (DAD1), DONGLE (DGL)and PLA1γ1 (Wasternack & Hause, 2013). However, in tomato no lipases involved inJA biosynthesis have been characterized yet.

In our experiment, PLA2 expression appeared to be down-regulated by T. urticaeas well as by A. lycopersici, but it did not change in response to T. evansi (not shownin FIGURE 4.2). Arabidopsis plants that were silenced for expression of PLDβ1 con-tained lower levels of PA and JA as well as JA-related defense gene expression,which consequently made them more susceptible to B. cinerea (Zhao et al., 2013),showing that PLDβ1 is necessary for the formation JA in Arabidopsis. On our toma-to array, expression of PLDβ1 was induced by T. urticae as well as by A. lycopersici,but not by T. evansi, which correlates well with the JA levels induced by these mites(FIGURE 4.1). Apart from PLAs and PLDs, also PLCs are known to play a role in theformation of PA and in tomato responses to pathogen attack (Munnik, 2001; Vossenet al., 2010). The PLCs on the array showed distinct expression patterns in responseto infestation with mites. Expression of PLC1 was suppressed by JA-inducing mites,but not by T. evansi, whereas for PLC2 the opposite pattern was observed, i.e.,induction by A. lycopersici, yet not by T. evansi. Tetranychus urticae also inducedexpression of PLC2, albeit not significantly. For PLC4 and PLC6 the expression lev-els did not change in response to mite infestations.

The first committed step in the octadecanoid pathway is carried out by theenzyme lipoxygenase (LOX), which catalyzes the insertion of molecular oxygen into

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FIGURE 4.3 The JA-pathway is differentially regulated by the three mite species. Shown is an overview of themost important genes that have been connected to the JA-defense signaling pathway. Bars represent the rela-tive average (+SEM) expression values of each gene. All values were divided by the lowest average value (suchthat the lowest average is always 1). C = Uninfested control plants, U = Tetranychus urticae. E = T. evansi, A =Aculops lycopersici. Asterisks indicate significant differences with uninfested control plants. Expression values aregiven for Prosystemin (Prosys; Solyc05g051750.2); Phospholipase Dβ1 (PLDβ1; Solyc08g080130.2);Lipoxygenase C (LOX-C; Solyc01g006540.2); Lipoxygenase D (LOX-D; Solyc03g122340.2); Allene OxideSynthase (AOS; Solyc04g079730.1); Allene Oxide Cyclase (AOC; Solyc02g085730.2); Jasmonate Zim-domainProtein 1 (JAZ1; Solyc12g009220.1); Jasmonate Zim-domain Protein 2 (JAZ2; Solyc03g122190.2); JasmonateZim-domain Protein 3 (JAZ3; Solyc01g005440.2); Jasmonate Zim-domain Protein 8 (JAZ8; Solyc08g036660.2);Polyphenol Oxidase D (PPO-D; Solyc08g074680.2); Polyphenol Oxidase E/F (PPO-E/F; Solyc08g074630.1);Proteinase Inhibitor I (PI-I; Solyc09g084470.2); Proteinase Inhibitor II (PI-IIf; Solyc03g020080.2); ProteinaseInhibitor II (PI-IIc; Solyc03g020050.2); Kunitz-type Proteinase Inhibitor (KPI; Solyc03g098790.1) which is similarto Cathepsin D inhibitor (CDI; AJ295638.1); Threonine Deaminase (TD; Solyc09g008670.2); LeucineAminopeptidase (LAP; Solyc12g010020.1); and Salicylic Acid Carboxyl Methyltransferase (SAMT;Solyc09g091550.2).


position 13 of LA, resulting in hydroperoxy-linolenic acid, which is then further con-verted by the enzymes AOS and allene oxide cyclase (AOC) into the JA-intermediateOPDA (Turner et al., 2002; Wasternack et al., 2006). Subsequently, OPDA is export-ed from the chloroplast and imported into the peroxisomes where it is converted bythe OPDA reductase (OPR3) into cyclopentane-1-octanoic acid, followed by threecycles of β-oxidation to yield the final product JA (Wasternack et al., 2006). In a nextstep, JA is then conjugated to isoleucine (Ile) by the enzyme jasmonate resistant 1(JAR1) (Suza et al., 2010), leading to the formation of JA-Ile, which is the bioactiveform of JA (Fonseca et al., 2009).

Clearly, the enzymes involved in JA-biosynthesis were differentially regulated bythe mites. Tomato possesses at least six lipoxygenases (LOXs) of which TomloxA,TomloxB and TomloxE are expressed in fruits during ripening, whereas TomloxC andTomloxD are expressed in leaves as well (Chen et al., 2004; Hu et al., 2013).

TomloxC expression levels did not change in response to mite infestations. In con-trast, TomloxD was significantly up-regulated by T. urticae as well as by A. lycoper-sici, but not by T. evansi. TomloxD is known to be expressed in leaves and is inducedby wounding and treatment with systemin or methyl jasmonate (Heitz et al., 1997).Similar to TomloxD, also AOS and AOC were up-regulated by T. urticae and A. lycop-ersici feeding. Despite the fact that feeding by T. evansi did not induce JA accumu-lation (FIGURE 4.1), it did up-regulate expression of AOS, to a similar extent as feed-ing by T. urticae did. Expression of AOC followed a similar trend, although T. evansidid not induce this gene significantly.

Taken together, these data show that both T. urticae and A. lycopersici activate theoctadecanoid pathway. In contrast, T. evansi may suppress TomloxD and thereby theflux through the pathway, thus preventing JA to accumulate. Alternatively, low expres-sion of TomloxD may be due to indirect feedback regulation by down-stream signalingcomponents. The induction of AOS in T. evansi-infested plants may indicate an attemptof the plant to compensate for the low JA levels, but this remains to be tested.

Activation of JA-regulated responses depends on the binding of JA-Ile to theSCFCOI1 (Skip/Cullin/F-box) receptor complex. In an un-induced situation, transcrip-tion is not activated due to the binding of the Jasmonate ZIM domain (JAZ) repres-sor proteins to downstream transcription factors, like MYC2, which themselves arebound to JA-responsive elements present in the promoters of JA-responsive genes(Wasternack & Hause, 2013).

JAZ proteins are known as suppressors of the JA-response since newly synthe-sized JAZ proteins, of which the expression is induced in response to JA, can estab-lish negative feedback loops by binding MYC2, thereby blocking expression ofdownstream JA-responsive genes (Chini et al., 2007). JAZ8 is also known as a sup-pressor of the JA-response, but appears to execute this function independent from

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the SCFCOI1 complex (Shyu et al., 2012). Upon stimulation of the JA-pathway, theJAZ proteins are ubiquitinated by the F-box protein CORONATINE INSENSITIVE1(COI1) complex and subsequently degraded, thereby un-repressing transcriptionfactors such as MYC and thereby releasing the transcription of defense-relatedgenes. Examples of such defense-related genes in tomato include Wound-InducedProteinase Inhibitor I and II (WIPI-I and II) (Graham et al., 1985a,b), JasmonicInducible Protein-21 (JIP-21) (Lisón et al., 2006), Polyphenol Oxidase-F (PPO-F)(Newman et al., 1993), Threonine Deaminase (TD) (Gonzales-Vigil et al., 2011) andLeucine Aminopeptidase (LAP) (Fowler et al., 2009). However, whether (all of) theseare under control of the JAZ proteins is as yet not clear.

Both T. urticae and A. lycopersici up-regulated expression of JAZ1 as well as JAZ8,and this response was enhanced in plants infested with both species simultaneously.Tetranychus evansi also induced expression of JAZ1 and JAZ8 although to a muchlesser extent than T. urticae or A. lycopersici. In contrast, the expression of JAZ2 andJAZ3 did not change significantly in response to mite feeding (FIGURE 4.3). Importantly,the effect of JAZ proteins on the JA-response is regulated on the protein level sinceinduction of JA-accumulation not only induces their expression, but also their bindingto the SCFCOI1 complex and, thus, their degradation. Hence, this implies that it is dif-ficult to draw conclusions solely based on the expression patterns of these genes.

Even though T. urticae and A. lycopersici induced JAZ expression to a similarextent, the defense genes responded very differently. Whereas T. urticae feedingcaused a significant up-regulation of JA-dependent defense genes, including PI-I,PI-IIc, PI-IIf and LAP, none of these were induced by A. lycopersici. Note that PI-IIfis also known as WIPI-II (Alba et al., submitted). Furthermore, the expression patternfor KPI (also known as JIP-21 or Cathepsin D inhibitor (CDI) (Lisón et al., 2006), PPO-E/F and TD showed a very similar trend, although for these genes there was toomuch variation to detect statistically significant differences (FIGURE 4.3). Notably, inco-infested plants, the expression levels of PI-I, PI-IIf, KPI, PPO-E/F, TD and LAP,but not PI-IIc, were reduced to half the levels induced in plants infested with T.urticae only (FIGURE 4.3). Hence, these results confirm previous findings that in plantsco-infested with A. lycopersici, T. urticae-induced JA-defense responses are sup-pressed, rather than not-induced. Moreover, this suppression occurs downstream ofJA-biosynthesis since in co-infested leaflets levels of JA remained up-regulated, forJA-Ile even to additive levels (FIGURE 4.1). With the exception of LAP, T. evansi did notcause a significant up-regulation of JA-dependent defense genes, thereby confirm-ing previous findings that T. evansi suppresses plant defenses (Sarmento et al.,2011). Strikingly, expression of PI-IIc and PPO-D was not suppressed by A. lycoper-sici in co-infested plants. Expression of Salicylic Acid carboxyl Methyl Transferase(SAMT) was induced by all three mite species, and, like PPO-D and PI-IIc, also this

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gene was not suppressed in co-infested plants. SAMT, which depends on JA-signal-ing, is known to be involved in the biosynthesis of the volatile methyl salicylate(MeSA) (Ament et al., 2004, 2010). Also Geranylgeranyl Pyrophosphate Synthase(GGPS1), which is involved in the biosynthesis of the volatile 4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT) and depends on both the JA- and SA-signaling pathway(Ament et al., 2006), was induced by T. urticae as well as by A. lycopersici, and lev-els in plants co-infested with both mite species were similar to those induced by T.urticae alone (not shown in FIGURE 4.3 as this gene depends on both JA and SA).

Hence, these results suggest that A. lycopersici does not suppress all JA-depend-ent genes. Possibly, suppression targets only a subset of genes, for instancebecause there is differential regulation of genes downstream of JA, as, for example,in Arabidopsis where there are two branches downstream of JA, i.e., the ERF and theMYC branch (Pieterse et al., 2012). Alternatively, it could be that some of these genesare regulated by other signals, additional to JA. Future experiments should reveal theeffect of A. lycopersici on the volatile profile emitted by tomato plants, as well as theconsequences for the third trophic level, i.e., attraction of natural enemies.

The phenylpropanoid pathwayIn CHAPTER 2, we found that both T. urticae and A. lycopersici induce accumulationof SA as well as accumulation of transcripts of the SA-marker gene PRP6. SA canbe synthesized in plants from chorismic acid via two distinct branches. However,note that several details of these pathways are unknown and can be different acrossplant species. The first branch runs via isochorismate synthase (ICS), which inArabidopsis and tomato is up-regulated upon pathogen infections and produces theSA precursor isochorismic acid (Wildermuth et al., 2001; Uppalapati et al., 2007). Inthe second branch, chorismic acid-derived cinnamic acid, which is produced fromphenylalanine via the enzyme phenylalanine ammonia lyase (PAL), is converted intoSA via either benzoate intermediates or coumaric acid (Boatwright et al., 2013).However, note that the steps of the second branch have not been experimentallyconfirmed in tomato (Blanco-Ulate et al., 2013).

Remarkably, even though T. urticae, as well as A. lycopersici, induced accumulationof SA (FIGURE 4.1), expression of ICS (Solyc06g071030 is tomato’s only ICS gene) didnot change in response to any of the mite treatments (not shown in FIGURE 4.4). In con-trast, expression of PAL was up-regulated by T. urticae as well as by A. lycopersici, butnot by T. evansi, suggesting that the up-regulation of SA in our tomato-mite system isconstrained by the activity of PAL, rather than that of ICS. However, it must be notedthat tomato possesses at least 17 loci annotated as PAL in the Heinz genome and nineadditional putative PAL loci organized in multiple clusters and hence there may be ahigh level of functional redundancy (Chang et al., 2008). Downstream of SA, the



expression of Pathogenesis-related Proteins (PRs) was up-regulated by both T. urticaeand A. lycopersici, including PR1, PR1b, PR4, β-glucanase (member of the PR-2 fam-ily), endochitinase (PR-3) and a thaumatin-like protein (PR-5). Co-infested plantsappeared to display additive responses of PR1 and PR1b compared to plants infest-ed with T. urticae alone, in accordance with previous results where we found a strongerresponse of PR-P6 in plants co-infested with T. urticae and A. lycopersici compared toplants infested with either T. urticae or A. lycopersici alone (CHAPTER 2).

PR-proteins are often used as markers for SA-defenses and are associated withresistance responses of plants to pathogens. For instance, β-1,3-glucanase and chiti-nase hydrolyze β-1,3-glucan and chitin, respectively, which are major components offungal and bacterial cell walls, and this is thought to inhibit pathogen growth (Van Loonet al., 2006). Also the peritrophic membrane of many arthropods contains chitin but per-foration of it due to induced plant chitinases, as can be obtained using insect chitinas-es (Kramer & Muthukrishnan, 1997) has not been demonstrated convincingly (Kitajimaet al., 2010). The osmotin precursor protein (NP24), a thaumatin-like protein, was alsoup-regulated by both T. urticae and A. lycopersici (not shown in FIGURE 4.4). NP24, whichwas previously found to be induced in tomato plants infected with Citrus Exocortis Viroid(Bellés et al., 1999), inhibits fungal growth on tomatoes (Pressey, 1997) and is possiblyunder control of ERF transcription factors (Hongxing et al., 2005). Furthermore, at leasteight peroxidases were induced by both T. urticae and A. lycopersici. Peroxidases havediverse functions: they produce reactive oxygen species, which are toxic to microorgan-isms, and they also play a role in the polymerization of cell wall compounds, like ligninand suberin (Passardi et al., 2005). Shown in FIGURE 4.4 is the Ep5C gene, which codesfor a secreted cationic peroxidase, and is induced by H2O2 during the oxidative burstfollowing Pseudomonas syringae infections (Coego et al., 2005). Ep5C is known as asusceptibility factor since plants with a functional Ep5C were more susceptible to P.syringae than tomatoes in which Ep5C was silenced (Coego et al., 2005). Hence, itwould be interesting to test the resistance of these tomatoes to A. lycopersici since theyinduced the highest expression of this gene and may take advantage of it. Taken togeth-er, the results so far confirm that tomatoes respond to T. urticae with a mixture of JA-and SA-dependent responses, whereas attack by A. lycopersici only up-regulates SA-mediated defenses. In contrast to the other two species, T. evansi did not, or only mar-ginally, induce JA- or SA-dependent defense responses.

The shikimate pathway, which precedes the phenylpropanoid pathway, does notonly generate SA but is also responsible for lignin, and flavonoid/benzonoid forma-tion. The first is a structural component of cell walls and the second constitutes alarge group of secondary metabolites predominantly associated with stress.Chorismate, the endproduct of the shikimate pathway, is produced from phospho-enolpyruvate and erythrose-4-phosphate and functions as the precursor for many

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compounds produced in the phenylpropanoid pathway (Weaver & Herrmann, 1997).In this pathway, chorismate is converted via the sequential actions of chorismatemutase (CMU), PAL, cinnamate 4-hydroxylase (C4H) and 4-coumarate:coenzyme Aligase (4CL) into 4-coumaroyl-CoA, which can then be further converted in separatesub-branches to produce either lignins, flavonoids or anthocyanins (FIGURE 4.3).

One out of the five 4CL genes on the array was induced in response to A. lycop-ersici, whereas the others did not change in any of the treatments. There were threegenes encoding Cinnamoyl-CoA-reductase (CCR), the first dedicated step in thelignin biosynthesis branch, of which one was significantly up-regulated by T. urticae(FIGURE 4.4). In contrast, the expression of genes coding for the second dedicatedenzyme cinnamyl alcohol dehydrogenase (CAD), did not change. Six genes on thearray, of the in total 25 putative loci present in the genome, were annotated as caf-feoyl-CoA O-methyltransferase (CCoAOMT) and all of these were up-regulated by A.lycopersici and, except for one, also by T. urticae, whereas T. evansi induced only oneof them (i.e., the one shown in FIGURE 4.4). CCoAOMT is associated with lignificationin tomato (Ye et al., 1997) and, because its activity is induced by fungal elicitors, ithas been proposed to play a role in defense by catalyzing the synthesis of polymersto reinforce the cell wall (Pakusch et al., 1989). Hence, this result suggests that plantslignify their cell walls to protect themselves against mite attack, a phenomenon whichactually has been described indeed for eriophyoid mites (Petanovic & Kielkiewicz,2010). Apart from lignins, 4-coumaroyl-CoA can also branch off to theflavonoids/benzenoids. In that case it is converted via the sequential actions of chal-cone synthase (CHS) and chalcone isomerase (CHI) into flavonoids, such as the fla-vanone naringenin (Petrussa et al., 2013). Naringenin can be further converted by fla-vanone 3-hydroxylase (F3H) to yield dihydrokaempferol which subsequently ishydroxylated by flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase(F3'5'H) to produce, respectively, the flavonoids dihydroquercetin and dihy-dromyricetin (Petrussa et al., 2013). Dihydroquercetin can then be further convertedby flavonol synthase (FLS) into quercetin (Olsen et al., 2010).

Tomato possesses two genes annotated as CHS (CHS1: Solyc09g091510 andCHS2: Solyc05g053550) of which the expression levels were lower in response to T.urticae and A. lycopersici compared to T. evansi. Significant down-regulation wasobserved for CHS1 in response to A. lycopersici, whereas CHS2 was significantlydown-regulated in response to infestation with both T. urticae and A. lycopersicisimultaneously (FIGURE 4.4). Three genes were annotated as CHI, which showed vari-able expression patterns in response to mite feeding. One of them, Solyc02g067870was slightly but significantly down-regulated by T. urticae (FIGURE 4.4). A second CHI(Solyc08g061480.2) was up-regulated by A. lycopersici, but not affected by T. urticaeor T. evansi (not shown in FIGURE 4.3).

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The tomato genome harbors eight F3H-like expressed genes, two of which wereon the array and of those one was up-regulated by A. lycopersici and T. urticae, butnot by T. evansi. The expression of flavonol synthase (FLS), of which tomato has fivehomologs, did not change much in response to any of the mites (FIGURE 4.4).


FIGURE 4.4 Regulation of the phenylpropanoid pathway by the three mite species. Shown is an overview of themost important genes of the phenylpropanoid pathway. Bars represent the relative average (+SEM) expressionvalues of each gene. All values were divided by the lowest average value (such that the lowest average is always1). C = Uninfested control plants, U = Tetranychus urticae. E = T. evansi, A = Aculops lycopersici. Asterisks indi-cate significant differences with uninfested control plants. Expression values are given for Phenylalanine ammonialyase (PAL; Solyc09g007900.2); Pathogenesis-related protein 1 (PR-1; Solyc00g174330.2); Pathogenesis-relat-ed protein 1b (PR-1b; Solyc00g174340.1); Pathogenesis-related protein-like protein (PR like; Solyc04g064880.2);Pathogenesis-related protein 4b (PR-4b; Solyc01g097240.2); Beta-glucanase (Solyc01g060020.2);Endochitinase (Solyc02g082920.2); Thaumatin-like protein (Solyc12g056360.1); Osmotin-like protein(Solyc08g080640.1); 4-coumarate CoA ligase (4CL; Solyc03g117870.2); Chalcone synthase 1 (CHS1;Solyc09g091510.2); Chalcone synthase 2 (CHS2; Solyc05g053550.2); Chalcone isomerase (CHI;Solyc02g067870.2); Cinnamoyl-CoA reductase (CCR; Solyc03g116910.2); Cinnamyl alcohol dehydrogenase(CAD; Solyc01g107590.2); Caffeoyl-CoA O-methyltransferase (CCoAOMT; Solyc02g093250.2); Flavanone 3-hydroxylase-like protein (F3H; Solyc03g080190.2); Flavonol synthase (FLS; Solyc06g083910.2); Dihydroflavonol4-reductase (DFR; Solyc02g085020.2); Anthocyanidin synthase (ANS; Solyc10g076660.1); and UDP flavonoid 3-O-glucosyltransferase (UFGT; Solyc10g083440.1).


Finally, dihydroflavonols can be converted to anthocyanins, in a series of reactionscatalyzed by dihydroflavonol reductase (DFR), anthocyanidin synthase (ANS) andUDP-glucose:flavonoid 3-O-glucosyl transferase (UFGT) (Petrussa et al., 2013).Expression levels of DFR and UFGT were significantly down-regulated in plants infest-ed with T. urticae and A. lycopersici simultaneously. The tomato genome possesseseight genes encoding ANS, of which four were on the array. Out of these four, one wasinduced by T. urticae (shown in FIGURE 4.4), two did not change in response to any ofthe mites, and one was down-regulated by T. urticae as well as by A. lycopersici.

Taken together, both A. lycopersici and T. urticae affect genes involved in flavonoidas well as anthocyanin biosynthesis, whereas expression levels did not change inresponse to T. evansi. However, the consequences of these effects remain inconclusivesince for many of these homologs it is unknown if they are tissue-specific and whichencode for the rate-limiting enzymes. Future work should include (1) a (targeted)metabolomics approach to reveal the consequences of these transcriptional changesfor flavonoid/anthocyanin levels, and also (2) the possible effects on mite performance.

Genes differentially regulated in Aculops lycopersici-infested plants incomparison to uninfested plantsGenes of which the expression was affected most strongly by A. lycopersici aredivided below in several categories i.e., coding for: (1) defense proteins, (2) proteinsinvolved in cell wall modification, (3) R-gene proteins and (4) other proteins.Furthermore, genes down-regulated by A. lycopersici are discussed (5). The com-plete list of the top-50 most strongly up- and down-regulated genes is shown inTABLE S4.1A and B, respectively.

Defense proteinsGenes from several of the major PR-gene families were among the top-50 most high-ly up-regulated genes, including members of the family PR-1 (PR-1b; #31 in TABLE

S4.1A), PR-2 (β-1,3-glucanase; #10) and PR-3 (chitinase; #16), (acidic chitinase; #39),(endochitinase; #49). Three peroxidases were highly up-regulated as well (#3, #6, #25).

The closest homolog in Arabidopsis (score: 398; E-value: 1e-111) of the highest A.lycopersici-induced peroxidase (#3) is a cell-wall bound peroxidase, namedAtPRX71, which plays a role in the lignification of cell walls (Shigeto et al., 2013).Furthermore, also a thaumatin-like protein (#43) as well as a gene annotated as Majorallergen Mal d 1 (#32) were highly up-regulated by A. lycopersici. The Major allergenMal d 1 is similar to a NCBI sequence annotated as Tomato Stress Induced-1 (TSI-1) (GB: Y15846.1), which is highly homologous to the potato (Solanum tuberosum)STH-2 gene (Matton et al., 1993) and inducible by SA and Fusarium oxysporum infec-tions (Sree Vidya et al., 1999).

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Cell wall modificationPlants possess a large array of enzymes involved in cell wall formation and modifi-cation but which can also affect disease susceptibility, for instance because theyinhibit the activity of phytopathogen-secreted cell-wall degrading enzymes (CWDEs).Examples of CWDEs that can be inhibited by plant proteins are polygalacturonases,pectinesterases, pectin lyases, pectate lyases, xylanases and endoglucanases(Juge, 2006; Cantu et al., 2008; Wolf et al., 2012).

Aculops lycopersici caused a significant 6-fold increase in the expression of a geneannotated as xylanase inhibitor (Solyc01g079940.2), and, notably, this response wastriggered by A. lycopersici only and not at all by T. urticae or T. evansi (note that thisgene is not listed in TABLE S4.1B as it represented number 63 and was therefore notin the top-50). Unfortunately, xylanase inhibitors have not been characterized yet intomato, and there are at least 18 loci annotated as xylanase inhibitor. It is likely thatmany of these genes serve primarily to regulate non-pathogenesis related develop-mental processes, which may involve cell wall modifications. Nevertheless, there areindications that xylanase inhibitors can also enhance the resistance to cell-walldegrading pathogens (Moscetti et al., 2013). Hence, given that xylans are a majorcomponent of plant cell walls, it could be that xylanases are important in pathogenic-ity, as shown for example for Botrytis cinerea (Brito et al., 2006). However, no genesannotated as xylanase were found in the A. lycopersici genome.

A gene that is related to the xylanase inhibitor is the xyloglucan-specific endoglu-canase inhibitor protein (XEGIP; GB: DQ056434), which was found to inhibit theactivity of the fungal xyloglucan-specific endoglucanase (XEG) (Qin et al., 2003).Although the levels were not significantly different from those in control plants, XEGIPwas most strongly induced by A. lycopersici when compared to the induction byother mite species. Also two other CWDE inhibitors, i.e., an inhibitor of polygalactur-onase (Solyc07g065090.1) and an inhibitor of pectinesterase (Solyc11g005820.1),were not regulated by A. lycopersici, nor by T. urticae or T. evansi.

Several concanavalin A-like lectin/glucanases have been found in the genome ofA. lycopersici, as well as in the genome of T. urticae, and some of these carry a sig-nal peptide, which suggests that in theory these mites could be secreting glucanas-es when piercing the plant cell to feed (see CHAPTER 6, General discussion). Takentogether, it could be that A. lycopersici secretes proteins that degrade polysaccha-rides in the plant cell wall to make penetration of it easier, analogous to what hasbeen described for pathogens and nematodes (Mitreva-Dautova et al., 2006).

Several cell-wall-modification-associated genes, including an unknown proteinwith an extensin-like region (#7) and a pectate lyase (#24) were strongly up-regulat-ed by A. lycopersici (TABLE S4.1B). Extensins are hydroxyproline-rich glycoproteinsthat form cross linked networks in the plant cell walls thereby strengthening it, which

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may well be part of a plant’s defense response (Showalter, 1993). The unknown pro-tein with an extensin-like region (#7) corresponds to an NCBI sequence calledLEMMI8 (GB: Z46674.1), which was found to be induced upon giant cell formationduring root-knot nematode (Meloidogyne) infections (Van der Eycken et al., 1996).LEMMI8 was also up-regulated by T. urticae, although only 4-fold in comparison tocontrol plants, but not by T. evansi. Hence, this suggests that A. lycopersici and gall-inducing nematodes, which both induce SA (Uehara et al., 2010), may have similareffects on cell wall metabolism.

The pectate lyase (#24) was also up-regulated by T. urticae (7-fold compared tocontrol plants), as well as by T. evansi, although less strongly by the latter (3-fold).Pectate lyases are involved in the degradation of the structural cell wall polysaccha-ride pectin. In addition, they cause the release of oligogalacturonides from the plantcell wall, which are perceived as elicitors by some plants leading to the activation ofinduced plant defenses (De Lorenzo et al., 1991; Doares et al., 1995). Pectate lyasescan also be produced and secreted by plant pathogens, thereby softening the leaftissue and facilitating pathogen growth (Marín-Rodríguez et al., 2002).

Taken together, whereas both A. lycopersici and T. urticae have a relatively strongeffect on genes involved in cell wall modification, this response is much less appar-ent in the case of T. evansi. Also, it remains to be investigated to what extent thesechanges are a response of the plant, for instance to repair damaged plant tissue, orwhether these changes are caused by the attacker to facilitate the infestation. Thexylanase inhibitor was the only CWDE inhibitor specifically induced by A. lycopersi-ci. Whether CWDEs cause pathogenicity of mites could be investigated by silencingtheir expression and assessing the consequences for mite performance and/or dam-age symptoms on tomato.

R-genesAmong the most strongly induced genes by A. lycopersici were several Resistance-genes (R-genes). R-proteins are sensors that recognize pathogen secreted proteins andare hard-wired to defenses, like the hypersensitive response, to limit the spread of thedisease (Bent & Mackey, 2007). Genes that share structural homology with R-genes thatwere induced included a disease resistance response protein (TABLE S4.1A; #1), a NBS-LRR class disease resistance protein (#36), and an EIX (Ethylene Inducing Xylanase 2)receptor (#11) (Ron & Avni, 2004). EIX receptor genes are homologous to the Ve and Cfresistance genes of tomato (Ron & Avni, 2004) and bind EIX, which is a fungal xylanaseelicitor (Bailey et al., 1990). It has been shown that EIX2, but not EIX1, is essential formediating defense responses in tobacco (N. tabacum) plants (Ron & Avni, 2004).Tetranychus urticae also induced the EIX2 gene, but not the xylanase inhibitor.

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Other genesOther interesting candidates on the top 50 list of up-regulated genes included threeWRKY transcription factors (TABLE S4.1A; #9; #15; #38), which play major roles in reg-ulating plant responses to pathogens (Pieterse et al., 2012), members of the 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene family (#22; #23; #30),involved in ethylene biosynthesis (Barry et al., 1996), and SAMT (#35), which convertsSA into methyl salicylate (Ament et al., 2010). Also a syntaxin (#20) was up-regulat-ed by A. lycopersici, as well as by T. urticae. BlastP analysis against the TAIR data-base, using the protein sequence obtained from the DNA sequence of this gene(Solyc01g006950.2) revealed that this gene has the highest sequence homology withAtSYP121 (score: 411; E-value: e-115). Plants in which this gene was mutated allowedfor a higher number of penetrations by barley powdery mildew (Blumeria graminis sp.hordei) (Collins et al., 2003), indicating that SYP121 plays a role in resistance at theplant cell wall.

Tomato genes down-regulated by Aculops lycopersiciAbout 10% of the top-50 of genes most strongly down-regulated by A. lycopersiciencoded proteins for chlorophyll metabolism, photosynthesis and/or carbon fixationincluding a protochlorophyllide reductase (TABLE S4.1B; #14), proteins related to pho-tosystem II (#45; #50) and light harvesting complexes (chlorophyll a-b binding pro-tein; #26; #31). Although not in the top-50, the expression of three Ribulose-1 5-bis-phosphate carboxylase (RuBisCO) genes was also significantly down-regulated byA. lycopersici. Down-regulation of photosynthesis is commonly observed in plantssubjected to biotic attack and this can be part of the plant’s JA-dependent defenseresponse (Creelman & Mullet, 1997; Bilgin et al., 2010). However, the same phenom-enon can also be due to the blockage of resource allocation leading to sucrose accu-mulation which is hard-wired to photosynthesis and down-regulates it, as observedfor some pathogens (Kocal et al., 2008) and nematodes (Cabello et al., 2014). Somepathogens hijack the plants sugar metabolism via secreted cytokinins (Walters &McRoberts, 2006), also typically produced by gall-inducing arthropods (Yamaguchiet al., 2012). Although A. lycopersici does not produce galls, it may secrete cytokininslike other eriophyoids have been suggested to do (De Lillo & Monfreda, 2004).

Comparison between transcriptional responses induced by Tetranychusurticae and Aculops lycopersiciSubsequently, we compared transcriptional responses induced by T. urticae and A.lycopersici by selecting the top-15 genes that were (a) most strongly up-regulatedrelative to the control by T. urticae but not regulated by A. lycopersici, (b) moststrongly up-regulated relative to the control by A. lycopersici, but not regulated by T.

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urticae, (c) most strongly up-regulated relative to the control by T. urticae and alsosignificantly up-regulated by A. lycopersici, or (d) most strongly down-regulated rel-ative to the control by T. urticae and also significantly down-regulated by A. lycoper-sici (TABLE S4.2A-D).

Among the genes specifically up-regulated by T. urticae were four proteinaseinhibitors (TABLE S4.2A; #2; #3; #7; #9), as well as a polyphenol oxidase (#10). In con-trast, genes induced by A. lycopersici, but not by T. urticae, included the xylanaseinhibitor described in the previous section (#2), an expansin (#3) and two GDSLesterases (#4; #6) (‘GDSL’ refers to a conserved protein motif). GDSL esterases/lipases are extracellular glycoproteins with multiple functions predominantly associ-ated with lipid metabolism (Chepyshko et al., 2012). To date, only a few membershave been characterized which play roles in plant development (Ling et al., 2006) andin plant immunity (Hong et al., 2008; Kim et al., 2013). In the tomato genome over a100 GDSL esterases/lipases are annotated, but the family is largely uncharacterized.Out of the 54 GDSL esterases/lipases on the array, eight were up-regulated by A.lycopersici, whereas six were down-regulated. Five of the A. lycopersici-down-regu-lated GDSL esterases were also suppressed by T. urticae, whereas four were inducedby both species. Only one GDSL esterase was regulated by T. evansi. Without indi-vidual characterization of these genes it is hard to speculate on their role in mite-induced plant responses.

A possibly relevant gene specifically up-regulated by A. lycopersici (3-fold) was agibberellin-regulated protein 2 (Solyc02g083880.2) (#20 and thus not listed in TABLE

S4.2B). Notably, two other gibberellin-regulated proteins were up-regulated by bothT. urticae and A. lycopersici, yet not by T. evansi, and the same was true for a geneannotated as gibberellin receptor GID1L2 (Solyc10g050880.1). Three gibberellin-reg-ulated proteins were down-regulated by T. urticae, yet not by A. lycopersici nor by T.evansi.

Since gibberellin and JA are known to antagonize each other’s action (Hou et al.,2013), it could be that gibberellin plays a role in the A. lycopersici-mediated inhibitionof the JA-response, analogous to the scenario suggested for the necrotrophic fun-gus Fusarium moniliforme which produces gibberellin, possibly to disable JA-medi-ated defenses (Navarro et al., 2008; cf. also FIGURE 4.2). Supporting this hypothesis,a putative ent-kaurene oxidase (Solyc04g083160.1), which in Arabidopsis is knownto be involved in biosynthesis of gibberellin (GA) (Helliwell et al., 1999), was 2-foldup-regulated by A. lycopersici. However, other genes involved in GA biosynthesis, forinstance the GA 20-oxidases (SlGA20ox1, -2, and -4), were not significantly regulat-ed by A. lycopersici indicating that more research, such as a direct determination ofGA levels, is required to make conclude on the effect of A. lycopersici on GA signal-ing. Yet, since GA also has been implicated in trichome branching and development

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(An et al., 2012) and considering the strong effect russet mites have on trichomes(Van Houten et al., 2013) it seems worthwhile to investigate this lead on the role ofGA in the tomato-russet mite interaction in more detail.

Among the genes most strongly up-regulated by T. urticae, but also by A. lycop-ersici, were SA-responsive genes, including PR1b and β-glucanase (#13; #6; respec-tively). Other genes that were up-regulated by both species included SAMT (#7) andan ACC-oxidase (#10) (TABLE S4.2C). Genes that were most strongly down-regulatedby T. urticae but also by A. lycopersici included photosynthesis-related genes (i.e.,photosystem I reaction center subunit VI; #13), genes related to cell-wall modifica-tion (2 XET/XTHs; #1; #5) and lignin biosynthesis (CCoAOMT; #12) as well as auxinsignaling (indole-3-acetic acid-amido synthetase GH3.8 and auxin response factor 9;#2; #7).

In rice, indole-3-acetic acid–amido synthetase was shown to prevent the accumu-lation of auxin and this consequently suppressed expression of expansins andenhanced plant resistance (Ding et al., 2008). Thus, down-regulation of indole-3-acetic acid-amido synthetase may have as a consequence that suppression ofexpansins is prevented and plant resistance is decreased. Of the 21 expansins onthe array, four were induced and one was down-regulated by A. lycopersici. Themost strongly A. lycopersici-induced expansin (TABLE S4.2B; #3) is highly homolo-gous to the expansin A8 gene, encoding an enzyme involved in cell wall extensionwhich, in Brassica oleracea, has been connected to auxin-dependent growthresponses in plants (Esmon et al., 2006). Possibly, up-regulation of this gene is relat-ed to a typical growth-related symptom of russet mite infestation, namely the russetmite-induced ‘curling’ of the leaves (FIGURE 1.5).

Comparison between transcriptional responses induced by Tetranychusurticae and T. evansiFinally, we compared the tomato transcriptional responses to feeding by the twosuppressor species T. evansi and A. lycopersici by selecting the top-15 genes thatwere (a) most strongly up-regulated compared to the control by T. evansi but not reg-ulated by A. lycopersici, (b) most strongly up-regulated compared to the control byA. lycopersici but not regulated by T. evansi, (c) most strongly up-regulated com-pared to the control by T. evansi and also significantly up-regulated by A. lycopersi-ci, or (d) strongest down-regulated compared to the control by T. evansi and also sig-nificantly down-regulated by A. lycopersici (TABLE S4.3A-D).

Strikingly, the magnitude of up- and down-regulation for most genes was muchlarger for A. lycopersici than for T. evansi (TABLE S4.3A-D). This can reflect a funda-mental difference between these two mites, but may also have been caused by dif-ferences in their densities (i.e., maybe at lower A. lycopersici densities the two

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responses would have been more similar). Among the genes specifically up-regulat-ed by T. evansi was a MYB transcription factor (TABLE S4.3A; #3), called Abscisic acidinduced MYB1 (AIM1: an R2R3MYB protein) (AbuQamar et al., 2009). AIM1 is homol-ogous to the Arabidopsis MYB78 (At5G49620) (score: 654; E-value: 3.5e-65) andMYB108/BOS1 (At3G06490) (score: 639; E-value: 2.2e-67) and mediates ABAresponses to Botrytis cinerea as well abiotic stresses such as salinity and oxidativestress in tomato (AbuQamar et al., 2009).

Among the genes up-regulated most strongly up-regulated by A. lycopersici, butnot by T. evansi, were several genes associated with SA-defenses, such as a WRKYtranscription factor (TABLE S4.3B; #6), a β-glucanase (#4), two peroxidases (#2; #13)and an unknown protein with an extensin-like region (#3), which is again the LEMMI8discussed previously (in the section Genes differentially regulated in A. lycopersici-infested plants in comparison to uninfested plants).

Among the genes most strongly up-regulated by T. evansi and also by A. lycoper-sici was a Ribonuclease T2 (TABLE S4.3C; #2). Ribonuclease T2, which is also up-reg-ulated by T. urticae, is associated with phosphate starvation, ethylene responses,and programmed cell death. Silencing Ribonuclease T2 delays leaf senescence andabscission (Lers et al., 2006). Also two cell-wall related genes, i.e., a pectinesterase(#4) and a pectate lyase (#9), as well as SAMT (#8) were up-regulated by T. evansiand A. lycopersici, albeit all of these to higher levels by A. lycopersici than by T. evan-si. The JA-biosynthesis enzyme AOS (#6), of which tomato has only a single copy inits genome, was induced up to similar levels by the three mite species. SAMT levelsinduced by T. urticae were approximately 2-fold higher compared to those inducedby A. lycopersici and approximately 5-fold higher compared to those induced by T.evansi, indicating that both ‘suppressor’ mites may partially down-regulate it’sexpression coinciding with lower levels of JA (FIGURE 4.1). In contrast, theRibonuclease T2 was induced almost 2-fold higher by T. urticae than by T. evansi but2-fold lower than by A. lycopersici. Furthermore, the expression patterns of thepectinesterase and the pectate lyase were induced to comparable levels by A. lycop-ersici and T. urticae, whereas their expression levels were 2- to 3-fold lower inresponse to T. evansi. Hence, the expression of these genes parallels the accumula-tion of both JA and SA, and may be (partly) dependent on JA-accumulation, as JAwas found to mediate modifications of the cell-wall pectin matrix, thereby increasingits defensive properties (Taurino et al., 2014).

Finally, genes that were down-regulated by both mite species included a cell-walllocated arabinogalactan protein (TABLE S4.3D; #8), as well as the auxin response fac-tor 9 (ARF9) (#1). ARF9 was found to be a repressor of auxin signaling (Zouine et al.,2014). Interestingly, it appeared that two of the four Auxin F-box 5 proteins (AFB5) onthe array (Solyc02g079190.2 and Solyc06g008780.1), both close homologs of the

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Arabidopsis auxin receptor TIR1 (At3G62980; score: 642; E-value: 0.0 and score:1290; E-value: 1.4e-139, respectively), were down-regulated by A. lycopersici but notby T. evansi, nor by T. urticae. AFB5 was discovered to function as part of an auxinco-receptor (Villalobos et al., 2012) and down-regulation of auxin signaling can bemediated by SA and is associated with anti-pathogen defenses (Wang et al., 2007;Kazan & Manners, 2009). Hence, it could be that in A. lycopersici-infested plantsauxin signaling is antagonized by SA. However, the relationship between auxin andanti-herbivore defenses still remains to be elucidated (Meldau et al., 2012).

ConclusionsTo obtain a broader overview of responses induced by mites we have compared thetranscriptional responses and phytohormonal rearrangements induced during feed-ing by three different mites species on tomato.

Tetranychus urticae feeding caused a 2- to 3-fold increase in the accumulation ofJA, JA-Ile and SA, which was accompanied by the induction of well-known JA- andSA-dependent defense genes, including wound-induced proteinase inhibitors andPR-proteins, in line with previous results (Kant et al., 2004, 2008; Alba et al., submit-ted). In total, T. urticae regulated 2014 genes (TABLE 4.1).

The phytohormonal and transcriptional profile in T. evansi-infested leaflets differedmarkedly from that in leaflets fed upon by T. urticae, even though the two species areclosely related. Tetranychus evansi did not induce the accumulation of JA, nor that ofABA, whereas levels of JA-Ile and SA were only slightly increased in comparison tocontrol plants. Most strikingly, T. evansi regulated only 274 genes (TABLE 4.1), whichis ca. 8× less than the number regulated by T. urticae and A. lycopersici. Furthermore,the magnitude of up and down-regulation in response to T. evansi was much lowerthan for the genes that respond to T. urticae and A. lycopersici feeding, which isremarkable given that T. evansi is known to cause ca. 2-fold more tissue damage ontomato compared to T. urticae after 7 days of infestation (Alba et al., submitted).Related to this, despite the fact that T. evansi causes more visual feeding damage tothe leaf blade, the undamaged tissues of T. evansi-infested leaflets stay longer greenand ‘healthy’ compared to those infested with T. urticae which senesce almost twiceas fast during the course of the infestation. Tetranychus urticae and T. evansi also dif-fer in the kind of visual feeding-damage symptoms they cause: whereas T. evansifeeding results in the formation of small, white-coloured chlorotic lesions, theselesions get surrounded by small areas of brownish and senesced tissue and some-times micro-oedema in the case of T. urticae, reminiscent of hypersensitive-likeresponses (Alba et al., submitted).

Aculops lycopersici belongs to the eriophyoid mites, which feed on epidermalcells. Interestingly, eriophyoid mites are thought to have evolved their stylet structure

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and support to feed on plants completely independent from the Tetranychidae(Lindquist et al., 1996). Aculops lycopersici induced the accumulation of JA-relatedphytohormones as well as that of SA and regulated a total of 2219 genes (TABLE 4.1)of which 50% was also regulated by T. urticae. Hence, the total number of genes reg-ulated by T. urticae and A. lycopersici was comparable, and even showed consider-able overlap, even though the effect on the JA-pathway was markedly different(FIGURE 4.3). Yet, there also appeared to be specificity in the response: for instance axylanase inhibitor was specifically induced by A. lycopersici and also some GA-relat-ed genes appeared to be specifically affected by A. lycopersici (FIGURE 4.2), possiblyindicating differential effects of these mites on tissue growth/cell proliferation.Experiments with plants in which the expression of A. lycopersici-induced genes aresilenced (e.g., the xylanase inhibitor) would be necessary to test to which extentthese genes affect the susceptibility of tomato to A. lycopersici and to which extentthese are related to the growth distortions that occur on tomato plants with high pop-ulation densities of A. lycopersici.

In summary, our visual observations underscore the transcriptome results that T.evansi and T. urticae have very different effects on their host plant: whereas T. evan-si has adapted in such a way that it is not, or hardly, recognized by the plant at all, T.urticae, as well as A. lycopersici, elicit strong responses in tomato, although they arenot the same. The question that remains is how T. evansi manages to suppress hostdefense responses.

Suppression of host plant responses has been described for species with lifestyles different from that of spider mites, for instance galling insects, like the tephri-tid fly Eurosta solidaginis and the gelechiid moth Gnorimoschema gallaesolidaginiswhich did not induce the accumulation of JA and SA in stems of Solidago altissimaplants (Tooker et al., 2008; Tooker & De Moraes, 2009). Similarly, also phloem-feed-ing aphids have been reported to elicit only weak responses in Nicotiana attenuata(Voelckel et al., 2004). It could be that, like gall-inducing pathogens (Deeken et al.,2006) and gall-inducing insects (Tooker et al., 2008; Compson et al., 2011), T. evan-si is somehow able to interfere with the plant’s source-sink relationships and maybecan block induced senescence thereby explaining why T. evansi attacked leafletsremain relatively ‘healthy and green’ as compared to leaflets that are attacked by the‘inducer’ species T. urticae.

It has been suggested that plants respond to herbivory by down-regulating theexpression of photosynthesis-related genes (Creelman & Mullet, 1997) and re-allocat-ing resources, i.e., transporting resources away from the herbivore-damaged tissuesto unattacked parts of the plants, like for instance the roots (Schwachtje et al., 2006).As shown in tomato, this process can be stimulated by JA (Gómez et al., 2010), butdoes not necessarily depend on it, as was shown in a study using N. attenuata

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(Schwachtje et al., 2006). However, according to an alternative second hypothesis, JAand herbivory may also cause an increased flow of resources from unattacked partsof a plant to the attacked parts to support or increase the production of defensesthere (Schultz et al., 2013; Ferrieri et al., 2013). Because these defenses are costly toproduce this may lead to a reduction of resources in the unattacked parts of the plant,as shown by Machado et al. (2013). Possibly T. urticae and A. lycopersici, which bothinduce strong accumulation of JA, stimulate re-allocation of resources, i.e., away fromthe attacked tissues, whereas in T. evansi-infested leaflets this process is blocked oreven reversed if such leaves become sinks. Hence, this could imply that the suppres-sion of JA and SA-related defenses is actually a side-product of a more general mech-anism by which T. evansi interferes with the source-sink physiology of its host.

Yet, following the second hypothesis of increased resource flow to the attackedtissues, it could also be that T. evansi suppresses defenses just to prevent the (local)depletion of resources from which not only the plant but also the mite ultimatelywould suffer. Induction of defenses in response to T. urticae and A. lycopersici would,according to this theory, increase resource flow but also cause the (local) depletionof resources, as they are used for the activation of defenses. Hence, this scenarioargues against the T. evansi ‘sink hypothesis’. To speculate a bit further on this, itcould also be that T. evansi somehow has evolved a way to optimize the balancebetween defense and availability of resources: i.e., it may increase (JA-dependent)sink strength, while at the same time suppressing JA-dependent downstreamresponses. Whether induction of JA can lead to an increase in sink strength could betested by making use of def-1 plants, which are deficient in the induced expressionof JA-dependent defense genes.

AcknowledgementsWe thank Harold Lemereis, Ludek Tikovsky and Thijs Hendrix for growing the toma-to plants and Michel de Vries for assistance with the LC-MS measurements.

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mpa

red

to c

ontr

ol (C

).#

Prim

ary

acce

ssio

nLo

cus

iden

tifie

r No

. mat

chin

g An

nota

tion*

*Av

erag

e re

lativ

e ex

pres

sion

(cv.

Hein

z ge

nom

e)pr

obes

*(C

) ± S

E(A

) ± S

EP

***

1BF

0977

28So

lyc01

g021

600.

21

Dise

ase

resi

stan

ce re

spon

se p

rote

in

1.0±

0.1

31.0

±4.

6<

0.00

1

2X7

9337

Solyc

05g0

0795

0.2

1Ri

bonu

clea

se T

21.

0±0.

220

.2±

2.5

0.00

1

3AK

3251

52So

lyc01

g105

070.

22

Pero

xidas

e 1.

0±0.

117

.6±

4.8

0.00

5

4TA

5390

0_40

81So

lyc02

g062

890.

11

Myo

-inos

itol t

rans

porte

r 2

1.10

.217

.7±

6.2

0.00

3

5BW

6928

67So

lyc05

g014

590.

21

Tran

scrip

tion

Fact

or

1.0±

0.2

14.3

±1.

20.

001

6TA

3839

2_40

81So

lyc03

g006

700.

22

Pero

xidas

e 1.

0±0.

214

.2±

5.4

0.01

3

7BI

2039

29So

lyc04

g071

070.

22

Unkn

own

Prot

ein

(Ext

ensi

n-lik

e re

gion

)1.

0±0.

213

.4±

6.8

0.01

5

8AI

7794

01So

lyc06

g034

370.

11

Pect

ines

tera

se

1.0±

0.2

12.8

±3.

30.

004

9TA

5533

4_40

81So

lyc02

g071

130.

21

WRK

Y tra

nscr

iptio

n fa

ctor

29

1.0±

0.1

12.3

±1.

90.

001

10M

8060

8So

lyc01

g060

020.

213

Beta

-glu

cana

se1.

0±0.

211

.9±

4.9

0.01

1

11TA

5661

7_40

81So

lyc07

g008

600.

12

LRR

rece

ptor

-like

ser

ine

(EIX

Rec

epto

r 2)

1.0±

0.1

11.8

±2.

20.

001

12BI

4211

64So

lyc03

g098

740.

11

Kuni

tz tr

ypsi

n in

hibi

tor

1.0±

0.0

10.7

±6.

10.

047

13AK

3261

43So

lyc06

g075

690.

22

Auxin

-reg

ulat

ed p

rote

in

1.0±

0.1

10.5

±2.

60.

001

14BI

4219

69So

lyc01

g080

570.

21

Inos

ine-

urid

ine

pref

errin

g nu

cleo

side

hyd

rola

se fa

mily

pro

tein

1.

0±0.

210

.4±

2.0

0.00

8

15AK

3250

41So

lyc08

g067

340.

23

WRK

Y tra

nscr

iptio

n fa

ctor

1.

0±0.

310

.1±

4.2

0.00

4

16CK

4686

96So

lyc04

g072

000.

21

Chiti

nase

1.

0±0.

19.

9±2.

70.

006

17DB

6871

20So

lyc02

g036

480.

11

Harp

in-in

duce

d pr

otei

n-lik

e 1.

0±0.

19.

6±1.

30.

002

18AK

3209

83So

lyc10

g078

230.

11

Cyto

chro

me

P450

1.10

.110

.0±

1.9

0.00

6

19AK

3289

76So

lyc01

g073

820.

21

CHP-

rich

zinc

finge

r pro

tein

-like

1.

0±0.

19.

4±1.

60.

002

20AK

3278

96So

lyc01

g006

950.

21

Synt

axin

1.

0±0.

29.

3±2.

20.

004

21AI

7812

81So

lyc09

g072

810.

21

Rece

ptor

like

kin

ase

1.0±

0.1

9.2±

1.4

0.00

2

22AK

3208

17So

lyc09

g010

000.

22

1-am

inoc

yclo

prop

ane-

1-ca

rbox

ylate

oxid

ase-

like

prot

ein

1.0±

0.2

8.9±

2.1

0.00

2

23BE

4516

16So

lyc11

g007

890.

11

1-am

inoc

yclo

prop

ane-

1-ca

rbox

ylate

oxid

ase

4 1.

0±0.

18.

6±1.

90.

006

24X5

5193

Solyc

02g0

9358

0.2

3Pe

ctat

e lya

se

1.0±

0.2

8.6±

2.0

0.00

3

25AK

3204

53So

lyc05

g052

280.

21

Pero

xidas

e 1.

0±0.

18.

4±2.

70.

006

26AK

3211

58So

lyc08

g068

680.

23

Deca

rbox

ylase

fam

ily p

rote

in

1.0±

0.2

8.4±

1.3

0.00

7

27TA

3674

2_40

81So

lyc09

g090

980.

21

Maj

or a

llerg

en M

al d

1

1.0±

0.1

8.3±

1.5

0.00

4

28AW

0399

18So

lyc01

g080

800.

21

PBSP

dom

ain-

cont

aini

ng p

rote

in

1.10

.18.

7±1.

60.

002

29BI

2053

17So

lyc12

g049

030.

11

Fatty

aci

d de

satu

rase

1.0±

0.3

8.0±

1.9

0.00

1

30X0

4792

Solyc

07g0

4953

0.2

31-

amin

ocyc

lopr

opan

e-1-

carb

oxyla

te o

xidas

e 1.

0±0.

27.

6±2.

00.

002

31Y0

8804

Solyc

00g1

7434

0.1

4Pa

thog

enes

is-r

elat

ed p

rote

in 1

b 1.

0±0.

27.

5±1.

40.

005

128



32AK

2471

06So

lyc09

g090

990.

21

Maj

or a

llerg

en M

al d

1

1.30

.49.

9±1.

80.

014

33AK

3225

67So

lyc04

g040

130.

11

Omeg

a-6

fatty

aci

d de

satu

rase

1.

0±0.

17.

4±1.

20.

005

34AI

4873

43So

lyc10

g005

320.

21

Tryp

toph

an s

ynth

ase

beta

cha

in 1

1.

0±0.

17.

4±3.

70.

036

35BI

2045

48So

lyc09

g091

550.

22

Salic

ylic

acid

car

boxy

l met

hyltr

ansf

eras

e 1.

0±0.

37.

3±1.

20.

013

36AK

2470

49So

lyc07

g056

200.

21

NBS-

LRR

clas

s di

seas

e re

sist

ance

pro

tein

1.

0±0.

17.

2±1.

10.

002

37AJ

0109

42So

lyc09

g075

820.

21

Solu

te c

arrie

r fam

ily fa

cilit

ated

glu

cose

tran

spor

ter m

embe

r 3

1.0±

0.1

7.1±

1.0

0.00

1

38AK

3285

88So

lyc04

g072

070.

21

WRK

Y tra

nscr

iptio

n fa

ctor

16

1.0±

0.1

7.1±

0.9

0.00

3

39TA

3858

5_40

81So

lyc05

g050

130.

22

Acid

ic c

hitin

ase

1.0±

0.2

7.0±

1.1

0.00

5

40AK

3238

31So

lyc08

g006

750.

21

Deca

rbox

ylase

fam

ily p

rote

in1.

0±0.

16.

9±2.

20.

011

41AK

3286

94So

lyc12

g045

030.

11

Shor

t-ch

ain

dehy

drog

enas

e1.

0±0.

26.

8±1.

7<

0.00

1

42AK

3231

16So

lyc04

g054

950.

22

Trop

inon

e re

duct

ase

I 1.

0±0.

16.

8±2.

10.

005

43BI

4229

97So

lyc12

g056

360.

11

Thau

mat

in-li

ke p

rote

in

1.0±

0.1

6.8±

0.8

0.00

2

44AK

3251

61So

lyc07

g053

420.

22

Ring

H2

finge

r pro

tein

1.0±

0.1

6.7±

1.1

0.00

7

45TA

3870

3_40

81So

lyc04

g048

900.

22

Calre

ticul

in 2

cal

cium

-bin

ding

pro

tein

1.

0±0.

16.

5±0.

60.

001

46TA

3655

7_40

81So

lyc02

g093

250.

21

Caffe

oyl-C

oA O

-met

hyltr

ansf

eras

e 1.

0±0.

16.

5±1.

00.

004

47AK

3243

73So

lyc01

g105

630.

21

Calm

odul

in

1.0±

0.1

6.5±

0.7

<0.

001

48CK

4687

10So

lyc09

g011

590.

23

Glut

athi

one

S-tra

nsfe

rase

-like

pro

tein

1.

0±0.

16.

5±0.

90.

003

49AK

3225

55So

lyc02

g082

920.

23

Endo

chiti

nase

(Chi

tinas

e)1.

0±0.

26.

5±1.

40.

001

50TA

5699

4_40

81So

lyc03

g093

890.

21

Myb

-rel

ated

tran

scrip

tion

fact

or

1.0±

0.1

6.4±

0.8

0.00

5

* E-

valu

e: m

atch

sm

alle

r or e

qual

to 1

E-20

(cor

resp

ondi

ng to

max

imal

ly th

ree

mis

mat

ches

with

targ

et s

eque

nce)

** S

eque

nces

with

out a

nnot

atio

n (‘u

nkno

wn

prot

eins

’) w

ere

excl

uded

***

Filte

r: ge

nes

sign

ifica

ntly

up-

regu

late

d (P

<0.

05, a

fter P

-val

ue a

djus

tmen

t); e

xpre

ssio

n le

vels

of A

rela

tive

to th

e co

ntro

l (A/

C) w

ere

sorte

d fro

m h

igh

to lo

w

129



TAB

LES

4.1B

List

of t

op 5

0 ge

nes

dow

n-re

gula

ted

by A

culo

psly

cope

rsic

i(A

) as

com

pare

d to

con

trol

(C).

#Pr

imar

y ac

cess

ion

Locu

s id

entif

ier

No. m

atch

ing

Anno

tatio

n**

Aver

age

rela

tive

expr

essi

on

(cv.

Hein

z ge

nom

e)pr

obes

*(C

) ± S

E(A

) ± S

EP

***

1AK

3246

67So

lyc11

g056

680.

11

LRR

rece

ptor

-like

ser

ine

7.0±

0.4

1.3±

0.2

0.00

2

2AK

3196

24So

lyc06

g072

710.

22

RNA

polym

eras

e si

gma

fact

or

4.7±

2.7

1.1±

0.2

0.02

0

3TA

4164

1_40

81So

lyc07

g007

260.

21

Met

allo

carb

oxyp

eptid

ase

inhi

bito

r 3.

9±0.

51.

0±0.

20.

012

4ES

8941

62So

lyc08

g005

680.

26

Unde

capr

enyl

pyro

phos

phat

e sy

ntha

se

5.3±

1.2

1.4±

0.3

0.00

6

5AK

3215

85So

lyc10

g008

720.

21

GDSL

est

eras

e5.

7±1.

11.

5±0.

40.

042

6AF

0220

22So

lyc03

g120

380.

21

Auxin

resp

onse

fact

or 9

4.

0±0.

81.

1±0.

30.

015

7AK

3231

65So

lyc09

g082

660.

21

Caffe

oyl-C

oA O

-met

hyltr

ansf

eras

e 7.

3±0.

62.

2±0.

40.

016

8AK

3208

70So

lyc07

g018

240.

11

Elon

gatio

n of

ver

y lo

ng c

hain

fatty

aci

ds p

rote

in 4

5.

3±0.

61.

6±0.

30.

019

9AK

3244

70So

lyc04

g050

730.

23

GDSL

est

eras

e4.

5±0.

81.

4±0.

30.

014

10GO

3753

39So

lyc08

g068

480.

11

Indo

le-3

-ace

tic a

cid-

amid

o sy

nthe

tase

GH3

.8

5.5±

1.2

1.7±

0.2

0.00

2

11AK

3261

26So

lyc09

g059

170.

11

Anth

ocya

nidi

n 3-

O-gl

ucos

yltra

nsfe

rase

5.

2±0.

91.

7±0.

40.

032

12AK

3199

52So

lyc03

g116

730.

25

Stea

royl-

CoA

9-de

satu

rase

3.

6±0.

61.

2±0.

30.

014

13AK

3228

11So

lyc01

g110

570.

21

Auxin

-indu

ced

SAUR

-like

pro

tein

5.

7±1.

72.

0±0.

50.

001

14AK

3195

68So

lyc10

g006

900.

22

Prot

ochl

orop

hyllid

e re

duct

ase

4.1±

0.9

1.4±

0.4

0.01

0

15AK

3237

49So

lyc10

g083

300.

11

Beta

-fru

ctof

uran

osid

ase

inso

lubl

e is

oenz

yme

2 4.

3±0.

81.

5±0.

40.

010

16AI

8995

66So

lyc01

g090

970.

21

Corti

cal c

ell-d

elin

eatin

g pr

otei

n 5.

1±1.

01.

8±0.

90.

047

17BP

9047

09So

lyc02

g080

210.

23

Pect

ines

tera

se

3.4±

0.7

1.2±

0.4

0.01

4

18BI

9319

68So

lyc05

g009

270.

22

Fatty

aci

d el

onga

se 3

-ket

oacy

l-CoA

syn

thas

e 3.

2±0.

41.

2±0.

20.

018

19AK

3209

97So

lyc02

g080

230.

21

Rop-

inte

ract

ive c

rib m

otif-

cont

aini

ng p

rote

in 1

4.

4±0.

51.

6±0.

30.

013

20AI

7719

73So

lyc12

g055

970.

11

Endo

gluc

anas

e 1

2.8±

0.5

1.0±

0.2

0.00

6

21TA

3593

4_40

81So

lyc02

g087

400.

11

Anky

rin re

peat

dom

ain

cont

aini

ng p

rote

in

3.4±

0.6

1.3±

0.3

0.00

8

22AK

3255

10So

lyc05

g010

320.

22

Chal

cone

--fla

vono

ne is

omer

ase

4.2±

0.2

1.6±

0.3

0.04

4

23AK

3236

69So

lyc08

g076

820.

22

BHLH

tran

scrip

tion

fact

or

3.6±

0.3

1.3±

0.3

0.02

0

24BI

9284

80So

lyc01

g110

340.

22

Endo

gluc

anas

e 1

3.3±

0.7

1.2±

0.2

0.03

7

25TA

3882

9_40

81So

lyc01

g006

330.

22

PAP

fibril

lin fa

mily

pro

tein

3.

7±0.

91.

4±0.

20.

022

26AK

3220

06So

lyc12

g011

280.

11

Chlo

roph

yll a

-b b

indi

ng p

rote

in

3.3±

0.9

1.3±

0.3

0.01

1

27AW

6240

57So

lyc04

g078

900.

21

Cyto

chro

me

P450

3.9±

0.8

1.5±

0.3

0.01

1

28DB

7185

13So

lyc12

g094

640.

11

Glyc

eral

dehy

de-3

-pho

spha

te d

ehyd

roge

nase

B

3.3±

0.3

1.3±

0.2

0.01

6

29BP

8966

62So

lyc02

g086

180.

21

Ster

ol C

-5 d

esat

uras

e3.

8±0.

71.

5±0.

2<

0.00

1

30TA

5569

0_40

81So

lyc05

g053

400.

11

Gluc

osylt

rans

fera

se

3.3±

0.3

1.3±

0.2

0.00

6

31AK

2465

35So

lyc12

g006

140.

11

Chlo

roph

yll a

-b b

indi

ng p

rote

in

3.2±

1.1

1.2±

0.4

0.00

8

130



32AK

3272

04So

lyc09

g091

510.

22

Chal

cone

syn

thas

e 4.

9±0.

71.

9±0.

40.

043

33AK

2474

38So

lyc10

g018

300.

11

Tran

sket

olas

e 1

3.2±

0.7

1.3±

0.3

0.01

6

34AK

3209

97So

lyc04

g081

300.

22

Endo

gluc

anas

e 1

3.2±

0.2

1.3±

0.2

0.01

5

35DB

6954

86So

lyc03

g025

190.

21

Mul

tidru

g re

sist

ance

pro

tein

mdt

K 5.

0±0.

52.

0±0.

40.

029

36AK

3251

10So

lyc04

g071

340.

22

Fruc

tose

-1 6

-bis

phos

phat

ase

clas

s 1

2.5±

0.8

1.0±

0.2

0.02

6

37TA

3689

6_40

81So

lyc04

g054

740.

27

Inos

itol-3

-pho

spha

te s

ynth

ase

3.4±

0.5

1.3±

0.2

0.00

4

38AW

6257

52So

lyc03

g083

100.

21

Calm

odul

in-b

indi

ng p

rote

in fa

mily

-like

3.

7±0.

61.

5±0.

10.

004

39TA

3641

7_40

81So

lyc08

g067

320.

11

Chlo

roph

yll a

3.4±

0.4

1.3±

0.3

0.00

5

40AK

3267

20So

lyc03

g019

790.

22

Alph

a-ga

lact

osid

ase

3.2±

0.4

1.3±

0.3

0.02

1

41AK

2474

84So

lyc01

g091

320.

21

Ster

ol 4

-alp

ha-m

ethy

l-oxid

ase

2 2.

5±0.

21.

0±0.

20.

024

42K0

3290

Solyc

08g0

6901

0.2

2Pe

ntat

ricop

eptid

e re

peat

-con

tain

ing

prot

ein

3.2±

0.5

1.3±

0.2

0.00

8

43DB

7078

84So

lyc09

g065

780.

21

Fatty

aci

d el

onga

se 3

-ket

oacy

l-CoA

syn

thas

e 2.

5±0.

81.

0±0.

10.

025

44AW

0392

65So

lyc01

g108

630.

22

Nitri

te re

duct

ase

2.5±

0.6

1.0±

0.2

0.00

5

45AI

7798

61So

lyc12

g099

650.

11

Phot

osys

tem

II 5

kDa

pro

tein

3.3±

0.8

1.4±

0.4

0.03

6

46AK

3253

98So

lyc05

g005

080.

25

Endo

-1 4

-bet

a-gl

ucan

ase

2.4±

0.3

1.0±

0.2

0.01

2

47AI

4826

04So

lyc03

g116

280.

22

1-am

inoc

yclo

prop

ane-

1-ca

rbox

ylate

oxid

ase

2.8±

0.3

1.2±

0.1

0.00

3

48BT

0137

70So

lyc04

g008

040.

22

mic

rotu

bule

ass

ocia

ted

prot

ein

Type

1

3.1±

0.5

1.3±

0.3

0.01

1

49TA

3971

6_40

81So

lyc06

g064

550.

25

Aspa

rtoki

nase

-hom

oser

ine

dehy

drog

enas

e 2.

9±0.

61.

2±0.

30.

015

50AK

3259

03So

lyc06

g060

340.

25

Chlo

ropl

ast p

hoto

syst

em II

-ass

ocia

ted

prot

ein

3.4±

0.4

1.4±

0.2

0.01

1

* Se

quen

ces

with

out a

nnot

atio

n (‘u

nkno

wn

prot

eins

’) w

ere

excl

uded

** E

-val

ue: m

atch

sm

alle

r or e

qual

to 1

E-20

(cor

resp

ondi

ng to

max

imal

ly th

ree

mis

mat

ches

with

targ

et s

eque

nce)

***

Filte

r: ge

nes

sign

ifica

ntly

dow

n-re

gula

ted

(P<

0.05

, afte

r P-v

alue

adj

ustm

ent);

exp

ress

ion

leve

ls o

f A re

lativ

e to

the

cont

rol (

A/C)

wer

e so

rted

from

low

to h

igh

131



TAB

LES

4.2A

List

of t

op 1

5 ge

nes

up-r

egul

ated

by

Tetr

anyc

hus

urtic

ae(U

) but

not

by

Acu

lops

lyco

pers

ici(

A) a

s co

mpa

red

to c

ontr

ol (C

).#

Prim

ary

acce

ssio

nLo

cus

iden

tifie

rNo

. An

nota

tion*

*Av

erag

e re

lativ

e ex

pres

sion

(cv.

Hein

z ge

nom

e)m

atch

ing

prob

es*

(C) ±

SE

(U) ±

SE

P**

*(A

) ± S

EP

(U+

A) ±

SEP

1BI

4227

99So

lyc10

g083

690.

21

Cyto

chro

me

P450

1.0±

0.2

22.4

±7.

90.

009

14.0

±7.

00.

072

54.4

±27

.60.

003

2AW

7382

22So

lyc03

g098

720.

21

Kuni

tz tr

ypsi

n in

hibi

tor

1.0±

0.2

17.0

±5.

80.

015

2.3±

0.6

0.05

33.

4±1.

10.

13

3TA

3683

1_40

81So

lyc03

g020

080.

21

Prot

eina

se in

hibi

tor I

I 1.

0±0.

215

.0±

5.8

0.00

42.

8±1.

40.

194.

6±1.

20.

012

4AK

3295

07So

lyc04

g064

880.

21

Path

ogen

esis

-rel

ated

pro

tein

-like

pro

tein

1.

0±0.

214

.7±

3.4

0.00

28.

9±5.

40.

080

52.7

±40

.20.

025

5TA

5314

9_40

81So

lyc01

g106

600.

22

Path

ogen

esis

-rel

ated

pro

tein

1

1.1±

0.1

9.8±

1.9

<0.

001

1.3±

0.3

0.58

7.6±

1.3

0.00

5

6AI

4834

84So

lyc01

g095

960.

22

Diac

ylglyc

erol

O-a

cyltr

ansf

eras

e 1.

0±0.

17.

5±0.

90.

003

1.4±

0.3

0.08

32.

5±0.

50.

12

7AK

2472

44So

lyc03

g020

060.

21

Prot

eina

se in

hibi

tor I

I 1.

0±0.

27.

5±1.

90.

006

1.3±

0.5

0.89

2.6±

0.9

0.02

3

8TA

3686

4_40

81So

lyc10

g083

700.

21

Cyto

chro

me

P450

1.0±

0.1

7.2±

2.8

0.02

44.

2±1.

60.

073

18.0

±9.

50.

013

9X9

4946

Solyc

03g0

2005

0.2

2Pr

otei

nase

inhi

bito

r II (

CEV1

57)

1.0±

0.1

6.7±

2.7

0.02

31.

6±0.

50.

276.

4±3.

20.

065

10TA

3997

0_40

81So

lyc08

g074

680.

22

Polyp

heno

l oxid

ase

1.0±

0.1

6.6±

1.0

0.01

21.

5±0.

40.

156.

7±1.

10.

008

11AK

3208

74So

lyc12

g045

020.

11

Cyto

chro

me

P450

1.2±

0.3

7.1±

0.9

0.01

04.

6±1.

20.

076

9.6±

0.5

0.00

4

12AK

3252

84So

lyc02

g087

070.

21

Pero

xidas

e fa

mily

pro

tein

1.

0±0.

15.

3±0.

60.

003

2.0±

0.3

0.06

65.

2±1.

10.

008

13AF

0926

55So

lyc06

g074

990.

11

Nitra

te tr

ansp

orte

r 1.

0±0.

15.

2±0.

90.

006

3.1±

0.8

0.07

48.

0±2.

20.

007

14AK

3204

68So

lyc03

g097

500.

22

Hydr

oxyc

inna

moy

l CoA

shi

kim

ate

1.0±

0.1

4.6±

0.5

0.00

43.

5±1.

60.

078

7.8±

1.9

0.00

9

15TA

4728

3_40

81So

lyc10

g076

680.

11

1-am

inoc

yclo

prop

ane-

1-ca

rbox

ylate

oxid

ase

1.0±

0.1

4.5±

1.4

0.01

21.

4±0.

40.

395.

0±2.

50.

075

* Se

quen

ces

with

out a

nnot

atio

n (‘u

nkno

wn

prot

eins

’) w

ere

excl

uded

** E

-val

ue: m

atch

sm

alle

r or e

qual

to 1

E-20

(cor

resp

ondi

ng to

max

imal

ly th

ree

mis

mat

ches

with

targ

et s

eque

nce)

***

Filte

r: ge

nes

sign

ifica

ntly

indu

ced

by U

(P<

0.05

, afte

r P-v

alue

adj

ustm

ent)

and

not r

egul

ated

by

A; e

xpre

ssio

n le

vels

of U

rela

tive

to th

e co

ntro

l (U/

C) w

ere

sorte

d fro

m h

igh

to lo

w

132



TAB

LES

4.2B

List

of t

op 1

5 ge

nes

up-r

egul

ated

by

Acu

lops

lyco

pers

ici(

A) b

ut n

ot b

y Te

tran

ychu

sur

ticae

(U) a

s co

mpa

red

to c

ontr

ol (C

).#

Prim

ary

acce

ssio

nLo

cus

iden

tifie

rNo

. An

nota

tion*

*Av

erag

e re

lativ

e ex

pres

sion

(cv.

Hein

z ge

nom

e)m

atch

ing

prob

es*

(C) ±

SE

(U) ±

SE

P**

*(A

) ± S

EP

(U+

A) ±

SEP

1BG

1270

02So

lyc08

g078

870.

13

Prol

ine-

rich

prot

ein

1.0±

0.2

2.7±

0.6

0.10

6.3±

1.4

0.01

46.

9±1.

60.

022

2AK

3239

35So

lyc01

g079

940.

23

Xyla

nase

inhi

bito

r 1.

0±0.

21.

6±0.

30.

176.

0±2.

10.

010

4.8±

0.4

0.00

7

3ES

8960

26So

lyc09

g018

020.

22

Expa

nsin

1.

1±0.

11.

0±0.

10.

706.

4±1.

70.

015

5.6±

1.7

0.02

8

4BW

6915

10So

lyc09

g063

060.

21

GDSL

est

eras

e1.

0±0.

11.

1±0.

10.

535.

8±2.

10.

048

3.3±

0.6

0.01

6

5TA

5459

0_40

81So

lyc06

g061

280.

21

Cinn

amoy

l-CoA

redu

ctas

e-lik

e pr

otei

n1.

0±0.

11.

4±0.

20.

135.

2±1.

50.

021

5.2±

1.0

0.00

7

6BT

0142

83So

lyc02

g071

620.

21

GDSL

est

eras

e1.

0±0.

11.

4±0.

20.

205.

1±0.

80.

001

5.2±

1.4

0.01

1

7AK

3217

80So

lyc08

g068

770.

11

N-ac

etylt

rans

fera

se

1.0±

0.2

3.6±

1.2

0.06

34.

9±0.

80.

024

7.1±

1.9

0.00

9

8AW

2238

74So

lyc12

g070

080.

11

N-ac

yleth

anol

amin

e am

idoh

ydro

lase

1.

0±0.

22.

8±0.

80.

053

4.2±

1.4

0.01

15.

7±1.

90.

043

9AW

6494

10So

lyc11

g067

190.

11

Fatty

acy

l coA

redu

ctas

e 1.

0±0.

11.

8±0.

40.

123.

9±0.

90.

005

11.8

±8.

90.

089

10AK

2473

96So

lyc08

g067

390.

21

Blig

ht-a

ssoc

iate

d pr

otei

n P1

2 1.

1±0.

21.

7±0.

30.

154.

0±0.

90.

021

3.2±

0.4

0.01

7

11BT

0129

62So

lyc05

g009

430.

21

Endo

nucl

ease

1.

0±0.

21.

5±0.

30.

253.

6±0.

50.

019

3.7±

0.7

0.01

6

12BI

2089

39So

lyc05

g007

300.

22

Rece

ptor

exp

ress

ion-

enha

ncin

g pr

otei

n 5

1.0±

0.1

1.5±

0.1

0.10

3.1±

0.6

0.01

92.

1±0.

20.

019

13AK

3210

18So

lyc09

g082

570.

21

Neur

ogen

ic lo

cus

notc

h pr

otei

n-lik

e 1.

0±0.

11.

5±0.

10.

079

3.1±

0.4

0.00

92.

7±0.

30.

007

14AF

5147

75So

lyc11

g065

760.

11

Thym

idin

e ki

nase

1.

0±0.

21.

7±0.

50.

083

3.1±

1.1

0.03

02.

1±0.

20.

12

15AK

3221

07So

lyc06

g072

550.

21

UPF0

497

mem

bran

e pr

otei

n 8

1.0±

0.3

1.7±

0.1

0.07

63.

0±0.

40.

007

3.0±

0.2

0.01

2

* Se

quen

ces

with

out a

nnot

atio

n (‘u

nkno

wn

prot

eins

’) w

ere

excl

uded

** E

-val

ue: m

atch

sm

alle

r or e

qual

to 1

E-20

(cor

resp

ondi

ng to

max

imal

ly th

ree

mis

mat

ches

with

targ

et s

eque

nce)

***

Filte

r: ge

nes

sign

ifica

ntly

indu

ced

by A

(P<

0.05

, afte

r P-v

alue

adj

ustm

ent)

and

not r

egul

ated

by

U; e

xpre

ssio

n le

vels

of A

rela

tive

to th

e co

ntro

l (A/

C) w

ere

sorte

d fro

m h

igh

to lo

w

133



TAB

LES

4.2C

Lis

t of

top

15

gene

s up

-reg

ulat

ed b

y bo

th A

culo

psly

cope

rsic

i(A

) and

Tet

rany

chus

urtic

ae(U

) as

com

pare

d to

con

trol

(C).

#Pr

imar

y ac

cess

ion

Locu

s id

entif

ier

No.

Anno

tatio

n**

Aver

age

rela

tive

expr

essi

on

(cv.

Hein

z ge

nom

e)m

atch

ing

prob

es*

(C) ±

SE

(U) ±

SE

P**

*(A

) ± S

EP

(U+

A) ±

SEP

1BE

3547

88So

lyc03

g098

760.

11

Kuni

tz-t

ype

prot

ease

inhi

bito

r-lik

e pr

otei

n 1.

0±0.

326

.3±

6.9

0.00

14.

4±1.

20.

017

9.9±

2.6

0.00

2

2AI

4873

43So

lyc10

g005

320.

21

Tryp

toph

an s

ynth

ase

beta

cha

in 1

1.

0±0.

122

.3±

0.8

<0.

001

7.4±

3.7

0.03

626

.9±

12.3

0.00

6

3BI

4219

69So

lyc01

g080

570.

21

Inos

ine-

urid

ine

pref

errin

g nu

cleo

side

hydr

olas

e fa

mily

pro

tein

1.

0±0.

219

.6±

0.9

<0.

001

10.4

±2.

00.

008

23.9

±5.

1<

0.00

1

4BW

6928

67So

lyc05

g014

590.

21

Tran

scrip

tion

Fact

or

1.0±

0.2

15.4

±2.

30.

002

14.3

±1.

20.

001

22.9

±3.

9<

0.00

1

5BI

9290

69So

lyc01

g105

450.

21

ABC

trans

porte

r G fa

mily

mem

ber 1

1 1.

0±0.

214

.8±

3.4

0.00

35.

0±0.

70.

003

9.9±

1.9

0.00

1

6M

8060

8So

lyc01

g060

020.

213

Beta

-glu

cana

se

1.0±

0.2

14.8

±5.

70.

004

11.9

±4.

90.

011

18.3

±2.

40.

002

7BI

2045

48So

lyc09

g091

550.

22

Salic

ylic

acid

car

boxy

l met

hyltr

ansf

eras

e 1.

0±0.

313

.3±

2.5

0.00

17.

3±1.

20.

013

13.6

±1.

60.

002

8X7

9337

Solyc

05g0

0795

0.2

1Ri

bonu

clea

se T

2 1.

0±0.

211

.3±

1.4

0.00

120

.2±

2.5

0.00

119

.7±

3.0

0.00

1

9AM

9497

88So

lyc02

g067

750.

21

Carb

onic

anh

ydra

se

1.0±

0.2

10.9

±5.

10.

012

3.3±

0.5

0.00

09.

2±3.

30.

025

10X0

4792

Solyc

07g0

4953

0.2

31-

amin

ocyc

lopr

opan

e-1-

carb

oxyla

te o

xidas

e 1.

0±0.

210

.7±

1.8

0.00

27.

6±2.

00.

002

16.1

±3.

70.

004

11TA

5341

2_40

81So

lyc02

g092

120.

21

Phyt

osul

foki

nes

3 1.

0±0.

110

.2±

0.7

<0.

001

6.4±

1.0

0.00

415

.9±

6.3

0.00

7

12BI

2053

17So

lyc12

g049

030.

11

Fatty

aci

d de

satu

rase

1.

0±0.

39.

8±0.

50.

002

8.0±

1.9

0.00

113

.6±

1.8

0.00

1

13Y0

8804

Solyc

00g1

7434

0.1

4Pa

thog

enes

is-r

elat

ed p

rote

in 1

b 1.

0±0.

29.

8±2.

30.

004

7.5±

1.4

0.00

516

.3±

2.7

0.00

1

14AK

3209

83So

lyc10

g078

230.

11

Cyto

chro

me

P450

1.1±

0.1

10.0

±1.

40.

004

10.0

±1.

90.

006

24.6

±8.

50.

002

15TA

3839

2_40

81So

lyc03

g006

700.

22

Pero

xidas

e 1.

0±0.

29.

1±2.

00.

003

14.2

±5.

40.

013

25.0

±5.

20.

003

* Se

quen

ces

with

out a

nnot

atio

n (‘u

nkno

wn

prot

eins

’) w

ere

excl

uded

** E

-val

ue: m

atch

sm

alle

r or e

qual

to 1

E-20

(cor

resp

ondi

ng to

max

imal

ly th

ree

mis

mat

ches

with

targ

et s

eque

nce)

***

Filte

r: ge

nes

sign

ifica

ntly

indu

ced

by U

as

wel

l as

by A

(P<

0.05

, afte

r P-v

alue

adj

ustm

ent);

exp

ress

ion

leve

ls o

f U re

lativ

e to

the

cont

rol (

U/C)

wer

e so

rted

from

hig

h to

low

134



TAB

LES

4.2D

List

of t

op 1

5 ge

nes

dow

n-re

gula

ted

by b

oth

Acu

lops

lyco

pers

ici(

A) a

nd T

etra

nych

usur

ticae

(U) a

s co

mpa

red

to c

ontr

ol (C

).#

Prim

ary

acce

ssio

nLo

cus

iden

tifie

rNo

. An

nota

tion*

*Av

erag

e re

lativ

e ex

pres

sion

(cv.

Hein

z ge

nom

e)m

atch

ing

prob

es*

(C) ±

SE

(U) ±

SE

P**

*(A

) ± S

EP

(U+

A) ±

SEP

1AK

3230

38So

lyc09

g008

320.

22

Xylo

gluc

an e

ndot

rans

gluc

osyla

se4.

8±1.

01.

4±0.

30.

004

2.0±

0.5

0.00

61.

0±0.

30.

005

2GO

3753

39So

lyc08

g068

480.

11

Indo

le-3

-ace

tic a

cid-

amid

o sy

nthe

tase

5.

5±1.

21.

7±0.

40.

003

1.7±

0.2

0.00

21.

0±0.

30.

044

3AK

3196

74So

lyc08

g063

090.

21

Delta

-6-d

esat

uras

e4.

6±0.

91.

5±0.

40.

018

2.3±

0.5

0.04

11.

0±0.

30.

008

4AK

3246

67So

lyc11

g056

680.

11

LRR

rece

ptor

-like

ser

ine

7.0±

0.4

2.3±

0.6

0.02

11.

3±0.

20.

002

1.0±

0.4

0.01

1

5AK

3194

83So

lyc07

g052

980.

24

Xylo

gluc

an e

ndot

rans

gluc

osyla

se4.

0±0.

61.

4±0.

30.

030

1.8±

0.3

0.00

01.

0±0.

40.

027

6AK

3221

23So

lyc01

g089

850.

21

Cycl

in-d

epen

dent

pro

tein

kin

ase

regu

lato

r 4.

0±0.

41.

5±0.

40.

010

2.1±

0.2

0.00

91.

0±0.

20.

019

7AF

0220

22So

lyc03

g120

380.

21

Auxin

resp

onse

fact

or 9

4.

0±0.

81.

5±0.

00.

018

1.1±

0.3

0.01

51.

0±0.

20.

001

8AK

3236

69So

lyc08

g076

820.

22

BHLH

tran

scrip

tion

fact

or

3.6±

0.3

1.4±

0.3

0.01

61.

3±0.

30.

020

1.0±

0.1

0.00

0

9AK

3228

11So

lyc01

g110

570.

21

Auxin

-indu

ced

SAUR

-like

pro

tein

5.

7±1.

72.

3±0.

90.

026

2.0±

0.5

0.00

11.

0±0.

20.

008

10AK

3209

04So

lyc02

g091

690.

23

BHLH

tran

scrip

tion

fact

or

3.3±

0.6

1.4±

0.2

0.04

11.

5±0.

20.

022

1.0±

0.1

0.00

5

11AI

8995

66So

lyc01

g090

970.

21

Corti

cal c

ell-d

elin

eatin

g pr

otei

n 5.

1±1.

02.

1±0.

50.

001

1.8±

0.9

0.04

71.

0±0.

30.

036

12AK

3231

65So

lyc09

g082

660.

21

Caffe

oyl-C

oA O

-met

hyltr

ansf

eras

e 7.

3±0.

63.

0±0.

60.

029

2.2±

0.4

0.01

61.

0±0.

30.

016

13DB

6901

87So

lyc06

g066

640.

21

Phot

osys

tem

I re

actio

n ce

nter

sub

unit

VI3.

0±0.

51.

2±0.

20.

001

1.4±

0.3

0.00

21.

0±0.

10.

005

14AK

3195

84So

lyc04

g074

640.

23

L-as

corb

ate

pero

xidas

e 3.

6±0.

61.

5±0.

30.

005

1.5±

0.5

0.02

91.

0±0.

20.

015

15AK

3209

97So

lyc02

g080

230.

21

Rop-

inte

ract

ive c

rib m

otif-

cont

aini

ng

prot

ein

1 4.

4±0.

51.

9±0.

30.

031

1.6±

0.3

0.01

31.

0±0.

20.

006

* Se

quen

ces

with

out a

nnot

atio

n (‘u

nkno

wn

prot

eins

’) w

ere

excl

uded

** E

-val

ue: m

atch

sm

alle

r or e

qual

to 1

E-20

(cor

resp

ondi

ng to

max

imal

ly th

ree

mis

mat

ches

with

targ

et s

eque

nce)

***

Filte

r: ge

nes

sign

ifica

ntly

dow

n-re

gula

ted

by U

as

wel

l as

by A

(P<

0.05

, afte

r P-v

alue

adj

ustm

ent);

exp

ress

ion

leve

ls o

f U re

lativ

e to

the

cont

rol (

U/C)

wer

e so

rted

from

low

to h

igh

135



TAB

LES

4.3A

List

of t

op 1

5 ge

nes

up-r

egul

ated

by

Tetr

anyc

hus

evan

si(E

) but

not

by

Acu

lops

lyco

pers

ici(

A) a

s co

mpa

red

to c

ontr

ol (C

). Te

tran

ychu

s ur

ticae

(U) i

s sh

own

as t

he b

ench

mar

k.#

Prim

ary

acce

ssio

nLo

cus

iden

tifie

rNo

. An

nota

tion*

*Av

erag

e re

lativ

e ex

pres

sion

(cv.

Hein

z ge

nom

e)m

atch

ing

prob

es*

(C) ±

SE

(E) ±

SE

P**

*(A

) ± S

EP

(U) ±

SE

P(U

+A)

± S

EP

1AW

7382

22So

lyc03

g098

720.

21

Kuni

tz tr

ypsi

n in

hibi

tor

1.0±

0.2

4.5±

1.2

0.01

02.

3±0.

60.

053

17.0

±5.

80.

015

3.4±

1.1

0.13

2U5

0152

Solyc

12g0

1002

0.1

2Le

ucyl

amin

opep

tidas

e 1.

0±0.

22.

6±1.

10.

032

1.6±

0.7

0.78

3.7±

1.2

0.04

81.

9±0.

70.

27

3EU

9347

34So

lyc12

g099

120.

11

MYB

tran

scrip

tion

fact

or

1.0±

0.1

1.4±

0.1

0.02

51.

1±0.

21.

01.

2±0.

20.

741.

2±0.

40.

95

4BE

4594

32So

lyc07

g008

210.

21

TPR

dom

ain

prot

ein

3.5±

0.7

4.8±

0.9

0.04

72.

5±0.

80.

322.

0±0.

20.

131.

0±0.

20.

042

5BI

9338

77So

lyc12

g005

310.

11

Auxin

-res

pons

ive G

H3-li

ke

1.2±

0.1

1.6±

0.2

0.00

61.

2±0.

10.

961.

0±0.

10.

321.

1±0.

10.

96

6BF

0964

97So

lyc09

g091

950.

11

Ethy

lene

-res

pons

ive tr

ansc

riptio

n

fact

or 1

1.

1±0.

11.

5±0.

10.

003

1.0±

0.2

0.78

1.3±

0.2

0.41

1.0±

0.1

0.62

7BP

8848

66So

lyc10

g007

680.

21

Regu

lato

r of c

hrom

osom

e

cond

ensa

tion

RCC1

1.

1±0.

01.

5±0.

00.

005

1.0±

0.0

0.16

1.0±

0.1

0.35

1.1±

0.1

0.98

8BM

4100

63So

lyc05

g008

040.

21

ATP-

depe

nden

t DNA

hel

icas

e

RECG

-like

1.0±

0.1

1.3±

0.1

0.01

81.

4±0.

10.

064

1.4±

0.0

0.07

21.

2±0.

10.

27

9TC

2098

57So

lyc03

g098

010.

21

Acid

pho

spha

tase

1.

5±0.

31.

9±0.

50.

008

1.0±

0.2

0.23

1.2±

0.2

0.52

1.1±

0.2

0.66

10AK

3246

39So

lyc01

g006

390.

21

Cyst

eine

-ric

h ex

tens

in-li

ke

prot

ein-

4 1.

1±0.

11.

3±0.

20.

018

1.0±

0.0

0.82

2.4±

0.3

0.00

31.

4±0.

30.

42

11DB

6857

72So

lyc05

g014

380.

21

Mul

tidru

g re

sist

ance

pro

tein

ABC

trans

porte

r fam

ily

1.0±

0.1

1.2±

0.0

0.03

21.

1±0.

10.

401.

2±0.

10.

181.

3±0.

20.

13

12AI

8989

32So

lyc04

g080

790.

22

BEL1

-like

hom

eodo

mai

n pr

otei

n 3

1.0±

0.1

1.2±

0.1

0.02

71.

3±0.

20.

201.

2±0.

10.

341.

3±0.

20.

26

13TA

5636

2_40

81So

lyc01

g098

690.

22

LRR

rece

ptor

-like

ser

ine

1.0±

0.1

1.2±

0.1

0.01

31.

5±0.

20.

053

1.4±

0.2

0.09

21.

5±0.

10.

011

14AK

3212

41So

lyc07

g045

160.

22

Phos

phof

ruct

okin

ase

fam

ily p

rote

in 1

.0±

0.3

1.2±

0.3

0.01

91.

3±0.

10.

231.

3±0.

20.

121.

3±0.

20.

22

15TA

4181

4_40

81So

lyc09

g042

750.

21

Acyl-

CoA

thio

este

rase

9

1.0±

0.1

1.2±

0.1

0.03

41.

3±0.

10.

311.

3±0.

10.

301.

2±0.

10.

42

* Se

quen

ces

with

out a

nnot

atio

n (‘u

nkno

wn

prot

eins

’) w

ere

excl

uded

** E

-val

ue: m

atch

sm

alle

r or e

qual

to 1

E-20

(cor

resp

ondi

ng to

max

imal

ly th

ree

mis

mat

ches

with

targ

et s

eque

nce)

***

Filte

r: ge

nes

sign

ifica

ntly

indu

ced

by E

(P<

0.05

, afte

r P-v

alue

adj

ustm

ent)

and

not b

y A;

exp

ress

ion

leve

ls o

f E re

lativ

e to

the

cont

rol (

E/C)

wer

e so

rted

from

hig

h to

low

136



TAB

LES

4.3B

List

of t

op 1

5 ge

nes

up-r

egul

ated

by

Acu

lops

lyco

pers

ici(

A) b

ut n

ot b

y Te

tran

ychu

sev

ansi

(E) a

s co

mpa

red

to c

ontr

ol (C

). Te

tran

ychu

s u

rtic

ae(U

) is

show

nas

the

ben

chm

ark.

#Pr

imar

y ac

cess

ion

Locu

s id

entif

ier

No.

Anno

tatio

n**

Aver

age

rela

tive

expr

essi

on

(cv.

Hein

z ge

nom

e)m

atch

ing

prob

es*

(C) ±

SE

(E) ±

SE

P**

*(A

) ± S

EP

(U) ±

SE

P(U

+A)

± S

EP

1TA

5390

0_40

81So

lyc02

g062

890.

11

Myo

-inos

itol t

rans

porte

r 2

1.1±

0.2

1.0±

0.1

1.0

17.7

±6.

20.

003

7.3±

1.4

0.00

316

.9±

2.2

0.00

1

2TA

3839

2_40

81So

lyc03

g006

700.

22

Pero

xidas

e1.

0±0.

21.

3±0.

20.

2414

.2±

5.4

0.01

39.

1±2.

00.

003

25.0

±5.

20.

003

3BI

2039

29So

lyc04

g071

070.

22

Unkn

own

Prot

ein

(Ext

ensi

n-lik

e

regi

on)

1.0±

0.2

2.1±

0.6

0.12

13.4

±6.

80.

015

3.5±

0.7

0.00

312

.8±

4.2

0.00

5

4M

8060

8So

lyc01

g060

020.

213

Beta

-glu

cana

se

1.0±

0.2

1.2±

0.2

0.60

11.9

±4.

90.

011

14.8

±5.

70.

004

18.3

±2.

40.

002

5BI

4211

64So

lyc03

g098

740.

11

Kuni

tz tr

ypsi

n in

hibi

tor

1.0±

0.0

1.1±

0.1

0.62

10.7

±6.

10.

047

8.7±

2.7

0.00

745

.5±

35.8

0.02

3

6AK

3250

41So

lyc08

g067

340.

23

WRK

Y tra

nscr

iptio

n fa

ctor

1.

0±0.

31.

4±0.

20.

2010

.1±

4.2

0.00

46.

5±1.

50.

002

9.2±

2.2

0.01

3

7DB

6871

20So

lyc02

g036

480.

11

Harp

in-in

duce

d pr

otei

n-lik

e1.

0±0.

11.

9±0.

40.

061

9.6±

1.3

0.00

27.

6±0.

50.

001

13.1

±3.

30.

003

8AK

3209

83So

lyc10

g078

230.

11

Cyto

chro

me

P450

1.1±

0.1

1.0±

0.2

1.0

10.0

±1.

90.

006

10.0

±1.

40.

004

24.6

±8.

50.

002

9AK

3278

96So

lyc01

g006

950.

21

Synt

axin

1.

0±0.

21.

8±0.

60.

219.

3±2.

20.

004

6.8±

1.2

<0.

001

13.4

±3.

80.

009

10AI

7812

81So

lyc09

g072

810.

21

Rece

ptor

like

kin

ase

1.0±

0.1

1.4±

0.4

0.52

9.2±

1.4

0.00

25.

4±0.

3<

0.00

110

.1±

1.5

0.00

1

11AK

3208

17So

lyc09

g010

000.

22

1-am

inoc

yclo

prop

ane-

1-ca

rbox

ylate

oxid

ase-

like

prot

ein

1.0±

0.2

1.5±

0.2

0.17

8.9±

2.1

0.00

24.

7±0.

90.

003

10.5

±2.

20.

003

12BE

4516

16So

lyc11

g007

890.

11

1-am

inoc

yclo

prop

ane-

1-

carb

oxyla

te o

xidas

e 4

1.0±

0.1

1.2±

0.1

0.29

8.6±

1.9

0.00

63.

1±0.

30.

002

7.2±

0.6

<0.

001

13AK

3204

53So

lyc05

g052

280.

21

Pero

xidas

e1.

0±0.

11.

2±0.

20.

178.

4±2.

70.

006

3.9±

0.9

0.00

28.

9±0.

8<

0.00

1

14TA

3674

2_40

81So

lyc09

g090

980.

21

Maj

or a

llerg

en M

al d

1

1.0±

0.1

1.2±

0.3

0.85

8.3±

1.5

0.00

46.

0±1.

00.

007

11.9

±2.

80.

004

15AW

0399

18So

lyc01

g080

800.

21

PBSP

dom

ain-

cont

aini

ng p

rote

in

1.1±

0.1

1.0±

0.2

0.84

8.7±

1.6

0.00

27.

6±1.

40.

004

17.0

±7.

50.

008

* Se

quen

ces

with

out a

nnot

atio

n (‘u

nkno

wn

prot

eins

’) w

ere

excl

uded

** E

-val

ue: m

atch

sm

alle

r or e

qual

to 1

E-20

(cor

resp

ondi

ng to

max

imal

ly th

ree

mis

mat

ches

with

targ

et s

eque

nce)

***

Filte

r: ge

nes

sign

ifica

ntly

indu

ced

by A

(P<

0.05

, afte

r P-v

alue

adj

ustm

ent)

and

not b

y E;

exp

ress

ion

leve

ls o

f A re

lativ

e to

the

cont

rol (

A/C)

wer

e so

rted

from

hig

h to

low

137



TAB

LES

4.3C

List

of t

op 1

5 ge

nes

up-r

egul

ated

by

both

Acu

lops

lyco

pers

ici(

A) a

nd T

etra

nych

usev

ansi

(E) a

s co

mpa

red

to c

ontr

ol (C

). Te

tran

ychu

s ur

ticae

(U) i

s sh

own

as t

he b

ench

mar

k.#

Prim

ary

acce

ssio

nLo

cus

iden

tifie

rNo

. An

nota

tion*

*Av

erag

e re

lativ

e ex

pres

sion

(cv.

Hein

z ge

nom

e)m

atch

ing

prob

es*

(C) ±

SE

(E) ±

SE

P**

*(A

) ± S

EP

(U) ±

SE

P(U

+A)

± S

EP

1BE

3547

88So

lyc03

g098

760.

11

Kuni

tz-t

ype

prot

ease

inhi

bito

r-

like

prot

ein

1.0±

0.3

6.3±

1.3

0.00

14.

4±1.

20.

017

26.3

±6.

90.

001

9.9±

2.6

0.00

2

2X7

9337

Solyc

05g0

0795

0.2

1Ri

bonu

clea

se T

21.

0±0.

26.

1±1.

90.

002

20.2

±2.

50.

001

11.3

±1.

40.

001

19.7

±3.

00.

001

3BW

6928

67So

lyc05

g014

590.

21

Tran

scrip

tion

Fact

or

1.0±

0.2

4.4±

1.0

0.00

214

.3±

1.2

0.00

115

.4±

2.3

0.00

222

.9±

3.9

<0.

001

4AI

7794

01So

lyc06

g034

370.

11

Pect

ines

tera

se

1.0±

0.2

3.8±

0.8

0.00

212

.8±

3.3

0.00

48.

1±0.

20.

001

15.3

±0.

4<

0.00

1

5AI

4873

43So

lyc10

g005

320.

21

Tryp

toph

an s

ynth

ase

beta

cha

in 1

1.0±

0.1

3.4±

0.2

0.00

27.

4±3.

70.

036

22.3

±0.

8<

0.00

126

.9±

12.3

0.00

6

6AJ

2710

93So

lyc04

g079

730.

12

cyto

chro

me

P450

(AOS

)1.

0±0.

13.

3±0.

70.

001

4.0±

0.4

0.00

74.

2±0.

70.

014

4.4±

0.7

0.00

6

7BI

9290

69So

lyc01

g105

450.

21

ABC

trans

porte

r G fa

mily

mem

ber 1

1 1.

0±0.

23.

0±0.

80.

006

5.0±

0.7

0.00

314

.8±

3.4

0.00

39.

9±1.

90.

001

8BI

2045

48So

lyc09

g091

550.

22

Salic

ylic

acid

car

boxy

l

met

hyltr

ansf

eras

e 1.

0±0.

32.

8±0.

60.

017

7.3±

1.2

0.01

313

.3±

2.5

0.00

113

.6±

1.6

0.00

2

9BI

2084

59So

lyc02

g093

580.

23

Pect

ate

lyase

1.0±

0.2

2.8±

0.6

0.00

18.

6±2.

00.

003

7.1±

0.8

0.00

111

.4±

1.9

0.00

1

10BF

0977

28So

lyc01

g021

600.

21

Dise

ase

resi

stan

ce re

spon

se

prot

ein

1.0±

0.1

2.6±

0.3

0.00

131

.0±

4.6

<0.

001

7.8±

0.4

<0.

001

29.4

±3.

4<

0.00

1

11AK

3261

43So

lyc06

g075

690.

22

Auxin

-reg

ulat

ed p

rote

in1.

0±0.

12.

4±0.

50.

024

10.5

±2.

60.

001

5.5±

0.9

0.00

113

.6±

3.8

<0.

001

12AK

3252

49So

lyc03

g013

160.

21

Amin

o ac

id tr

ansp

orte

r fam

ily

prot

ein

1.0±

0.1

2.3±

0.6

0.01

13.

1±0.

30.

009

4.5±

0.4

<0.

001

4.1±

0.7

0.00

4

13AK

3272

64So

lyc01

g109

710.

24

Unive

rsal

stre

ss p

rote

in1.

0±0.

22.

3±0.

30.

026

4.2±

0.9

0.01

34.

1±0.

40.

004

7.1±

2.0

<0.

001

14EG

5535

51So

lyc08

g036

660.

21

Prot

ein

TIFY

5A

1.0±

0.1

2.3±

0.2

0.01

26.

0±1.

00.

003

7.2±

1.2

0.00

211

.0±

2.0

0.00

1

15AK

3276

84So

lyc01

g107

390.

21

Auxin

-res

pons

ive G

H3 p

rodu

ct

1.0±

0.1

2.3±

0.4

0.03

83.

8±0.

70.

008

7.7±

0.8

<0.

001

12.0

±5.

50.

019

* Se

quen

ces

with

out a

nnot

atio

n (‘u

nkno

wn

prot

eins

’) w

ere

excl

uded

** E

-val

ue: m

atch

sm

alle

r or e

qual

to 1

E-20

(cor

resp

ondi

ng to

max

imal

ly th

ree

mis

mat

ches

with

targ

et s

eque

nce)

***

Filte

r: ge

nes

sign

ifica

ntly

indu

ced

by A

as

wel

l as

by E

(P<

0.05

, afte

r P-v

alue

adj

ustm

ent);

exp

ress

ion

leve

ls o

f E re

lativ

e to

the

cont

rol (

E/C)

wer

e so

rted

from

hig

h to

low

138



TAB

LES

4.3D

List

of t

op 1

5 ge

nes

dow

n-re

gula

ted

by b

oth

Acu

lops

lyco

pers

ici(

A) a

nd T

etra

nych

usev

ansi

(E) a

s co

mpa

red

to c

ontr

ol (C

). Te

tran

ychu

s ur

ticae

(U) i

s sh

own

as t

he b

ench

mar

k.#

Prim

ary

acce

ssio

nLo

cus

iden

tifie

rNo

. An

nota

tion*

*Av

erag

e re

lativ

e ex

pres

sion

(cv.

Hein

z ge

nom

e)m

atch

ing

prob

es*

(C) ±

SE

(E) ±

SE

P**

*(A

) ± S

EP

(U) ±

SE

P(U

+A)

± S

EP

1AF

0220

22So

lyc03

g120

380.

21

Auxin

resp

onse

fact

or 9

4.0±

0.8

2.1±

0.4

0.00

71.

1±0.

30.

015

1.5±

0.0

0.01

81.

0±0.

20.

001

2AK

3202

14So

lyc03

g123

410.

11

Oxal

ate

oxid

ase-

like

germ

in 1

713.

5±0.

82.

0±0.

60.

004

2.0±

0.8

0.04

71.

5±0.

60.

043

1.0±

0.1

0.00

4

3TC

2133

26So

lyc01

g088

250.

22

IQ-d

omai

n 11

2.

5±0.

41.

8±0.

20.

019

1.2±

0.1

0.00

31.

1±0.

00.

009

1.0±

0.2

0.00

2

4AK

3208

70So

lyc07

g018

240.

11

Elon

gatio

n of

ver

y lo

ng c

hain

fatty

acid

s pr

otei

n 4

5.3±

0.6

3.8±

0.4

0.01

81.

6±0.

30.

019

2.5±

0.8

0.01

51.

0±0.

20.

028

5AK

3201

74So

lyc06

g061

070.

25

Glyc

ine

clea

vage

sys

tem

H p

rote

in 1

2.6±

0.3

1.9±

0.2

0.04

71.

4±0.

20.

013

1.5±

0.3

0.03

11.

0±0.

30.

041

6TA

4016

4_40

81So

lyc02

g084

610.

11

LRR

rece

ptor

-like

ser

ine

3.4±

0.2

2.4±

0.2

0.03

11.

6±0.

20.

002

1.6±

0.2

0.00

21.

0±0.

30.

018

7AK

3210

42So

lyc04

g082

710.

21

Cath

epsi

n B-

like

cyst

eine

prot

eina

se 3

1.

8±0.

11.

3±0.

10.

009

1.1±

0.0

0.00

31.

1±0.

20.

097

1.0±

0.1

0.00

3

8GO

3762

69So

lyc01

g091

530.

21

Fasc

iclin

-like

ara

bino

gala

ctan

prot

ein

13

3.0±

0.2

2.2±

0.2

<0.

001

1.3±

0.2

0.01

31.

6±0.

10.

003

1.0±

0.2

0.01

4

9BT

0128

74So

lyc05

g043

330.

21

GDSL

est

eras

e3.

1±0.

72.

3±0.

50.

015

1.5±

0.3

0.01

21.

8±0.

50.

043

1.0±

0.3

0.00

7

10AI

8964

55So

lyc07

g047

620.

11

Pent

atric

opep

tide

repe

at-

cont

aini

ng p

rote

in1.

4±0.

11.

0±0.

10.

038

1.0±

0.1

0.01

51.

1±0.

00.

076

1.0±

0.1

0.07

0

11AK

3247

15So

lyc04

g077

780.

24

LIM

dom

ain

prot

ein

2.6±

0.2

2.0±

0.3

0.04

41.

5±0.

20.

018

1.6±

0.3

0.02

61.

0±0.

10.

014

12AK

3195

09So

lyc04

g015

040.

22

Pept

idyl-

prol

yl ci

s-tra

ns is

omer

ase

2.7±

0.4

2.1±

0.2

0.04

51.

4±0.

30.

032

1.6±

0.3

0.03

01.

0±0.

20.

020

13BG

1291

33So

lyc08

g005

440.

22

Cc-n

bs-lr

r res

ista

nce

prot

ein

1.3±

0.1

1.0±

0.1

0.00

71.

0±0.

10.

030

1.0±

0.1

0.03

41.

1±0.

10.

19

14AK

3220

96So

lyc05

g005

540.

21

BURP

dom

ain-

cont

aini

ng p

rote

in

2.0±

0.3

1.5±

0.3

0.04

81.

0±0.

10.

015

1.1±

0.2

0.01

91.

2±0.

30.

12

15AK

3215

15So

lyc07

g054

210.

21

Prot

ochl

orop

hyllid

e re

duct

ase

-

ike

prot

ein

3.3±

0.3

2.5±

0.2

0.01

91.

5±0.

30.

015

1.7±

0.2

0.03

41.

0±0.

30.

011

* Se

quen

ces

with

out a

nnot

atio

n (‘u

nkno

wn

prot

eins

’) w

ere

excl

uded

** E

-val

ue: m

atch

sm

alle

r or e

qual

to 1

E-20

(cor

resp

ondi

ng to

max

imal

ly th

ree

mis

mat

ches

with

targ

et s

eque

nce)

***

Filte

r: ge

nes

sign

ifica

ntly

dow

n-re

gula

ted

by A

as

wel

l as

by E

(P<

0.05

, afte

r P-v

alue

adj

ustm

ent);

exp

ress

ion

leve

ls o

f E re

lativ

e to

the

cont

rol (

E/C)

wer

e so

rted

from

low

to h

igh

139



UvA-DARE (Digital Academic Repository) Consequences of russet … · spider mites, but also have a...

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Transcript of UvA-DARE (Digital Academic Repository) Consequences of russet … · spider mites, but also have a...