• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of plantsigLink to Publisher's site
Plant Signal Behav. 2006 Nov-Dec; 1(6): 305–311.
PMCID: PMC2634245

The Arabidopsis thaliana Transcription Factor AtMYB102 Functions in Defense Against the Insect Herbivore Pieris rapae

Abstract

In Arabidopsis thaliana the R2R3-MYB transcription factor family consists of over 100 members and is implicated in many biological processes, such as plant development, metabolism, senescence, and defense. The R2R3-MYB transcription factor gene AtMYB102 has been shown to respond to salt stress, ABA, JA, and wounding, suggesting that AtMYB102 plays a role in the response of plants to dehydration after wounding. Here, we studied the role of AtMYB102 in the response of A. thaliana to feeding by larvae of the white cabbage butterfly Pieris rapae. A. thaliana reporter lines expressing GUS under control of the AtMYB102 promoter revealed that AtMYB102 is expressed locally at the feeding sites of herbivore-damaged leaves, but not systemically in uninfested plant parts. Knockout AtMYB102 transposon-insertion mutant plants (myb102) allowed a faster development of P. rapae caterpillars than wild-type Col-0 plants. Moreover, the number of caterpillars that had developed into pupae within 14 days was significantly higher on myb102, indicating that in wild-type plants AtMYB102 contributes to basal resistance against P. rapae feeding. Microarray analysis of wild-type Col-0 and AtMYB102 overexpressing 35S::MYB102 plants revealed a large number of differentially expressed genes. Besides several defense-related genes, a relatively large number of genes is associated with cell wall modifications.

Key Words: Pieris rapae, insect resistance, Arabidopsis, MYB transcription factor, AtMYB102, jasmonic acid

Introduction

Plants possess a broad range of defense mechanisms to effectively combat invasion by microbial pathogens or attack by herbivorous insects. These mechanisms include preexisting physical and chemical barriers, as well as inducible defense responses that become activated upon pathogen infection or insect herbivory.1,2 A concerted action of these defensive activities helps the plant to minimize damage caused by the attacker. Many studies have indicated that jasmonic acid (JA) and its derivatives are the most important regulators of induced resistance against herbivore attack.3,4 A classic example is the observation that following attack by insect herbivores, tomato leaves accumulate JA resulting in the activation of genes encoding proteinase inhibitors that inhibit digestive serine proteinases of herbivorous insects and reduce further insect feeding.5,6 Genetic evidence demonstrates that JAs also play an important role in induced defense against different types of herbivores in Arabidopsis thaliana.712 Analysis of the transcriptome of A. thaliana upon infestation by larvae of the cabbage white butterfly, Pieris rapae, revealed that the majority of the induced changes in gene expression is regulated by JA.9,13,14 Among the JA-responsive genes that are activated, several encode transcription factors, including members of the MYB-transcription factor family. However, their role in induced resistance against insects is to a large extent unknown.

MYB genes encode transcription factor proteins that share the conserved MYB DNA-binding domain,15 and were first identified as oncogenes derived from retroviruses in animal cells.16 MYB proteins are categorized into subfamilies depending on the number of conserved MYB domain repeats. MYB proteins from animals generally contain three MYB repeats, which are referred to as R1, R2 and R3. Most of the MYB-like genes in plants have only the R2 and R3 repeats. An inventory of the A. thaliana genome revealed that this plant species contains approximately 125 R2R3-MYB genes.17,18 R2R3-MYB proteins in plants have been implicated in a range of activities, such as plant secondary metabolism, regulation of cell death, stress tolerance (reviewed in ref. 17), and pathogen resistance, but the functions of most of them have not been determined. The family of R2R3-MYB-like transcription factors has repeatedly been implicated in JA-dependent defense responses. For instance, the OsLTR1 gene from rice regulates JA-dependent defense responses, whereas AtMYB15, AtMYB34, AtMYB51 and AtMYB75 are associated with the wound response or resistance against insect herbivores.1921 Furthermore, Mengiste et al22 demonstrated a role for the R2R3-MYB transcription factor protein BOS1 (AtMYB108) in resistance against the necrotrophic pathogens Botrytis cinerea and Alternaria brassicicola, both of which are sensitive to JA-dependent defense responses.23,24 Pathogen-induced expression of AtMYB108 was impaired in the JA-response mutant coi1, indicating that AtMYB108 is regulated by JA. Interestingly, AtMYB108 knockout mutants were not only impaired in resistance against necrotrophic pathogens, but also displayed impaired tolerance against water deficit and salt stress.22 These observations suggest that AtMYB108 is a central player in multiple stress responses in A. thaliana.

Another R2R3-MYB transcription factor family member, AtMYB102, was identified from an A. thaliana transcription factor collection.25 The gene is upregulated in A. thaliana upon treatment with ABA, JA, or a combined treatment of osmotic stress and wounding.26 Plant responses that are triggered by feeding insects partly overlap with those activated upon dehydration stress and wounding.14 This prompted us to study the role of AtMYB102 in the response of A. thaliana to feeding larvae of the specialist herbivore P. rapae. Here, we provide evidence that AtMYB102 plays a role in resistance against these tissue-chewing caterpillars.

Materials and Methods

Cultivation of plants.

Seeds of Arabidopsis thaliana accession Col-0, reporter line MYB102::GUS,14 knockout mutant myb102 (transposon insertion line Sm_3_41654 obtained from the EXOTIC collection of the Nottingham Arabidopsis Stock Centre; ref. 27) and AtMYB102 overexpressing 35S::MYB102 plants (line 2.3; ref. 28) were sown in quartz sand. All genotypes were in the Col-0 background. Two-week-old seedlings were transferred to 60-mL pots containing a sand/potting soil mixture that was autoclaved twice for 20 min. Plants were cultivated in a growth chamber with an 8-hr day (200 µE.m−2.sec−1 at 24°C) and 16-hr night (20°C) cycle at 70% relative humidity for another 3 weeks. Plants were watered every other day and received half-strength Hoagland nutrient solution29 containing 10 µM Sequestreen (CIBA-Geigy, Basel, Switzerland), once a week. For the microarray analysis, Col-0 and 35S::MYB102 plants were grown until they had reached the same developmental stage (fully mature rosettes that had not started bolting; five weeks for Col-0 and eight weeks for 35S::MYB102) in potting soil in a growth chamber with an 8-hr day (24°C) and a 16-hr night (20°C) cycle at 70% relative humidity. Instead of line 2.3 (which was used for the insect bioassays), 35S::MYB102 line 8.4 was used for the microarray analysis. Both AtMYB102 overexpressing lines showed similar levels of AtMYB102 mRNA.28

Insect bioassay.

Tissue-chewing larvae of the small cabbage white butterfly Pieris rapae were reared on Brussels sprout plants (Brassica oleracea gemmifera cv. Cyrus) in a growth chamber with a 16-hr day and 8-hr night cycle (21°C; 50–70% relative humidity) as described previously.30 Infestation of A. thaliana plants was carried out by transferring first-instar larvae to 5-week-old plants using a fine paintbrush. To study P. rapae performance, a single first-instar larva was transferred to each of 20 Col-0, myb102, or 35S::MYB102 plants. At seven and ten days, the fresh weight of each larva was determined, as described.31 After ten days, the first larvae started to pupate. Therefore, fresh weight was determined only up to ten days of feeding. To examine effects on caterpillar development, the percentage of caterpillars that had pupated within 14 days after hatching was determined.

Verification of transposon insertion site in myb102.

Verification of the transposon insertion in transposon insertion line Sm_3_41654 (myb102) was performed according to the guidelines from the Genome Laboratory of the John Innes Centre from which Sm_3_41654 was obtained. In brief, seedlings from Col-0 and Sm_3_41654 were grown for ten days on Murashige and Skoog (MS) medium and subsequently harvested for isolation of genomic DNA. Line Sm_3_41654 contains a transposon insertion at the end of the first exon of the AtMYB102 gene (At4g21440), 124 bp downstream of the translation start site. To verify disruption of the AtMYB102 gene, AtMYB102-specific primers were designed ~500 bp up and downstream of the predicted transposon insertion site (At4g21440; AtMYB102-FW (in 5′ UTR) 5′-TCA ATC CCC ATT CTA AGT AGC TTC TTT C-′3; AtMYB102-RV (in 3rd exon) 5′-CGT ATA GCT GCC ACA AAC GTA AAA ATA A-'3). In addition a transposon-specific primer was used (Spm32; 5′-TAC GAA TAA GAG CGT CCA TTT TAG AGT GA-'3). Gene-specific primers for AtTUB8 (At5g23860; AtTUB8-FW; 5′-TCT CTA TGA CAT TTG CTT CAG AA-'3; AtTUB8-RV: 5′-ACG TTG TTT GGG ATC CAT TCC AC-'3) were used as an internal control. The following PCR program was used for all PCR reactions: 94°C for 3 min; 30 cycles of 94°C for 30 sec, 58°C for 45 sec, and 72°C for 1 min. A PCR with the two AtMYB102-specific primers and the transposon-specific primer will discriminate between wild-type Col-0, and heterozygous and homozygous transposon-inserted plants. Wild-type plants will yield a single band of ~850 bp (amplified from primers AtMYB102-FW and AtMYB102-RV), plants homozygous for the transposon insertion will yield a single band of ~550 bp (amplified from Spm32 and either AtMYB102-FW or AtMYB102-RV). When the AtMYB102 gene is disrupted by the transposon insertion, the distance between the AtMYB102-FW and AtMYB102-RV primers will be too large to amplify a PCR product under the PCR conditions used. Heterozygous plants will yield both the ~550- and ~850-bp PCR products.

Quantitative real-time PCR.

Q-RT-PCR analysis was performed basically as described previously.32 Two micrograms of RNA was digested with Turbo DNA-free™ (Ambion, Huntingdon, UK) according to the manufacturer's instructions. To check for genomic DNA contamination, a PCR with primers designed on intron sequences of AtACT7 (At5g09810; ACT7-FOR: 5′-GAC ATG GAA AAG ATA TGG CAT CAC AC-3′; ACT7-REV: 5′-AGA TCC TTC CTG ATA TCG ACA TCA C-3′) was carried out. Subsequently, DNA-free total RNA was converted into cDNA using oligo-dT20 primers (Invitrogen, Breda, the Netherlands), 10 mM dNTPs, and SuperScript™ III Reverse Transcriptase (Invitrogen, Breda, the Netherlands) according to the manufacturer's instructions. Efficiency of cDNA synthesis was assessed by Q-RT-PCR, using primers of the constitutively expressed gene AtUBI10 (At4g05320; UBI10-FOR: 5′ AAA GAG ATA ACA GGA ACG GAA ACA TAG T-3′; UBI10-REV: 5′-GGC CTT GTA TAA TCC CTG ATG AAT AAG-3′). Gene-specific primers were designed for AtMYB102 (At4g21440; AtMYB102-FOR: 5′-GTT GCC AGA AGA ACG GAC TC-3′; AtMYB102-REV: 5′-GGG AGG GTT CTC CAG TTA CC-3′). Q-RT-PCR analysis was done in optical 96-well plates with a MyIQ™ SingleColor Real-Time PCR Detection System (Bio-Rad, Veenendaal, the Netherlands), using SYBR® Green to monitor dsDNA synthesis. Each reaction contained 1 µL of cDNA, 0.5 µL of each of the two gene-specific primers (10 pmol.µL−1), and 10 µL of 2x IQ SYBR® Green Supermix reagent (Bio-Rad, Veenendaal, the Netherlands) in a final volume of 20 µl. The following PCR program was used for all Q-RT-PCR reactions: 95°C for 3 min; 40 cycles of 95°C for 30 sec, 59.5°C for 30 sec, and 72°C for 30 sec. CT (threshold cycle) values were calculated using Optical System Software, version 1.0 for MyIQ™ (Bio-Rad, Veenendaal, the Netherlands). Subsequently, CT values were normalized for differences in dsDNA synthesis using the AtUBI10 CT values. Normalized transcript levels of AtMYB102 were compared to untreated controls and the fold change in expression level was calculated after 24 hr of feeding by P. rapae.

GUS assay.

Larvae of P. rapae were transferred to five-week-old MYB102::GUS plants. After 24 hr of caterpillar feeding, leaf tissue was harvested and GUS activity assessed by transferring the leaves to GUS staining solution (1 mM X-Gluc, 100 mM NaPi buffer, pH 7.0, 10 mM EDTA, 0.1% (v/v) Triton X-100, 1 mM potassium ferrocyanide and 1 mM potassium ferricyanide). After overnight incubation at 37°C, the leaves were destained by repeated washes in 70% ethanol and evaluated for staining intensity as described.33

Microarray preparation.

The Arabidopsis 6K microarray used consisted of 6008 cDNA fragments, of which 5834 were from the Incyte Unigene collection (Arabidopsis Gem I; Incyte, Palo Alto, CA), and 408 positive and negative controls, of which 384 were from the Lucidea Microarray ScoreCard v1.1 (GE Healthcare Bio-Sciences, Little Chalfont, UK). cDNA fragments were spotted in duplicate and distantly from each other; for details see the VIB MicroArray Facility (MAF) website (www.microarrays.be).

Sample preparation, target labeling and hybridizations.

For isolation of RNA from leaf tissue, shoots of untreated wild-type Col-0 and 35S::MYB102 line 8.4 plants were harvested. Total RNA (5 µg) of each sample was reverse transcribed to double-stranded cDNA and further amplified. Subsequent Cy5 and Cy3 (GE Healthcare Bio-Sciences) labeling, hybridization, post-hybridization washing and scanning were performed as described previously.34 All protocols for Cy3 and Cy5 labeling, hybridization and scanning can be accessed through the VIB MAF (www.microarrays.be/service.htm/protocols).

Experimental design and statistical analysis of the microarray data.

We constructed an experimental design consisting of two replicated dye-swap Arabidopsis 6K microarray experiments, in which two independent pools of untreated wild-type Col-0 and 35S::MYB102 line 8.4 shoots were directly compared. The expression data were analyzed in two steps: (1) a within-slide analysis aimed at removing variation associated with differential dye responses to binding and scanning, as noise; and (2) a between-slide analysis aimed at estimating the mean differences between treatments (i.e., genotypes) and their standard error. For the within-slide analysis we used the robust scatter plot smoother LOWESS35 as implemented in Genstat,36 where the response variable is the log2 ratio of the artefact-removed total foreground Cy5 (R) and Cy3 (G) fluorescence intensities (M) measured at the 6008 Arabidopsis spots and a total of 24 negative control spots containing a Bacillus subtilis-specific cDNA. The fraction of the data used for estimating the local LOWESS fit was set at 20%. Once the adjusted log2 ratios (M') for each gene were obtained, adjusted log2R and log2G signal intensities were calculated. Positive signals were selected as described previously34 based on the 48 adjusted log2R and log2G signal intensities of the 24 negative controls spots printed in duplicate. One hundred eighty five genes were below the signal threshold in all observations for each genotype and were subsequently removed from the data set.

For the between-slide analysis, we applied an ANOVA model to the 46584 LOWESS fits to the spot measurements as described previously.37 Briefly, to account for the multiple sources of variation in the microarray experiment, we applied the linear normalization ANOVA model of the form:

response = µ + array + dye + genotype + gene + (gene.array) + (gene.dye) + (gene.genotype) + error

where the response variable represents the 46584 corrected log2-transformed Cy3 and Cy5 fluorescence intensity measurements. Array models the hybridization effects of each of the four microarrays, dye models the effects of each of the two dyes, genotype represents the effect of each of the two genotypes, gene represents the effect of each of the 5823 genes, gene.array represents the spot effect, gene.dye represents the gene-specific dye effects and gene.genotype represents the interaction between the genotypes and the genes, which are the effects of interest. Nonzero differences in the genotype by gene effects for a given gene indicate differential expression. To determine which of these differences in the genotype by gene interactions are significantly different from zero, we estimated 95.0% confidence intervals for these differences employing a bootstrap analysis of the residuals as described in reference 37 and based on 1000 bootstrap data sets. Since e0.48 = 1.395 means a fold-change of approximately 40 % appears significant at the 0.05 level. No further multiple testing has been taken into account.

Functional analysis.

Clones were annotated using the MIPS Arabidopsis thaliana Genome Database (MatDB; http://mips.gsf.de/proj/thal/db/index.html). Biological function and predicted subcellular localization of the proteins was assessed using the same Internet facilities and the Gene Ontology tool at TAIR.

Results

AtMYB102 expression upon herbivore attack.

The transcription factor gene AtMYB102 is induced by dehydration and wounding.26 Because herbivore-damaged plants also suffer from water loss, we investigated the role of AtMYB102 in defense against caterpillar feeding. Wild-type A. thaliana Col-0 plants were infested with larvae of P. rapae and the expression of AtMYB102 was analyzed 24 hr later. Q-RT-PCR analysis of AtMYB102 mRNA levels showed a 2.3-fold induction of AtMYB102 in P. rapae-damaged tissue compared to untreated Col-0 plants (Fig. 1A), indicating that insect feeding induced the expression of AtMYB102. This result was confirmed by data from a previously published whole-genome GeneChip array experiment,13 in which AtMYB102 mRNA levels were increased at both 12 hr and 24 hr after infestation by P. rapae (Fig. 1B).

Figure 1
Pieris rapae-induced expression of AtMYB102. (A) Q-RT-PCR analysis of AtMYB102 mRNA levels in Col-0 plants 24 hr after feeding by first-instar larvae of P. rapae. Uninfested control is set at 1. (B) Relative level of AtMYB102 mRNA in Col-0 plants 12 and ...

To further study the herbivore-induced expression of AtMYB102, we made use of a transgenic AtMYB102::GUS reporter line, containing a translational fusion of the uidA reporter gene with the promoter of the AtMYB102 gene.28 Figure 1C shows that β-glucuronidase (GUS) activity was induced around the feeding sites of P. rapae. All together, these results indicate that wounding caused by feeding of P. rapae triggers the expression of AtMYB102, predominantly in the cells surrounding the feeding sites.

Role of AtMYB102 in resistance against P. rapae.

To investigate the role of AtMYB102 in resistance against P. rapae, a defective Suppressor-mutator (dSpm) transposon insertion line with a dSpm element inserted in the first exon of the AtMYB102 gene 124 bp from the translation start site (line Sm_3_41654 designated myb102; Fig. 2A), and a 35S::MYB102 overexpressing line (line 2.3; ref. 28) were used. To confirm disruption of AtMYB102 in the myb102 knockout mutant, two PCR reactions were performed on Col-0 and myb102 genomic DNA. One reaction with non-target AtTUB8 gene-specific primers, and one reaction with a transposon-specific primer (Spm32) and two AtMYB102-specific primers (AtMYB102-FW and AtMYB102-RV located ~500 bp up and downstream of the transposon insertion, respectively). The AtTUB8 primers amplified a similar PCR product of ~500 bp on Col-0 and myb102 genomic DNA, confirming that equal amounts of template genomic DNA were used in the PCR reactions (Fig. 2A; TUB). The PCR reaction with the two AtMYB102-specific primers and the Spm32 transposon-specific primer amplified a ~850-bp fragment on Col-0 DNA, which corresponds to the ~850-bp region of the AtMYB102 gene that is located between the AtMYB102 primers (Fig. 2A; MYB102). A similar PCR reaction with myb102 genomic DNA yielded a ~550-bp fragment, which corresponds to the ~550-bp region between the Spm32 binding site in the dSpm transposon and the AtMYB102-specific primer binding site in the AtMYB102 gene. The ~850-bp AtMYB102 DNA fragment was not amplified on myb102 DNA because this fragment contains the dSpm transposon and is, thus, too large to be amplified under the PCR conditions used. Overexpression of AtMYB102 in 35S::MYB102 line 2.3 was confirmed by northern blot analysis of RNA that was isolated from uninduced wild-type and transgenic plants (Fig. 2B).

Figure 2
Molecular analysis of knockout mutant myb102 and AtMYB102 overexpressor 35S::MYB102 lines. (A) Structure of the AtMYB102 gene and position of the transposon insertion in the myb102 knockout mutant. Exons are indicated as gray boxes. The nucleotide numbers ...

To study herbivore performance in the knockout mutant and the overexpressor in comparison to wild-type Col-0, five-week-old plants were each infested with one first-instar P. rapae larva. Subsequently, larval performance was monitored over a period up to ten days by determining larval weight gain. In addition, we determined the percentage of larvae that pupated within 14 days of infestation. Figure 3A shows that on days seven and ten, the weight of the larvae that fed on myb102 was significantly higher (approx. 1.5-fold) than that of the larvae feeding on wild-type Col-0 plants. This increased caterpillar weight was associated with a greater percentage of larvae that had entered pupation by day 14. About 50% of the larvae feeding from myb102 plants had developed into pupae on day 14, while only 5% of the larvae feeding from wild-type Col-0 plants had pupated at that time (Fig. 3B). Surprisingly, overexpression of AtMYB102 did not result in a reduction of larval performance. Caterpillar growth on 35S::MYB102 plants did not differ significantly from that on Col-0 plants (Fig. 3A). Also the percentage of larvae feeding from 35S::MYB102 plants that had pupated by day 14 did not differ significantly from those feeding on wild-type Col-0 plants (Fig. 3B). These data indicate that AtMYB102 contributes to basal resistance against P. rapae feeding. However, overexpression of AtMYB102 does not increase resistance above the basal level.

Figure 3
Effect of herbivore-induced resistance on P. rapae performance. (A) Growth of P. rapae larvae on wild-type Col-0, mutant myb102, and AtMYB102 overexpressing 35S::MYB102 plants. Larval fresh weight (FW) was measured after seven and ten days of feeding. ...

Expression profiling of 35S::MYB102 plants.

To study downstream effects of upregulation of AtMYB102 by P. rapae we performed a microarray experiment to identify the genes that are affected by overexpression of the transcription factor gene AtMYB102. To this end, wild-type Col-0 and 35S::MYB102 line 8.428 plants were compared in a replicated, dye-swapped microarray experiment using a cDNA micro-array consisting of 6008 A. thaliana cDNA fragments. To identify genes of which the expression was substantially affected we applied two selection criteria: i) the changes in gene expression should be statistically significant, and ii) the changes should be above the arbitrarily chosen level of 2-fold. Although statistically significant changes in gene expression below the threshold level of 2-fold may be biologically relevant, we have chosen to disregard all changes below this threshold level to limit the number of false positives. Overexpression of AtMYB102 significantly increased the expression (>2-fold) of 151 genes, while 117 genes showed an at least 2-fold reduction (supplementary data Table S1). Among the genes that are upregulated in the 35S::MYB102 plants, were AtTHI2.1 (At1g72260) encoding an anti-microbial thionin,38 an ethylene biosynthesis gene (At2g19590) encoding a putative 1-aminocyclopropane-1-carboxylate oxidase, an anti-microbial thaumatin-like gene (At4g38660) with high homology to AtTLP1 that is activated in A. thaliana upon colonization of the roots by resistance-inducing rhizobacteria,39 and AtVSP1 (At5g24780), encoding a vegetative storage protein.40 Recently, AtVSP2 (At5g24770), which shares 82% amino acid sequence identity with AtVSP1, was shown to possess anti-insect properties that could be contributed to the acid phospatase activity of the protein.41 When incorporated into the diets of different insects, AtVSP2 significantly delayed development of the insects and increased their mortality.

We categorized the differentially expressed genes according to biological function (Fig. 4A) and predicted subcellular localization (Fig. 4B) using internet tools from the MIPS Arabidopsis thaliana Genome Database (MatDB; http://mips.gsf.de/proj/thal/db/index.html) and the Gene Ontology tool at TAIR (http://arabidopsis.org/tools/bulk/go/index.jsp). Classification according to biological functions indicates that a substantial percentage of the differentially expressed genes in the 35S::MYB102 overexpressing line encode proteins involved in metabolism. However, this is not surprising because of all annotated genes in the A. thaliana genome, metabolism is the largest category of genes with known biological function. Moreover, 10% of the genes up and downregulated by overexpression of AtMYB102 have been shown to be involved in stress and defense reactions. Classification according to predicted subcellular localization of the proteins revealed that a large proportion of the upregulated genes encode proteins that are thought to function in the cell wall or at the plasma membrane. Among the upregulated genes are several that code for cell wall-modifying proteins, such as EXPANSIN4, 8, 10 and 11, and pectolytic enzymes (Table 1).

Figure 4
Biological function and predicted cellular localization of the proteins encoded by the differentially expressed genes in 35S::MYB102 plants. (Supplementary Table S1). Selected genes showing a statistically significant and at least 2-fold difference in ...
Table 1
Fold-change ratio of upregulated genes that are associated with modification of the cell wall upon overexpression of AtMYB102 in A. thaliana

Discussion

Large scale expression analysis of the A. thaliana MYB transcription factor gene family revealed that several MYB transcription factor genes are regulated by JA.18 Because JA plays an important role in insect resistance, several members of the MYB transcription factor family may play a role in insect resistance. Previously, a few of them have already been implicated in resistance against insect herbivores. For instance, manipulation of transcript levels of the A. thaliana MYB transcription factor gene ATR1 (ALTERED TRYPTOPHAN REGULATION 1; AtMYB3417), which encodes a MYB transcription factor that regulates the expression of genes involved in the tryptophan pathway,42 severely altered the production of indolic glucosinolate anti-herbivore compounds.20 In another study, a transgenic line of A. thaliana constitutively expressing PAP1 (PRODUCTION OF ANTHOCYANIN PIGMENT 1; AtMYB7517), which encodes a MYB transcription factor that activates the phenylpropanoid biosynthetic pathway,43 showed a significantly increased resistance to feeding by fall armyworms (Spodoptera frugiperda).21 In this study we showed that damage caused by P. rapae induced the expression of AtMYB102 around the feeding sites. On knockout mutant myb102 plants, P. rapae caterpillars developed significantly faster than on Col-0 plants, indicating that in wild-type plants, AtMYB102 plays a role in defense against this herbivore. Overexpression of the AtMYB102 gene did not further enhance the level of resistance against P. rapae feeding, suggesting that the level of AtMYB102 that is induced upon herbivore feeding is already fully effective.

Transcript profiling of wild-type Col-0 and AtMYB102 overexpressing plants revealed enhanced expression of several defense-related genes in 35S::AtMYB102 plants, including AtVSP1, which possesses acid phosphatase activity that is associated with enhanced insect resistance.41 Furthermore, a relatively large number of the genes that were upregulated in the AtMYB102 overexpressor encode proteins that are predicted to exert their function in the cell wall or the plasma membrane. Several of these genes are involved in cell wall remodeling. Our findings that AtMYB102 plays a role in resistance against P. rapae, and regulates genes that are associated with cell wall modification, raises the question to what extent a causal relationship exist between these two processes. The speed of tissue consumption by P. rapae suggests that the cell wall modifications that are induced upon activation of AtMYB102 are unlikely to contribute to inhibition of growth of the caterpillars. Hence, the AtMYB102-mediated cell wall modifications may reflect repair mechanisms that are initiated upon wounding and dehydration. However, knockout mutant myb102 clearly allows a faster development of P. rapae larvae, indicating that AtMYB102-regulated genes contribute to resistance against this herbivore. Hence, future research will be focused on understanding the role of AtMYB102 in resistance against insect feeding.

Supplementary Material

Supplementary Material:

Acknowledgements

The authors acknowledge Leo Koopman, Frans van Aggelen and André Gidding for insect rearing. This research was supported, in part, by the European Commission [REGIA EU contract no. QLG2-1999-00876 (S.C.M.S.)] and grants 865.03.002 (M.D.) and 865.04.002 (C.M.J.P.) from the Earth and Life Sciences Foundation (ALW), which is subsidized by the Netherlands Organization of Scientific Research (NWO).

Note

Note

Supplemental Table S1 can be found at: www.landesbioscience.com/supplement/devosPSB1-6-sup.pdf

Footnotes

Previously published online as a Plant Signaling & Behavior E-publication: http://www.landesbioscience.com/journals/psb/abstract.php?id=3512

References

1. Slusarenko AJ, Fraser RSS, Van Loon LC. Mechanisms of Resistance to Plant Diseases. Dordrecht: Kluwer Academic Publishers; 2000.
2. Schoonhoven LM, Van Loon JJA, Dicke M. Insect-Plant Biology. Oxford: Oxford University Press; 2005.
3. Kessler A, Baldwin IT. Plant responses to insect herbivory: The emerging molecular analysis. Annu Rev Plant Biol. 2002;53:299–328. [PubMed]
4. Dicke M, Hilker M. Induced plant defences: From molecular biology to evolutionary ecology. Basic Appl Ecol. 2003;4:3–14.
5. Howe GA. Jasmonates as signals in the wound response. J Plant Growth Regul. 2005;23:223–237.
6. Ryan CA. The systemin signaling pathway: Differential activation of plant defensive genes. Biochim Biophys Acta. 2000;1477:112–121. [PubMed]
7. Ellis C, Karafyllidis L, Turner JG. Constitutive activation of jasmonate signaling in Arabidopsis mutant correlates with enhanced resistance to Erysiphe cichoracearum, Pseudomonas syringae, and Myzus persicae. Mol Plant-Microbe Interact. 2002;15:1025–1030. [PubMed]
8. McConn J, Creelman RA, Bell E, Mullet JE, Browse J. Jasmonate is essential for insect defense in Arabidopsis. Proc Natl Acad Sci USA. 1997;94:5457–5473. [PMC free article] [PubMed]
9. Reymond P, Bodenhausen N, Van Poecke RMP, Krishnamurthy V, Dicke M, Farmer EE. A conserved transcriptional pattern in response to a specialist and a generalist herbivore. Plant Cell. 2004;16:3132–3147. [PMC free article] [PubMed]
10. Stotz HU, Koch T, Biedermann A, Weniger K, Boland W, Mitchell-Olds T. Evidence for regulation of resistance in Arabidopsis to Egyptian cotton worm by salicylic and jasmonic acid signaling pathways. Planta. 2002;214:648–652. [PubMed]
11. Stintzi A, Weber H, Reymond P, Browse J, Farmer EE. Plant defense in the absence of jasmonic acid: The role of cyclopentenones. Proc Natl Acad Sci USA. 2001;98:12837–12842. [PMC free article] [PubMed]
12. Van Poecke RMP, Dicke M. Indirect defence of plants against herbivores: Using Arabidopsis thaliana as a model plant. Plant Biol. 2004;6:387–401. [PubMed]
13. De Vos M, Van Oosten VR, Van Poecke RMP, Van Pelt JA, Pozo MJ, Mueller MJ, Buchala AJ, Métraux JP, Van Loon LC, Dicke M, Pieterse CMJ. Signal signature and transcriptome changes of Arabidopsis during pathogen and insect attack. Mol Plant-Microbe Interact. 2005;18:923–937. [PubMed]
14. Reymond P, Weber H, Damond M, Farmer EE. Differential gene expression in response to mechanical wounding and insect feeding in Arabidopsis. Plant Cell. 2000;12:707–719. [PMC free article] [PubMed]
15. Jin H, Martin C. Multifunctionality and diversity within the plant MYB-gene family. Plant Mol Biol. 1999;41:577–585. [PubMed]
16. Klempnauer KH, Gonda JJ, Bishop JM. Nucleotide sequence of the retroviral leukemia gene v-myb and its cellular pregenitor c-myb: The architecture of a tranduced oncogene. Cell. 1982;31:453–463. [PubMed]
17. Stracke R, Werber M, Weisshaar B. The R2R3-MYB gene family in Arabidopsis thaliana. Curr Opinion Plant Biol. 2001;4:447–456. [PubMed]
18. Yanhui C, Xiaoyuan Y, Kun H, Meihua L, Jigang L, Zhaofeng G, Zhiqiang L, Yunfei Z, Xiaoxiao W, Xiaoming Q, Yunping S, Li Z, Xiaohui D, Jingchu L, Xing-Wang D, Zhangliang C, Hongya G, Li-Jia Q. The MYB transcription factor superfamily of Arabidopsis: Expression analysis and phylogenetic comparison with the rice MYB family. Plant Mol Biol. 2006;60:107–124. [PubMed]
19. Cheong YH, Chang HS, Gupta R, Wang X, Zhu T, Luan S. Transcriptional profiling reveals novel interactions between wounding, pathogen, abiotic stress, and hormonal responses in Arabidopsis. Plant Physiol. 2002;129:661–677. [PMC free article] [PubMed]
20. Celenza JL, Quiel JA, Smolen GA, Merrikh H, Silvestro AR, Normanly J, Bender J. The Arabidopsis ATR1 Myb transcription factor controls indolic glucosinolate homeostasis. Plant Physiol. 2005;137:253–262. [PMC free article] [PubMed]
21. Johnson ET, Dowd PF. Differentially enhanced insect resistance, at a cost, in Arabidopsis thaliana constitutively expressing a transcription factor of defensive metabolites. J Agric Food Chem. 2004;52:5135–5138. [PubMed]
22. Mengiste T, Chen X, Salmeron J, Dietrich R. The BOTRYTIS SUSCEPTIBLE1 gene encodes an R2R3MYB transcription factor protein that is required for biotic and abiotic stress responses in Arabidopsis. Plant Cell. 2003;15:2551–2565. [PMC free article] [PubMed]
23. Thomma BPHJ, Eggermont K, Penninckx IAMA, Mauch-Mani B, Vogelsang R, Cammue BPA, Broekaert WF. Separate jasmonate-dependent and salicylate-dependent defense-response pathways in Arabidopsis are essential for resistance to distinct microbial pathogens. Proc Natl Acad Sci USA. 1998;95:15107–15111. [PMC free article] [PubMed]
24. Ton J, Van Pelt JA, Van Loon LC, Pieterse CMJ. Differential effectiveness of salicylate-dependent and jasmonate/ethylene-dependent induced resistance in Arabidopsis. Mol Plant-Microbe Interact. 2002;15:27–34. [PubMed]
25. Quaedvlieg N, Dockx J, Keultjes G, Kock P, Wilmering J, Weisbeek P, Smeekens S. Identification of a light-regulated MYB gene from an Arabidopsis transcription factor gene collection. 1996;32:987–993. [PubMed]
26. Denekamp M, Smeekens SC. Integration of wounding and osmotic stress signals determines the expression of the AtMYB102 transcription factor gene. Plant Physiol. 2003;132:1415–1423. [PMC free article] [PubMed]
27. Tissier AF, Marillonnet S, Klimyuk V, Patel K, Torres MA, Murphy G, Jones JDG. Multiple independent defective suppressor-mutator transposon insertions in Arabidopsis: A tool for functional genomics. Plant Cell. 1999;11:1841–1852. [PMC free article] [PubMed]
28. Denekamp M. Arabidopsis thaliana MYB transcription factor functional genomics. The Netherlands: Utrecht University; 2001. Ph. D. Thesis.
29. Hoagland DR, Arnon DI. The water culture method for growing plants without soil. Calif Agric Exp Stn Bull. 1938;347:36–39.
30. Van Poecke RMP, Posthumus MA, Dicke M. Herbivore-induced volatile production by Arabidopsis thaliana leads to attraction of the parasitoid Cotesia rubecula: Chemical, behavioral, and gene-expression analysis. J Chem Ecol. 2001;27:1911–1928. [PubMed]
31. De Vos M, Van Zaanen W, Koornneef A, Korzelius JP, Dicke M, Van Loon LC, Pieterse CMJ. Herbivore-induced resistance against microbial pathogens in Arabidopsis. Plant Physiol. 2006;142:352–363. [PMC free article] [PubMed]
32. Czechowski T, Bari RP, Stitt M, Scheible WR, Udvardi MK. Real-time RT-PCR profiling of over 1400 Arabidopsis transcription factors: Unprecedented sensitivity reveals novel root-and shoot-specific genes. Plant J. 2004;38:366–379. [PubMed]
33. Spoel SH, Koornneef A, Claessens SMC, Korzelius JP, Van Pelt JA, Mueller MJ, Buchala AJ, Métraux JP, Brown R, Kazan K, Van Loon LC, Dong X, Pieterse CMJ. NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense pathways through a novel function in the cytosol. Plant Cell. 2003;15:760–770. [PMC free article] [PubMed]
34. Vuylsteke M, Van Eeuwijk F, Van Hummelen P, Kuiper M, Zabeau M. Genetic analysis of variation in gene expression in Arabidopsis thaliana. Genetics. 2005;171:1267–1275. [PMC free article] [PubMed]
35. Yang YH, Dudoit S, Luu P, Lin DM, Peng V, Ngai J, Speed TP. Normalization for cDNA microarray data: A robust composite method addressing single and multiple slide systematic variation. Nucl Acids Res. 2002;30:e15. [PMC free article] [PubMed]
36. Payne RW, Lane PW. Genstat® Release Reference Manual. Part 3. VSN Oxford, UK: Procedure Library PL16; 2005.
37. Kerr MK, Martin M, Churchill GA. Analysis of variance for gene expression microarray data. J Comput Biol. 2000;7:819–837. [PubMed]
38. Epple P, Apel K, Bohlmann H. Overexpression of an endogenous thionin enhances resistance of Arabidopsis against Fusarium oxysporum. Plant Cell. 1997;9:509–520. [PMC free article] [PubMed]
39. Léon-Kloosterziel KM, Verhagen BWM, Keurentjes JJB, Van Pelt JA, Rep M, Van Loon LC, Pieterse CMJ. Colonization of the Arabidopsis rhizosphere by fluorescent Pseudomonas spp. activates a root-specific, ethylene-responsive PR-5 gene in the vascular bundle. Plant Mol Biol. 2005;57:731–748. [PubMed]
40. Berger S, Bell E, Sadka A, Mullet JE. Arabidopsis thaliana Atvsp is homologous to soybean VspA and VspB, genes encoding vegetative storage protein acid phosphatases, and is regulated similarly by methyl jasmonate, wounding, sugars, light and phosphate. Plant Mol Biol. 1995;27:933–942. [PubMed]
41. Liu Y, Ahn JE, Datta S, Salzman RA, Moon J, Huyghues-Despointes B, Pittendrigh B, Murdock LL, Koiwa H, Zhu-Salzman K. Arabidopsis vegetative storage protein is an anti-insect acid phosphatase. Plant Physiol. 2005;139:1545–1556. [PMC free article] [PubMed]
42. Bender J, Fink GR. A Myb homologue, ATR1, activates tryptophan gene expression in Arabidopsis. Proc Natl Acad Sci USA. 1998;95:5655–5660. [PMC free article] [PubMed]
43. Borevitz JO, Xia Y, Blount J, Dixon RA, Lamb C. Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis. Plant Cell. 2000;12:2383–2394. [PMC free article] [PubMed]

Articles from Plant Signaling & Behavior are provided here courtesy of Landes Bioscience
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...