Arabidopsis basic helix-loop-helix transcription factors MYC2, MYC3, and MYC4 regulate glucosinolate biosynthesis, insect performance, and feeding behavior

Abstract

Arabidopsis thaliana plants fend off insect attack by constitutive and inducible production of toxic metabolites, such as glucosinolates (GSs). A triple mutant lacking MYC2, MYC3, and MYC4, three basic helix-loop-helix transcription factors that are known to additively control jasmonate-related defense responses, was shown to have a highly reduced expression of GS biosynthesis genes. The myc2 myc3 myc4 (myc234) triple mutant was almost completely devoid of GS and was extremely susceptible to the generalist herbivore Spodoptera littoralis. On the contrary, the specialist Pieris brassicae was unaffected by the presence of GS and preferred to feed on wild-type plants. In addition, lack of GS in myc234 drastically modified S. littoralis feeding behavior. Surprisingly, the expression of MYB factors known to regulate GS biosynthesis genes was not altered in myc234, suggesting that MYC2/MYC3/MYC4 are necessary for direct transcriptional activation of GS biosynthesis genes. To support this, chromatin immunoprecipitation analysis showed that MYC2 binds directly to the promoter of several GS biosynthesis genes in vivo. Furthermore, yeast two-hybrid and pull-down experiments indicated that MYC2/MYC3/MYC4 interact directly with GS-related MYBs. This specific MYC-MYB interaction plays a crucial role in the regulation of defense secondary metabolite production and underlines the importance of GS in shaping plant interactions with adapted and nonadapted herbivores.

Figure 1.

Aliphatic and Indolic GS Biosynthetic Pathway in A. thaliana. The biosynthesis of Met-derived aliphatic and Trp-derived indolic GSs is depicted. A. thaliana genes involved in distinct and common steps are indicated as well as genes involved in primary sulfate assimilation. MYB TFs known to regulate the expression of aliphatic- or indole-GS genes are indicated in blue. Genes in red were differentially expressed between Col-0 and myc234 plants (this study). The figure is adapted from Sønderby et al. (2010b) and Yatusevich et al. (2010).

Figure 2.

Expression of GS Pathway Genes in Response to Herbivory. Expression of nine GS pathway genes was measured in different genotypes by real-time PCR in untreated plants (white bars) and in plants challenged for 48 h with S. littoralis larvae (black bars). Values are the mean (±se) of three biological replicates. myc234 stands for the myc2 myc3 myc4 triple mutant. Different letters within each treatment indicate significant differences at P < 0.05 (Tukey’s highly significant difference test); capital letters compare with each other, and lowercase letters compare with each other. Asterisks denote statistically significant differences between indicated samples (**P < 0.01 and *P < 0.05; Student's t test).

Figure 3.

Quantification GSs in Response to Herbivory. Levels of four abundant GS were quantified in different plant genotypes. Plants were challenged for 2 d with S. littoralis larvae (black bars). Unchallenged plants were used as controls (white bars). Values are the mean (±se) of four biological replicates. Significant differences between control and treated plants are indicated (Student's t test; * P < 0.05, **P < 0.01, and ***P < 0.001). 4MSOB, 4-methylsulfinylbutyl-GS; 8MSOO, 8-methylsulfinyloctyl-GS; 4MTB, 4-methylthiobutyl-GS; I3M, indol-3-ylmethyl-GS. quadGS stands for the cyp79b2 cyp79b3 myb28 myb29 quadruple mutant. FW, fresh weight.

Figure 4.

Performance of a Generalist and a Specialist Herbivore on GS Mutants. Larval performance was tested in a no-choice test on wild-type plants, on JA signaling myc234 and coi1-1 mutants, and on the GS-deficient quadGS mutant. Freshly hatched S. littoralis (A) or P. brassicae (B) larvae were placed on 3-week-old plants and larval weight (mean ± se) was measured after 7 d of feeding. Different letters indicate significant differences at P < 0.05 (Tukey’s highly significant difference test).

Figure 5.

Contrasting Feeding Preferences of S. littoralis and P. brassicae Larvae in Dual-Choice Tests. (A) Dual-choice tests between Col-0 and myc234 intact plants. Newly hatched larvae were allowed to feed for 7 d on both genotypes, and the picture was taken at the end of the feeding period. (B) and (C) Dual-choice tests between Col-0 and JA signaling or GS biosynthesis mutants. Feeding preference of S. littoralis (B) and P. brassicae (C) larvae on two leaves of each genotype was measured in Petri dishes after 4 h. Values are the mean (±se) of 25 to 30 independent samples. Asterisks indicate statistically significant differences between the tested genotypes (Student’s t test; *P < 0.05, **P < 0.01, and ***P < 0.001).

Figure 6.

S. littoralis and P. brassicae Prefer to Feed on the Leaf Margin of GS-Lacking Plants. (A) Two neonate S. littoralis larvae were placed on a 4-week-old plant and allowed to feed for 36 h. The picture is taken at the end of the feeding period. Arrows indicate feeding sites. (B) Number of feeding sites on inner and outer leaf blade was scored on each eaten leaf. (C) Experiment with P. brassicae was done similarly except that one neonate larva was used per plant and feeding time was reduced to 24 h. Values are the mean (±se) from 28 plants for each A. thaliana genotype. Asterisks indicate statistically significant differences between inner and outer lamina (Student’s t test; *P < 0.05, **P < 0.01, and ***P < 0.001). Experiments were repeated three times independently with similar results.

Figure 7.

The JID Domain of MYC2, MYC3, and MYC4 Is Sufficient for Interaction with MYB Proteins. MYC2^93-160 (top panel), MYC3^82-141 (middle panel), and MYC4^99-150 (bottom panel) derivatives in GAL4-BD were tested for interaction with MYB28, MYB29, MYB34, MYB51, MYB76, and MYB122 in GAL4-AD. JAZ1 and JAZ9 were used as positive interaction controls. Yeast cells cotransformed with pGBKT7g- MYC2^93-160, pGBKT7g- MYC3^82-141, or pGBKT7g- MYC4^99-150 (bait), and pGADT7-MYBs (prey) were selected and subsequently grown on yeast synthetic dropout lacking Leu and Trp (-2) or on selective media lacking Ade, His, Leu, and Trp (-4) to test protein interactions. pGBKT7g-MYC2^93-160, pGBKT7g MYC3^82-141, and pGBKT7g MYC4^99-150 cotransformations with the pGADT7g vector were included as negative controls.

Figure 8.

MYC2, MYC3, and MYC4 Interact with MYB28 and MYB34 in Pull-Down Assays. Immunoblots with anti-GFP antibody of recovered MYC2-GFP, MYC3-GFP, and MYC4-GFP after pull-down reactions using crude protein extracts from 35S:MYC2-GFP, 35S:MYC3-GFP, 35S:MYC4-GFP, or the wild type (Col-0) A. thaliana plants and resin-bound recombinant MBP-MYB proteins. Input lanes show the level of expression of recombinant proteins in transgenic and control plants. Coomassie blue staining shows the amount of recombinant proteins used (CB).

Figure 9.

MYC2 Binds to the Promoter of GS Biosynthesis Genes in Vivo. ChIP-Seq was conducted on MYC2 using a MYC2-FLAG construct under native expression (Hou et al., 2010). Reads from high-throughput sequencing of immunoprecipitation from JA-treated plants (IP) and the mock experiment (mock) are shown. Screenshots from in-house genome browser (Anno-J, www.annoj.org) include four genes involved in the core biosynthetic pathways for indole-GSs ([A], CYP79B3; [B], SOT16) and aliphatic-GSs ([C], IPMDH1; [D], BCAT4).

Figure 10.

A Working Model for Regulation of GS Accumulation in A. thaliana. (A) In unstimulated plants, JAZ repressors bind to the JID domain of bHLH MYC2/MYC3/MYC4 TFs through their Jas domain and inhibit the interaction between these MYCS and GS-related R2R3-MYB TFs. This attenuates the transcriptional activity of MYC-MYB complexes at the promoters of GS pathway genes. Competitive binding to JID domain by JAZs and MYBs might allow some basal transcriptional activity and therefore could explain the presence of constitutive GS levels in wild-type plants. (B) Upon herbivory and activation of the JA pathway, JAZ repressors are degraded by the SCF^COI1 complex. A strong interaction between MYCs and MYBs leads to enhanced transcription of GS pathway genes and results in elevated GS levels. In addition, increased expression of MYC2 and MYB34 (red) might enhance the amount of MYC-MYB complexes and potentiate GS biosynthesis. The exact composition of MYC-MYB complexes is not known, but homo- and heterodimerization of MYC2/MYC3/MYC4 have been reported (Fernández-Calvo et al., 2011). Direct binding of GS-related MYBs to the promoter of GS genes has not yet been demonstrated, but several GS genes contain MBS in their promoter. G-box, MYC binding site; MBS, MYB binding site.