MYC2 Termination of Jasmonate Signaling via anJan 04, 2019 · promoters. Therefore, MYC2 and MTB proteins form an autoregulatory negative feedback circuit to . terminate JA signaling

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RESEARCH ARTICLE 1

2

MYC2 Regulates the Termination of Jasmonate Signaling via an 3

Autoregulatory Negative Feedback Loop4

Yuanyuan Liua,b,1

, Minmin Dub,1

, Lei Dengb,1

, Jiafang Shenc, Mingming Fang

b, Qian Chen

c, 5

Yanhui Lud, Qiaomei Wang

a,2, Chuanyou Li

b,e,2, and Qingzhe Zhai

b,2 6

7 aKey Laboratory of Horticultural Plant Growth, Development and Quality Improvement, Ministry of 8

Agriculture, Department of Horticulture, Zhejiang University, Hangzhou 310058, China9 bState Key Laboratory of Plant Genomics, National Centre for Plant Gene Research (Beijing), 10

Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, 11

China 12 cState Key Laboratory of Crop Biology, College of Agronomy, Shandong Agricultural University, 13

Taian, Shandong 271018, China 14 dInstitute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, China 15 eUniversity of Chinese Academy of Sciences, Beijing, 100049, China 16 1These authors contributed equally to this work. 17 2Address correspondence to [email protected], [email protected], or [email protected] 18

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Short title: MYC2 regulates the termination of JA signaling 20

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One sentence summary: The jasmonate-inducible transcription factor MYC2 activates the 22

expression of three MYC2-TARGETED BHLH (MTB) proteins, which function as competitive 23

repressors of MYC2-mediated transcription of jasmonate-responsive genes in tomato. 24

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The author responsible for distribution of materials integral to the findings presented in this article in 26

accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Chuanyou 27

Li ([email protected]). 28

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ABSTRACT 30

In tomato (Solanum lycopersicum), as in other plants, the immunity hormone jasmonate (JA) triggers 31

genome-wide transcriptional changes in response to pathogen and insect attack. These changes are 32

largely regulated by the basic helix-loop-helix (bHLH) transcription factor MYC2. The function of 33

MYC2 depends on its physical interaction with the MED25 subunit of the Mediator transcriptional 34

co-activator complex. Although much has been learned about the MYC2-dependent transcriptional 35

activation of JA-responsive genes, relatively less studied is the termination of JA-mediated 36

transcriptional responses and the underlying mechanisms. Here, we report an unexpected function of 37

MYC2 in regulating the termination of JA signaling through activating a small group of JA-inducible 38

bHLH proteins, termed MYC2-TARGETED BHLH 1 (MTB1), MTB2, and MTB3. MTB proteins 39

negatively regulate JA-mediated transcriptional responses via their antagonistic effects on the 40

functionality of the MYC2–MED25 transcriptional activation complex. MTB proteins impair the 41

formation of the MYC2–MED25 complex and compete with MYC2 to bind to its target gene 42

Plant Cell Advance Publication. Published on January 4, 2019, doi:10.1105/tpc.18.00405

©2019 American Society of Plant Biologists. All Rights Reserved

mailto:[email protected]

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promoters. Therefore, MYC2 and MTB proteins form an autoregulatory negative feedback circuit to 43

terminate JA signaling in a highly organized manner. We provide examples demonstrating that gene 44

editing tools such as CRISPR/Cas9 open up new avenues to exploit MTB genes for crop protection. 45

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INTRODUCTION 47

As a major immunity hormone, jasmonate (JA) promotes plant defense in response to 48

mechanical wounding, insect attack, and pathogen infection. In addition, JA acts as a growth 49

hormone to repress vegetative growth and promote reproductive development (Browse, 2009; 50

Wasternack and Hause, 2013; Chini et al., 2016; Goossens et al., 2016; Zhai et al., 2017b; 51

Howe et al., 2018). Underling these juxtaposing physiological functions, JA orchestrates a 52

genome-wide transcriptional program controlling resource allocation between growth- and 53

defense-related processes, thus optimizing plant fitness according to the rapidly changing and 54

often hostile environment (Guo et al., 2018a, 2018b; Major et al., 2017). 55

Decades of studies in the model systems of Arabidopsis thaliana and tomato (Solanum 56

lycopersicum) have elucidated a core JA signaling pathway consisting of multiple 57

interconnected protein–protein interaction modules that govern the transcriptional state of 58

hormone-responsive genes. Among the most studied JA-inducible transcription factors is the 59

basic helix-loop-helix (bHLH) protein MYC2, which acts as a master regulator of diverse 60

aspects of JA responses both in Arabidopsis (Boter et al., 2004; Lorenzo et al., 2004; 61

Dombrecht et al., 2007; Kazan and Manners, 2013; Zhai et al., 2013; Zhai et al., 2017b) and 62

in tomato (Yan et al., 2013; Du et al., 2017; Zhai et al., 2017b). In the resting (i.e., 63

“repressed”) stage of JA signaling, a group of JASMONATE-ZIM DOMAIN (JAZ) proteins 64

physically recruit the general co-repressor TOPLESS (TPL) to form a repression complex, 65

thereby preventing MYC2 and its close homologs MYC3, MYC4, MYC5 from activating the 66

expression of JA-responsive genes (Chini et al., 2007; Thines et al., 2007; Yan et al., 2007; 67

Pauwels et al., 2010; Cheng et al., 2011; Fernández-Calvo et al., 2011; Niu et al., 2011; Qi et 68

al., 2015). In the presence of the bioactive JA ligand jasmonoyl-isoleucine (JA-Ile), JAZ 69

proteins form a JA-Ile-dependent co-receptor complex with CORONATINE-INSENSITIVE 70

1 (COI1), the F-box subunit of the SCFCOI1

ubiquitin ligase (Xie et al., 1998; Devoto et al., 71

2002; Xu et al., 2002; Chini et al., 2007; Thines et al., 2007; Yan et al., 2007; Fonseca et al., 72

2009; Sheard et al., 2010). The SCFCOI1

-dependent degradation of JAZ repressors leads to the 73

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“de-repression” of MYC2 (Chini et al., 2007; Thines et al., 2007; Yan et al., 2007; Fonseca et 74

al., 2009; Sheard et al., 2010). In turn, free MYC2 forms a transcriptional activation complex 75

with the MED25 subunit of the plant Mediator transcriptional co-activator complex to 76

activate the expression of JA-responsive genes (Çevik et al., 2012; Chen et al., 2012; An et 77

al., 2017; Zhai et al., 2017a). 78

We recently showed that, in addition to interacting with MYC2 for the assembly of the 79

pre-initiation complex (PIC), the multitalented Mediator subunit MED25 physically interacts 80

with and coordinates the actions of multiple regulators during different stages of JA signaling. 81

For example, MED25 physically brings the hormone receptor COI1 to promoters of MYC2 82

target genes during the resting stage and facilitates COI1-dependent degradation of JAZ 83

repressors in the presence of JA-Ile (An et al., 2017). Furthermore, upon hormone elicitation, 84

MED25 physically recruits the epigenetic regulator HISTONE ACETYLTRANSFERASE 85

OF THE CBP FAMILY1 (HAC1), which selectively regulates hormone-induced acetylation 86

of lysine 9 of histone H3 (H3K9ac) in MYC2 target promoters (An et al., 2017). All of these 87

mechanistically related functions of MED25 favor the hormone-induced activation of MYC2 88

function. Together, these findings indicate a central role of the MYC2–MED25 complex in 89

the activation of JA-responsive genes. 90

Although JA-mediated defense responses facilitate the adaptation of plants to a wide 91

range of biotic and abiotic stresses, these responses can be detrimental if they continue 92

excessively and are not terminated in a timely manner. Emerging evidence suggests that 93

plants have evolved diverse and sophisticated mechanisms that ensure the proper termination 94

of JA-mediated defense responses. For example, an evolutionarily conserved metabolic 95

network converts the bioactive JA-Ile into an inactive or less active form (Miersch et al., 96

2008; Kitaoka et al., 2011; Koo et al., 2011; VanDoorn et al., 2011; Heitz et al., 2012), thus 97

providing an efficient route to switch off the JA signaling (Koo and Howe, 2012). Most JAZ 98

genes contain a highly conserved Jas intron, whose alternative splicing generates a repertoire 99

of JAZ splice variants lacking the intact C-terminal Jas motif (Yan et al., 2007; Chung and 100

Howe, 2009; Chung et al., 2010; Moreno et al., 2013). These dominant JAZ splicing variants 101

are unable to recruit SCFCOI1

but still retain the ability to repress MYC2 and their interacting 102

transcription factors, thus contributing to the desensitization of JA signaling (Zhang et al., 103

4

2017). In addition, the Arabidopsis JAZ8 and JAZ13 are non-canonical JAZ repressors that 104

are less sensitive to hormone-induced degradation and can recruit the co-repressor TPL in a 105

NOVEL INTERACTOR OF JAZ (NINJA)-independent manner (Shyu et al., 2012; Thireault 106

et al., 2015). Furthermore, the bHLH subclade IIId of MYC-related DNA-binding proteins, 107

termed JASMONATE-ASSOCIATED MYC2-LIKE 1 (JAM1), JAM2, and JAM3, play a 108

negative role in JA-mediated defense responses via competing with the DNA-binding 109

capacity of MYC2 (Nakata et al., 2013; Sasaki-Sekimoto et al., 2013; Song et al., 2013; 110

Fonseca et al., 2014). Collectively, these observations support that the appropriate 111

termination of JA-mediated defense response is an integral part of JA signaling. 112

Here, we report the mechanistic action of a small group of JA-inducible bHLH proteins, 113

termed MYC2-TARGETED BHLH 1 (MTB1), MTB2, and MTB3, in terminating JA 114

signaling. Interestingly, these MTB proteins function as negative regulators of JA signaling 115

and are direct transcriptional targets of the master activator MYC2. We demonstrate that 116

MTB proteins impair the formation of the MYC2–MED25 transcriptional activation complex 117

and competes with MYC2 to bind to its target gene promoters, thus de-activating 118

MYC2-dependent gene transcription. Therefore, MYC2 and MTB proteins form a negative 119

feedback circuit to terminate JA signaling. 120

121

RESULTS 122

Tomato MED25 Acts as a Co-activator of MYC2 123

High similarity between tomato and Arabidopsis MED25 proteins (Supplemental Figure 1A) 124

prompted us to test whether tomato MED25 acts as a co-activator of MYC2, which controls a 125

transcriptional regulatory cascade involved in JA-mediated plant immunity (Du et al., 2017). 126

In yeast two-hybrid (Y2H) assays, we found that MED25 interacts with the transcriptional 127

activation domain (TAD) of MYC2 (Figure 1A). To confirm the physical interaction between 128

MED25 and MYC2, we performed in vitro pull-down experiments using a purified 129

maltose-binding protein (MBP)-tagged MED25 fragment from amino acid 243 to 806 130

(MBP-MED25) and glutathione S-transferase (GST)-tagged MYC2 (GST-MYC2). As shown 131

in Figure 1B, GST-MYC2 pulled down MBP-MED25, indicating that MED25 interacts with 132

MYC2 in vitro. 133

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Since the TAD of MYC2 is also required for the binding of JAZ repressors (Du et al., 134

2017), we investigated the possibility that MED25 competes with JAZ repressors to bind to 135

MYC2. Yeast three-hybrid (Y3H) assays revealed MED25–MYC2 interaction on the 136

synthetic defined (SD) medium lacking Ade, His, Trp and Leu (SD/-4); however, the 137

induction of JAZ7 expression on SD medium lacking Ade, His, Trp, Leu and Met (SD/-5) led 138

to a dramatic reduction in MED25–MYC2 interaction (Figure 1C), suggesting that JAZ7 139

interferes with MED25–MYC2 interaction. On the other hand, JAZ7–MYC2 interaction was 140

detected on SD/-4 medium, and this interaction was dramatically reduced by inducing the 141

expression of MED25 on SD/-5 medium (Figure 1C), suggesting that MED25 also impairs 142

JAZ7–MYC2 interaction. In parallel Y3H experiments, we showed that induction of MBP 143

expression did not affect the MED25–MYC2 interaction and the JAZ7–MYC2 interaction 144

(Figure 1C). Together, these results support that MED25 and JAZ7 compete with each other 145

to bind to MYC2. 146

To substantiate the above observations, we performed in vitro pull-down experiments 147

using a constant protein concentration of Histidine (His)-MYC2 and increasing protein 148

concentration of MBP-MED25 or MBP-JAZ7. Results showed that the ability of His-MYC2 149

to pull down MBP-MED25 decreased as the amount of MBP-JAZ7 increased (Figure 1D, left 150

panel). Similarly, the ability of His-MYC2 to pull down MBP-JAZ7 decreased as the amount 151

of MBP-MED25 increased (Figure 1D, right panel). As a negative control, we showed that 152

the ability of His-MYC2 to pull down MBP-MED25 or MBP-JAZ7 was not obviously 153

affected by increasing the amount of MBP (Figure 1D). These results corroborate that 154

MED25 and JAZ7 compete with each other to bind to MYC2. 155

To explore the significance of MED25–MYC2 interaction in JA signaling, we generated 156

plants expressing antisense (AS) MED25 (MED25-AS) in which the expression level of 157

endogenous MED25 was substantially reduced compared with that in wild-type (WT) plants 158

(Supplemental Figure 1B). The MED25-AS transgenic and WT plants were then subjected to 159

a standard wounding assay. Reverse transcription-quantitative polymerase chain reaction 160

(RT-qPCR) analysis revealed that wound-induced expression of MYC2 direct target genes 161

TOMATO LIPOXYGENASE D (TomLoxD) and JA2L (Yan et al., 2013; Du et al., 2014; Du et 162

al., 2017) was significantly reduced in MED25-AS plants compared with the WT (Figure 1E). 163

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Similarly, wound-induced expression of the defense marker genes THREONINE 164

DEAMINASE (TD) (Chen et al., 2005) and PROTEINASE INHIBITOR II (PI-II) (Ryan and 165

Pearce, 1998; Ryan, 2000) was also significantly reduced in MED25-AS plants compared 166

with the WT (Figure 1E). These results indicate that MED25 plays a positive role in 167

wound-induced activation of JA-responsive MYC2 target genes. 168

169

Overexpression of MYC2 Attenuates, Rather than Enhances, JA-Mediated Defense 170

Responses 171

The above results showed that MED25 competitively binds to MYC2, thereby activating 172

JA-responsive gene expression upon wound-induced JA-Ile accumulation and subsequent 173

JAZ degradation. Therefore, we hypothesized that the overexpression of MYC2 enhances 174

JA-mediated defense responses. To test this hypothesis, we generated several transgenic lines 175

overexpressing green fluorescent protein (GFP)-tagged MYC2; the expression of MYC2-GFP 176

was elevated in MYC2-OE plants (Supplemental Figure 1C). Contrary to our hypothesis, 177

wound-induced expression levels of TomLoxD, JA2L, TD, and PI-II were decreased in 178

MYC2-OE plants compared with WT plants (Figure 1F), indicating that the overexpression of 179

MYC2 weakens, rather than enhances, JA-mediated defense responses. 180

As a first step to understand why MYC2-OE plants are defective in wound-induced 181

defense gene expression, we compared wound-induced JA accumulation between MYC2-OE 182

plants and their WT counterparts. Wound-induced JA accumulation was significantly reduced 183

in MYC2-OE plants compared to the WT (Supplemental Figure 2A), suggesting that 184

MYC2-OE plants are defective in wound-induced JA production. In addition, exogenous 185

methyl jasmonate (MeJA)-induced expression of TomLoxD, JA2L, TD, and PI-II was also 186

significantly decreased in MYC2-OE plants compared to the WT (Supplemental Figure 2B), 187

suggesting that MYC2-OE plants are also defective in JA signaling. Together, these 188

observations led us to a hypothesis that, in addition to regulating the activation of JA 189

signaling, MYC2 might also autoregulate the inactivation and/or termination of JA signaling 190

via a hitherto unknown mechanism. 191

192

MTB1–3 Are Directly Targeted by MYC2 193

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To understand how MYC2 regulates the termination of JA-mediated defense responses, we 194

attempted to identify MYC2-targeted transcription factors (MTFs) (Du et al., 2017) that 195

negatively regulate JA signaling. From our genome-wide binding profile of MYC2 (Du et al., 196

2017), we identified three MTB proteins, including MTB1 (bHLH113, Solyc01g096050), 197

MTB2 (bHLH133, Solyc05g050560), and MTB3 (bHLH138, Solyc06g0839800). 198

Phylogenetic analysis revealed that MTB1–3 proteins are homologs of the Arabidopsis 199

JASMONATE-ASSOCIATED MYC2-LIKE (JAM) proteins (Nakata et al., 2013; 200

Sasaki-Sekimoto et al., 2013; Song et al., 2013; Fonseca et al., 2014; Goossens et al., 2017) 201

(Supplemental Figure 3) and they share high sequence similarity with MYC2 (Sun et al., 202

2015) (Supplemental Figure 3). In addition to tomato and Arabidopsis, MYC2- and MTB-like 203

proteins are also present in other plant species including Oryza sativa, Zea mays, Populus 204

trichocarpa, Nicotiana tabacum, Catharanthus roseaus, Musa acuminate, Malus domestica 205

and Hevea brasiliensis (Supplemental Figure 3A and Supplemental Data set 1). 206

Consistent with the recent chromatin immunoprecipitation (ChIP)-sequencing data 207

(Supplemental Figure 4) (Du et al., 2017), our ChIP-qPCR assays using MYC2-GFP plants 208

(Supplemental Figure 1C) (Du et al., 2017) revealed an enrichment of MYC2-GFP 209

recombinant protein on the G-box (CACATG) motifs in MTB gene promoters (Figures 2A 210

and 2B). Electrophoretic mobility shift assays (EMSAs) showed that MBP-MYC2 211

recombinant protein bound a DNA probe containing the G-box motif but failed to bind a 212

DNA probe in which the G-box motif was mutated (Figure 2C), indicating that MYC2 213

specifically binds the G-box motif of MTB promoters. Collectively, these results demonstrate 214

that MTB1–3 are direct transcriptional targets of MYC2. 215

We then compared the temporal expression patterns of MTB1–3 and MYC2 in response 216

to wounding. RT-qPCR assays revealed that, although wounding induced the expression of 217

both MYC2 and MTB genes in WT plants, the timing of their induction was distinct; while the 218

expression of MYC2 peaked at 0.5 h after wounding, the expression of MTB genes peaked at 219

1 h after wounding (Figure 2D). The wound-induced expression kinetics of MTB1–3 and 220

MYC2 were overall conserved for their Arabidopsis homologs in response to MeJA (Hickman 221

et al., 2017). Notably, wound-induced expression of MYC2 and MTB genes was largely 222

abolished in the jai1 mutant, which harbors a null mutation of the JA-Ile receptor protein of 223

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tomato (Li et al., 2004), indicating that wound-induced expression of MTB genes depends on 224

COI1. Additionally, wound-induced expression of all three MTB genes was significantly 225

reduced in RNA interference (RNAi)-mediated MYC2 known-down plants (MYC2-RNAi-3#) 226

(Yan et al., 2013; Du et al., 2017) and MED25-AS-5# plants compared with the WT (Figure 227

2E), indicating that wound-induced expression of MTB genes requires MYC2- and 228

MED25-dependent JA signaling. 229

230

MTB1–3 Negatively Regulate Diverse Aspects of JA Responses 231

To determine the function of MTB genes in JA signaling, we generated MTB-RNAi transgenic 232

tomato plants in which the expression of MTB1, MTB2, and MTB3 was downregulated 233

(Supplemental Figure 5A). We also generated MTB1-OE plants overexpressing MTB1 cDNA 234

fused with GFP under the control of the 35S promoter (Supplemental Figures 5B and 5C). 235

Two lines of MTB-RNAi plants (MTB-RNAi-2#, MTB-RNAi-8#) and two lines of MTB1-OE 236

plants (MTB1-OE-5#, and MTB1-OE-6#) were selected for further analyses. In a standard 237

wounding response assay of tomato seedlings, wound-induced expression levels of TomLoxD, 238

JA2L, TD, and PI-II were increased in MTB-RNAi plants and decreased in MTB1-OE plants 239

compared with WT plants (Figure 3A), indicating that MTB proteins negatively regulate the 240

wound response of tomato plants. 241

In the context that JA plays a critical role in regulating the plant wound response 242

(Schilmiller and Howe, 2005; Sun et al., 2011; Campos et al., 2014; Zhai et al., 2017b; Howe 243

et al., 2018), we reasoned that MTB proteins might attenuate the wound response by 244

negatively regulating the JA signaling. Indeed, MeJA-induced expression levels of TomLoxD, 245

JA2L, TD, and PI-II were increased in MTB-RNAi plants and decreased in MTB1-OE plants 246

compared with WT plants (Figure 3B), confirming that MTB proteins negatively regulate 247

MeJA-induced expression of these defense genes. 248

In line with previous observations (Li et al., 2004; Qi et al., 2011; Chen et al., 2011), we 249

found that exogenous application of JA to WT seedlings led to anthocyanin accumulation 250

(Figure 3C) and root growth inhibition (Figure 3D) in a dose-dependent manner. Notably, 251

JA-induced anthocyanin accumulation was significantly increased in MTB-RNAi plants and 252

significantly decreased in MTB1-OE plants compared with WT plants (Figure 3C). Similarly, 253

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JA-induced root growth inhibition was significantly enhanced in MTB-RNAi plants and 254

significantly attenuated in MTB1-OE plants compared with WT plants (Figure 3D). 255

Collectively, these results support that MTB proteins negatively regulate diverse aspects of 256

JA responses. 257

To explore whether MTB proteins play a role in plant defense responses to herbivorous 258

insects, we examined the expression pattern of MTB1–3 in response to insect attack. For these 259

experiments, 18-day-old WT seedlings were pre-challenged with newly hatched cotton 260

bollworm (Helicoverpa armigera) larvae for 1 h; afterwards, seedlings were either 261

continuously challenged for another 1 h or relieved of challenging by removing the insects 262

from plants. RT-qPCR assays revealed that, the expression of MTB1–3 was low at steady 263

state and was induced to high levels at 1 h upon larvae challenge (Supplemental Figure 6A). 264

Interestingly, the expression of the MTB genes was retained at similar levels when seedlings 265

were continuously challenged by the larvae for another 1 h (2C) or not (2R) (Supplemental 266

Figure 6A). 267

Next, we examined the performance of MTB-RNAi and MTB1-OE plants to herbivorous 268

insects. For these experiments, 5-week-old plants of each genotype and their WT counterparts 269

were challenged with larvae. After the termination of the feeding trial, the expression of 270

defense-related gene PI-II was measured in the remaining leaf tissues of damaged plants. 271

Results showed that the expression of PI-II was significantly higher in herbivore-damaged 272

MTB-RNAi leaves and markedly reduced in herbivore-damaged MTB1-OE leaves compared 273

with herbivore-damaged WT leaves (Figure 3E). In turn, the average weight of larvae reared 274

on MTB-RNAi plants was significantly lower and that of larvae reared on MTB1-OE plants 275

was 2.0-fold greater than that of larvae reared on WT plants (Figure 3F). These results 276

demonstrate that MTB proteins negatively regulate plant defense responses against chewing 277

insects. 278

We have recently shown that MYC2-dependent JA-signaling plays a critical role in 279

regulating plant resistance to the necrotrophic pathogen Botrytis cinerea (Du et al., 2017). We 280

found that the expression of MTB1–3 was induced by B. cinerea infection (Supplemental 281

Figure 6B). To test that MTB proteins may play a role in plant resistance to this pathogen, 282

leaves of MTB-RNAi and MTB1-OE plants were inoculated with B. cinerea spore suspensions 283

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for 24 h and the expression of pathogen-related marker gene PR-STH2 (Marineau et al., 1987; 284

Matton and Brisson, 1989; Despres et al., 1995; Du et al., 2017) was examined. The 285

expression of PR-STH2 was significantly increased in B. cinerea-infected MTB-RNAi leaves 286

and markedly reduced in B. cinerea-infected MTB1-OE leaves compared with that in WT 287

(Figure 3G). Consistently, MTB-RNAi plants exhibited increased resistance against B. cinerea 288

compared with WT plants, whereas MTB1-OE plants displayed more severe disease symptom 289

compared with WT plants, as revealed by the size of the necrotic lesions (Figure 3H). These 290

results demonstrate that MTB proteins negatively regulate plant resistance against B. cinerea 291

infection. 292

Previous studies revealed that JA induces plant susceptibility to the bacterium strain 293

Pseudomonas syringae pv. tomato (Pst) DC3000 by antagonizing salicylic acid 294

(SA)-mediated plant defense responses (Kloek et al., 2001; Zhao et al., 2003; Brooks et al., 295

2005; Pieterse et al., 2012). The negative roles of MTB proteins in JA signaling promoted us 296

to test whether MTB proteins are involved in the Pst DC3000-induced plant defense. 297

RT-qPCR assays revealed that MTB genes were induced by Pst DC3000 infection 298

(Supplemental Figure 6C). We examined the response of MTB-RNAi plants and MTB1-OE 299

plants to infection by Pst DC3000. As shown in Figure 3I, Pst DC3000-induced expression of 300

PR1b (Zhao et al., 2003) showed a dramatic reduction in MTB-RNAi plants and a slight yet 301

significant increase in MTB1-OE plants compared with WT plants. Consistent with this 302

pattern, MTB-RNAi plants were more susceptible than WT to Pst DC3000 infection, whereas 303

MTB1-OE plants were more resistant than WT to Pst DC3000 infection, as revealed by 304

pathogen growth (Figure 3J). These results suggest that MTB proteins play a positive role in 305

plant resistance against Pst DC3000 infection. 306

Taken together, our results support that, in contrast to MYC2, which positively regulates 307

JA signaling, MTB proteins play a negative role in regulating JA signaling. 308

309

MTB Proteins Interact with Most Tomato JAZ Proteins via a Conserved 310

JAZ-Interacting Domain (JID) 311

The negative regulation of JA-mediated defense responses by MTB proteins prompted us to 312

compare the structural domains of MTB proteins with that of MYC2, which positively 313

11

regulates JA-mediated defense responses in tomato (Du et al., 2017). Sequence comparisons 314

revealed that MTB proteins contained a conserved JAZ-interacting domain (JID) 315

(Fernández-Calvo et al., 2011), and key amino acids required for interacting with JAZ 316

proteins (Zhang et al., 2015) were largely conserved among MTB proteins, MYC2, and the 317

Arabidopsis homologs AtMYC2 and AtMYC3 (Supplemental Figure 7A). Consistent with 318

these data, Y2H assays revealed that MTB1 interacted with 10 of the 11 JAZ proteins 319

encoded by the tomato genome, with JAZ9 being the only exception; this interaction between 320

MTB1 and JAZ proteins occurred via the N-terminal fragment of MTB1 containing JID and 321

TAD (Supplemental Figure 7B). These results are in line with the observations that the 322

Arabidopsis JAM proteins are targets of JAZ repressors (Song et al., 2013; Fonseca et al., 323

2014). The interaction between MTB1 and JAZ proteins were further validated in pull-down 324

experiments using transgenic plants expressing c-myc-tagged MTB1 (MTB1-myc) protein 325

(Supplemental Figure 8 and purified epitope-tagged JAZ proteins. Ten MBP-JAZ fusions or 326

GST-JAZ fusions, but not GST-JAZ9, pulled down MTB1-myc (Supplemental Figure 7C). 327

Thus, the interaction of MTB proteins with most of the JAZ proteins in tomato suggests that, 328

like MYC2 (Du et al., 2017), these bHLH proteins act at a high hierarchical position in the JA 329

signaling pathway (i.e., immediately downstream of the COI1-JAZ co-receptor complex). 330

331

MTB Proteins Lack a Canonical MED25-Interacting Domain (MID) and Repress 332

TomLoxD Transcription 333

Amino acid sequence comparisons of MTB1–3, MYC2 and its Arabidopsis homologs 334

AtMYC2 and AtMYC3 revealed sequence variation in the putative TAD of MTB proteins 335

(Supplemental Figure 7A). Previously, TAD of MYC2 was shown to interact with MED25 in 336

Arabidopsis (Chen et al., 2012; An et al., 2017). In Y2H assays, MYC2, but not MTB1, 337

interacted with MED25 (Figures 1A and 4A). Consistent with these results, in an in vitro 338

pull-down assay, recombinant GST-MYC2, but not GST-MTB1, pulled down MBP-MED25 339

(Figure 4B). Together, these observations indicated that the MED25-interacting domain (MID) 340

of MYC2 must be localized within its TAD. Domain mapping with Y2H assays revealed that 341

the deletion of a 12-amino-acid (aa) fragment from the N-terminus of MYC2 TAD abolished 342

MED25–MYC2 interaction (Figure 4C), indicating that this 12-aa fragment represents the 343

12

MID of MYC2 (Figure 4C). Notably, the MID-corresponding region in MTB1 (MTB1132–144

)344

exhibits a high degree of sequence variation compared to the MID of MYC2 (Figure 4C), 345

referred to here as altered MID (AMID). Notably, sequence alignment indicated that the MID 346

is generally conserved in MYC2-like proteins and that the AMID is conserved in MTB-like 347

proteins in different plant species (Supplemental Figure 3B). 348

Notably, deletion of MID abolished the interaction of MYC2 with all of the JAZ 349

proteins in Y2H assays (Supplemental Figure 9A). Protein gel analysis indicated that 350

MYC2△MID and all JAZ fusion proteins were expressed in yeast cells (Supplemental Figure 351

9B), indicating that the absence of interaction between MYC2△MID and JAZ proteins 352

cannot be due to a lack of protein expression. These results support that the MID is also 353

involved in MYC2–JAZ interaction. In parallel Y2H experiments, we found that deletion of 354

the AMID also abolished the interaction of MTB1 with JAZ proteins (Supplemental Figures 355

9C and 9E), protein gel analysis indicated that MTB1, MTB1△AMID and all JAZ fusion 356

proteins were expressed in yeast cells (Supplemental Figures 9D and 9F), indicating that the 357

absence of interaction between MTB1△AMID and JAZ proteins cannot be due to a lack of 358

protein expression. These results support that the AMID is involved in MTB1–JAZ 359

interaction. 360

Because MTB proteins lacked a canonical MID and negatively regulated JA signaling, 361

we hypothesized that these bHLH proteins function as transcriptional repressors of 362

JA-responsive genes. To test this hypothesis, we used a yeast assay (Zhai et al., 2013) to 363

examine the transcriptional activation capability of MTB1 and MYC2. Results showed that 364

MYC2 functioned as a strong transcriptional activator, whereas MTB1 did not (Figure 4D). 365

Next, we examined the effect of MTB1 and MYC2 on transient expression of the firefly 366

luciferase (LUC) gene driven by the JA-responsive TomLoxD promoter in tobacco (Nicotiana 367

benthamiana) leaves (Yan et al., 2013). We previously demonstrated that the JA biosynthetic 368

gene TomLoxD is a direct target of MYC2 (Yan et al., 2013; Du et al., 2017). In line with our 369

previous observation (Yan et al., 2013), when the PTomLoxD:LUC reporter was co-expressed 370

with MYC2-GFP in N. benthamiana leaves, LUC activity was greatly increased compared 371

with that in the empty vector control (Figures 4E–4G). By contrast, co-expression of 372

MTB1-GFP and PTomLoxD:LUC reporter construct resulted in reduced LUC activity (Figures 373

13

4E–4G). When MTB1-GFP and MYC2-GFP were simultaneously co-expressed with the 374

PTomLoxD:LUC reporter, MYC2-mediated enhancement of LUC activity was significantly 375

inhibited (Figures 4E–4G). Together, these data substantiate that, unlike the transcriptional 376

activator MYC2, MTB1 likely functions as a transcriptional repressor of JA-responsive genes 377

and counteracts the function of MYC2 in regulating these genes. 378

379

MTB1 Physically Interacts with MYC2 and Interferes with the MED25–MYC2 380

Interaction 381

Compared with MYC2 and other bHLH proteins, MTB proteins contain a conserved HLH 382

domain (Supplemental Figure 7A), which is believed to form homo- and heterodimers 383

(Carretero-Paulet et al., 2010; Fernández-Calvo et al., 2011; Goossens et al., 2016). This was 384

evident in our in vitro pull-down assays, in which both GST-MYC2 and GST-MTB1 pulled 385

down recombinant MBP-MYC2 (Figure 5A) as well as recombinant MBP-MTB1 (Figure 386

5B). These results suggest that, like MYC2, the bHLH protein MTB1 forms homodimers 387

with itself and heterodimers with MYC2 in vitro. Moreover, MTB1-myc fusion proteins from 388

protein extracts of MTB1-myc-11# plants (Supplemental Figure 8) were pulled down by both 389

MBP-MTB1 and MBP-MYC2 (Figure 5C), corroborating that MTB1 forms homodimers 390

with itself and heterodimers with MYC2. 391

The heterodimerization of MTB1 with MYC2 raised the possibility that MTB1 disrupts 392

the interaction of MYC2 with its co-activator MED25, which plays a critical role in 393

MYC2-regulated transcriptional activation of JA-responsive genes (Chen et al., 2012; An et 394

al., 2017). Y3H assays detected MED25–MYC2 interaction on SD/-4 medium (Figure 5D). 395

However, the induction of MTB1 expression on SD/-5 medium significantly impaired the 396

MED25–MYC2 interaction (Figure 5D); by contrast, the induction of MBP expression on 397

SD/-5 medium showed negligible effects on the MED25–MYC2 interaction (Figure 5D). 398

These results suggest that MTB1 competitively inhibits MED25–MYC2 interaction in yeast 399

cells. These observations were further confirmed by pull-down experiments in which both 400

His-MYC2 and MBP-MED25 fusion proteins were maintained at a constant amount in each 401

sample, and the concentration of GST-MTB1 was increased in a gradient. In this assay, the 402

ability of His-MYC2 to pull down MBP-MED25 decreased as the amount of GST-MTB1 403

14

increased (Figure 5E); as a control, we showed that the ability of His-MYC2 to pull down 404

MBP-MED25 was not affected as the amount of GST increased (Figure 5E). These results 405

corroborate our hypothesis that MTB1 interferes with MED25–MYC2 interaction. 406

The interruption of MED25–MYC2 interaction due to MTB1 further suggested the 407

possibility that MTB1 affects the enrichment of MED25 on MYC2 target gene promoters. To 408

test this possibility, we generated several transgenic lines expressing MED25-GFP 409

(Supplemental Figure 10), and subsequently transferred the MED25-GFP transgene into 410

MTB-RNAi and MTB1-OE genetic backgrounds by crossing MED25-GFP-13# plants with 411

both MTB-RNAi-8# and MTB1-OE-11# plants. ChIP-qPCR analysis revealed the enrichment 412

of MED25-GFP on the G-box of TomLoxD and JA2L promoters upon wounding (Figures 5F–413

5H). Notably, the enrichment of MED25-GFP on these promoters was significantly increased 414

in MTB-RNAi-8# plants and significantly reduced in MTB1-OE-11# plants compared with 415

WT plants (Figures 5F–5H). These results indicate that MTB1 impairs MYC2-dependent 416

recruitment of MED25 to MYC2 target promoters. 417

418

MTB1 Antagonizes MYC2 for DNA Binding 419

Sequence alignment revealed that the basic domain is highly conserved between MTB 420

proteins and MYC2 as well as other bHLH proteins (Supplemental Figure 7A). Given that the 421

basic domain is involved in DNA binding (Carretero-Paulet et al., 2010; Fernández-Calvo et 422

al., 2011; Goossens et al., 2016), we hypothesized that MTB proteins bind to the same 423

promoter region as MYC2. ChIP-qPCR assays revealed that, similar to MYC2-GFP, 424

MTB1-GFP was preferentially enriched on the G-box motifs of TomLoxD and JA2L 425

promoters instead of on the upstream promoter regions and coding sequences of these genes 426

(Figures 6A–6C). We then compared the wound-induced binding kinetics of MTB1-GFP and 427

MYC2-GFP to the G-box regions of TomLoxD and JA2L gene promoters. As shown in 428

Figures 6D and 6E, the enrichment of MYC2-GFP on the G-box motifs of TomLoxD and 429

JA2L reached a peak at 0.5 h after wounding, whereas that of MTB1-GFP on the same 430

promoter regions reached a peak at 1 h after wounding, indicating that the enrichment of 431

MTB proteins at its target sites occurs later than that of MYC2. 432

To test whether MTB proteins antagonize the binding of MYC2 to its target promoters, 433

15

we transferred the MYC2-GFP transgene into the backgrounds of MTB-RNAi-8# and 434

MTB1-OE-11# through crossing. The results of ChIP-qPCR assays revealed that, upon 435

wounding, the enrichment of MYC2-GFP on the G-box of TomLoxD and JA2L was 436

significantly increased in MTB-RNAi-8# plants compared with WT plants (Figure 6F) but 437

significantly decreased in MTB1-OE-11# plants compared with WT plants (Figure 6G). We 438

then performed EMSAs to examine the effect of MTB1 on the binding of MYC2 to a DNA 439

probe containing the G-box motif of the TomLoxD promoter (Yan et al., 2013; Du et al., 440

2017). In these experiments, fixed amounts of MBP-MYC2 recombinant protein and the 441

DNA probe were added to an increasing amount of MBP-MTB1 recombinant protein. As 442

shown in Figure 6H, the amount of MBP-MYC2-bound DNA probe decreased as the amount 443

of MBP-MTB1 fusion protein increased, revealing a competition effect of MBP-MTB1 on 444

the DNA-binding ability of MBP-MYC2. As a control, we showed that MBP protein did not 445

influence the DNA binding ability of MBP-MYC2 (Figure 6H). Taken together, these data 446

demonstrate that MTB proteins antagonize MYC2 by binding to its target gene promoters. 447

448

MTB Genes Represent a Potential Crop Protection Tool 449

Our results showed that MYC2 and its direct transcriptional targets, MTB proteins, form a 450

feedback loop to terminate the JA responses (Figure 7A). Considering that MTB proteins 451

negatively regulate JA signaling, we reasoned that the manipulation of MTB genes via gene 452

editing would provide an effective tool for crop protection against insect attack in an 453

environmentally friendly manner. We used the CRISPR/Cas9 gene editing system (Deng et 454

al., 2018) to generate mtb1-c single mutant and mtb1mtb2-c double mutant plants 455

(Supplemental Figures 11A–11D). Sequence analyses indicated that mtb1-c carries a 5-bp 456

deletion in the MTB1 open reading frame (ORF), which leads to frame shift and the 457

generation of a premature stop codon TAA (Supplemental Figures 11A and 11B). In the mtb1 458

mtb2-c double mutant, the MTB1 ORF contains the same mutation as that in mtb1-c 459

(Supplemental Figures 11A and 11B); and the MTB2 ORF contains a T insertion at 460

nucleotide 490, which also leads to frame shift and the generation of a premature stop codon 461

TAA (Supplemental Figures 11C and 11D). RT-qPCR assays indicated that the MTB1 and/or 462

MTB2 transcript levels do not show obvious change in mtb1-c or mtb1 mtb2-c compared with 463

16

the WT (Supplemental Figure 11E). However, protein gel analysis using anti-MTB1 464

antibodies failed to detect MTB1 protein accumulation in the mtb1-c single mutant and the 465

mtb1 mtb2-c double mutant (Supplemental Figure 11F). 466

Five-week-old mtb1-c, mtb1mtb2-c, and WT plants were challenged with newly hatched 467

H. armigera larvae. After terminating the feeding trial, the expression of defense-related gene 468

PI-II was measured in the remaining leaf tissues of damaged plants. The expression of PI-II 469

was significantly higher in herbivore-damaged mtb1-c and mtb1mtb2-c leaves compared with 470

herbivore-damaged WT leaves (Figure 7B). Consistent with this, the average weight of larvae 471

reared on mtb1-c and mtb1mtb2-c plants was significantly lower than that of larvae reared on 472

WT plants (Figure 7C), indicating that these mutants are more resistant to herbivore attack 473

than WT plants. Apart from resistance to insect attack, mtb1-c and mtb1mtb2-c plants did not 474

exhibit visible difference from the WT plants in term of overall plant growth, fertility and 475

fruit set (Supplemental Figure 12). These results suggest that MTB genes have a great 476

potential for application in crop protection. 477

478

DISCUSSION 479

Although it is well recognized that the JA signaling pathway is subject to multiple layers of 480

regulation to accurately control the amplitude, duration, and timing of defense- and 481

growth-related responses, an understanding of the underlying mechanism(s) has been lacking. 482

Here, we describe a highly organized feedback circuit that controls the accurate termination 483

of JA signaling in tomato. This study not only provides mechanistic insights into the broader 484

role of JA in controlling physiological trade-offs, but also promises to open new avenues for 485

the application of these basic insights in the field of crop protection. 486

487

MYC2 and MTB Proteins Form an Autoregulatory Feedback Loop to Terminate JA 488

Signaling 489

We provide evidence that JA-induced expression of the three MTB genes (MTB1–3) depends 490

on the MYC2–MED25 transcriptional activation complex. First, wound-induced expression 491

of these MTB genes was delayed compared with that of MYC2. Second, wound-induced 492

expression of MTB genes was impaired in MYC2- or MED25-knockdown plants. Third, 493

17

results of ChIP-qPCR analysis and EMSA indicated that MYC2 directly bound the MTB 494

promoters. These results demonstrated that the wound-induced expression of MTB genes was 495

directly controlled by the MYC2–MED25 transcriptional activation complex. Considering 496

that the MYC2–MED25 complex plays a central role in the initiation and amplification of 497

JA-mediated transcriptional responses (Chen et al., 2012; An et al., 2017; Du et al., 2017), 498

our results suggest that the activation of MTB genes is a default mechanism that inactivates 499

the MYC2–MED25 transcriptional activation complex. In other words, the formation of the 500

MYC2–MTB feedback loop is already pre-programmed during the induction phase of JA 501

signaling. Thus, in addition to controlling the initiation and amplification of JA-mediated 502

transcriptional responses, the MYC2–MED25 transcriptional activation complex also 503

executes intrinsic termination of JA-mediated defense responses. Therefore, MYC2 and MTB 504

proteins form an autoregulatory feedback loop, which temporally and spatially terminates JA 505

signaling. 506

Considering that MTB1–3 operate as a termination step of the JA signaling, it is 507

reasonable to speculate that their induction may only be seen when the invading insects are 508

removed from the plant. To test this, we designed experiments to examine whether the 509

induction of MTB1–3 is absent when plants are continuously challenged with insects. 510

Contrary to this speculation, our insect feeding experiments indicated that the MTB1–3 511

induction is still present in conditions of continuous insect feeding (Supplemental Figure 6A). 512

These, together with the fact that the induction of MTB1–3 depends on the MYC2–MED25 513

transcriptional activation complex, reinforce our scenario that the operation of these 514

termination regulators is already pre-programmed during the induction phase of JA signaling. 515

It is generally believed that, comparing to insect attack or mechanical wounding, 516

pathogen infection could be more continuous and durable. Further support to our hypothesis 517

that the operation of MTB1–3 is a pre-programmed process came from our physiological 518

assays with different pathogens. Our results indicated that MTB1–3 were induced by B. 519

cinerea infection and they played a negative role in plant resistance to this necrotrophic 520

pathogen, which is controlled by JA-inducible defenses. 521

In addition, MTB1–3 were also induced by Pst DC3000 infection and they played a 522

positive role in plant resistance to this hemi-biotrophic pathogen, which is controlled by 523

18

SA-inducible defenses. In the context that Pst DC3000 infection leads to increased 524

accumulation of endogenous JA (Spoel et al., 2003; Van der Does et al., 2013) and that this 525

pathogen activates the JA signaling by producing the JA-Ile mimic coronatine (reviewed in 526

Pieterse et al., 2012; Zhang et al., 2017), it is easy to understand that this hemi-biotrophic 527

invader also induces MTB1-3 expression. Considering that the antagonism between SA and 528

JA plays a central role in the modulation of the plant immune signaling network (Kloek et al., 529

2001; Zhao et al., 2003; Brooks et al., 2005; Pieterse et al., 2012; Zhang et al., 2017), it is 530

reasonable to speculate that MTB1-3 may play a role in SA-mediated suppression of JA 531

signaling. In this context, our finding that MTB1-3 are direct transcriptional targets of MYC2 532

is consistent with the observation that the SA-mediated inhibition of the JA pathway is 533

executed downstream of the SCFCOI1

-JAZ co-receptor complex through targeting534

JA-responsive transcription factors (Van der Does, et al., 2013). 535

Notably, the wound-triggered induction of MTB genes was delayed relative to that of 536

MYC2, indicating that the negative feedback regulation by MTB proteins is temporally 537

delayed. Characteristic delays are also observed in other negative feedback loops, including 538

the de novo synthesis of stabilized JAZ repressors (Yan et al., 2007; Chung and Howe, 2009; 539

Chung et al., 2010; Moreno et al., 2013) and induction of JA-Ile catabolic pathways (Miersch 540

et al., 2008; Kitaoka et al., 2011; Koo et al., 2011; VanDoorn et al., 2011; Heitz et al., 2012). 541

We speculate that the delayed termination of JA signaling ensures the complete execution of 542

an already-initiated JA response. In future studies, it is critical to determine whether the 543

MYC2–MTB circuit operates synergistically with or independent of the induction of 544

stabilized JAZ repressors or JA-Ile catabolic pathways to de-activate JA-mediated defense 545

responses. 546

547

MTB Proteins Impinge Their Effect on the MYC2–MED25 Transcriptional Activation 548

Complex 549

Like the master transcriptional activator MYC2, MTB1 physically interacted with most of the 550

JAZ proteins in tomato via the conserved JID, implying that MTB proteins act at a high 551

hierarchical level (i.e., immediately downstream of the hormone co-receptor complex) to 552

de-activate JA signaling. Our results revealed two mechanisms by which MTB proteins 553

19

imposed their negative effect on the MYC2–MED25 transcriptional activation complex. First, 554

MTB1 physically interacted with MYC2 and competitively inhibited the interaction of 555

MYC2 with its co-activator MED25; because the MYC2–MED25 complex is essential for 556

MYC2-dependent PIC formation, the interference of MTB1 determines the transcriptional 557

output of the MYC2–MED25 transcriptional activation complex (Chen et al., 2012; An et al., 558

2017; Du et al., 2017). In this regard, the action mechanism of MTB proteins is different from 559

the Arabidopsis JAM proteins, which did not show physical interaction with AtMYC2 in Y2H 560

and co-IP assays (Song et al., 2013; Fonseca et al., 2014). Second, MTB1 directly bound the 561

G-box motifs of MYC2 target genes via its conserved basic domain and competitively 562

inhibited the binding of MYC2 to its target gene promoters. 563

Arabidopsis MYC2 forms a homotetramer, and the tetramerization of AtMYC2 enhances 564

its DNA-binding and transcriptional activation capacity (Lian et al., 2017). Taking these data 565

into consideration, it is reasonable to speculate that MTB repressors might also form 566

homotetramers, which would further inhibit both the DNA-binding and transcriptional 567

activation capacity of MYC2. Consistent with this speculation, our sequence analysis 568

revealed that several key residues of the AtMYC2 tetramer interface (Lian et al., 2017) were 569

conserved in SlMYC2 as well as all three SlMTB proteins (Supplemental Figure 5A). Further 570

structural investigations should reveal key insights into the mechanisms by which MTB 571

transcriptional repressors counteract the function of MYC transcriptional activators in 572

regulating JA-responsive gene transcription. 573

Importantly, our results revealed that the MID couples the MYC2 interactions with both 574

its co-activator MED25 and its repressors JAZ proteins. This finding, which is consistent 575

with our recent observation that MED25 and JAZ1 form a ternary complex with MYC2 and 576

thereby fine-tunes the transcriptional output of the JA signaling in Arabidopsis (An et al., 577

2017), supports a scenario that the MID of MYC2 is evolved to keep the activation and 578

repression of this master transcriptional regulator in-check. Consistent with their negative 579

role in regulating JA signaling, MTB1–3 do not harbor a canonical MID, but rather harbor an 580

AMID, and therefore fail to physically interact with the MED25 co-activator. Intriguingly, we 581

found that, as with the MID plays a critical role for the MYC2–JAZ interaction, the AMID 582

also plays a critical role for the MTB1–JAZ interaction. We predict that these findings will 583

20

stimulate future research to elucidate the functional relevance of “repressor–repressor 584

interactions” in JA signaling or other signal transduction pathways. In the context that both 585

MTB genes and JAZ genes are induced by wounding and JA, it is possible that JAZ proteins 586

might “help” MTB proteins to repress gene expression and to terminate JA signaling. At this 587

stage, however, we could not rule out an opposite possibility that MTB–JAZ interaction 588

might result in a positive effect on JA signaling (i.e., represses a repressor may lead to a 589

positive effect). Anyway, we believe that our findings provide a starting point to address these 590

possibilities. For example, we could manipulate the AMID and test the resulting effects on 591

the function of MTB1. Another interesting direction for future exploration is to identify the 592

co-factors (i.e., co-repressors) that are involved in the action of MTB proteins and to test 593

whether the AMID plays an important role for the interaction of MTB proteins with these 594

co-factors. 595

596

METHODS 597

Plant Materials and Growth Conditions 598

Tomato (Solanum lycopersicum) cv M82 was used as the WT for all plant materials except 599

for jai1, mtb1-c and mtb1 mtb2-c. For jai1, cv Castlemart (CM) was used as the WT, and for 600

mtb1-c and mtb1 mtb2-c, cv Ailsa Craig (AC) was used as the WT. The following tomato 601

genotypes were used in this study: MED25-AS, MED25-GFP, MYC2-OE (MYC2-GFP) (Du 602

et al., 2017), MYC2-RNAi (Du et al., 2017), MTB1-OE (MTB1-GFP or MTB1-myc), 603

MTB-RNAi, mtb1-c, and mtb1mtb2-c. The MYC2-GFP and MED25-GFP transgenes were 604

introduced into the MTB-RNAi and MTB1-OE backgrounds via crossing. Homozygous plants 605

were selected by genotyping. The original jai1 mutant, which is in the genetic background of 606

cv Micro-Tom (Li et al., 2004), was backcrossed into the CM background through at least 607

five generations. Homozygous jai1 plants were identified as described previously (Li et al., 608

2004). Tomato seeds were placed on moistened filter paper for 48 h for germination. Tomato 609

seedlings were transferred to growth chambers and maintained under a long-day photoperiod 610

(16 h light/8 h dark) with a white light intensity of 200 mM photons m-2

s-1

at 25°C during the611

subjective day and at 18°C during the subjective night. N. benthamiana plants were grown at 612

28°C under a long-day photoperiod. 613

21

614

Plasmid Construction and Plant Transformation 615

To generate the MED25-AS construct, the CDS of MED25 was PCR amplified, cloned into 616

pENTR in the reverse orientation using a pENTR Directional TOPO Cloning Kit (Invitrogen), 617

and recombined with the binary vector PGWB2 (35S promoter). To generate the 618

35Spro:MED25-GFP construct, MED25 CDS was cloned into pENTR and recombined with 619

the binary vector PGWB5 (35S promoter, c-GFP). To generate the 35Spro:MTB1-GFP 620

construct, MTB1 CDS was PCR amplified, cloned into pENTR, and recombined with the 621

binary vector PGWB5 (35S promoter, c-GFP). To generate the 35Spro:MTB1-myc construct, 622

pENTR-MTB1 was recombined with the binary vector PGWB17 (35S promoter, c-4myc). To 623

generate the MTB-RNAi construct, fragments of the open reading frames of MTB1 (1401–624

1600 bp), MTB2 (81–280 bp), and MTB3 (601–800 bp) were PCR amplified and fused to 625

generate a 658 bp PCR product containing the attB1 and attB2 sites. The fused PCR product 626

was cloned into pHellsgate2 (Invitrogen). All DNA constructs were generated following 627

standard molecular biology protocols and using Gateway (Invitrogen) technology (Nakagawa 628

et al., 2007). Primers used for plasmid construction are listed in Supplemental Table 1. All 629

constructs were introduced into tomato cv M82 via Agrobacterium-mediated transformation 630

(Du et al., 2014). Transformants were selected based on their resistance to hygromycin B or 631

kanamycin. Homozygous T2 or T3 transgenic plants were used for phenotypic and molecular 632

characterization. 633

634

Generation of mtb1-c and mtb1mtb2-c Using CRISPR/Cas9 Technology 635

A 19 bp fragment of the MTB1 CDS (240–258 bp) was used as the targeting sequence for 636

genome editing of MTB1. To synthesize two target guide RNA (gRNA) sequences, PCR 637

reactions were carried out using forward and reverse primers containing the gRNAs 638

(Supplemental Table 1) and pHSE401 vector as the template. The tomatoU6-26-MTB1-gRNA 639

cassette and the CRISPR/Cas9 binary vector pCBC-DT1T2_tomatoU6 were digested with 640

BsaI, and the cassette was cloned into the binary vector to generate 641

pCBC-DT1T2_tomatoU6-MTB1. To generate CRISPR/Cas9 construct carrying two gRNAs 642

targeting MTB1 and MTB2, 19 bp fragments of each CDS (MTB1, 240–258 bp; MTB2, 473–643

22

491 bp) were used as target gRNAs. The pCBC-DT1T2_tomatoU6-MTB1MTB2 construct 644

was generated in the same way as the pCBC-DT1T2_tomatoU6-MTB1 construct. The final 645

binary vectors were introduced into tomato cv AC via Agrobacterium-mediated 646

transformation (Du et al., 2014). CRISPR/Cas9-induced mutations were genotyped by PCR 647

amplification and DNA sequencing. Primers used for plasmid construction are listed in 648

Supplemental Table 1. Cas9-free T2 plants carrying mutations were identified for further 649

experiments. 650

651

Y2H Assays 652

Y2H assays were performed using the MATCHMAKER GAL4 Two-Hybrid System 653

(Clontech). To verify the interaction of MED25 with MYC2 and MTB1, MYC2 CDS, MTB1 654

CDS and MYC2 derivatives were fused to GAL4 AD in pGADT7, and MED25 CDS was 655

fused to GAL4 BD in pGBKT7. To verify the interaction of JAZ proteins with MYC2 and 656

MTB1, MYC2 CDS, MTB1 CDS, MYC2 derivatives and MTB1 derivatives were fused to 657

GAL4 AD in pGADT7, and CDS of JAZ genes were fused to GAL4 BD in pGBKT7. Primers 658

used for plasmid construction are listed in Supplemental Table 1. Constructs used to test 659

protein–protein interactions were co-transformed into yeast (Saccharomyces cerevisiae) strain 660

AH109. Co-transformation of empty pGBKT7 and pGADT7 vectors was used as a negative 661

control. The presence of transgenes in yeast cells was confirmed by growing these cells on 662

plates containing solid synthetic defined (SD) medium lacking Leu and Trp (SD/-2). To 663

assess protein–protein interactions, transformed yeast cells were suspended in liquid SD/-2 664

medium to an optical density at 600 nm absorbance (OD600) of 1.0. Samples (5 µL) of 665

suspended yeast cells were spread on plates containing SD medium lacking Ade, His, Leu, 666

and Trp (SD/-4). To detect protein–protein interactions, plates were examined after 3 days of 667

incubation at 30°C. 668

669

Y3H Assays 670

Y3H assays were performed based on the MATCHMAKER GAL4 Two-Hybrid System 671

(Clontech). To construct pBridge-MED25-JAZ7, MED25 CDS was cloned into the multiple 672

cloning site (MCS) I of pBridge vector (Clontech) fused to the GAL4 BD domain, and JAZ7 673

23

CDS was cloned into MCS II of the pBridge vector and expressed as the “bridge” protein 674

only in the absence of methionine. Constructs used for testing protein–protein interactions 675

were co-transformed into yeast strain AH109. The presence of transgenes was confirmed by 676

growing the yeast cells on SD/-2 medium. Transformed yeast cells were spread on plates 677

containing SD/-4 medium to assess the MYC2–MED25 interaction without the expression of 678

JAZ7 and on plates containing SD/-Ade/-His/-Leu/-Trp/-Met (SD/-5) medium to induce JAZ7 679

expression. Interactions were observed after 3 days of incubation at 30°C. To construct 680

pBridge-JAZ7-MED25, JAZ7 and MED25 CDSs were cloned into MCS I and MCS II, 681

respectively, of pBridge vector, and MED25 was expressed as the “bridge” protein to test its 682

effect on MYC2–JAZ7 interaction. To construct pBridge-MED25-MTB1, MED25 and MTB1 683

CDSs were cloned into MCS I and MCS II, respectively, of pBridge vector, and MTB1 was 684

expressed as the “bridge” protein to test its effect on the MYC2–MED25 interaction. To 685

construct pBridge-MED25-MBP, MED25 and MBP CDSs were cloned into MCS I and MCS 686

II, respectively, of pBridge vector, and MBP was expressed as the “bridge” protein to test its 687

effect on the MYC2–MED25 interaction. To construct pBridge-JAZ7-MBP, JAZ7 and MBP 688

CDSs were cloned into MCS I and MCS II, respectively, of pBridge vector, and MBP was 689

expressed as the “bridge” protein to test its effect on the MYC2–JAZ7 interaction. The 690

experimental procedures were the same as those described above. 691

692

Transactivation Activity Assay in Yeast 693

Full-length CDSs of MYC2 and MTB1 were fused to the GAL4 BD in pGBKT7. The resulting 694

constructs were then co-transformed with pGADT7 into the yeast strain AH109. The 695

MATCHMAKER GAL4-based Two-Hybrid System 3 (Clontech) was used for the 696

transactivation activity assay. Transformed yeast cells were suspended in liquid SD/-2 697

medium to an OD600 = 0.6, and 5 µL of each dilution was spread onto plates containing SD/-4 698

medium. 699

700

Pull-Down Assays 701

To produce MBP-JAZ and GST-JAZ fusion proteins, full-length JAZ CDSs were PCR 702

amplified and cloned into pMAL-c2X and pGEX-4T-3, respectively. The recombinant vectors 703

24

were transformed into Escherichia coli BL21 (DE3) cells. The MBP-JAZ and GST-JAZ 704

fusion proteins were expressed by adding 0.5 mM isopropyl--D-thiogalactoside (IPTG) and 705

then purified using the amylose resin (NEB) or GST Bind™ Resin (Millipore), respectively. 706

A similar approach was used to produce MBP-MTB1, GST-MTB1, MBP-MYC2, 707

GST-MYC2, and MBP-MED25243–806

(MBP-MED25) fusion proteins. To produce His-MYC2 708

fusion protein, full-length CDS of MYC2 was PCR amplified and cloned into pCold™ TF 709

DNA (TAKARA). Primers used for plasmid construction are listed in Supplemental Table 1. 710

To detect MYC2–MED25 interaction using in vitro pull-down assays, GST-MYC2 and 711

MBP-MED25 fusion proteins were affinity purified. For each reaction, 15 μL of agarose 712

beads bound with 1 μg of GST-MYC2 was incubated with 1 μg of MBP-MED25 in 1 mL of 713

reaction buffer (25 mM Tris-HCl [pH 7.5], 100 mM NaCl, 1 mM DTT, and Roche protease 714

inhibitor cocktail) at 4°C for 2 h. Subsequently, beads were collected and washed three times 715

with washing buffer (25 mM Tris-HCl [pH 7.5], 150 mM NaCl, and 1 mM DTT). After 716

washing, samples were denatured using sodium dodecyl sulfate (SDS) loading buffer and 717

separated using SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The MBP-MED25 718

fusion protein was detected by immunoblotting with anti-MBP antibody (NEB). Purified GST 719

was used as a negative control. Five microliter aliquots of GST and GST-MYC2 fusion 720

proteins were separated by SDS-PAGE, and the staining of polyacrylamide gels with 721

Coomassie Brilliant Blue (CBB) was used as a loading control. To detect MTB1–MED25, 722

MTB1–MTB1, and MTB1–MYC2 interactions using in vitro pull-down experiments, 723

sequential manipulations were similar to those described above. 724

To detect whether the effect of JAZ7 on MYC2–MED25 interaction was 725

concentration-dependent, in vitro pull-down assays were carried out by adding 1 μg each of 726

purified His-MYC2 and MBP-MED25 proteins to increasing concentrations of MBP-JAZ7 727

fusion protein and MBP. The Ni-NTA His Bind Resin (Millipore) were used to pull down 728

proteins. Sequential manipulation was performed as described above. The effect of MED25 729

on MYC2–JAZ7 interaction and the effect of MTB1 on MYC2–MED25 interaction was 730

detected similarly. 731

To detect the MTB1–JAZ interaction, pull-down assays were conducted using protein 732

extracts of 10-day-old MTB1-myc tomato seedlings. Seedlings were ground in liquid nitrogen 733

25

and homogenized in extraction buffer containing 50 mM Tris-HCl (pH 7.4), 80 mM NaCl, 10% 734

glycerol, 0.1% Tween 20, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF), 50 mM 735

MG132 (Sigma-Aldrich), and protease inhibitor cocktail (Roche). After centrifugation at 736

16,000 g and 4°C, the supernatant was collected. One milligram of total protein extract was 737

incubated with resin-bound MBP-JAZ and GST-JAZ fusion proteins for 2 h at 4°C with 738

rotation. Purified MBP and GST proteins were used as negative controls. After washing, 739

samples were denatured using SDS loading buffer and separated by SDS-PAGE. The 740

MTB1-myc protein was detected using anti-myc antibody (Abmart). Five microliter aliquots 741

of MBP-JAZ and GST-JAZ fusion proteins were separated by SDS-PAGE, and the staining of 742

polyacrylamide gels with CBB was used as a loading control. To detect MTB1–MTB1 and 743

MTB1–MYC2 interactions, pull-down assays were conducted by incubating 1 mg of total 744

protein extract of 10-day-old MTB1-myc tomato seedlings with resin-bound MBP-MTB1 and 745

MBP-MYC2 fusion proteins, respectively, for 2 h at 4°C with rotation. Purified MBP protein 746

was used as a negative control. Sequential manipulation was similar to that described above. 747

748

Plant Treatment and Gene Expression Analysis 749

For wounding treatment, eighteen-day-old seedlings were wounded with a hemostat across 750

the midrib of all leaflets of the lower and upper leaves. The same leaflets were wounded 751

again, proximal to the petiole. Plants with wounded leaves were incubated under continuous 752

light. Five whole leaves were harvested at each sampling time point and used for extracting 753

total RNA. For MeJA treatment, eighteen-day-old seedlings were enclosed in a Lucite box 754

(10 × 32 × 60 cm) containing 5 µL of MeJA applied to cotton wicks that were spaced evenly 755

within the box. Plants exposed to MeJA vapor were harvested at each sampling time point 756

and used for extracting total RNA (Li et al., 2004). RNA extraction and RT-qPCR analysis 757

were performed as previously described (Du et al., 2014). Expression levels of target genes 758

were normalized relative to that of the tomato ACTIN2 gene. Primers used to quantify gene 759

expression levels are listed in Supplemental Table 1. 760

761

Anthocyanin Content Measurement 762

Anthocyanin content was measured as previously described (Deikman and Hammer, 1995). 763

26

Seven-day-old tomato seedlings (50 mg) grown on 0.5 × MS medium with 0, 5, or 20 μM JA 764

were placed into 1 mL extract buffer (Propanol:HCl:H2O, 18:1:81, v:v:v). Then the sample 765

was boiled for 3 minutes and incubated overnight at room temperature. Absorbance values 766

(A535 and A650) of the extraction solution were measured using spectrophotometer. The 767

anthocyanin content is presented as (A535-A650)/g fresh weight. 768

769

Root Length Measurement 770

Root lengths of seven-day-old tomato seedlings for each genotype grown on 0.5 × MS 771

medium with 0, 0.5, 1 or 2 μM JA were measured and presented. 772

773

Botrytis cinerea Inoculation Assays 774

B. cinerea isolate B05.10 was grown on 2 × V8 agar (36% V8 juice, 0.2% CaCO3, and 2%775

Bacto-agar) for 14 d at 20°C under a 12-h photoperiod prior to spore collection. Spore 776

suspensions were prepared by harvesting the spores in 1% Sabouraud Maltose Broth (SMB), 777

filtering them through nylon mesh to remove hyphae, and adjusting the concentration to 106

778

spores/mL (Mengiste et al., 2003). B. cinerea inoculation of tomato plants was performed as 779

previously described (El Oirdi et al., 2011; Yan et al., 2013; Zhai et al., 2013; Du et al., 2017; 780

Lian et al., 2018), with minor modifications. For the pathogenicity test, detached leaves from 781

5-week-old tomato plants were placed in Petri dishes containing 0.8% agar medium (agar782

dissolved in sterile water), with the petiole embedded in the medium. Each leaflet was spotted 783

with a single 5 µL droplet of B. cinerea spore suspension at a concentration of 106 spores/mL.784

The trays were covered with lids and kept under the same conditions used for plant growth. 785

Photographs were taken after 3 d, and the lesion sizes were recorded. 786

For RT-qPCR, inoculations were performed in planta: leaves of 5-week-old plants were 787

spotted with a 5 µL B. cinerea spore suspension (106 spores/mL). The plants were then788

incubated in a growth chamber with high humidity. A similar experiment was performed 789

using SMB-spotted plants as a control. Spotted leaves were harvested 24 h after inoculation 790

for RT-qPCR. Error bars represent the SD of three biological replicates. Each biological 791

replicate (sample) consisted of the pooled leaves of three spotted plants from one tray 792

(different genotypes were grown together in a randomized design per tray). Biological 793

27

replicates (trays) were grown at different locations in growth chambers and treated separately. 794

795

Pst DC3000 Infection Assays 796

Pst DC3000 (Melotto et al., 2006) was cultured at 28°C in King's B medium (10 g/L tryptone, 797

5 g/L yeast extract, and 5 g/L NaCl) containing 50 mg/mL rifampicin until OD600nm of 0.8 798

was reached. The bacteria were collected, centrifuged at 2,500 g for 10 min, washed twice, 799

and resuspended in 10 mM MgCl2 solution containing 0.02% Silwet L-77. For vacuum 800

infiltration, 5-week-old plants were vacuum-infiltrated with Pst DC3000 at a concentration of 801

0.5 x 105 c.f.u/mL, and then kept under high humidity until disease symptoms developed. 802

Leaves infiltrated with 10 mM MgCl2 containing 0.02% Silwet L-77 were used as controls. 803

Spotted leaves were harvested 24 h after inoculation for RT-qPCR experiments. Error bars 804

represent the SD of three biological replicates. Each biological replicate (sample) consisted of 805

the pooled leaves of three spotted plants from one tray (different genotypes were grown 806

together in a randomized design per tray). Biological replicates (trays) were grown at 807

different locations in growth chambers and treated separately. Disease symptoms were 808

observed at 3 days after inoculation (DAI). For quantification of Pst DC3000 growth, discs 809

from the infected leaves were collected at 3 DAI and ground in 10 mM MgCl2 with a 810

Microfuge tube glass pestle. The samples were diluted 1:10 serially and spotted on solid 811

King's B medium. After growth at 28 °C for 2 days the colony forming unit (c.f.u) was 812

counted. 813

814

JA Quantification 815

For JA content measurement, 18-day-old seedlings were wounded with a hemostat across the 816

midrib of all leaflets of the lower and upper leaves. The same leaflets were wounded again, 817

proximal to the petiole. Plants with wounded leaves were incubated under continuous light. 818

Approximately 600 mg leaf tissue (fresh weight) from five different plants was pooled for JA 819

quantification as described previously (Fu et al., 2012). Leaf tissues were also harvested from 820

unwounded plants as controls. 821

822

ChIP-qPCR Assay 823

28

Leaves of 18-day-old MTB1-GFP, MYC2-GFP, MYC2-GFP/MTB-RNAi, 824

MYC2-GFP/MTB1-OE, MED25-GFP, MED25-GFP/MTB-RNAi, and 825

MED25-GFP/MTB1-OE seedlings were wounded for the indicated times. Two grams of 826

wounded or unwounded (control) leaves of each sample were harvested and cross-linked in 1% 827

formaldehyde at room temperature for 10 min, followed by neutralization with 0.125 M 828

glycine. The chromatin–protein complex was isolated, resuspended in lysis buffer (50 mM 829

HEPES [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% SDS, 1% Triton X-100, 0.1% sodium 830

deoxycholate, 1 mM PMSF, and 1 × Roche protease inhibitor mixture), and sheared by 831

sonication to reduce the average DNA fragment size to ~500 bp. Then, 50 μL of sheared 832

chromatin was saved for use as input control. Anti-GFP antibody (Abcam) was incubated 833

with Dynabeads™ Protein G (Invitrogen) at 4°C for at least 6 h and added to the remaining 834

chromatin for overnight incubation at 4°C. The immunoprecipitated chromatin–protein 835

complex was sequentially washed with low-salt buffer (20 mM Tris-HCl [pH 8.0], 2 mM 836

EDTA, 150 mM NaCl, 0.5% Triton X-100, and 0.2% SDS), high-salt buffer (20 mM 837

Tris-HCl [pH 8.0], 2 mM EDTA, 500 mM NaCl, 0.5% Triton X-100, and 0.2% SDS), LiCl 838

buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 0.25 M LiCl, 0.5% Nonidet P-40, and 0.5% 839

sodium deoxycholate), and TE buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA). After 840

washing, the immunoprecipitated chromatin was eluted with elution buffer (1% SDS and 0.1 841

M NaHCO3). Protein–DNA crosslinks were reversed by incubating the immunoprecipitated 842

complexes with 20 µL of 5 M NaCl at 65°C overnight. DNA was recovered using QIAquick 843

PCR Purification Kit (Qiagen) and analyzed by qPCR. The enrichment of target gene 844

promoters is shown as the percent of the input DNA, which was calculated by determining 845

the immunoprecipitation efficiency at TomLoxD and JA2L loci as the ratio of the amount of 846

immunoprecipitated DNA to the normalized amount of starting material (% of input DNA). 847

ACTIN2 was used as a non-specific target gene. Primers for qPCR are listed in Supplemental 848

Table 1. 849

850

EMSA 851

Full-length CDSs of MYC2 and MTB1 were PCR amplified and cloned into pMAL-c2X. The 852

recombinant MBP-fusion proteins were expressed in E. coli BL21 (DE3) cells and purified to 853

29

homogeneity using an amylase resin column. Oligonucleotide probes were synthesized and 854

labeled with biotin at the 5 ends (Invitrogen). EMSAs were performed as previously 855

described (Chen et al., 2011; Du et al., 2014). Briefly, biotin-labeled probes were incubated 856

with MBP-fusion proteins at room temperature for 20 min, and free and bound probes were 857

separated via PAGE. Mutated MTB probes, in which the specific transcription factor-binding 858

motif 5-CACATG-3 was replaced by 5-AAAAAA-3, and mutated TomLoxD probes, in 859

which the specific transcription factor-binding motif 5-ACCATGTG-3 was deleted, were 860

used as negative controls. Probes used for EMSA are listed in Supplemental Table 1. 861

862

Transient Expression Assays 863

Transient expression assays were performed in N. benthamiana leaves as previously 864

described (Matsui et al., 2008; Shang et al., 2010; Chen et al., 2011). The TomLoxD promoter 865

was PCR amplified, cloned into pENTR using a pENTR Directional TOPO Cloning Kit 866

(Invitrogen), and then fused with the LUC reporter gene into the plant binary vector pGWB35 867

using Gateway cloning (Nakagawa et al., 2007) to generate the PTomLoxD:LUC reporter 868

construct. MYC2-GFP (Du et al., 2014) and MTB1-GFP were used as effector constructs. 869

Agrobacterium cells transformed with different constructs were incubated, harvested, and 870

resuspended in infiltration buffer (10 mM MES, 0.2 mM acetosyringone, and 10 mM MgCl2) 871

to a final concentration of OD600 = 0.5. Equal volumes of transformed Agrobacterium cells 872

were mixed in different combinations and co-infiltrated into N. benthamiana leaves with a 873

needleless syringe. Infiltrated plants were incubated at 28°C for 72 h before CCD imaging. A 874

low-light cooled CCD imaging apparatus (NightOWL II LB983 with Indigo software) was 875

used to capture images showing LUC expression and to quantify LUC luminescence intensity. 876

Leaves were sprayed with 100 mM luciferin and incubated in the dark for 3 min before 877

luminescence detection. Five independent determinations were performed. 878

879

Insect Feeding Trials 880

Cotton bollworm (Helicoverpa armigera) larvae were hatched at 26°C, as recommended by 881

the supplier (Yanhui Lu, Institute of Plant Protection, Chinese Academy of Agricultural 882

Sciences). Hatched larvae were reared on an artificial diet for 3 days followed by no diet for 2 883

30

days, before transfer to tomato plants. Fifteen third-instar H. armigera larvae were reared on 884

5 tomato plants per genotype in three independent replicates. The average weight of larvae at 885

the beginning of the feeding trial was ∼5 mg. After the termination of the 4 d feeding trial, 886

the weight gain of larvae was measured, and the expression of PI-II in the remaining leaf 887

tissues was examined by RT-qPCR. 888

For monitoring insect attack-induced MTB expression, 18-day-old tomato seedlings that 889

were exposed to third-instar H. armigera larvae were divided into three groups, and the 890

expression of MTBs in the remaining leaf tissues was examined by RT-qPCR. In the first 891

group, 6 attacked tomato plants were harvested after 1 h of feeding. In the second group, 6 892

attacked tomato plants were incubated with the insects for 1 h and the insects were removed 893

for an additional 1 h and then leaves were harvested. In the third group, 6 attacked tomato 894

plants were harvested after 2 h of continuous feeding. 895

896

Antibody Generation 897

The region of MTB1 (amino acids 196–400, MTB1196–400

) was PCR-amplified from WT898

cDNA using gene-specific primers (Supplemental Table 1). The resultant PCR product was 899

cloned into vector pET28a (Novagen) to express the His-MTB1196–400

protein fusion in900

Escherichia coli BL21 (DE3). The recombinant fusion protein was purified with 901

nickel-nitrilotriacetic acid (Ni-NTA) His•Bind Resin (Novagen) and used to raise polyclonal 902

antibodies in mouse. Anti-MTB1 antibodies were used in protein gel blotting at a final 903

dilution of 1:2,000. 904

905

Accession Numbers 906

Sequence data from this article can be found in the Sol Genomics Network Initiative under 907

the following accession numbers: MTB1 (Solyc01g096050), MTB2 (Solyc05g050560), MTB3 908

(Solyc06g083980), MYC2 (Solyc08g076930), MED25 (Solyc12g070100), JAZ1 909

(Solyc07g042170), JAZ2 (Solyc12g009220), JAZ3 (Solyc03g122190), JAZ4 910



(Soly08g036620), JAZ11 (Solyc08g036660), TomLoxD (Solyc03g122340), JA2L 913

31

(Solyc07g063410), TD (Solyc09g008670), PI-II (NP_001234627.1), PR-STH2 914

(Solyc05g054380), PR1b (Solyc09g007010) and ACTIN2 (Solyc11g005330). 915

916

Supplemental Data 917

Supplemental Figure 1. Generation of MED25-AS and MYC2-OE (MYC2-GFP) Plants. 918

Supplemental Figure 2. MYC2-OE Plants Show Decreased JA Accumulation in Response to 919

Wound and Reduced Defense Gene Expression in Response to MeJA Treatment. 920

Supplemental Figure 3. Conservation of MYC2- and MTB-related bHLH Proteins from 921

Tomato, Arabidopsis and other Plants. 922

Supplemental Figure 4. MTB1–3 Are Direct Transcriptional Targets of MYC2. 923

Supplemental Figure 5. Generation of MTB-RNAi and MTB1-OE (MTB1-GFP) Plants. 924

Supplemental Figure 6. Expression of MTB1–3 in Response to Different Stimuli. 925

Supplemental Figure 7. MTB Proteins Interact with Most of the JAZ Proteins in Tomato. 926

Supplemental Figure 8. Generation of MTB1-OE (MTB1-myc) Plants. 927

Supplemental Figure 9. MID of MYC2 and AMID of MTB1 Are Important for Their 928

Interactions with JAZ Proteins. 929

Supplemental Figure 10. Generation of MED25-GFP Plants. 930

Supplemental Figure 11. Construction of mtb1-c and mtb1 mtb2-c Plants Using the 931

CRISPR/Cas9 System. 932

Supplemental Figure 12. Growth and Reproductive Phenotypes of WT, mtb1-c and mtb1 933

mtb2-c Plants. 934

Supplemental Table 1. List of the DNA Primers Used in This Study. 935

Supplemental Dataset 1. Text File of the Alignment Used for the Phylogenetic Analysis in 936

Supplemental Figure 3. 937

Supplemental Dataset 2. Statistical Analysis 938

939

ACKNOWLEDGMENTS 940

We thank Prof. Xia Cui for providing the pHSE401_tomatoU6 and the 941

pCBC-DT1T2_tomatoU6 vector. This work was supported by the National Key R&D 942

Program of China (2017YFD0200400), the Ministry of Agriculture of China 943

32

(2016ZX08009003-001), the National Natural Science Foundation of China (31730010, 944

31672157), the National Basic Research Program of China (Grant 2015CB942900) and the 945

Tai-Shan Scholar Program from the Shandong Provincial Government. 946

947

AUTHOR CONTRIBUTIONS 948

C.L., Q.Z., Q.W., and M.D. designed the research. Y.L., M.D., L.D., J.S., and Q.C.949

performed the research. Y.L., M.D., Q.Z., and C.L. analyzed data. Q.Z. and C.L. wrote the 950

paper. 951

952

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1226

FIGURE LEGENDS 1227

Figure 1. MED25 and JAZ7 Compete to Interact with MYC2. 1228

(A) Yeast two-hybrid (Y2H) assays examining interactions between the GAL4 DNA 1229

activation domain (AD) fusions of MYC2 or MYC2 variants and GAL4 DNA-binding 1230

domain (BD) fusion of MED25. The transformed yeast cells were plated on SD medium 1231

lacking His, Ade, Leu, and Trp (SD/-4). The schematic diagram shows the MYC2 domain 1232

constructs. The conserved JAZ-interacting domain (JID), transcription activation domain 1233

(TAD), basic (b) domain, and helix-loop-helix (HLH) domain are indicated with green, red, 1234

blue, and pink boxes, respectively. (B) In vitro pull-down assays to verify the interaction 1235

between MED25 and MYC2. Purified MBP-MED25 was incubated with GST or GST-MYC2 1236

for the GST pull-down assay and detected by immunoblotting using anti-MBP antibody. The 1237

positions of purified GST and GST-MYC2 separated by SDS-PAGE are marked with 1238

asterisks on the Coomassie Brilliant Blue (CBB) stained gel. (C) Y3H assays showing that 1239

JAZ7 and MED25 compete with each other to interact with MYC2. Yeast cells 1240

co-transformed with AD-MYC2 and pBridge-MED25-JAZ7/MBP (top three rows) or with 1241

42

AD-MYC2 and pBridge-JAZ7-MED25/MBP (bottom three rows) were grown on SD/-4 1242

medium to assess MYC2–MED25 interaction or MYC2–JAZ7 interaction, respectively. The 1243

co-transformed yeast cells were grown on SD/-5 medium to induce the expression of 1244

JAZ7/MBP (top three rows) or MED25/MBP (bottom three rows). (D) In vitro pull-down 1245

assays testing for JAZ7 and MED25 competition to interact with MYC2. Fixed amounts of 1246

His-MYC2 and MBP-MED25 fusion proteins were incubated with an increasing amount of 1247

MBP-JAZ7 fusion protein and MBP protein (left), and fixed amounts of His-MYC2 and 1248

MBP-JAZ7 fusion proteins were incubated with an increasing amount of MBP-MED251249

fusion protein and MBP protein (right). Protein samples were immunoprecipitated with 1250

anti-His antibody and immunoblotted with anti-MBP antibody. CBB staining shows the 1251

amount of recombinant proteins loaded on the gel. Asterisks indicate the specific bands of 1252

recombinant proteins. (E and F) RT-qPCR assays characterizing wound-induced expression 1253

of TomLoxD, JA2L, TD, and PI-II in wild-type (WT) and MED25-antisense (MED25-AS) 1254

plants (E) and in WT and MYC2-OE plants (F). Eighteen-day-old seedlings of the indicated 1255

tomato genotypes with two fully expanded leaves were mechanically wounded using a 1256

hemostat on both leaves for the indicated times before extracting total RNAs for RT-qPCR 1257

assays. Data represent mean ± standard deviation (SD) (N = 3). Asterisks indicate significant 1258

difference from the wild type according to Student’s t-test at *P < 0.05, **P < 0.01, ***P < 1259

0.001 (Supplemental Data set 2). 1260

1261

Figure 2. MTB1–3 Are Direct Transcriptional Targets of MYC2. 1262

(A) Schematic representation of MTB1, MTB2, and MTB3 genes showing the primers and1263

probe used for ChIP-qPCR assay and EMSA. (B) ChIP-qPCR analysis of MYC2 enrichment 1264

on the chromatin of MTB1, MTB2, and MTB3. Chromatin of MYC2-GFP-9# plants was 1265

immunoprecipitated using anti-GFP antibody, and the immunoprecipitated DNA was 1266

quantified by qPCR. The enrichment of target gene promoters is displayed as a percentage of 1267

input DNA. Data represent mean ± SD (N = 3). ACTIN2 was used as a non-specific target. (C) 1268

EMSA showing that MBP-MYC2 directly binds to the promoters of MTB1, MTB2, and 1269

MTB3. The MBP protein was incubated with the labeled probe to serve as a negative control; 1270

mutated probes were used as a negative control. Ten- and 20-fold excess of unlabeled or 1271

43

mutated probes were used for competition. Ten- and 20-fold excess of MBP protein was used 1272

for competition. Mu, mutated probe in which the G-box motif 5ʹ-CACATG-3ʹ was replaced 1273

with 5ʹ-AAAAAA-3ʹ. (D and E) RT-qPCR assays showing wound-induced expression of 1274

MYC2, MTB1, MTB2, and MTB3 in WT and jai1 plants (D), MTB1, MTB2, and MTB3 in WT, 1275

MYC2-RNAi-3#, and MED25-AS-5# plants (E). Eighteen-day-old seedlings of the indicated 1276

tomato genotypes with two fully expanded leaves were mechanically wounded using a 1277

hemostat on both leaves for the indicated times before extracting total RNAs for RT-qPCR 1278

assays. Data represent mean ± SD (N = 3). Asterisks indicate significant difference from the 1279

wild type according to Student’s t-test at **P < 0.01, ***P < 0.001 (Supplemental Data set 1280

2). 1281

1282

Figure 3. MTB1–3 Negatively Regulate Diverse Aspects of JA Responses. 1283

(A) RT-qPCR assays of the expression of TomLoxD, JA2L, TD, and PI-II in WT, 1284

MTB-RNAi-2#, MTB-RNAi-8#, MTB1-OE-5#, and MTB1-OE-6# plants in response to 1285

mechanical wounding (A) and MeJA treatment (B). For (A), 18-day-old seedlings of the 1286

indicated tomato genotypes with two fully expanded leaves were mechanically wounded 1287

using a hemostat on both leaves for the indicated times before extracting total RNAs for 1288

RT-qPCR assays. For (B), 18-day-old seedlings of the indicated tomato genotypes with two 1289

fully expanded leaves were exposed to MeJA vapor for the indicated times before extracting 1290

total RNAs for RT-qPCR assays. (C) Anthocyanin contents of the 7-day-old seedlings in WT, 1291

MTB-RNAi-2#, MTB-RNAi-8#, MTB1-OE-5#, and MTB1-OE-6# plants grown on MS 1292

medium containing indicated concentrations of JA. FW, fresh weight. (D) Root growth 1293

inhibition assay of 7-day-old seedlings of WT, MTB-RNAi-2#, MTB-RNAi-8#, MTB1-OE-5#, 1294

and MTB1-OE-6# plants grown on MS medium supplied with indicated concentrations (µM) 1295

of JA. Data represent mean ± SD (N = 20). (E) Expression of PI-II in WT, MTB-RNAi-2#, 1296

MTB-RNAi-8#, MTB1-OE-5#, and MTB1-OE-6# plants subjected to cotton bollworm 1297

(Helicoverpa armigera) larvae. Plants were harvested at the indicated time points during the 1298

feeding trial for RNA extraction and RT-qPCR analysis. (F) Average weight of larvae 1299

recovered at the end of day 4 of the feeding trial using whole plants of WT, MTB-RNAi-2#, 1300

MTB-RNAi-8#, MTB1-OE-5#, and MTB1-OE-6# genotypes. Data represent mean ± SD (N = 1301

44

15). Each dot represents the weight of an individual larvae. (G) Expression of PR-STH2 in 1302

WT, MTB-RNAi-2#, MTB-RNAi-8#, MTB1-OE-5#, and MTB1-OE-6# plants treated with B. 1303

cinerea suspensions. Five-week-old plants were spotted with 5 µL spore suspension (106

1304

spores/mL). Leaves were harvested 24 h after inoculation for RNA extraction and RT-qPCR 1305

analysis. (H) Growth of B. cinerea in WT, MTB-RNAi-2#, MTB-RNAi-8#, MTB1-OE-5#, and 1306

MTB1-OE-6# plants. Detached leaves from 5-week-old tomato plants were spotted with a 5 1307

µL spore suspension (106 spores/mL). The lesion areas were analyzed at 3 DAI. Data1308

represent mean ± SD (N = 9). (I) Expression of PR1b in WT, MTB-RNAi-2#, MTB-RNAi-8#, 1309

MTB1-OE-5#, and MTB1-OE-6# plants infected with Pst DC3000. Five-week-old plants 1310

were vacuum infiltrated with Pst DC3000 (0.5 × 10-5

c.f.u/mL). Leaves were harvested 24 h1311

after inoculation for RNA extraction and RT-qPCR analysis. (J) Growth of Pst DC3000 in 1312

WT, MTB-RNAi-2#, MTB-RNAi-8#, MTB1-OE-5#, and MTB1-OE-6# plants. c.f.u, 1313

colony-forming units. Data represent mean ± SD (N = 6). Asterisks indicate significant 1314

difference from the wild type according to Student’s t-test at *P < 0.05. For (A), (B), (C), (E), 1315

(G) and (I), data represent mean ± SD (N = 3). For (A–J), Asterisks indicate significant1316

difference from the wild type according to Student’s t-test at *P < 0.05, **P < 0.01, ***P < 1317

0.001 (Supplemental Data set 2). 1318

1319

Figure 4. MTB Proteins Lack a Canonical MED25-Interacting Domain (MID) and Act as 1320

Transcriptional Repressors. 1321

(A) Y2H assays of the interaction of MED25 with MYC2 and MTB1. Full-length coding1322

sequence (CDS) of MED25 was fused with the BD in pGBKT7, and full-length CDS of 1323

MYC2 or MTB1 was fused with the AD in pGADT7. Transformed yeast cells were grown on 1324

SD/-4 to determine protein–protein interactions. (B) In vitro pull-down assays of the 1325

interaction of MED25 with MYC2 and MTB1. Purified MBP-MED25 was incubated with 1326

GST, GST-MYC2, or GST-MTB1 for the GST pull-down assay and detected by 1327

immunoblotting using anti-MBP antibody. The positions of various purified proteins 1328

separated by SDS-PAGE are marked with asterisks on CBB stained gel. (C) Y2H assays of 1329

MYC2 interaction with MED25 through MID. According to the sequence alignment of the 1330

transcriptional activation domain (TAD) of MYC2 and MTB1, MYC2 TAD was divided into 1331

45

three fragments labeled as MID, MYC2 (186–202), and MYC2 (203–212). Full-length MYC2 1332

CDS and MYC2 variants lacking the MID, MYC2 (186–202), and MYC2 (203–212) 1333

fragments were fused with the AD in pGADT7, and full-length MED25 CDS was fused with 1334

the BD in pGBKT7. Transformed yeast cells were grown on SD/-4 medium to determine 1335

protein–protein interactions. Co-transformation of the AD vector with MED25 was used as a 1336

negative control. (D) Yeast assays used to detect the transcriptional activity of MYC2 and 1337

MTB1. Yeast cells co-transformed with BD-MYC2 (or BD-MTB1) and AD vector were grown 1338

on SD/-4 medium. (E–G) Transient expression assays to detect the effect of MTB1 on the 1339

transcriptional activity of MYC2. Schematic representation of effector constructs and firefly 1340

luciferase (LUC) reporter construct used in transient expression assays (E). Quantification of 1341

luminescence intensity of LUC in N. benthamiana leaves 72 h after co-infiltration with 1342

PTomLoxD:LUC reporter construct and effector constructs or infiltration of only the 1343

PTomLoxD:LUC reporter construct (control) (F). RT-qPCR analysis of MYC2 and MTB1 1344

expression in N. benthamiana leaves infiltrated with the indicated constructs (G). For (F) and 1345

(G), data represent mean ± SD (N = 5). Statistically significant differences between control 1346

and test samples were determined using Student’s t-test and are indicated using asterisks (*P 1347

< 0.05; Supplemental Data set 2). 1348

1349

Figure 5. MTB1 Interacts with MYC2 and Disrupts MED25–MYC2 Interaction. 1350

(A) In vitro pull-down assays of MYC2 formation of homo- and heterodimers. Purified1351

MBP-MYC2 was incubated with GST, GST-MYC2, or GST-MTB1 for the GST pull-down 1352

assay and detected by immunoblotting using anti-MBP antibody. (B and C) Pull-down assays 1353

of MTB1 formation of homo- and heterodimers. Purified MBP-MTB1 was incubated with 1354

GST, GST-MTB1, or GST-MYC2 for the GST pull-down assay and detected by 1355

immunoblotting using anti-MBP antibody (B), and protein extracts prepared from MTB1-myc 1356

seedlings were incubated with MBP, MBP-MTB1, or MBP-MYC2 for the MBP pull-down 1357

assay and detected by immunoblotting using anti-myc antibody (C). For (A–C), positions of 1358

various purified proteins separated by SDS-PAGE are indicated with asterisks on CBB 1359

stained gels. (D) Y3H assays showing that MTB1 interferes with MYC2–MED25 interaction. 1360

Yeast cells co-transformed with pGADT7-MYC2 and pBridge-MED25-MTB1/MBP were 1361

46

plated on SD/-4 medium to assess the MYC2–MED25 interaction and on SD/-5 medium to 1362

induce MTB1/MBP. (E) In vitro pull-down assays of MTB1 interference with MED25–1363

MYC2 interaction. Fixed amounts of His-MYC2 and MBP-MED25 fusion proteins were 1364

incubated with an increasing amount of GST-MTB1 fusion protein or GST protein. Protein 1365

samples were immunoprecipitated with anti-His antibody and immunoblotted with anti-MBP 1366

antibody. (F) Schematic diagram of PCR amplicons of TomLoxD and JA2L used for 1367

ChIP-qPCR. Positions of the transcription start site (TSS) and transcription termination site 1368

(TTS) are indicated. (G and H) ChIP-qPCR assays of that MTB-RNAi (G) and MTB1-OE (H) 1369

impairment of the enrichment of MED25 on the G-boxes of TomLoxD and JA2L promoters 1370

upon wounding. MED25-GFP-13# and MED25-GFP-13#/MTB-RNAi-8# plants (G) and 1371

MED25-GFP-13# and MED25-GFP-13#/MTB1-OE-11# plants (H) were treated with 1372

mechanical wounding for 1 h before cross-linking, and chromatin of each sample was 1373

immunoprecipitated using anti-GFP antibody. Immunoprecipitated DNAs were quantified by 1374

qPCR. The enrichment of target gene promoters is displayed as a percentage of input DNA. 1375

ACTIN2 was used as a non-specific control. Data represent mean ± SD (N = 3). Statistically 1376

significant differences between MED25-GFP-13# and other genotypes were determined 1377

using Student’s t-test and are indicated using asterisks (*P < 0.05, **P < 0.01; Supplemental 1378

Data set 2). 1379

1380

Figure 6. MTB1 Antagonizes MYC2 for DNA Binding. 1381

(A) Schematic representation of TomLoxD and JA2L showing the amplicons and probe used1382

for ChIP-qPCR assay and EMSA. Positions of the transcription start site (TSS) and 1383

transcription termination site (TTS) are indicated. (B and C) ChIP-qPCR analysis of the 1384

enrichment of MYC2 (B) and MTB1 (C) on the chromatin of TomLoxD and JA2L in 1385

MYC2-GFP-9# and MTB1-GFP-5# plants, respectively. (D and E) ChIP-qPCR analysis of 1386

the enrichment of MYC2 (D) and MTB1 (E) on the G-box regions of TomLoxD and JA2L in 1387

MYC2-GFP-9# and MTB1-GFP-5# plants, respectively, upon wounding. Plants were treated 1388

with mechanical wounding for the indicated times before cross-linking. (F and G) 1389

ChIP-qPCR assays of the impaired enrichment of MYC2 on the G-boxes of TomLoxD and 1390

JA2L due to MTB-RNAi in MYC2-GFP-9# and MYC2-GFP-9#/MTB-RNAi-8# plants (F) and 1391

47

MTB1-OE in MYC2-GFP-9# and MYC2-GFP-9#/MTB1-OE-11# plants (G) upon wounding. 1392

Plants were treated with mechanical wounding for 1 h before cross-linking. For (B–G), 1393

chromatin of each sample was immunoprecipitated using anti-GFP antibody and quantified 1394

by qPCR. The enrichment of target gene promoters is displayed as a percentage of input 1395

DNA. Data represent mean ± SD (N = 3). For (D–G), ACTIN2 was used as a non-specific 1396

control. For (F) and (G), asterisks indicate significant differences detected using Student’s 1397

t-test (*P < 0.05, **P < 0.01; Supplemental Data set 2) when compared with MYC2-GFP. (H)1398

EMSA showing that MBP-MTB1 interferes with MBP-MYC2 to bind to DNA probes from 1399

the TomLoxD promoter in vitro. Biotin-labeled probes were incubated with a fixed amount of 1400

MBP-MYC2 and an increasing amount of MBP-MTB1 or MBP, and the free and bound 1401

DNAs were separated on an acrylamide gel. The MBP protein was incubated with the labeled 1402

probe to serve as a negative control; mutated probes were used as a negative control. Mu, 1403

mutated probe in which the G-box motif 5ʹ-ACCATGTG-3ʹ was deleted. 1404

1405

Figure 7. MTB Genes Represent a Tool for Crop Protection. 1406

(A) Proposed mechanism by which MYC2 and MTB proteins form a feedback loop to1407

terminate JA signaling. MYC2 and MED25 activate the expression of MTB genes; in turn, 1408

MTB proteins interfere with the MED25–MYC2 interaction and DNA-binding activity of 1409

MYC2 to terminate JA-triggered defense responses. (B) Expression of PI-II in WT, mtb1-c 1410

and mtb1 mtb2-c plants exposed to cotton bollworm (Helicoverpa armigera) larvae. Plants 1411

were harvested at the indicated time points during the feeding trial for RNA extraction and 1412

RT-qPCR analysis. (C) Average weight (top) and images (bottom) of larvae recovered at the 1413

end of day 4 of the feeding trial using whole plants of WT, mtb1-c, and mtb1 mtb2-c. Data 1414

represent mean ± SD (N = 15). Each dot denotes the weight of an individual larva. Asterisks 1415

indicate significant difference from the WT according to Student’s t-test at *P < 0.05 1416

(Supplemental Data set 2). 1417

1418

1419

Figure 1. MED25 and JAZ7 Compete to Interact with MYC2. (A) Yeast two-hybrid (Y2H) assaysexamining interactions between theGAL4 DNA activation domain (AD)fusions of MYC2 or MYC2 variants andGAL4 DNA-binding domain (BD)fusion of MED25. The transformed yeastcells were plated on SD medium lackingHis, Ade, Leu, and Trp (SD/-4). Theschematic diagram shows the MYC2domain constructs. The conserved JAZ-interacting domain (JID), transcriptionactivation domain (TAD), basic (b)domain, and helix-loop-helix (HLH)domain are indicated with green, red,blue, and pink boxes, respectively. (B) Invitro pull-down assays to verify theinteraction between MED25 and MYC2.Purified MBP-MED25 was incubatedwith GST or GST-MYC2 for the GSTpull-down assay and detected byimmunoblotting using anti-MBPantibody. The positions of purified GSTand GST-MYC2 separated by SDS-PAGE are marked with asterisks on theCoomassie Brilliant Blue (CBB) stainedgel. (C) Y3H assays showing that JAZ7and MED25 compete with each other tointeract with MYC2. Yeast cells co-

transformed with AD-MYC2 and pBridge-MED25-JAZ7/MBP (top three rows) or with AD-MYC2 and pBridge-JAZ7-MED25/MBP (bottom three rows) were grown on SD/-4 medium to assess MYC2–MED25 interaction or MYC2–JAZ7 interaction, respectively. The co-transformed yeast cells were grown on SD/-5 medium to induce the expression of JAZ7/MBP (top three rows) or MED25/MBP (bottom three rows). (D) In vitro pull-down assays testing for JAZ7 and MED25 competition to interact with MYC2. Fixed amounts of His-MYC2 and MBP-MED25 fusion proteins were incubated with an increasing amount of MBP-JAZ7 fusion protein and MBP protein (left), and fixed amounts of His-MYC2 and MBP-JAZ7 fusion proteins were incubated with an increasing amount of MBP-MED25 fusion protein and MBP protein (right). Protein samples were immunoprecipitated with anti-His antibody and immunoblotted with anti-MBP antibody. CBB staining shows the amount of recombinant proteins loaded on the gel. Asterisks indicate the specific bands of recombinant proteins. (E and F) RT-qPCR assays characterizing wound-induced expression of TomLoxD, JA2L, TD, and PI-II in wild-type (WT) and MED25-antisense (MED25-AS) plants (E) and in WT and MYC2-OE plants (F). Eighteen-day-old seedlings of the indicated tomato genotypes with two fully expanded leaves were mechanically wounded using a hemostat on both leaves for the indicated times before extracting total RNAs for RT-qPCR assays. Data represent mean ± standard deviation (SD) (N = 3). Asterisks indicate significant difference from the wild type according to Student’s t-test at *P < 0.05, **P < 0.01, ***P < 0.001 (Supplemental Data set 2).

Figure 2. MTB1–3 Are Direct Transcriptional Targets of MYC2. (A) Schematic representation of MTB1, MTB2, and MTB3 genes showing the primers and probe used for ChIP-qPCR assay andEMSA. (B) ChIP-qPCR analysis of MYC2 enrichment on the chromatin of MTB1, MTB2, and MTB3. Chromatin of MYC2-GFP-9# plants was immunoprecipitated using anti-GFP antibody, and the immunoprecipitated DNA was quantified by qPCR. Theenrichment of target gene promoters is displayed as a percentage of input DNA. Data represent mean ± SD (N = 3). ACTIN2 wasused as a non-specific target. (C) EMSA showing that MBP-MYC2 directly binds to the promoters of MTB1, MTB2, and MTB3.The MBP protein was incubated with the labeled probe to serve as a negative control; mutated probes were used as a negativecontrol. Ten- and 20-fold excess of unlabeled or mutated probes were used for competition. Ten- and 20-fold excess of MBP proteinwas used for competition. Mu, mutated probe in which the G-box motif 5ʹ-CACATG-3ʹ was replaced with 5ʹ-AAAAAA-3ʹ. (D andE) RT-qPCR assays showing wound-induced expression of MYC2, MTB1, MTB2, and MTB3 in WT and jai1 plants (D), MTB1,MTB2, and MTB3 in WT, MYC2-RNAi-3#, and MED25-AS-5# plants (E). Eighteen-day-old seedlings of the indicated tomatogenotypes with two fully expanded leaves were mechanically wounded using a hemostat on both leaves for the indicated timesbefore extracting total RNAs for RT-qPCR assays. Data represent mean ± SD (N = 3). Asterisks indicate significant difference fromthe wild type according to Student’s t-test at **P < 0.01, ***P < 0.001 (Supplemental Data set 2).

Figure 3. MTB1–3 Negatively Regulate Diverse Aspects of JA Responses. (A) RT-qPCR assays of the expression ofTomLoxD, JA2L, TD, and PI-II in WT, MTB-RNAi-2#, MTB-RNAi-8#, MTB1-OE-5#, andMTB1-OE-6# plants in response tomechanical wounding (A) and MeJAtreatment (B). For (A), 18-day-old seedlingsof the indicated tomato genotypes with twofully expanded leaves were mechanicallywounded using a hemostat on both leaves forthe indicated times before extracting totalRNAs for RT-qPCR assays. For (B), 18-day-old seedlings of the indicated tomatogenotypes with two fully expanded leaveswere exposed to MeJA vapor for theindicated times before extracting total RNAsfor RT-qPCR assays. (C) Anthocyanincontents of the 7-day-old seedlings in WT,MTB-RNAi-2#, MTB-RNAi-8#, MTB1-OE-5#, and MTB1-OE-6# plants grown on MSmedium containing indicated concentrationsof JA. FW, fresh weight. (D) Root growthinhibition assay of 7-day-old seedlings ofWT, MTB-RNAi-2#, MTB-RNAi-8#, MTB1-OE-5#, and MTB1-OE-6# plants grown onMS medium supplied with indicatedconcentrations (µM) of JA. Data representmean ± SD (N = 20). (E) Expression of PI-IIin WT, MTB-RNAi-2#, MTB-RNAi-8#,MTB1-OE-5#, and MTB1-OE-6# plantssubjected to cotton bollworm (Helicoverpaarmigera) larvae. Plants were harvested at

the indicated time points during the feeding trial for RNA extraction and RT-qPCR analysis. (F) Average weight of larvae recovered at the end of day 4 of the feeding trial using whole plants of WT, MTB-RNAi-2#, MTB-RNAi-8#, MTB1-OE-5#, and MTB1-OE-6# genotypes. Data represent mean ± SD (N = 15). Each dot represents the weight of an individual larvae. (G) Expression of PR-STH2 in WT, MTB-RNAi-2#, MTB-RNAi-8#, MTB1-OE-5#, and MTB1-OE-6# plants treated with B. cinerea suspensions. Five-week-old plants were spotted with 5 µL spore suspension (106 spores/mL). Leaves were harvested 24 h after inoculation for RNA extraction and RT-qPCR analysis. (H) Growth of B. cinerea in WT, MTB-RNAi-2#, MTB-RNAi-8#, MTB1-OE-5#, and MTB1-OE-6# plants. Detached leaves from 5-week-old tomato plants were spotted with a 5 µL spore suspension (106 spores/mL). The lesion areas were analyzed at 3 DAI. Data represent mean ± SD (N = 9). (I) Expression of PR1b in WT, MTB-RNAi-2#, MTB-RNAi-8#, MTB1-OE-5#, and MTB1-OE-6# plants infected with Pst DC3000. Five-week-old plants were vacuum infiltrated with Pst DC3000 (0.5 × 10-5 c.f.u/mL). Leaves were harvested 24 h after inoculation for RNA extraction and RT-qPCR analysis. (J) Growth of Pst DC3000 inWT, MTB-RNAi-2#, MTB-RNAi-8#, MTB1-OE-5#, and MTB1-OE-6# plants. c.f.u, colony-forming units. Data represent mean ± SD(N = 6). Asterisks indicate significant difference from the wild type according to Student’s t-test at *P < 0.05. For (A), (B), (C), (E),(G) and (I), data represent mean ± SD (N = 3). For (A–J), Asterisks indicate significant difference from the wild type according toStudent’s t-test at *P < 0.05, **P < 0.01, ***P < 0.001 (Supplemental Data set 2).

Figure 4. MTB Proteins Lack a Canonical MED25-Interacting Domain (MID) and Act as Transcriptional Repressors. (A) Y2H assays of the interaction of MED25with MYC2 and MTB1. Full-length codingsequence (CDS) of MED25 was fused with theBD in pGBKT7, and full-length CDS of MYC2or MTB1 was fused with the AD in pGADT7.Transformed yeast cells were grown on SD/-4to determine protein–protein interactions. (B)In vitro pull-down assays of the interaction ofMED25 with MYC2 and MTB1. PurifiedMBP-MED25 was incubated with GST, GST-MYC2, or GST-MTB1 for the GST pull-downassay and detected by immunoblotting usinganti-MBP antibody. The positions of variouspurified proteins separated by SDS-PAGE aremarked with asterisks on CBB stained gel. (C)Y2H assays of MYC2 interaction with MED25through MID. According to the sequencealignment of the transcriptional activationdomain (TAD) of MYC2 and MTB1, MYC2TAD was divided into three fragments labeledas MID, MYC2 (186–202), and MYC2 (203–212). Full-length MYC2 CDS and MYC2variants lacking the MID, MYC2 (186–202),and MYC2 (203–212) fragments were fusedwith the AD in pGADT7, and full-lengthMED25 CDS was fused with the BD inpGBKT7. Transformed yeast cells were grownon SD/-4 medium to determine protein–proteininteractions. Co-transformation of the ADvector with MED25 was used as a negativecontrol. (D) Yeast assays used to detect thetranscriptional activity of MYC2 and MTB1.Yeast cells co-transformed with BD-MYC2 (orBD-MTB1) and AD vector were grown on SD/-4 medium. (E–G) Transient expression assaysto detect the effect of MTB1 on thetranscriptional activity of MYC2. Schematicrepresentation of effector constructs and fireflyluciferase (LUC) reporter construct used in

transient expression assays (E). Quantification of luminescence intensity of LUC in N. benthamiana leaves 72 h after co-infiltration with PTomLoxD:LUC reporter construct and effector constructs or infiltration of only the PTomLoxD:LUC reporter construct (control) (F). RT-qPCR analysis of MYC2 and MTB1 expression in N. benthamiana leaves infiltrated with the indicated constructs (G). For (F) and (G), data represent mean ± SD (N = 5). Statistically significant differences between control and test samples were determined using Student’s t-test and are indicated using asterisks (*P < 0.05; Supplemental Data set 2).

Figure 5. MTB1 Interacts with MYC2 and Disrupts MED25–MYC2 Interaction. (A) In vitro pull-down assays of MYC2formation of homo- and heterodimers.Purified MBP-MYC2 was incubated withGST, GST-MYC2, or GST-MTB1 for theGST pull-down assay and detected byimmunoblotting using anti-MBP antibody.(B and C) Pull-down assays of MTB1formation of homo- and heterodimers.Purified MBP-MTB1 was incubated withGST, GST-MTB1, or GST-MYC2 for theGST pull-down assay and detected byimmunoblotting using anti-MBP antibody(B), and protein extracts prepared fromMTB1-myc seedlings were incubated withMBP, MBP-MTB1, or MBP-MYC2 for theMBP pull-down assay and detected byimmunoblotting using anti-myc antibody(C). For (A–C), positions of various purifiedproteins separated by SDS-PAGE areindicated with asterisks on CBB stained gels.(D) Y3H assays showing that MTB1interferes with MYC2–MED25 interaction.Yeast cells co-transformed with pGADT7-MYC2 and pBridge-MED25-MTB1/MBPwere plated on SD/-4 medium to assess the

MYC2–MED25 interaction and on SD/-5 medium to induce MTB1/MBP. (E) In vitro pull-down assays of MTB1 interference with MED25–MYC2 interaction. Fixed amounts of His-MYC2 and MBP-MED25 fusion proteins were incubated with an increasing amount of GST-MTB1 fusion protein or GST protein. Protein samples were immunoprecipitated with anti-His antibody and immunoblotted with anti-MBP antibody. (F) Schematic diagram of PCR amplicons of TomLoxD and JA2L used for ChIP-qPCR. Positions of the transcription start site (TSS) and transcription termination site (TTS) are indicated. (G and H) ChIP-qPCR assays of that MTB-RNAi (G) and MTB1-OE (H) impairment of the enrichment of MED25 on the G-boxes of TomLoxD and JA2L promoters upon wounding. MED25-GFP-13# and MED25-GFP-13#/MTB-RNAi-8# plants (G) and MED25-GFP-13# and MED25-GFP-13#/MTB1-OE-11# plants (H) were treated with mechanical wounding for 1 h before cross-linking, and chromatin of each sample was immunoprecipitated using anti-GFP antibody. Immunoprecipitated DNAs were quantified by qPCR. The enrichment of target gene promoters is displayed as a percentage of input DNA. ACTIN2 was used as a non-specific control. Data represent mean ± SD (N = 3). Statistically significant differences between MED25-GFP-13# and other genotypes were determined using Student’s t-test and are indicated using asterisks (*P < 0.05, **P < 0.01; Supplemental Data set 2).

Figure 6. MTB1 Antagonizes MYC2 for DNA Binding. (A) Schematic representation ofTomLoxD and JA2L showing theamplicons and probe used for ChIP-qPCR assay and EMSA. Positions of thetranscription start site (TSS) andtranscription termination site (TTS) areindicated. (B and C) ChIP-qPCRanalysis of the enrichment of MYC2 (B)and MTB1 (C) on the chromatin ofTomLoxD and JA2L in MYC2-GFP-9#and MTB1-GFP-5# plants, respectively.(D and E) ChIP-qPCR analysis of theenrichment of MYC2 (D) and MTB1 (E)on the G-box regions of TomLoxD andJA2L in MYC2-GFP-9# and MTB1-GFP-5# plants, respectively, uponwounding. Plants were treated withmechanical wounding for the indicatedtimes before cross-linking. (F and G)ChIP-qPCR assays of the impairedenrichment of MYC2 on the G-boxes ofTomLoxD and JA2L due to MTB-RNAiin MYC2-GFP-9# and MYC2-GFP-9#/MTB-RNAi-8# plants (F) and MTB1-OE in MYC2-GFP-9# and MYC2-GFP-9#/MTB1-OE-11# plants (G) uponwounding. Plants were treated with

mechanical wounding for 1 h before cross-linking. For (B–G), chromatin of each sample was immunoprecipitated using anti-GFP antibody and quantified by qPCR. The enrichment of target gene promoters is displayed as a percentage of input DNA. Data represent mean ± SD (N = 3). For (D–G), ACTIN2 was used as a non-specific control. For (F) and (G), asterisks indicate significant differences detected using Student’s t-test (*P < 0.05, **P < 0.01; Supplemental Data set 2) when compared with MYC2-GFP. (H) EMSA showing that MBP-MTB1 interferes with MBP-MYC2 to bind to DNA probes from the TomLoxD promoter in vitro. Biotin-labeled probes were incubated with a fixed amount of MBP-MYC2 and an increasing amount of MBP-MTB1 or MBP, and the free and bound DNAs were separated on an acrylamide gel. The MBP protein was incubated with the labeled probe to serve as a negative control; mutated probes were used as a negative control. Mu, mutated probe in which the G-box motif 5ʹ-ACCATGTG-3ʹ was deleted.

Figure 7. MTB Genes Represent a Tool for Crop Protection. (A) Proposed mechanism by which MYC2 and MTB proteins form a feedback loop to terminate JA signaling. MYC2 and MED25activate the expression of MTB genes; in turn, MTB proteins interfere with the MED25–MYC2 interaction and DNA-bindingactivity of MYC2 to terminate JA-triggered defense responses. (B) Expression of PI-II in WT, mtb1-c and mtb1 mtb2-c plantsexposed to cotton bollworm (Helicoverpa armigera) larvae. Plants were harvested at the indicated time points during the feedingtrial for RNA extraction and RT-qPCR analysis. (C) Average weight (top) and images (bottom) of larvae recovered at the end of day4 of the feeding trial using whole plants of WT, mtb1-c, and mtb1 mtb2-c. Data represent mean ± SD (N = 15). Each dot denotes theweight of an individual larva. Asterisks indicate significant difference from the WT according to Student’s t-test at *P < 0.05(Supplemental Data set 2).

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DOI 10.1105/tpc.18.00405; originally published online January 4, 2019;Plant Cell

Wang, Chuanyou Li and Qingzhe ZhaiYuanyuan Liu, Minmin Du, Lei Deng, Jiafang Shen, Mingming Fang, Qian Chen, Yanhui Lu, Qiaomei

Feedback LoopMYC2 Regulates the Termination of Jasmonate Signaling via an Autoregulatory Negative

This information is current as of May 9, 2021

Supplemental Data /content/suppl/2019/01/04/tpc.18.00405.DC1.html

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MYC2 Termination of Jasmonate Signaling via anJan 04, 2019 · promoters. Therefore, MYC2 and MTB proteins form an autoregulatory negative feedback circuit to . terminate JA signaling