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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
25
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
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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
46
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
(Solyc12g049400), JAZ5 (Solyc03g118540), JAZ6 (Solyc01g005440), JAZ7 911
(Solyc11g011030), JAZ8 (Solyc06g068930), JAZ9 (Solyc08g036640), JAZ10 912
(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
REFERENCES 953
An, C., Li, L., Zhai, Q., You, Y., Deng, L., Wu, F., Chen, R., Jiang, H., Wang, H., Chen, 954
Q., and Li, C. (2017). Mediator subunit MED25 links the jasmonate receptor to 955
transcriptionally active chromatin. Proc. Natl. Acad. Sci. USA 114: E8930–E8939. 956
Boter, M., Ruiz-Rivero, O., Abdeen, A., and Prat, S. (2004). Conserved MYC transcription 957
factors play a key role in jasmonate signaling both in tomato and Arabidopsis. Genes 958
Dev. 18: 1577–1591. 959
Brooks, D.M., Bender, C.L., and Kunkel, B.N. (2005). The Pseudomonas syringae 960
phytotoxin coronatine promotes virulence by overcoming salicylic acid-dependent 961
defences in Arabidopsis thaliana. Mol. Plant Pathol. 6: 629-639. 962
Browse, J. (2009). Jasmonate passes muster: a receptor and targets for the defense hormone. 963
Annu. Rev. Plant Biol. 60: 183–205. 964
Campos, M. L., Kang, J. H., and Howe, G. A. (2014). Jasmonate-triggered plant immunity. 965
J. Chem. Ecol. 40: 657–670.966
Carretero-Paulet, L., Galstyan, A., Roig-Villanova, I., Martinez-Garcia, J.F., 967
Bilbao-Castro, J.R., and Robertson, D.L. (2010). Genome-wide classification and 968
evolutionary analysis of the bHLH family of transcription factors in Arabidopsis, 969
poplar, rice, moss, and algae. Plant Physiol. 153: 1398–1412. 970
Çevik, V., Kidd, B.N., Zhang, P., Hill, C., Kiddle, S., Denby, K.J., Holub, E.B., Cahill, 971
D.M., Manners, J.M., Schenk, P.M., Beynon, J., and Kazan, K. (2012).972
MEDIATOR25 acts as an integrative hub for the regulation of jasmonate-responsive 973
33
gene expression in Arabidopsis. Plant Physiol. 160: 541–555. 974
Chen, H., Wilkerson, C.G., Kuchar, J.A., Phinney, B.S., and Howe, G.A. (2005). 975
Jasmonate-inducible plant enzymes degrade essential amino acids in the herbivore 976
midgut. Proc. Natl. Acad. Sci. USA 102: 19237–19242. 977
Chen, Q., Sun, J., Zhai, Q., Zhou, W., Qi, L., Xu, L., Wang, B., Chen, R., Jiang, H., Qi, 978
J., Li, X., Palme, K., and Li, C. (2011). The basic helix-loop-helix transcription 979
factor MYC2 directly represses PLETHORA expression during jasmonate-mediated 980
modulation of the root stem cell niche in Arabidopsis. Plant Cell 23: 3335–3352. 981
Chen, R., Jiang, H., Li, L., Zhai, Q., Qi, L., Zhou, W., Liu, X., Li, H., Zheng, W., Sun, J., 982
and Li, C. (2012). The Arabidopsis mediator subunit MED25 differentially regulates 983
jasmonate and abscisic acid signaling through interacting with the MYC2 and ABI5 984
transcription factors. Plant Cell 24: 2898–2916. 985
Cheng, Z., Sun, L., Qi, T., Zhang, B., Peng, W., Liu, Y., and Xie, D. (2011). The bHLH 986
transcription factor MYC3 interacts with the Jasmonate ZIM-domain proteins to 987
mediate jasmonate response in Arabidopsis. Mol. Plant 4: 279–288. 988
Chini, A., Gimenez-Ibanez, S., Goossens, A., and Solano, R. (2016). Redundancy and 989
specificity in jasmonate signalling. Curr. Opin. Plant Biol. 33: 147–156. 990
Chini, A., Fonseca, S., Fernández, G., Adie, B., Chico, J.M., Lorenzo, O., Garcia-Casado, 991
G., Lopez-Vidriero, I., Lozano, F.M., Ponce, M.R., Micol, J.L., and Solano, R. 992
(2007). The JAZ family of repressors is the missing link in jasmonate signalling. 993
Nature 448: 666–671. 994
Chung, H.S., and Howe, G.A. (2009). A critical role for the TIFY motif in repression of 995
jasmonate signaling by a stabilized splice variant of the JASMONATE ZIM-domain 996
protein JAZ10 in Arabidopsis. Plant Cell 21: 131–145. 997
Chung, H.S., Cooke, T.F., Depew, C.L., Patel, L.C., Ogawa, N., Kobayashi, Y., and Howe, 998
G.A. (2010). Alternative splicing expands the repertoire of dominant JAZ repressors 999
of jasmonate signaling. Plant J. 63: 613–622. 1000
Deikman, J., and Hammer, P.E. (1995). Induction of anthocyanin accumulation by 1001
Cytokinins in Arabidopsis thaliana. Plant Physiol. 108: 47-57. 1002
Deng, L., Wang, H., Sun, C., Li, Q., Jiang, H., Du, M., Li, C.B., and Li, C. (2018). 1003
34
Efficient generation of pink-fruited tomatoes using CRISPR/Cas9 system. J Genet 1004
Genomics. 45: 51–54. 1005
Despres, C., Subramaniam, R., Matton, D.P., and Brisson, N. (1995). The Activation of 1006
the Potato PR-10a Gene Requires the Phosphorylation of the Nuclear Factor PBF-1. 1007
Plant Cell 7: 589-598. 1008
Devoto, A., Nieto-Rostro, M., Xie, D., Ellis, C., Harmston, R., Patrick, E., Davis, J., 1009
Sherratt, L., Coleman, M., and Turner, J.G. (2002). COI1 links jasmonate 1010
signalling and fertility to the SCF ubiquitin-ligase complex in Arabidopsis. Plant J. 32: 1011
457–466. 1012
Dombrecht, B., Xue, G.P., Sprague, S.J., Kirkegaard, J.A., Ross, J.J., Reid, J.B., Fitt, 1013
G.P., Sewelam, N., Schenk, P.M., Manners, J.M., and Kazan, K. (2007). MYC2 1014
differentially modulates diverse jasmonate-dependent functions in Arabidopsis. Plant 1015
Cell 19: 2225–2245. 1016
Du, M., Zhai, Q., Deng, L., Li, S., Li, H., Yan, L., Huang, Z., Wang, B., Jiang, H., Huang, 1017
T., Li, C.B., Wei, J., Kang, L., Li, J., and Li, C. (2014). Closely related NAC 1018
transcription factors of tomato differentially regulate stomatal closure and reopening 1019
during pathogen attack. Plant Cell 26: 3167–3184. 1020
Du, M., Zhao, J., Tzeng, D.T.W., Liu, Y., Deng, L., Yang, T., Zhai, Q., Wu, F., Huang, Z., 1021
Zhou, M., Wang, Q., Chen, Q., Zhong, S., Li, C.B., and Li, C. (2017). MYC2 1022
orchestrates a hierarchical transcriptional cascade that regulates jasmonate-mediated 1023
plant immunity in tomato. Plant Cell 29: 1883–1906. 1024
El Oirdi, M., El Rahman, T. A., Rigano, L., El Hadrami, A., Rodriguez, M. C., Daayf, F., 1025
Vojnov, A., and Bouarab, K. (2011). Botrytis cinerea manipulates the antagonistic 1026
effects between immune pathways to promote disease development in tomato. Plant 1027
Cell 23: 2405–2421. 1028
Fernández-Calvo, P., Chini, A., Fernández-Barbero, G., Chico, J.M., Gimenez-Ibanez, 1029
S., Geerinck, J., Eeckhout, D., Schweizer, F., Godoy, M., Franco-Zorrilla, J.M., 1030
Pauwels, L., Witters, E., Puga, M.I., Paz-Ares, J., Goossens, A., Reymond, P., De 1031
Jaeger, G., and Solano, R. (2011). The Arabidopsis bHLH transcription factors 1032
MYC3 and MYC4 are targets of JAZ repressors and act additively with MYC2 in the 1033
35
activation of jasmonate responses. Plant Cell 23: 701–715. 1034
Fonseca, S., Chini, A., Hamberg, M., Adie, B., Porzel, A., Kramell, R., Miersch, O., 1035
Wasternack, C., and Solano, R. (2009). (+)-7-iso-Jasmonoyl-L-isoleucine is the 1036
endogenous bioactive jasmonate. Nature Chem. Biol. 5: 344–350. 1037
Fonseca, S., Fernández-Calvo, P., Fernández, G.M., Diez-Diaz, M., Gimenez-Ibanez, S., 1038
Lopez-Vidriero, I., Godoy, M., Fernández-Barbero, G., Van Leene, J., De Jaeger, 1039
G., Franco-Zorrilla, J.M., and Solano, R. (2014). bHLH003, bHLH013 and 1040
bHLH017 are new targets of JAZ repressors negatively regulating JA responses. PloS 1041
One 9: e86182. 1042
Fu, J., Chu, J., Sun, X., Wang, J., and Yan, C. (2012). Simple, rapid, and simultaneous 1043
assay of multiple carboxyl containing phytohormones in wounded tomatoes by 1044
UPLC-MS/MS using single SPE purification and isotope dilution. Anal. Sci. 28: 1045
1081–1087. 1046
Goossens, J., Fernández-Calvo, P., Schweizer, F., and Goossens, A. (2016). Jasmonates: 1047
signal transduction components and their roles in environmental stress responses. 1048
Plant Mol. Biol. 91: 673–689. 1049
Goossens, J., Mertens, J., and Goossens, A. (2017). Role and functioning of bHLH 1050
transcription factors in jasmonate signalling. J. Exp. Bot. 68: 1333–1347. 1051
Guo, Q., Major, I.T., and Howe, G.A. (2018a). Resolution of growth–defense conflict: 1052
mechanistic insights from jasmonate signaling. Curr. Opin. Plant Biol. 44: 72–81. 1053
Guo, Q., Yoshida, Y., Major, I.T., Wang, K., Sugimoto, K., Kapali, G., Havko, 1054
N.E., Benning, C., Howe, G.A. (2018b). JAZ repressors of metabolic defense1055
promote growth and reproductive fitness in Arabidopsis. Proc. Natl. Acad. Sci. U S A 1056
115: E10768–E10777. 1057
Heitz, T., Widemann, E., Lugan, R., Miesch, L., Ullmann, P., Desaubry, L., Holder, E., 1058
Grausem, B., Kandel, S., Miesch, M., Werck-Reichhart, D., and Pinot, F. (2012). 1059
Cytochromes P450 CYP94C1 and CYP94B3 catalyze two successive oxidation steps 1060
of plant hormone Jasmonoyl-isoleucine for catabolic turnover. J. Biol. Chem. 287: 1061
6296–6306. 1062
36
Hickman, R., Van Verk, M.C., Van Dijken, A.J.H., Mendes, M.P., Vroegop-Vos, I.A., 1063
Caarls, L., Steenbergen, M., Van der Nagel, I., Wesselink, G.J., Jironkin, A., 1064
Talbot, A., Rhodes, J., De Vries, M., Schuurink, R.C., Denby, K., Pieterse, C.M.J., 1065
and Van Wees, S.C.M. (2017). Architecture and Dynamics of the Jasmonic Acid 1066
Gene Regulatory Network. Plant Cell 29: 2086–2105. 1067
Howe, G.A., Major, I.T., and Koo, A.J. (2018). Modularity in Jasmonate Signaling for 1068
Multistress Resilience. Annu. Rev. Plant Biol. 69: 387–415. 1069
Kazan, K., and Manners, J.M. (2013). MYC2: the master in action. Mol. Plant 6: 686-703. 1070
Kitaoka, N., Matsubara, T., Sato, M., Takahashi, K., Wakuta, S., Kawaide, H., Matsui, 1071
H., Nabeta, K., and Matsuura, H. (2011). Arabidopsis CYP94B3 encodes 1072
jasmonyl-L-isoleucine 12-hydroxylase, a key enzyme in the oxidative catabolism of 1073
jasmonate. Plant Cell Physiol. 52: 1757–1765. 1074
Kloek, A.P., Verbsky, M.L., Sharma, S.B., Schoelz, J.E., Vogel, J., Klessig, D.F., and 1075
Kunkel, B.N. (2001). Resistance to Pseudomonas syringae conferred by an 1076
Arabidopsis thaliana coronatine-insensitive (coi1) mutation occurs through two 1077
distinct mechanisms. Plant Cell 26: 509–522. 1078
Koo, A.J., and Howe, G.A. (2012). Catabolism and deactivation of the lipid-derived 1079
hormone jasmonoyl-isoleucine. Front. Plant Sci. 3:19. 1080
Koo, A.J., Cooke, T.F., and Howe, G.A. (2011). Cytochrome P450 CYP94B3 mediates 1081
catabolism and inactivation of the plant hormone jasmonoyl-L-isoleucine. Proc. Natl. 1082
Acad. Sci. USA 108: 9298–9303. 1083
Li, L., Zhao, Y., McCaig, B.C., Wingerd, B.A., Wang, J., Whalon, M.E., Pichersky, E., 1084
and Howe, G.A. (2004). The tomato homolog of CORONATINE-INSENSITIVE1 is 1085
required for the maternal control of seed maturation, jasmonate-signaled defense 1086
responses, and glandular trichome development. Plant Cell 16: 126–143. 1087
Lian, J., Han, H., Zhao, J., and Li, C. (2018). In-vitro and in-planta Botrytis cinerea 1088
inoculation assays for tomato. Bio-protocol 8: e2810. 1089
Lian, T.F., Xu, Y.P., Li, L.F., and Su, X.D. (2017). Crystal structure of tetrameric 1090
Arabidopsis MYC2 reveals the mechanism of enhanced interaction with DNA. Cell 1091
Rep. 19: 1334–1342. 1092
37
Lorenzo, O., Chico, J.M., Sanchez-Serrano, J.J., and Solano, R. (2004). 1093
JASMONATE-INSENSITIVE1 encodes a MYC transcription factor essential to 1094
discriminate between different jasmonate-regulated defense responses in Arabidopsis. 1095
Plant Cell 16: 1938–1950. 1096
Major, I.T., Yoshida, Y., Campos, M.L., Kapali, G., Xin, X.F., Sugimoto, K., de Oliveira 1097
Ferreira, D., He, S.Y., and Howe, G.A. (2017). Regulation of growth–defense 1098
balance by the JASMONATE ZIM-DOMAIN (JAZ)-MYC transcriptional module. 1099
New Phytol. 215: 1533–1547. 1100
Marineau, C., Matton, D.P., and Brisson, N. (1987). Differential accumulation of potato 1101
tuber mRNAs during the hypersensitive response induced by arachidonic acid elicitor. 1102
Plant Mol. Biol. 9: 335–342. 1103
Matsui, K., Umemura, Y., and Ohme-Takagi, M. (2008). AtMYBL2, a protein with a 1104
single MYB domain, acts as a negative regulator of anthocyanin biosynthesis in 1105
Arabidopsis. Plant J. 55: 954–967. 1106
Matton, D.P., and Brisson, N. (1989). Cloning, expression, and sequence conservation of 1107
pathogenesis-related gene transcripts of potato. Mol. Plant Microbe Interact. 2: 325–1108
331. 1109
Melotto, M., Underwood, W., Koczan, J., Nomura, K., and He, S. Y. (2006). Plant stomata 1110
function in innate immunity against bacterial invasion. Cell 126: 969–980. 1111
Miersch, O., Neumerkel, J., Dippe, M., Stenzel, I., and Wasternack, C. (2008). 1112
Hydroxylated jasmonates are commonly occurring metabolites of jasmonic acid and 1113
contribute to a partial switch-off in jasmonate signaling. New Phytol. 177: 114–127. 1114
Moreno, J.E., Shyu, C., Campos, M.L., Patel, L.C., Chung, H.S., Yao, J., He, S.Y., and 1115
Howe, G.A. (2013). Negative feedback control of jasmonate signaling by an 1116
alternative splice variant of JAZ10. Plant Physiol. 162: 1006–1017. 1117
Nakagawa, T., Kurose, T., Hino, T., Tanaka, K., Kawamukai, M., Niwa, Y., Toyooka, K., 1118
Matsuoka, K., Jinbo, T., and Kimura, T. (2007). Development of series of gateway 1119
binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant 1120
transformation. J. Biosci. Bioeng. 104: 34–41. 1121
38
Nakata, M., Mitsuda, N., Herde, M., Koo, A.J., Moreno, J.E., Suzuki, K., Howe, G.A., 1122
and Ohme-Takagi, M. (2013). A bHLH-type transcription factor, ABA-INDUCIBLE 1123
BHLH-TYPE TRANSCRIPTION FACTOR/JA-ASSOCIATED MYC2-LIKE1, acts 1124
as a repressor to negatively regulate jasmonate signaling in arabidopsis. Plant Cell 25: 1125
1641–1656. 1126
Niu, Y., Figueroa, P., and Browse, J. (2011). Characterization of JAZ-interacting bHLH 1127
transcription factors that regulate jasmonate responses in Arabidopsis. J. Exp. Bot. 62: 1128
2143–2154. 1129
Pauwels, L., Barbero, G.F., Geerinck, J., Tilleman, S., Grunewald, W., Perez, A.C., 1130
Chico, J.M., Bossche, R.V., Sewell, J., Gil, E., Garcia-Casado, G., Witters, E., 1131
Inze, D., Long, J.A., De Jaeger, G., Solano, R., and Goossens, A. (2010). NINJA 1132
connects the co-repressor TOPLESS to jasmonate signalling. Nature 464: 788–791. 1133
Pieterse, C.M.J., Van der Does, D., Zamioudis, C., Leon-Reyes, A., and Van Wees, 1134
S.C.M.1135
(2012). Hormonal modulation of plant immunity. Annu. Rev. Cell Dev. Biol. 28: 1136
489– 1137
521. 1138
Qi, T., Wang, J., Huang, H., Liu, B., Gao, H., Liu, Y., Song, S., and Xie, D. (2015). 1139
Regulation of Jasmonate-Induced Leaf Senescence by Antagonism between bHLH 1140
Subgroup IIIe and IIId Factors in Arabidopsis. Plant Cell 27:1634–1649. 1141
Ryan, C.A. (2000). The systemin signaling pathway: differential activation of plant defensive 1142
genes. Biochim. Biophys. Acta. 1477: 112–121. 1143
Ryan, C.A., and Pearce, G. (1998). Systemin: a polypeptide signal for plant defensive genes. 1144
Annu. Rev. Cell Dev. Biol. 14: 1–17. 1145
Sasaki-Sekimoto, Y., Jikumaru, Y., Obayashi, T., Saito, H., Masuda, S., Kamiya, Y., 1146
Ohta, H., and Shirasu, K. (2013). Basic helix-loop-helix transcription factors 1147
JASMONATE-ASSOCIATED MYC2-LIKE1 (JAM1), JAM2, and JAM3 are 1148
negative regulators of jasmonate responses in Arabidopsis. Plant Physiol. 163: 291–1149
304. 1150
Schilmiller, A.L., and Howe, G.A. (2005). Systemic signaling in the wound response. Curr. 1151
39
Opin. Plant Biol. 8: 369–377. 1152
Shang, Y., Yan, L., Liu, Z.Q., Cao, Z., Mei, C., Xin, Q., Wu, F.Q., Wang, X.F., Du, S.Y., 1153
Jiang, T., Zhang, X.F., Zhao, R., Sun, H.L., Liu, R., Yu, Y.T., and Zhang, D.P. 1154
(2010). The Mg-chelatase H subunit of Arabidopsis antagonizes a group of WRKY 1155
transcription repressors to relieve ABA-responsive genes of inhibition. Plant Cell 22: 1156
1909–1935. 1157
Sheard, L.B., Tan, X., Mao, H., Withers, J., Ben-Nissan, G., Hinds, T.R., Kobayashi, Y., 1158
Hsu, F.F., Sharon, M., Browse, J., He, S.Y., Rizo, J., Howe, G.A., and Zheng, N. 1159
(2010). Jasmonate perception by inositol-phosphate-potentiated COI1-JAZ 1160
co-receptor. Nature 468, 400–405. 1161
Shyu, C., Figueroa, P., Depew, C.L., Cooke, T.F., Sheard, L.B., Moreno, J.E., Katsir, L., 1162
Zheng, N., Browse, J., and Howe, G.A. (2012). JAZ8 lacks a canonical degron and 1163
has an EAR motif that mediates transcriptional repression of jasmonate responses in 1164
Arabidopsis. Plant Cell 24: 536–550. 1165
Song, S., Qi, T., Fan, M., Zhang, X., Gao, H., Huang, H., Wu, D., Guo, H., and Xie, D. 1166
(2013). The bHLH subgroup IIId factors negatively regulate jasmonate-mediated 1167
plant defense and development. PLoS Genet. 9: e1003653. 1168
Spoel, S.H., et al. (2003). NPR1 modulates cross-talk between salicylate- and jasmonate- 1169
dependent defense pathways through a novel function in the cytosol. Plant Cell 15: 1170
760–770. 1171
Sun, H., Fan, H., and Ling, H. (2015). Genome-wide identification and characterization of 1172
the bHLH gene family in tomato. BMC Genomics 16: 9. 1173
Sun, J. Q., Jiang, H. L., and Li, C. Y. (2011). Systemin/Jasmonate-mediated systemic 1174
defense signaling in tomato. Mol. Plant 4: 607–615. 1175
Thines, B., Katsir, L., Melotto, M., Niu, Y., Mandaokar, A., Liu, G., Nomura, K., He, 1176
S.Y., Howe, G.A., and Browse, J. (2007). JAZ repressor proteins are targets of the 1177
SCFCOI1
complex during jasmonate signalling. Nature 448: 661–665. 1178
Thireault, C., Shyu, C., Yoshida, Y., St Aubin, B., Campos, M.L., and Howe, G.A. (2015). 1179
Repression of jasmonate signaling by a non-TIFY JAZ protein in Arabidopsis. Plant J. 1180
82: 669–679. 1181
40
VanDoorn, A., Bonaventure, G., Schmidt, D.D., and Baldwin, I.T. (2011). Regulation of 1182
jasmonate metabolism and activation of systemic signaling in Solanum nigrum: COI1 1183
and JAR4 play overlapping yet distinct roles. New Phytol. 190: 640–652. 1184
Van der Does, D., Leon-Reyes, A., Koornneef, A., Van Verk, M.C., Rodenburg, N., 1185
Pauwels, L., Goossens, A., Körbes, A.P., Memelink, J., Ritsema, T., Van Wees, 1186
S.C.M., and Pieterse, C.M.J. (2013). Salicylic acid suppresses jasmonic acid1187
signaling downstream of SCFCOI1-JAZ by targeting GCC promoter motifs via 1188
transcription factor ORA59. Plant Cell 25: 744–761. 1189
Wasternack, C., and Hause, B. (2013). Jasmonates: biosynthesis, perception, signal 1190
transduction and action in plant stress response, growth and development. An update 1191
to the 2007 review in Annals of Botany. Ann. Bot. 111: 1021–1058. 1192
Xie, D., Feys, B.F., James, S., Nieto-Rostro, M., and Turner, J.G. (1998). COI1: an 1193
Arabidopsis gene required for jasmonate-regulated defense and fertility. Science 280: 1194
1091–1094. 1195
Xu, L., Liu, F., Lechner, E., Genschik, P., Crosby, W.L., Ma, H., Peng, W., Huang, D., 1196
and Xie, D. (2002). The SCFCOI1
ubiquitin-ligase complexes are required for1197
jasmonate response in Arabidopsis. Plant Cell 14: 1919–1935. 1198
Yan, L., Zhai, Q., Wei, J., Li, S., Wang, B., Huang, T., Du, M., Sun, J., Kang, L., Li, C.B., 1199
and Li, C. (2013). Role of tomato lipoxygenase D in wound-induced jasmonate 1200
biosynthesis and plant immunity to insect herbivores. PLoS Geneti. 9: e1003964. 1201
Yan, Y., Stolz, S., Chetelat, A., Reymond, P., Pagni, M., Dubugnon, L., and Farmer, E.E. 1202
(2007). A downstream mediator in the growth repression limb of the jasmonate 1203
pathway. Plant Cell 19: 2470–2483. 1204
Zhai, Q., Li, L., An, C., and Li, C. (2017a). Conserved Function of Mediator in Regulating 1205
Nuclear Hormone Receptor Activation between Plants and Animals. Plant Signal. 1206
Behav. doi: 10.1080/15592324.2017.1403709. 1207
Zhai, Q., Yan, C., Li, L., Xie, D., and Li, C. (2017b). Jasmonates. In Hormone Metabolism 1208
and Signaling in Plants, J. Li, C. Li, and S.M. Smith, eds (London: ESLEVIER. 1209
Zhai, Q., Yan, L., Tan, D., Chen, R., Sun, J., Gao, L., Dong, M.Q., Wang, Y., and Li, C. 1210
(2013). Phosphorylation-coupled proteolysis of the transcription factor MYC2 is 1211
41
important for jasmonate-signaled plant immunity. PLoS Genet. 9: e1003422. 1212
Zhang, F., Ke, J., Zhang, L., Chen, R., Sugimoto, K., Howe, G.A., Xu, H.E., Zhou, M., 1213
He, S.Y., and Melcher, K. (2017). Structural insights into alternative 1214
splicing-mediated desensitization of jasmonate signaling. Proc. Natl. Acad. Sci. USA 1215
114: 1720–1725. 1216
Zhang, F., Yao, J., Ke, J., Zhang, L., Lam, V.Q., Xin, X., Zhou, X., Chen, J., Brunzelle, J., 1217
Griffin, P.R., Zhou, M., Xu, H., Melcher, K., and He, S. (2015). Structural basis of 1218
JAZ repression of MYC transcription factors in jasmonate signalling. Nature 525: 1219
269–273. 1220
Zhang, L., Zhang, F., Melotto, M., Yao, J., He, S.Y. (2017) Jasmonate signaling and 1221
manipulation by pathogens and insects. J Exp Bot 68: 1371–1385. 1222
Zhao, Y., Thilmony, R., Bender, C.L., Schaller, A., He, S.Y., and Howe, G.A. (2003). 1223
Virulence systems of Pseudomonas syringae pv. tomato promote bacterial speck 1224
disease in tomato by targeting the jasmonate signaling pathway. Plant J. 36: 485–499. 1225
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).
Parsed CitationsAn, C., Li, L., Zhai, Q., You, Y., Deng, L., Wu, F., Chen, R., Jiang, H., Wang, H., Chen, Q., and Li, C. (2017). Mediator subunit MED25 linksthe jasmonate receptor to transcriptionally active chromatin. Proc. Natl. Acad. Sci. USA 114: E8930–E8939.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Boter, M., Ruiz-Rivero, O., Abdeen, A., and Prat, S. (2004). Conserved MYC transcription factors play a key role in jasmonate signalingboth in tomato and Arabidopsis. Genes Dev. 18: 1577–1591.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Brooks, D.M., Bender, C.L., and Kunkel, B.N. (2005). The Pseudomonas syringae phytotoxin coronatine promotes virulence byovercoming salicylic acid-dependent defences in Arabidopsis thaliana. Mol. Plant Pathol. 6: 629-639.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Browse, J. (2009). Jasmonate passes muster: a receptor and targets for the defense hormone. Annu. Rev. Plant Biol. 60: 183–205.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Campos, M. L., Kang, J. H., and Howe, G. A. (2014). Jasmonate-triggered plant immunity. J. Chem. Ecol. 40: 657–670.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Carretero-Paulet, L., Galstyan, A., Roig-Villanova, I., Martinez-Garcia, J.F., Bilbao-Castro, J.R., and Robertson, D.L. (2010). Genome-wide classification and evolutionary analysis of the bHLH family of transcription factors in Arabidopsis, poplar, rice, moss, and algae.Plant Physiol. 153: 1398–1412.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Çevik, V., Kidd, B.N., Zhang, P., Hill, C., Kiddle, S., Denby, K.J., Holub, E.B., Cahill, D.M., Manners, J.M., Schenk, P.M., Beynon, J., andKazan, K. (2012). MEDIATOR25 acts as an integrative hub for the regulation of jasmonate-responsive gene expression in Arabidopsis.Plant Physiol. 160: 541–555.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Chen, H., Wilkerson, C.G., Kuchar, J.A., Phinney, B.S., and Howe, G.A. (2005). Jasmonate-inducible plant enzymes degrade essentialamino acids in the herbivore midgut. Proc. Natl. Acad. Sci. USA 102: 19237–19242.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Chen, Q., Sun, J., Zhai, Q., Zhou, W., Qi, L., Xu, L., Wang, B., Chen, R., Jiang, H., Qi, J., Li, X., Palme, K., and Li, C. (2011). The basichelix-loop-helix transcription factor MYC2 directly represses PLETHORA expression during jasmonate-mediated modulation of theroot stem cell niche in Arabidopsis. Plant Cell 23: 3335–3352.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Chen, R., Jiang, H., Li, L., Zhai, Q., Qi, L., Zhou, W., Liu, X., Li, H., Zheng, W., Sun, J., and Li, C. (2012). The Arabidopsis mediator subunitMED25 differentially regulates jasmonate and abscisic acid signaling through interacting with the MYC2 and ABI5 transcription factors.Plant Cell 24: 2898–2916.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Cheng, Z., Sun, L., Qi, T., Zhang, B., Peng, W., Liu, Y., and Xie, D. (2011). The bHLH transcription factor MYC3 interacts with theJasmonate ZIM-domain proteins to mediate jasmonate response in Arabidopsis. Mol. Plant 4: 279–288.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Chini, A., Gimenez-Ibanez, S., Goossens, A., and Solano, R. (2016). Redundancy and specificity in jasmonate signalling. Curr. Opin.Plant Biol. 33: 147–156.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Chini, A., Fonseca, S., Fernández, G., Adie, B., Chico, J.M., Lorenzo, O., Garcia-Casado, G., Lopez-Vidriero, I., Lozano, F.M., Ponce,M.R., Micol, J.L., and Solano, R. (2007). The JAZ family of repressors is the missing link in jasmonate signalling. Nature 448: 666–671.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Chung, H.S., and Howe, G.A. (2009). A critical role for the TIFY motif in repression of jasmonate signaling by a stabilized splice variantof the JASMONATE ZIM-domain protein JAZ10 in Arabidopsis. Plant Cell 21: 131–145.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Chung, H.S., Cooke, T.F., Depew, C.L., Patel, L.C., Ogawa, N., Kobayashi, Y., and Howe, G.A. (2010). Alternative splicing expands therepertoire of dominant JAZ repressors of jasmonate signaling. Plant J. 63: 613–622.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Deikman, J., and Hammer, P.E. (1995). Induction of anthocyanin accumulation by Cytokinins in Arabidopsis thaliana. Plant Physiol. 108:47-57.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Deng, L., Wang, H., Sun, C., Li, Q., Jiang, H., Du, M., Li, C.B., and Li, C. (2018). Efficient generation of pink-fruited tomatoes usingCRISPR/Cas9 system. J Genet Genomics. 45: 51–54.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Despres, C., Subramaniam, R., Matton, D.P., and Brisson, N. (1995). The Activation of the Potato PR-10a Gene Requires thePhosphorylation of the Nuclear Factor PBF-1. Plant Cell 7: 589-598.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Devoto, A., Nieto-Rostro, M., Xie, D., Ellis, C., Harmston, R., Patrick, E., Davis, J., Sherratt, L., Coleman, M., and Turner, J.G. (2002).COI1 links jasmonate signalling and fertility to the SCF ubiquitin-ligase complex in Arabidopsis. Plant J. 32: 457–466.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Dombrecht, B., Xue, G.P., Sprague, S.J., Kirkegaard, J.A., Ross, J.J., Reid, J.B., Fitt, G.P., Sewelam, N., Schenk, P.M., Manners, J.M.,and Kazan, K. (2007). MYC2 differentially modulates diverse jasmonate-dependent functions in Arabidopsis. Plant Cell 19: 2225–2245.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Du, M., Zhai, Q., Deng, L., Li, S., Li, H., Yan, L., Huang, Z., Wang, B., Jiang, H., Huang, T., Li, C.B., Wei, J., Kang, L., Li, J., and Li, C.(2014). Closely related NAC transcription factors of tomato differentially regulate stomatal closure and reopening during pathogenattack. Plant Cell 26: 3167–3184.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Du, M., Zhao, J., Tzeng, D.T.W., Liu, Y., Deng, L., Yang, T., Zhai, Q., Wu, F., Huang, Z., Zhou, M., Wang, Q., Chen, Q., Zhong, S., Li, C.B.,and Li, C. (2017). MYC2 orchestrates a hierarchical transcriptional cascade that regulates jasmonate-mediated plant immunity intomato. Plant Cell 29: 1883–1906.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
El Oirdi, M., El Rahman, T. A., Rigano, L., El Hadrami, A., Rodriguez, M. C., Daayf, F., Vojnov, A., and Bouarab, K. (2011). Botrytis cinereamanipulates the antagonistic effects between immune pathways to promote disease development in tomato. Plant Cell 23: 2405–2421.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Fernández-Calvo, P., Chini, A., Fernández-Barbero, G., Chico, J.M., Gimenez-Ibanez, S., Geerinck, J., Eeckhout, D., Schweizer, F.,Godoy, M., Franco-Zorrilla, J.M., Pauwels, L., Witters, E., Puga, M.I., Paz-Ares, J., Goossens, A., Reymond, P., De Jaeger, G., and Solano,R. (2011). The Arabidopsis bHLH transcription factors MYC3 and MYC4 are targets of JAZ repressors and act additively with MYC2 inthe activation of jasmonate responses. Plant Cell 23: 701–715.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Fonseca, S., Chini, A., Hamberg, M., Adie, B., Porzel, A., Kramell, R., Miersch, O., Wasternack, C., and Solano, R. (2009). (+)-7-iso-Jasmonoyl-L-isoleucine is the endogenous bioactive jasmonate. Nature Chem. Biol. 5: 344–350.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Fonseca, S., Fernández-Calvo, P., Fernández, G.M., Diez-Diaz, M., Gimenez-Ibanez, S., Lopez-Vidriero, I., Godoy, M., Fernández-Barbero, G., Van Leene, J., De Jaeger, G., Franco-Zorrilla, J.M., and Solano, R. (2014). bHLH003, bHLH013 and bHLH017 are newtargets of JAZ repressors negatively regulating JA responses. PloS One 9: e86182.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Fu, J., Chu, J., Sun, X., Wang, J., and Yan, C. (2012). Simple, rapid, and simultaneous assay of multiple carboxyl containingphytohormones in wounded tomatoes by UPLC-MS/MS using single SPE purification and isotope dilution. Anal. Sci. 28: 1081–1087.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Goossens, J., Fernández-Calvo, P., Schweizer, F., and Goossens, A. (2016). Jasmonates: signal transduction components and theirroles in environmental stress responses. Plant Mol. Biol. 91: 673–689.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Goossens, J., Mertens, J., and Goossens, A. (2017). Role and functioning of bHLH transcription factors in jasmonate signalling. J. Exp.Bot. 68: 1333–1347.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Guo, Q., Major, I.T., and Howe, G.A. (2018a). Resolution of growth–defense conflict: mechanistic insights from jasmonate signaling.Curr. Opin. Plant Biol. 44: 72–81.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Guo, Q., Yoshida, Y., Major, I.T., Wang, K., Sugimoto, K., Kapali, G., Havko, N.E., Benning, C., Howe, G.A. (2018b). JAZ repressors ofmetabolic defense promote growth and reproductive fitness in Arabidopsis. Proc. Natl. Acad. Sci. U S A 115: E10768–E10777.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Heitz, T., Widemann, E., Lugan, R., Miesch, L., Ullmann, P., Desaubry, L., Holder, E., Grausem, B., Kandel, S., Miesch, M., Werck-Reichhart, D., and Pinot, F. (2012). Cytochromes P450 CYP94C1 and CYP94B3 catalyze two successive oxidation steps of planthormone Jasmonoyl-isoleucine for catabolic turnover. J. Biol. Chem. 287: 6296–6306.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Hickman, R., Van Verk, M.C., Van Dijken, A.J.H., Mendes, M.P., Vroegop-Vos, I.A., Caarls, L., Steenbergen, M., Van der Nagel, I.,Wesselink, G.J., Jironkin, A., Talbot, A., Rhodes, J., De Vries, M., Schuurink, R.C., Denby, K., Pieterse, C.M.J., and Van Wees, S.C.M.(2017). Architecture and Dynamics of the Jasmonic Acid Gene Regulatory Network. Plant Cell 29: 2086–2105.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Howe, G.A., Major, I.T., and Koo, A.J. (2018). Modularity in Jasmonate Signaling for Multistress Resilience. Annu. Rev. Plant Biol. 69:387–415.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Kazan, K., and Manners, J.M. (2013). MYC2: the master in action. Mol. Plant 6: 686-703.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Kitaoka, N., Matsubara, T., Sato, M., Takahashi, K., Wakuta, S., Kawaide, H., Matsui, H., Nabeta, K., and Matsuura, H. (2011). ArabidopsisCYP94B3 encodes jasmonyl-L-isoleucine 12-hydroxylase, a key enzyme in the oxidative catabolism of jasmonate. Plant Cell Physiol. 52:1757–1765.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Kloek, A.P., Verbsky, M.L., Sharma, S.B., Schoelz, J.E., Vogel, J., Klessig, D.F., and Kunkel, B.N. (2001). Resistance to Pseudomonassyringae conferred by an Arabidopsis thaliana coronatine-insensitive (coi1) mutation occurs through two distinct mechanisms. PlantCell 26: 509–522.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Koo, A.J., and Howe, G.A. (2012). Catabolism and deactivation of the lipid-derived hormone jasmonoyl-isoleucine. Front. Plant Sci. 3:19.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Koo, A.J., Cooke, T.F., and Howe, G.A. (2011). Cytochrome P450 CYP94B3 mediates catabolism and inactivation of the plant hormonejasmonoyl-L-isoleucine. Proc. Natl. Acad. Sci. USA 108: 9298–9303.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Li, L., Zhao, Y., McCaig, B.C., Wingerd, B.A., Wang, J., Whalon, M.E., Pichersky, E., and Howe, G.A. (2004). The tomato homolog ofCORONATINE-INSENSITIVE1 is required for the maternal control of seed maturation, jasmonate-signaled defense responses, andglandular trichome development. Plant Cell 16: 126–143.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Lian, J., Han, H., Zhao, J., and Li, C. (2018). In-vitro and in-planta Botrytis cinerea inoculation assays for tomato. Bio-protocol 8: e2810.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Lian, T.F., Xu, Y.P., Li, L.F., and Su, X.D. (2017). Crystal structure of tetrameric Arabidopsis MYC2 reveals the mechanism of enhancedinteraction with DNA. Cell Rep. 19: 1334–1342.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Lorenzo, O., Chico, J.M., Sanchez-Serrano, J.J., and Solano, R. (2004). JASMONATE-INSENSITIVE1 encodes a MYC transcription factoressential to discriminate between different jasmonate-regulated defense responses in Arabidopsis. Plant Cell 16: 1938–1950.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Major, I.T., Yoshida, Y., Campos, M.L., Kapali, G., Xin, X.F., Sugimoto, K., de Oliveira Ferreira, D., He, S.Y., and Howe, G.A. (2017).Regulation of growth–defense balance by the JASMONATE ZIM-DOMAIN (JAZ)-MYC transcriptional module. New Phytol. 215: 1533–1547.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Marineau, C., Matton, D.P., and Brisson, N. (1987). Differential accumulation of potato tuber mRNAs during the hypersensitiveresponse induced by arachidonic acid elicitor. Plant Mol. Biol. 9: 335–342.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Matsui, K., Umemura, Y., and Ohme-Takagi, M. (2008). AtMYBL2, a protein with a single MYB domain, acts as a negative regulator ofanthocyanin biosynthesis in Arabidopsis. Plant J. 55: 954–967.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Matton, D.P., and Brisson, N. (1989). Cloning, expression, and sequence conservation of pathogenesis-related gene transcripts ofpotato. Mol. Plant Microbe Interact. 2: 325–331.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Melotto, M., Underwood, W., Koczan, J., Nomura, K., and He, S. Y. (2006). Plant stomata function in innate immunity against bacterialinvasion. Cell 126: 969–980.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Miersch, O., Neumerkel, J., Dippe, M., Stenzel, I., and Wasternack, C. (2008). Hydroxylated jasmonates are commonly occurringmetabolites of jasmonic acid and contribute to a partial switch-off in jasmonate signaling. New Phytol. 177: 114–127.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Moreno, J.E., Shyu, C., Campos, M.L., Patel, L.C., Chung, H.S., Yao, J., He, S.Y., and Howe, G.A. (2013). Negative feedback control ofjasmonate signaling by an alternative splice variant of JAZ10. Plant Physiol. 162: 1006–1017.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Nakagawa, T., Kurose, T., Hino, T., Tanaka, K., Kawamukai, M., Niwa, Y., Toyooka, K., Matsuoka, K., Jinbo, T., and Kimura, T. (2007).Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation.J. Biosci. Bioeng. 104: 34–41.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Nakata, M., Mitsuda, N., Herde, M., Koo, A.J., Moreno, J.E., Suzuki, K., Howe, G.A., and Ohme-Takagi, M. (2013). A bHLH-typetranscription factor, ABA-INDUCIBLE BHLH-TYPE TRANSCRIPTION FACTOR/JA-ASSOCIATED MYC2-LIKE1, acts as a repressor tonegatively regulate jasmonate signaling in arabidopsis. Plant Cell 25: 1641–1656.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Niu, Y., Figueroa, P., and Browse, J. (2011). Characterization of JAZ-interacting bHLH transcription factors that regulate jasmonateresponses in Arabidopsis. J. Exp. Bot. 62: 2143–2154.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Pauwels, L., Barbero, G.F., Geerinck, J., Tilleman, S., Grunewald, W., Perez, A.C., Chico, J.M., Bossche, R.V., Sewell, J., Gil, E., Garcia-Casado, G., Witters, E., Inze, D., Long, J.A., De Jaeger, G., Solano, R., and Goossens, A. (2010). NINJA connects the co-repressorTOPLESS to jasmonate signalling. Nature 464: 788–791.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Pieterse, C.M.J., Van der Does, D., Zamioudis, C., Leon-Reyes, A., and Van Wees, S.C.M.
2012. . Hormonal modulation of plant immunity. Annu. Rev. Cell Dev. Biol. 28: 489– 521. Qi, T., Wang, J., Huang, H., Liu, B., Gao, H., Liu,Y., Song, S., and Xie, D. (2015). Regulation of Jasmonate-Induced Leaf Senescence by Antagonism between bHLH Subgroup IIIe andIIId Factors in Arabidopsis. Plant Cell 27:1634–1649. Ryan, C.A. (2000). The systemin signaling pathway: differential activation of plantdefensive genes. Biochim. Biophys. Acta. 1477: 112–121. Ryan, C.A., and Pearce, G. (1998). Systemin: a polypeptide signal for plantdefensive genes. Annu. Rev. Cell Dev. Biol. 14: 1–17. Sasaki-Sekimoto, Y., Jikumaru, Y., Obayashi, T., Saito, H., Masuda, S., Kamiya, Y.,Ohta, H., and Shirasu, K. (2013). Basic helix-loop-helix transcription factors JASMONATE-ASSOCIATED MYC2-LIKE1 (JAM1), JAM2, andJAM3 are negative regulators of jasmonate responses in Arabidopsis. Plant Physiol. 163: 291–304. Schilmiller, A.L., and Howe, G.A.(2005). Systemic signaling in the wound response. Curr. Opin. Plant Biol. 8: 369–377. Shang, Y., Yan, L., Liu, Z.Q., Cao, Z., Mei, C., Xin,Q., Wu, F.Q., Wang, X.F., Du, S.Y., Jiang, T., Zhang, X.F., Zhao, R., Sun, H.L., Liu, R., Yu, Y.T., and Zhang, D.P. (2010). The Mg-chelataseH subunit of Arabidopsis antagonizes a group of WRKY transcription repressors to relieve ABA-responsive genes of inhibition. Plant
Cell 22: 1909–1935. Sheard, L.B., Tan, X., Mao, H., Withers, J., Ben-Nissan, G., Hinds, T.R., Kobayashi, Y., Hsu, F.F., Sharon, M., Browse,J., He, S.Y., Rizo, J., Howe, G.A., and Zheng, N. (2010). Jasmonate perception by inositol-phosphate-potentiated COI1-JAZ co-receptor.Nature 468, 400–405. Shyu, C., Figueroa, P., Depew, C.L., Cooke, T.F., Sheard, L.B., Moreno, J.E., Katsir, L., Zheng, N., Browse, J., andHowe, G.A. (2012). JAZ8 lacks a canonical degron and has an EAR motif that mediates transcriptional repression of jasmonateresponses in Arabidopsis. Plant Cell 24: 536–550. Song, S., Qi, T., Fan, M., Zhang, X., Gao, H., Huang, H., Wu, D., Guo, H., and Xie, D.(2013). The bHLH subgroup IIId factors negatively regulate jasmonate-mediated plant defense and development. PLoS Genet. 9:e1003653. Spoel, S.H., et al. (2003). NPR1 modulates cross-talk between salicylate- and jasmonate- dependent defense pathwaysthrough a novel function in the cytosol. Plant Cell 15: 760–770. Sun, H., Fan, H., and Ling, H. (2015). Genome-wide identification andcharacterization of the bHLH gene family in tomato. BMC Genomics 16: 9. Sun, J. Q., Jiang, H. L., and Li, C. Y. (2011).Systemin/Jasmonate-mediated systemic defense signaling in tomato. Mol. Plant 4: 607–615. Thines, B., Katsir, L., Melotto, M., Niu, Y.,Mandaokar, A., Liu, G., Nomura, K., He, S.Y., Howe, G.A., and Browse, J. (2007). JAZ repressor proteins are targets of the SCFCOI1complex during jasmonate signalling. Nature 448: 661–665. Thireault, C., Shyu, C., Yoshida, Y., St Aubin, B., Campos, M.L., and Howe,G.A. (2015). Repression of jasmonate signaling by a non-TIFY JAZ protein in Arabidopsis. Plant J. 82: 669–679. VanDoorn, A.,Bonaventure, G., Schmidt, D.D., and Baldwin, I.T. (2011). Regulation of jasmonate metabolism and activation of systemic signaling inSolanum nigrum: COI1 and JAR4 play overlapping yet distinct roles. New Phytol. 190: 640–652. Van der Does, D., Leon-Reyes, A.,Koornneef, A., Van Verk, M.C., Rodenburg, N., Pauwels, L., Goossens, A., Körbes, A.P., Memelink, J., Ritsema, T., Van Wees, S.C.M.,and Pieterse, C.M.J. (2013). Salicylic acid suppresses jasmonic acid signaling downstream of SCFCOI1-JAZ by targeting GCC promotermotifs via transcription factor ORA59. Plant Cell 25: 744–761. Wasternack, C., and Hause, B. (2013). Jasmonates: biosynthesis,perception, signal transduction and action in plant stress response, growth and development. An update to the 2007 review in Annalsof Botany. Ann. Bot. 111: 1021–1058. Xie, D., Feys, B.F., James, S., Nieto-Rostro, M., and Turner, J.G. (1998). COI1: an Arabidopsis generequired for jasmonate-regulated defense and fertility. Science 280: 1091–1094. Xu, L., Liu, F., Lechner, E., Genschik, P., Crosby, W.L.,Ma, H., Peng, W., Huang, D., and Xie, D. (2002). The SCFCOI1 ubiquitin-ligase complexes are required for jasmonate response inArabidopsis. Plant Cell 14: 1919–1935. Yan, L., Zhai, Q., Wei, J., Li, S., Wang, B., Huang, T., Du, M., Sun, J., Kang, L., Li, C.B., and Li, C.(2013). Role of tomato lipoxygenase D in wound-induced jasmonate biosynthesis and plant immunity to insect herbivores. PLoS Geneti.9: e1003964. Yan, Y., Stolz, S., Chetelat, A., Reymond, P., Pagni, M., Dubugnon, L., and Farmer, E.E. (2007). A downstream mediator inthe growth repression limb of the jasmonate pathway. Plant Cell 19: 2470–2483. Zhai, Q., Li, L., An, C., and Li, C. (2017a). ConservedFunction of Mediator in Regulating Nuclear Hormone Receptor Activation between Plants and Animals. Plant Signal. Behav. doi:10.1080/15592324.2017.1403709. Zhai, Q., Yan, C., Li, L., Xie, D., and Li, C. (2017b). Jasmonates. In Hormone Metabolism and Signaling inPlants, J. Li, C. Li, and S.M. Smith, eds (London: ESLEVIER. Zhai, Q., Yan, L., Tan, D., Chen, R., Sun, J., Gao, L., Dong, M.Q., Wang, Y.,and Li, C. (2013). Phosphorylation-coupled proteolysis of the transcription factor MYC2 is important for jasmonate-signaled plantimmunity. PLoS Genet. 9: e1003422. Zhang, F., Ke, J., Zhang, L., Chen, R., Sugimoto, K., Howe, G.A., Xu, H.E., Zhou, M., He, S.Y., andMelcher, K. (2017). Structural insights into alternative splicing-mediated desensitization of jasmonate signaling. Proc. Natl. Acad. Sci.USA 114: 1720–1725. Zhang, F., Yao, J., Ke, J., Zhang, L., Lam, V.Q., Xin, X., Zhou, X., Chen, J., Brunzelle, J., Griffin, P.R., Zhou, M., Xu,H., Melcher, K., and He, S. (2015). Structural basis of JAZ repression of MYC transcription factors in jasmonate signalling. Nature 525:269–273. Zhang, L., Zhang, F., Melotto, M., Yao, J., He, S.Y. (2017) Jasmonate signaling and manipulation by pathogens and insects. JExp Bot 68: 1371–1385. Zhao, Y., Thilmony, R., Bender, C.L., Schaller, A., He, S.Y., and Howe, G.A. (2003). Virulence systems ofPseudomonas syringae pv. tomato promote bacterial speck disease in tomato by targeting the jasmonate signaling pathway. Plant J. 36:485–499.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
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
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