the ring-type e3 ubiquitin ligase jul1 targets the vq-motif … · 2019-03-05 · 95 jav1...

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Short title: E3 ligase JUL1 targets JAV1 in JA signaling 1

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The Ring-type E3 Ubiquitin Ligase JUL1 Targets the VQ-motif Protein JAV1 3

to Coordinate Jasmonate Signaling 4

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Mohamed R. M. Alia1, Takuya Uemuraa1, Abdelaziz Ramadanb, Kyoko 6

Adachia, Keiichirou Nemotob, Akira Nozawab, Ryosuke Hoshinoa, Hiroshi 7

Abec, Tatsuya Sawasakib and Gen-ichiro Arimuraa* 8 aDepartment of Biological Science & Technology, Faculty of Industrial Science & 9

Technology, Tokyo University of Science, Tokyo, Japan 10 bProteo-Science Center, Ehime University, Matsuyama, Japan 11 cExperimental Plant Division, RIKEN BioResource Center, Tsukuba, Japan 12

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Author contributions: MRMA, TU, AR, AN, TS, and GA designed the research. 14

MRMA, TU, AR, KA, KN, AN, RH, and HA performed experiments. MRMA, TU, 15

AR, AN, and GA analyzed the data. MRMA, TU, and GA wrote the paper. 16

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One-sentence summary: The JAV1/JUL1 system acts specifically in coordination 18

of plant defenses without interfering with plant development or growth. 19

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*Corresponding Author: 22

Gen-ichiro Arimura 23

Department of Biological Science & Technology, Faculty of Industrial Science & 24

Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo 25

125-8585, Japan 26

Tel: 81 3 5876 1467 27

Fax: 81 3 5876 1639 28

E-mail: [email protected] 29

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Plant Physiology Preview. Published on March 5, 2019, as DOI:10.1104/pp.18.00715

Copyright 2019 by the American Society of Plant Biologists

https://plantphysiol.orgDownloaded on November 17, 2020. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

https://plantphysiol.org

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1These authors contributed equally to the article. 31



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ABSTRACT 32

Jasmonates regulate plant defense and development. In Arabidopsis (Arabidopsis 33

thaliana), JASMONATE-ASSOCIATED VQ-MOTIF GENE 1 (JAV1/VQ22) is a 34

repressor of jasmonate-mediated defense responses and is degraded through the 35

ubiquitin-26S proteasome system after herbivory. We found that JAV1-36

ASSOCIATED UBIQUITIN LIGASE 1 (JUL1), a RING-type E3 ubiquitin ligase, 37

interacted with JAV1. JUL1 interacted with JAV1 in the nucleus to ubiquitinate 38

JAV1, leading to proteasomal degradation of JAV1. The transcript levels of JUL1 39

and JAV1 were coordinately and positively regulated by the CORONATINE 40

INSENSITIVE1 (COI1)-dependent signaling pathway in the jasmonate signaling 41

network, but in a manner that was not dependent on COI1-mediated signaling upon 42

herbivory by Spodoptera litura. Gain or loss of function of JUL1 modulated the 43

expression levels of the defensin gene PDF1.2 in leaves, conferring on the plants 44

various defense properties against the generalist herbivore S. litura. Because 45

neither the JUL1 mutant nor over-expression lines showed any obvious 46

developmental defects, we concluded that the JAV1/JUL1 system functions as a 47

specific coordinator of reprogramming of plant defense responses. All together, our 48

findings offer insight into the mechanisms by which the JAV1/JUL1 system acts 49

specifically to coordinate plant defense responses without interfering with plant 50

development or growth. 51

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Keywords: Arabidopsis, herbivory, jasmonates, JAV1, JUL1, ring-type E3 ligase 53



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INTRODUCTION 54

Jasmonates, a lipid-derived class of plant hormones, promote gene regulation for 55

defense responses in plants. Jasmonates, including jasmonic acid (JA) and its active 56

derivative jasmonyl-isoleucine (JA-Ile), play a vital role in defending plants against 57

herbivorous insects and necrotrophic pathogens (McConn et al., 1997; Browse, 58

2009; Pieterse et al., 2012; Okada et al., 2015; Chini et al., 2016), and mediate 59

many developmental processes in root growth, stamen development and senescence 60

(Wasternack and Hause, 2013). Following JA signaling via the JA-Ile receptor 61

CORONATINE INSENSITIVE 1 (COI1), jasmonate-ZIM domain proteins (JAZs), 62

act as negative regulators of JA signalling (Chini et al., 2007; Thines et al., 2007). 63

JA-associated degradation of JAZs leads to transcriptional reprogramming of a vast 64

array of genes controlled by factors such as MYC2 transcription factors (TFs), 65

leading to the activation of defense responses and modulation of numerous 66

processes of plant growth and development, e.g., sexual maturation, trichome 67

initiation and root growth (Browse, 2009; Fernández-Calvo et al., 2011; Qi et al., 68

2011; Song et al., 2011; Song et al., 2013; Wasternack and Hause, 2013; Goossens 69

et al., 2017). Activation of JA signaling adversely affects plant growth through a 70

trade-off between JA-induced defense responses and gibberellin (GA)-promoted 71

development of Arabidopsis (Arabidopsis thaliana) plants, in which angiosperm 72

plants prioritize JA-mediated defense over growth (Yang et al., 2012; Guo et al., 73

2018). It appears that JA signaling controls plant growth by suppressing the GA-74

mediated degradation of DELLA, a GA-responsive repressor of TFs (including 75

phytochrome interacting factors (PIFs)) (Yang et al., 2012). Moreover, it appears 76

that the plant growth-defense trade-off is promoted via relief of transcription 77

repression rather than diversion of photoassimilates from growth to defense 78

(Campos et al., 2016). 79

Hu et al. (2013) identified JASMONATE-ASSOCIATED VQ-MOTIF GENE 1 80

(JAV1, also known as VQ22) as a novel repressor of JA signaling in Arabidopsis. 81

JAV1 is a member of a family of plant-specific proteins with the conserved short 82

amino acid sequence motif FxxhVQxhTG, where ‘x’ represents any amino acid and 83

‘h’ represents a hydrophobic residue (Jing and Lin, 2015). This family plays 84



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various roles in plant defense responses, stress tolerance, and growth and 85

development (Jing and Lin, 2015). JAV1 has been shown to specifically down-86

regulate jasmonate-mediated defense responses against a necrotrophic pathogen 87

(Botrytis cinerrea) and an herbivore (Spodoptera exigua larvae), without adversely 88

impacting plant growth or development (Hu et al., 2013; Zhu and Zhu, 2013). 89

Additionally, the JAV1-JAZ8-WRKY51 complex represses JA biosynthesis in 90

healthy plants, but herbivory triggers calmodulin-dependent phosphorylation of 91

JAV1, which leads to JAV1 degradation and consequently to release of WRKY51, 92

thereby activating JA biosynthesis for plant defense (Yan et al., 2018). 93

Like JAZ1 degradation, JAV1 degradation requires JA signaling, which mediates 94

JAV1 degradation through the ubiquitin-26S proteasome system (UPS), and 95

thereby leads to the activation of defense genes, including PDF1.2 (Hu et al., 2013). 96

JAV1 is able to interact with not only WRKY51, but also WRKY28, a positive 97

regulator of PDF1.2 expression. However, the E3 ligase(s) specific to JAV1 98

degradation remain to be identified. 99

Ubiquitination is a post-translational modification process that functions in all 100

eukaryotes, and is achieved by the consecutive action of three enzymes: ubiquitin 101

(Ub)-activating enzyme (E1), Ub-conjugating enzyme (E2), and Ub ligase (E3). 102

The Arabidopsis thaliana genome contains more than 1400 genes encoding E3 103

ubiquitin ligases (Vierstra, 2009). These E3 ubiquitin ligases play critical roles in 104

determining the substrate specificity of UPS for various target proteins, and the 105

large number of E3 ligases implies their specific recognition of target substrates 106

(Sadanandom et al., 2012). E3 ubiquitin ligases are classified into three families: 107

the “homology to E6-AP C-terminus” (HECT) E3 ligases, the “really interesting 108

new gene” (RING) type of E3 ligases, and the U-box family members (Chen and 109

Hellmann, 2013). RING type E3 ligase family members can either ubiquitinate 110

substrates independently or function as part of a multi-subunit complex, which in 111

plants includes Skp1-Cullin-F-box (SCF), VHL-Elongin-CUL2/5, Cullin 3 112

(CUL3)-Bric a brac, Tramtrack, Broad complex/Pox virus and Zinc finger 113

(BTB/POZ), UV-Damaged DNA-Binding protein 1 (CUL4-DDB1) and Anaphase 114

Promoting Complex (APC) as independent complexes (Sadanandom et al., 2012). 115



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There are more than 460 proteins in the RING-type protein family in Arabidopsis 116

(Stone et al., 2005; Lee and Kim, 2011). These E3 ligases act in plant biotic stress 117

tolerance (Marino et al., 2012; Duplan and Rivas, 2014) and play essential roles in 118

the JA signaling pathway (Zhang et al., 2012; Nagels Durand et al., 2016; 119

Miricescu et al., 2018). However, the substrate targets of only a few E3 ligases 120

have been identified. In the current study, we characterized the features of a RING-121

type E3 ligase that interacts with JAV1 in Arabidopsis. This E3 ligase, which we 122

named JUL1 (JAV1-ASSOCIATED UBIQUITIN LIGASE 1), was found to play a 123

primary role in UPS for JAV1 degradation, leading to positive regulation of 124

defense responses. 125

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RESULTS 129

Screening of JAV1-Interacting Ubiquitin Ligases 130

To construct a protein library of putative Arabidopsis RING-type E3 ligases, we 131

prepared 210 cDNA clones from the RAFL library (Ramadan et al., 2015), as 132

shown in Supplemental Table S1. Transcription templates with an N-terminal 133

FLAG-tag sequence were used to synthesize proteins using a cell-free protein 134

synthesis system. To identify characteristic(s) of E3 ligase(s) that interact with 135

JAV1 from the library proteins, we used the AlphaScreen-based detection system. 136

Among the 210 synthesized proteins, JAV1 interacted most strongly with an E3 137

ligase (at5g10650), hereafter designated JUL1 (Fig. 1A). JUL1 has 3 predicted 138

nuclear localization signals and a RING domain at the C-terminus (Fig. 1B). 139

Among the crustacean homologues exhibiting E-value <10-5, none of the proteins 140

whose functions have been predicted or characterized have sequence similarities to 141

JUL1 (Fig. 1C). For instance, the well-characterized E3 ligases RFI2 (Chen and Ni, 142

2006) and AIPR1 (Ryu et al., 2010) did not have similarity to JUL1. Instead, since 143

JUL1 showed high similarity to a predicted E3 ligase (at5g24870) (58% identity) 144

that was not present in our RAFL library, we performed the AlphaScreen assay 145

again for interaction between JAV1 and the JUL1 homologue. However, the JAV1 146

and JUL1 homologues did not show interaction (Supplemental Fig. S1). Likewise, 147

the JAV1 homologue at4g15120 did not interact with JUL1. 148

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JUL1 and JAV1 Interact in the Nucleus 150

JAV1 is a nuclear protein (Hu et al., 2013). We therefore assessed the subcellular 151

localization of JUL1 using a JUL1-GFP (green fluorescent protein) fusion protein 152

expressed in Arabidopsis mesophyll protoplast cells (Fig. 2A). In contrast to the 153

ubiquitous localization of the native GFP protein, which served as a control, the 154

fusion protein showed nuclear localization. 155

Moreover, in order to assess the interaction in vivo between JUL1 and JAV1 156

proteins, a bimolecular fluorescence complementation (BiFC) assay was conducted. 157

JUL1 fused to the N-terminal portion of yellow fluorescent protein (YFP) (JUL1-158

nYFP) and JAV1 fused to the C-terminal portion of YFP (JAV1-cYFP) were 159



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transiently expressed in Arabidopsis mesophyll protoplast cells (Fig. 2B). The 160

fluorescent signals of these recombinant YFP proteins were detected in the nuclei 161

of Arabidopsis protoplast cells only when MG132, a proteasome inhibitor, was 162

applied to the cells (Fig. 2B). However, fluorescence was not detected when JAV1-163



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cYFP was coexpressed with a JUL1-ring domain mutant (JUL1RM) fused to the N-164

terminal portion of YFP. These results indicated that persistent access of JUL1 to 165

JAV1 caused rapid UPS-dependent JAV1 degradation. 166

Moreover, immunoprecipitation assays confirmed that V5-tagged JAV1 interacted 167

with FLAG-tagged JUL1 but not JUL1RM in the presence of MG132 (Fig. 2C). 168

169

Ubiquitin Ligase Activity of JUL1 170

To assess the auto-ubiquitination activity of JUL1 in vitro, biotinylated JUL1 171

protein (Bio-JUL1) was incubated with HA-tagged ubiquitin (HA-Ub), in either the 172

presence or absence of UBCH5b (Fig. 3A). UBCH5b is an E2 ubiquitin ligase that 173

is involved in the polyubiquitination of a suite of E3 ligases, including K48 (David 174

et al., 2011). Immunoprecipitation with anti-biotin and subsequent anti-HA 175

antibody detection revealed that Bio-JUL1 underwent auto-ubiquitination in the 176

presence of UBCH5b. Moreover, when biotinylated JAV1 protein (Bio-JAV1) was 177

incubated with FLAG-tagged ubiquitin (FLAG-Ub), UBCH5b and JUL1, the Bio-178

JAV1 protein was ubiquitinated, as was the positive control, biotinylated P53 179

protein (Bio-P53), when it was incubated with FLAG-Ub, UBCH5b (E2) and 180



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MDM2, a human p53-specific E3 ligase (Fig. 3B). In contrast, when biotinylated 181

Escherichia coli dihydrofolate reductase (Bio-DHFR) was used as substrate, it was 182

not ubiquitinated by either MDM2 or JUL1. 183

JAV1 contains two lysine residues, Lys52 and Lys179. The level of ubiquitination of 184

JAV1 by JUL1 decreased when a JAV1 mutant, in which either Lys52 or Lys179 was 185

replaced by Thr, was used as the substrate in the ubiquitination assay (Fig. 3C). The 186

ubiquitination signal was more markedly decreased when a JAV1 mutant in which 187

both of the Lys residues were replaced with Thr was used as the substrate. 188

Next, in vivo degradation of JAV1 by the JUL1-UPS system was evaluated. FLAG-189

tagged JAV1 protein (JAV1-FLAG) was expressed with V5-tagged JUL1 or 190

JUL1RM (JUL1-V5 or JUL1RM-V5) in WT leaf protoplast cells (Fig. 4A). 191

Irrespective of the presence or absence of MG132, the levels of the transiently 192

expressed JAV1 proteins were well maintained when JUL1RM-V5 was co-193

expressed. In contrast, JAV1 protein levels decreased when JUL1-V5 was co-194

expressed in the absence of MG132. In the presence of MG132, JAV1 protein 195

levels tended to recover slightly in comparison to those in the absence of MG132. 196

Such JAV1 degradation in the absence of MG132 was also observed in WT and 197



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coi1-1 protoplast cells (Supplemental Fig. S2), indicating that COI1-dependent JA 198

signaling is not required for JUL1 ubiquitination activity towards JAV1. 199

In additional assays, JAV1-FLAG was expressed in leaf protoplast cells prepared 200

from Arabidopsis WT and the jul1 mutant line (see below) (Fig. 4B). Irrespective 201

of the presence or absence of MG132, the JAV1 protein levels were elevated in the 202

jul1 cells compared the WT cells (Fig. 4B). Moreover, JAV1-FLAG was expressed 203

in leaf protoplast cells prepared from Arabidopsis WT and a transgenic line 204

overexpressing JUL1 (JUL1-OX1; see below) (Fig. 4C). In comparison to the level 205

in WT, the JAV1 protein level was decreased in the JUL1-OX1 cells incubated in 206

the absence of MG132, but not in those incubated in the presence of MG132. These 207

findings confirmed that JUL1 contributes to JAV1 degradation through the UPS in 208

leaf cells. 209



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Finally, we evaluated the level of the JAV1 protein in Arabidopsis T87 cells 210

constitutively overexpressing JAV1, with or without JUL1, using an anti-JAV1 211

antibody, and found a decreased level of JAV1 protein in the cells expressing both 212

JAV1 and JUL1 in comparison to the cells expressing JAV1 without JUL1 (Fig. 5A). 213

In contrast, the JAV1 protein level was elevated in the leaves of the jul1 mutant, in 214

comparison to that in WT leaves (Fig. 5B). 215

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Regulation of Transcript Levels of JUL1 and JAV1 via the COI1 System 217

Transcript levels of JUL1, JAV1 and PDF1.2 (JA-associated defense gene) were 218

elevated in WT leaves treated with methyl jasmonate (MeJA) or damaged by larvae 219

of the generalist herbivore S. litura (Fig. 6). In contrast, the coronatine-insensitive 220

1 mutant (coi1-1) showed defective elevation of these transcript levels in leaves 221

after MeJA treatment. Upon herbivory, however, the transcript levels of JUL1 and 222

JAV1 in coi1-1 leaves showed no significant defect of elevation in comparison to 223

the levels in WT leaves, whereas the PDF1.2 transcript level was significantly 224

lower in coi1-1 leaves in comparison to that in WT leaves. 225



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Intriguingly, it should also be noted that the transcript level of JAV1 was higher in 226

unstressed coi1-1 leaves than in WT leaves. 227

228

Phenotypic Characterization of JUL1 Mutants and JUL1-overexpressing 229

Lines 230

To assess the function of JUL1 in plants, we generated two JUL1-overexpressing 231

lines (JUL1-OX1 and -OX2), exhibiting 96-fold and 101-fold increased levels of 232

JUL1 expression, respectively, compared to the level in WT leaves (Fig. 7D). 233

These transgenic lines, as well as the jul1 and jav1 knockdown mutants 234

(Supplemental Fig. S3), showed no obvious differences in plant growth, 235

development or morphology, including root growth, vegetative stage development, 236

or seed number, with or without MeJA treatment, in comparison to WT plants (Fig. 237

7A, Fig. 7B, Supplemental Fig. S4 and Supplemental Fig. S5). 238

The defense ability of these lines was changed compared to that of WT plants. JA-239

inducible PDF1.2 was highly expressed in the JUL1-OX line and jav1 mutant 240

leaves (Fig. 7C). Another JA-inducible gene, VSP2, was highly expressed in jav1 241

mutant leaves but only tended to be slightly more highly expressed in the JUL1-OX 242



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line leaves compared to WT leaves (Fig. 7C). Conversely, the salicylate (JA-243

antagonist)-inducible PR1 (Spoel et al., 2003; Beckers and Spoel, 2006) was highly 244

expressed only in jul1 mutant leaves (Fig. 7C). 245



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Next, to confirm the plant defense properties of those lines against larvae of the 246

generalist herbivore Spodoptera litura, we assessed the growth of larvae on the 247

potted plants. The larvae on JUL1-OX and jav1 plants exhibited lower weight gain 248

for up to 3 days after the larvae were released, compared to that exhibited by the 249



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larvae on WT plants (Fig. 7D). In turn, the larvae on jul1 mutant plants exhibited 250

higher weight gain after 2 days and 3 days, compared to the weight gains exhibited 251

by the larvae on WT plants. The feeding behaviour observed at 1 day showed a 252

similar trend to the leaf damage area when JUL1-OX and jav1 leaves were 253

challenged with S. litura larvae for 2 h (Supplemental Fig. S6). Moreover, similar 254

trends were observed for the leaf lesion sizes after infection with the necrotic 255

pathogen Botrytis cinerea for 3 days (Fig. 7E). 256

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DISCUSSION 259

Arabidopsis JAV1 has been reported to be a negative regulator of JA-dependent 260

defense responses that acts to coordinate the primary COI1-JAZ signaling pathway 261

(Hu et al., 2013; Zhu and Zhu, 2013). JAV1 can form a complex with JAZ8 and 262

TFs, including WRKY51, in order to suppress JA biosynthesis genes under normal 263

conditions. However, in response to herbivory, all of these molecules are 264

dissociated from the complex, JAV1 is then degraded via the UPS and the free 265

WRKY51 is able to access JA biosynthesis-related genes (Yan et al., 2018). In the 266

current study, JUL1 was found to have the characteristics of a RING-type E3 267

ubiquitin ligase that catalyzes ubiquitination of JAV1 as a substrate, thereby 268

leading to the degradation of JAV1 via the UPS (Fig. 4 and Fig. 5). According to 269

data from (Hu et al., 2013) and our preliminary AlphaScreen-based assays for 270

interactions between JAV1 and 566 TF proteins (Supplemental Table. S2), it 271

appears that the JAV1/JUL1 system is able to control a suite of WRKY family (e.g., 272

WRKY28, WRKY48 and WRKY51) members involved in promoting the 273

transcription of defense-related genes, including PDF1.2 (Zhu and Zhu, 2013). 274

Such a regulatory system involving JAV1 is in accord with the basic nature of the 275

VQ family, whose members specifically interact with the WRKY domain of group 276

I and IIc WRKY transcription factors (Cheng et al., 2012). Although Arabidopsis 277

SIB1 (VQ23)/SIB2 (VQ16) and VQ10 have been shown to contribute to activation 278

of WRKY33 and WRKY8 in plant defense responses (Lai et al., 2011; Chen et al., 279

2018), respectively, an array of other Arabidopsis VQ proteins, including not only 280

JAV1 but also VQ21/MKS1, VQ12 and VQ29, act as negative regulators of plant 281

defense responses (Andreasson et al., 2005; Petersen et al., 2010; Hu et al., 2013; 282

Wang et al., 2015). 283

WRKYs function as positive (WRKY28) or negative regulators (WRKY48 and 284

WRKY51) of defense genes (Xing et al., 2008; Gao et al., 2011; Wu et al., 2011). 285

Based on these facts, and given our finding here that both loss of JAV1 function 286

and a gain of JUL1 function result in enhanced expression of PDF1.2 in leaves (Fig. 287

7D), we conclude that the orchestration of WRKY-based transcriptional machinery, 288

according to the level of JAV1-based defense, is ultimately related to the positive 289



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regulation of the defense response. However, this should be flexible to enable 290

adjustment to the circumstances of the plants, for example, whether they are being 291

stressed, in which case the transcriptional modes can be drastically reprogrammed 292

in plant cells. 293

It also appeared that JUL1 and JAV1 are constitutively expressed during several 294

stages of Arabidopsis development, based on the microarray database 295

GENEVESTIGATOR (Zimmermann et al., 2004) (Supplemental Fig. S7). 296

Remarkably, in the leaf senescence stage, transcripts of JUL1 and JAV1 showed 297

high and low levels, respectively, indicating that JUL1 might play a role in 298

senescence. Moreover, both JUL1 and JAV1 transcript levels were found to be 299

attenuated in coi1-1 leaves as compared to WT leaves in response to MeJA (Fig. 6), 300

indicating that those genes are positively regulated by a COI1-dependent signaling 301

cascade. Intriguingly, when plants were challenged with herbivores, however, 302

neither JUL1 nor JAV1 transcripts appeared to be induced in a COI1-dependent 303

manner. Taking all these results together, we suggest that COI1-independent 304

factor(s) may strongly contribute to the regulation of JUL1 and JAV1 transcript 305

levels in response to herbivory. Interestingly, the level of JAV1 transcripts was 306

found to be negatively regulated by the COI1-dependent signaling cascade (Fig. 6). 307

Our observation of the negative impact of COI1 on JAV1 expression is in accord 308

with data in the microarray database GENEVESTIGATOR (Zimmermann et al., 309

2004) (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE21762) showing 310

that JAV1 transcript levels are higher in coi1-2 leaves than in WT leaves under 311

normal growth conditions (Supplemental Fig. S8). However, what this regulation 312

implies for the JAV1/JUL1 module remains to be clarified. 313

RING-type E3 ligases play vital roles in the regulation of physiological and 314

morphological processes in plants in response to various internal and external 315

stimuli (Duplan and Rivas, 2014; Shabek and Zheng, 2014; Ding et al., 2015; 316

Lazaro et al., 2015; Zhang et al., 2015). Nonetheless, to the best of our knowledge, 317

RGLGs 3/4 and KEG are the only JA-associated RING-type E3 ligases reported 318

thus far (Zhang et al., 2012; Pauwels et al., 2015). However, it was recently 319

reported that RGLG3 and RGLG4 can ubiquitinate GRXS17, and thereby control 320



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the redox state of proteins, and that the RGLG system is not strongly relevant to the 321

JA response module, in contrast to the findings in the previous report (Nagels 322

Durand et al., 2016). The findings of the current study clearly showed that JUL1 is 323

a novel RING-type E3 involved in JA signaling. Notably, some RING-type E3 324

ligases have been shown to have a diverse range of substrate targets. One of the 325

most striking examples is CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), 326

which is involved in a wide range of plant growth and developmental processes 327

(Kim et al., 2017). It thus remains possible that JUL1 has multiple substrate targets 328

involved in an array of functions in plant defense, stress tolerance and other 329

physiological processes, and this issue should be assessed in the future. 330

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332

MATERIALS AND METHODS 333

Plant Materials 334

Arabidopsis thaliana ecotype Col-0, its mutants (SALK_146039C [jav1], 335

SALK_101465C [jul1]), and transgenic plants overexpressing JUL1 (see below) 336

were grown in plastic pots for 4-5 weeks in a growth chamber at 22 ± 1°C with a 337

photoperiod of 14 h (80 µE m–2 s–1). The coi1-1 seeds were germinated on 1/2 338

Murashige and Skoog (MS) medium supplemented with 0.5% (w/v) sucrose, 0.8% 339

(w/v) agar and 50 µM MeJA (Wako Pure Chemical Industrials, Ltd., Osaka, Japan) 340

to screen the individuals showing normal root growth for 2 weeks (Xie et al., 1998). 341

Homozygous SALK lines were confirmed according to the method described in 342

http://signal.salk.edu/tdnaprimers.2.html. 343

344

Primers 345

Primers used for all the polymerase chain reactions in this study are listed in 346

Supplemental Table S3. 347

348

Phylogeny Analysis 349

The phylogenetic tree was generated using the “one click” mode of Phylogeny.fr 350

pipeline (http://www.phylogeny.fr/; Dereeper et al., (2008)). 351



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352

Generation of Transgenic Arabidopsis Plants 353

The full-length coding region of JUL1 (at5g10650) was inserted into binary 354

plasmid pMDC32 (2x Cauliflower mosaic virus 35S promoter [35SP]::the Gateway 355

cassette [GW] region::nopaline synthase terminator [NOST]) using the Gateway 356

cloning system (Thermo Fisher Scientific, Waltham, MA, USA). The resulting 357

plasmid, pMDC32-JUL1, was transformed into Agrobacterium tumefaciens strain 358

EHA105 by electroporation. Col-0 wild-type (WT) Arabidopsis plants that had 359

been grown for 6-7 weeks were transformed via the floral-dip transformation 360

method (Clough and Bent, 1998). Transgenic T1 seeds from each transformant were 361

tested for germination on 1/2 Murashige and Skoog (MS) medium supplemented 362

with 30 mg L–1 hygromycin. T2 seeds were harvested from each individual. T2 363

plants that showed a segregation ratio of about 3:1 were tested for hygromycin-364

resistance again. T3 homozygous plant lines were used for further analyses. 365

366

Chemical and Herbivore Treatment 367

For chemical treatment, potted Arabidopsis plants were evenly sprayed with 1 mL 368

of aqueous solution (0.5% ethanol) of MeJA (0.2 mM) and incubated in a growth 369

chamber at 22 ± 1°C (14 h photoperiod at a light intensity of 80 µE m–2 s–1) for 4 h. 370

Spedoptera litura were reared on an artificial diet (Insecta LF, Nihon Nosan Nogyo 371

Ltd, Tokyo, Japan) in the laboratory at 24 ± 1°C with a photoperiod of 16 h. For 372

herbivore treatment, four third-instar larvae were released on potted Arabidopsis 373

plants in a growth chamber at 22 ± 1°C (14 h photoperiod at a light intensity of 80 374

µE m–2 s–1) for 24 h. 375

376

Cell-free Protein Synthesis 377

Cell-free protein synthesis was carried out according to the method described 378

previously (Ramadan et al., 2015; Takahashi et al., 2016). Primers used are listed in 379

Supplemental Table S3. 380

381

AlphaScreen System 382



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We performed the AlphaScreen-based protein-protein interaction assay to screen 383

the interaction of 210 Arabidopsis RING-type E3 ligases (Ramadan et al., 2015) 384

and 566 Arabidopsis TFs (Nozawa et al., 2009) with JAV1. Assays for interaction 385

between JUL1 and the JAV1 homologue (at4g15120) or between the JUL1 386

homologue (at5g24870) and JAV1 were also carried out. Assays were performed in 387

10 µL of 100 mM Tris-HCl (pH 8.0), 0.1% Tween 20, 1 mg/mL BSA 388

(AlphaScreen buffer), and 1 μL of biotinylated JAV1 (or JAV1 homologue) and 389

FLAG-adapted E3 ligase (either JUL1 or a JUL1 homologue) or TFs per well at 390

25°C for 1 h in a 384-well OptiPlate (PerkinElmer, Waltham, MA, USA). In accord 391

with the AlphaScreen IgG (Protein A) detection kit (PerkinElmer) instruction 392

manual, 10 μL of detection mixture containing AlphaScreen buffer, 5 μg/mL anti-393

FLAG M2 antibody (Sigma-Aldrich, St. Louis, MO, USA), 0.1 μL of streptavidin-394

coated donor beads, and 0.1 μL of Protein A-coated acceptor beads were added to 395

each well of a 384-well OptiPlate, followed by an additional incubation at 25°C for 396

1 h. Luminescence was analyzed using the AlphaScreen detection program 397

(PerkinElmer). All data represent the average of three independent experiments, 398

and the background was controlled using a dihydrofolate reductase (DHFR) from 399

Escherichia coli. 400

401

Mutagenesis 402

JAV1 point mutations (K52 and K179) were obtained by inverse polymerase chain 403

reaction (PCR) using In-Fusion® HD Cloning Plus (Takara Bio Inc., Kusatsu, 404

Japan), primers (JAV1-K1-fw, JAV1-K1-rv; JAV2-K1-fw and JAV1-K2-rv 405

[Supplemental Table S3]), and JAV1 cDNA as template. 406

Similarly, JUL1 protein mutations were obtained at C493, H495 and H498 of the 407

RING domain using primers (JUL1-RING-M-F, JUL1-RING-M-R [Supplemental 408

Table S3]) and JUL1 cDNA as template, to obtain the JUL1 RING domain mutant 409

(JUL1RM) based on Stone et al. (2005). 410

411

Subcellular Localization and BiFC Assays in Protoplast Cells 412



22

The full-length coding regions of JUL1 and JAV1 (except for the stop codons) were 413

cloned into Gateway destination plasmid pGWB451 (35SP::GW::GFP) (Nakagawa 414

et al., 2007), resulting in the construction of the plasmid pGW451-JUL1-GFP. This 415

plasmid, plus pGW452 (35SP::GFP::GW), which served as a control, were 416

transiently transformed into Arabidopsis mesophyll protoplasts as previously 417

described (Yoo et al., 2007; Wu et al., 2009). The transformed protoplasts were 418

incubated for 18-20 h in the dark at 25°C, and GFP fluorescence was observed in 419

the protoplasts under a BZ-X700 fluorescence microscope (Keyence Co., Osaka, 420

Japan). 421

The full-length coding regions of JUL1, JUL1RM (see below) and JAV1 were 422

inserted (separately) into the Gateway destination vector 35SPΩ-GW-V5-nYFP, 423

along with the N-terminal fragment of YFP, and 35SPΩ-GW-V5-cYFP with the C-424

terminal fragment of YFP, respectively (Nemoto et al., 2017). Ten micrograms of 425

each plasmid were co-transformed into Arabidopsis protoplast cells. The 426

transformed protoplasts were incubated for 14 h in the dark at 25°C, and then 50 427

µM MG132 (Wako) in 0.1% DMSO or 0.1% DMSO solution (control) was 428

applied. Following an additional 6-h incubation in the dark at 25°C, the protoplast 429

cells were incubated in WI solution (5 mM MES (pH 5.7), 0.4 M mannitol and 20 430

mM KCl) with 1 µg mL-1 DAPI (Sigma-Aldrich) for 5 min before observation. The 431

resultant YFP fluorescence was observed in the protoplasts under a fluorescence 432

microscope (BZ-X700). 433

434

In vitro Ubiquitination Assay 435

The ubiquitination assays for JUL1 were carried out as previously described 436

(Ramadan et al., 2015). The assays were performed in a 10 μL reaction mixture 437

containing 1 μL of recombinant MDM2 (AF527840, mouse double minute 2 438

homolog) or JUL1 protein, 1 μL of recombinant substrate (P53 439

[AB082923, p53 tumor suppressor targeted by MDM2 E3 ubiquitin ligase], JAV1 440

or JAV1 mutant [see above]), 1.2 μM UbcH5b (Enzo Life Sciences, Farmingdale, 441

NY, USA), 20 mM Tris-HCl pH 7.5, 0.2 mM DTT, 5 mM MgCl2, 10 μM zinc 442

acetate, 3 mM ATP, 1 mg/mL bovine serum albumin (BSA), and 400 nM FLAG-443



23

tagged or HA-tagged human ubiquitin (FLAG-Ub or HA-Ub) (Boston Biochem, 444

Cambridge, MA, USA) at 37°C for 3 h. The reactions were terminated by addition 445

of 5 μL of 3x SDS sample buffer and boiling for 5 min, and the products were then 446

analysed by 10% SDS-PAGE followed by immunoblotting (see below) using anti-447

FLAG-HRP (Sigma-Aldrich) or anti-HA-HRP (clone 3F10) antibody (Roche 448

Applied Science, Indianapolis, IN, USA). 449

450

In vivo Protein Degradation Assay 451

Polyethylene glycol-mediated DNA transfection of Arabidopsis mesophyll 452

protoplasts from WT, jul1, and JUL1-OX1 backgrounds was performed as 453

previously described (Yoo et al., 2007; Wu et al., 2009) with slight modifications. 454

The full-length coding regions of JAV1 (except for the stop codons) were cloned 455

into Gateway destination plasmid p35SΩ::GW::3×FLAG::NOST (Nemoto et al., 456

2017), resulting in the construction of the plasmid p35SΩ::JAV1 tagged with 457

3×FLAG at the C-terminus (JAV1-FLAG)::NOST. Likewise, the full-length coding 458

regions of JUL1 and JUL1RM (except for the stop codons) were cloned into 459

Gateway destination plasmid p35SΩ::GW::V5::NOST (Nemoto et al., 2017), 460

resulting in the construction of the plasmid p35SΩ::JUL1 tagged with V5 at the C-461

terminus (JUL1-V5)::NOST and p35SΩ::JUL1RM tagged with V5 at the C-terminus 462

(JUL1RM-V5)::NOST, respectively. Thirty micrograms of JAV1-FLAG vector, 463

along with 6 µg of pGWB452 (35SP::GFP::GW) vector (Nemoto et al., 2017), 464

were transformed into 2 × 105 WT, jul1, or JUL1-OX1 cells. Otherwise, thirty 465

micrograms of JAV1-FLAG vector and 20 µg of JUL1-V5 vector, along with 6 µg 466

of pGWB452 (35SP::GFP::GW) vector, were transformed into 2 × 105 WT cells. 467

Following incubation overnight in the dark at 22 ± 1°C, 6 hours before harvest, 468

protoplasts were treated with 50 μM MG132 in 0.1% DMSO or with 0.1% DMSO 469

(control). Total proteins were extracted from the protoplasts with 3× sample buffer. 470

JAV1-FLAG protein was analyzed by immunoblotting using anti-FLAG HRP (see 471

below). 472

For co-immunoprecipitation assays, 30 µg of JAV1-V5 vector (p35SΩ::JAV1 473

tagged with V5 at the C-terminus) and 20 µg of JUL1-FLAG vector (p35SΩ::JUL1 474



24

tagged with 3×FLAG at the C-terminus) were transformed into 1× 106 WT cells, 475

the protoplasts were incubated overnight and treated with 50 μM MG132 for 6 476

hours before being homogenized with IP lysis/wash buffer (Thermo Fisher 477

Scientific) supplemented with 50 μM MG132 and 1% Protease Inhibitor Cocktail 478

(Sigma-Aldrich). Immunoprecipitation of JUL1-FLAG protein from the protoplast 479

lysate was performed using a Pierce Crosslink Magnetic IP/Co-IP Kit (Thermo 480

Fisher Scientific) and anti-FLAG (DYKDDDDK) monoclonal primary antibody 481

(1E6; Wako) following the manufacturer’s protocol. After the beads were washed 482

twice with IP lysis/wash buffer, the immunoprecipitated proteins were eluted with 483

50 μL elution buffer. JAV1-V5 protein was analyzed by immunoblotting using 484

anti-V5 HRP (see below). RbcL was visualized as a loading control using Ponceau-485

S staining solution. 486

487

Generation and Analysis of Transgenic Arabidopsis T87 Cultured Cells 488

Expressing JAV1-GFP and JUL1 489

The full-length coding region of JAV1 (at3g22160) was inserted into binary 490

plasmid pGWB5 (35SP::GW:NOST) to construct pGWB5-JAV1. Additionally, the 491

expression cassette of JUL1 in pMDC32-JUL1 was subcloned into pGWB5-JAV1 492

to construct pGWB5-JAV1-JUL1. pGWB5-JAV1 and pGWB5-JAV1-JUL1 were 493

transformed into A. tumefaciens strain GV3103 by electroporation. Arabidopsis 494

T87 cells were transformed and cultured in JPL medium supplemented with 10 mg 495

L–1 hygromycin according to the method described previously (Ogawa et al., 2008). 496

Total proteins were extracted from the transgenic cells with extraction buffer (50 497

mM HEPES (pH 7.4), 5 mM ethylenediaminetetraacetic acid (EDTA), 5 mM 498

ethylene glycol tetraacetic acid (EGTA), 50 mM β-glycerophosphate, 10 mM 499

Na3VO4, 10 mM NaF, 2 mM dithiothreitol (DTT), and 1％ Triton-X100). JAV1-500

GFP protein was analyzed by immunoblotting using anti-JAV1 antibody (see 501

below). Data were normalized by transcript levels of the hygromycin 502

phosphotransferase (HPT) gene. 503

504

Immunoblot Analysis 505



25

Ten- to fifteen-microliter samples of the boiled proteins in SDS sample buffer were 506

subjected to 10% SDS-PAGE and the proteins were transferred to a PVDF 507

membrane (ATTO, Tokyo, Japan) using the Trans-Blot®Turbo™ Blotting System 508

(Bio-Rad). Immunoblotting was carried out with anti-FLAG-HRP-conjugated 509

antibody (Wako Pure Chemical Industries, Ltd, Osaka, Japan), anti-V5-HRP-510

conjugated antibody (Wako Pure Chemical Industries, Ltd, Osaka, Japan) or anti-511

GFP chicken IgY primary antibody (GeneTex Inc., San Antonio, TX, USA) and 512

then with anti-chicken IgY-HRP-conjugated secondary antibody (Promega, 513

Madison, WI, USA). Detection was performed using Immobilon Western 514

Chemiluminescent HRP Substrate (ECL, Millipore, Billerica, MA, USA), 515

according to the manufacturer’s procedure. The signals were visualized using 516

ImageQuant LAS 4000 mini (GE Healthcare, Buckinghamshire, UK). 517

Immunoblotting for JAV1 was carried out with anti-JAV1 antibody obtained by 518

immunizing rabbits with a peptide antigen (the peptide sequence: 519

CWQYNFQPHAPLQPPQR; Cosmo Bio, Tokyo Japan). Unlabeled anti-rabbit IgG 520

(#3678, Cell Signaling Technology, Danvers, MA) and HRP-linked anti-mouse 521

IgG (#7076) antibodies were used as the secondary and tertiary antibodies, 522

respectively. The membranes were soaked with SuperSignal West Femto 523

Maximum Sensitivity Substrate (Thermo Fisher Scientific, Waltham, MA, USA), 524

and the signals were detected with an ImageQuant LAS-4000 imaging system (GE 525

Healthcare, Buckinghamshire, UK). 526

527

RNA Extraction, cDNA Synthesis and Reverse Transcription Quantitative 528

Polymerase Chain Reaction (RT-qPCR) 529

Approximately 100 mg of leaf tissues or 1 ml of cell culture was homogenized in 530

liquid nitrogen, and total RNA was isolated and purified using Sepasol®-RNA I 531

Super G (Nacalai Tesque, Kyoto, Japan) following the manufacturer’s protocol. 532

First-strand cDNA synthesis and qPCR were performed according to the method 533

described previously (Uemura et al., 2018). Relative transcript abundances were 534

determined after normalization of raw signals with the transcript abundance of the 535

housekeeping gene ACT8 (At1g49240). We did not use samples and data when 536



26

sufficient amounts of RNA were not isolated from leaves or when abnormal 537

quantification cycle (Cq) values for the actin gene were obtained. Finally, replicate 538

analyses were conducted with 5 or 6 independent samples, except for the data in 539

Fig. 5B (3 independent samples). 540

541

Root Length Measurement 542

Plant seedlings (14 days old) were grown on 1/2 MS medium with or without 50 543

µM MeJA. Root lengths were determined using ImageJ software (version 1.50i; 544

Schneider et al. (2012)). 545

546

Herbivore Assay 547

We performed assays to assess the growth of S. litura larvae at 22 ± 1°C (14 h 548

photoperiod at 80 µE m–2 s–1). Second-instar larvae were initially weighed (1.6-2.1 549

mg), and each larva was released onto a potted plant for 3 days. The net body 550

weight that S. litura larvae gained each of the following three days was determined. 551

When a larva died or was lost during the assay, we excluded that sample, and final 552

replicate analyses were conducted with 12-15 independent samples. 553

For leaf-consumption assays, five second-instar larvae, starved overnight, were 554

released onto the shoots of potted plants for 2 h. The leaves were then scanned, and 555

the total leaf area and the consumed leaf area were determined using ImageJ. 556

Replicate analyses were conducted with 8-10 independent samples. 557

558

Pathogen Infection Assay 559

B. cinerea (strain IuRy-1) was grown on potato sucrose agar under black light at 560

25˚C for 1 week to promote the formation of conidia. For inoculation, 10 µL of 561

conidial spore suspension (2x105 conidia mL-1, in 2.5% glucose) was spotted onto 562

the left and right sides of the upper surface of a leaf of the potted plants. The 563

inoculated plants were covered with a plastic sheet to enable the maintenance of 564

humidity. The plants were placed in a growth chamber at 24 ± 1°C for 3 days. The 565

leaf lesion area was analyzed using ImageJ. 566

567



27

Statistical Analyses 568

We performed t-tests for pairwise analysis and one-way ANOVA with Holm's 569

sequential Bonferroni post-hoc test or Tukey’s HSD test using the program 570

(http://astatsa.com/OneWay_Anova_with_TukeyHSD/) for comparing multiple 571

samples. 572

573

Accession Numbers 574

The sequence and detailed information for the genes in this study can be found in 575

the ABRC database under the accession numbers listed in Supplemental Table S3. 576

577

Supplemental Data 578

Supplemental Figure S1. Interactions between JUL1 and JAV1 homologue 579

(at4g15120) or between JAV1 and JUL1 homologue (at5g24870). 580

Supplemental Figure S2. COI1-independent protein levels of JAV1 regulated by 581

the JUL1-26S proteasome system. 582

Supplemental Figure S3. Molecular analysis of T-DNA insertion mutants and 583

gene expression profiling in the mutants. 584

Supplemental Figure S4. Root length of the seedlings of wild type (WT), JUL1-585

overexpressing lines (JUL1-OX1 and LUL1-OX2), and jul1 and jav1 mutants with 586

or without methyl jasmonate (MeJA) treatment. 587

Supplemental Figure S5. Leaf-consumption of S. litura larvae. 588

Supplemental Figure S6. Transition of transcript levels of JUL1 and JAV1 during 589

the Arabidopsis life cycle, based on the GENEVESTIGATOR database. 590

Supplemental Figure S7. Transcript levels of JAV1 in Arabidopsis wild-type 591

(WT) and coi1-2 mutant leaves 4 h after application of methyl jasmonate (MeJA), 592

based on the GENEVESTIGATOR database. 593

Supplemental Table S1. AlphaScreen-based binding intensity to JAV1. 594

Supplemental Table S2. AlphaScreen-based binding strength of transcription 595

factors to JAV1. 596

Supplemental Table S3. Primers. 597

598



28

599

ACKNOWLEDGMENTS 600

The plasmids pGWB451 and pGW452 were kindly provided by Dr. Tsuyoshi 601

Nakagawa (Shimane Univ.). This work was financially supported in part by a JSPS 602

KAKENHI to GA (16K07407) and MEXT Grants-in-Aid for Scientific Research 603

on Innovative Areas to GA (18H04630 and 18H04786). 604

605

606

Figure legends 607

Figure 1. Screening and structure of JUL1. Luminescence-based protein-protein 608

interaction detection (AlphaScreen) was used for high throughput screening of 210 609

FLAG-conjugated putative Arabidopsis E3 ligases for interaction with biotinylated-610

JAV1 (Bio-JAV1). The data for the top 34 E3 ligases that interacted strongly with 611

Bio-JAV1 are shown (see Supplemental Table S2 for the full set of data). 612

Luminescence intensities were normalized by those of Escherichia coli 613

dihydrofolate reductase. Data represent the mean of three replicates. (B) JUL1 614

protein is schematically represented with the nuclear localization signals and RING 615

domain and lysine residues. (C) Phylogenetic tree inferred from deduced amino 616

acid sequences of JUL11 and other proteins from several plant taxa including 617

Arabidopsis. BRAD, Brachypodium distachyon; GLYMA, Glycine max; OS, Oryza 618

sativa; PHYPADRAFT, Physcomitrella patens; POPTR, Populus trichocarpa; SB, 619

Sorghum bicolor; SOLYC, Solanum lycopersicum; VIT, Vitis vinifera. 620

621

Figure 2. Subcellular localization and interaction of JAV1 and JUL1. (A) The 622

plasmid containing the CaMV 35S promoter (35SP)::GFP or JUL1 fused to GFP 623

(JUL1-GFP), was used to transform Arabidopsis leaf protoplast cells. (B) 624

Bimolecular fluorescence complementation (BiFC) analysis of JUL1-JAV1 625

interactions. The plasmid constructs of 35SP::JUL1 or the JUL1-ring domain 626

mutant (JUL1RM) fused to the N-terminal fragment of YFP (JUL1-nYFP or 627

JUL1RM-nYFP) and JAV1 fused to the C-terminal fragment of YFP (JAV1-cYFP), 628

as shown in the top panel, were co-transformed into the Arabidopsis leaf protoplast 629



29

cells. The protoplasts were treated with a potent proteasome inhibitor, MG132 630

(+MG132), or not treated with the inhibitor (-MG132). V5, V5 epitope tag. (C) The 631

plasmid constructs of 35SPΩ::V5-tagged JAV1 (JAV1-V5) and FLAG-tagged 632

JUL1 (JUL1-FLAG) or JUL1RM (JUL1RM-FLAG) were co-transformed into 633

Arabidopsis leaf protoplast cells, and the cells were then treated with MG132 634

during the incubation. The extracted JAV1 and JUL1 or JUL1RM proteins (input) 635

were immunoprecipitated with anti-FLAG antibody, subjected to SDS-PAGE, and 636

detected using anti-V5. Three independent experiments (Expt. 1, 2 and 3) were 637

performed. IB, Immuno-blotting; IP, Immunoprecipitation. 638

639

Figure 3. In vitro ubiquitination and degradation of JUL1 on JAV1 as substrate. (A) 640

Auto-ubiquitination activity of JUL1. Biotinylated JUL1 proteins (Bio-JUL1) were 641

incubated with HA-tagged ubiquitin (HA-Ub) in the presence or absence of 642

UBCH5b (E2). Biotinylated human dihydrofolate reductase (Bio-DHFR) served as 643

a negative control. (B) Ubiquitination activity of JUL1 on JAV1 substrate. JUL1 or 644

MDM2 (a human p53-specific E3 ligase), which served as a positive control, was 645

incubated in the presence or absence of the FLAG-tagged ubiquitin (FLAG-Ub), 646

UBCH5b (E2) and biotinylated substrate proteins (Bio-p53 or Bio-JAV1). Bio-647

DHFR served as a substrate-negative control. For these assays, the incubated 648

reaction mixtures were immunoprecipitated using streptavidin Alexa Fluor 647 649

(anti-Biotin), subjected to SDS-PAGE, and probed with anti-HA-HRP (HA-Ub) or 650

anti-FLAG-HRP (FLAG-Ub) antibody. IB, Immuno-blotting; IP, 651

immunoprecipitation. (c) Wild type of the biotinylated JAV1 protein (Bio-JAV1) 652

and its mutants, in which either the Lys52 or Lys179 residue or both Lys residues in 653

the VQ motif domain were replaced with Thr (Bio-JAV1K52T, Bio-JAV1K179T, and 654

Bio-JAV1K52T-K179T), were incubated with UBCH5b (E2) or FLAG-tagged JUL1 655

(FLAG-JUL1). Assays in the absence of either UBCH5b or Bio-JAV1 served as 656

controls. The incubation mixtures were immunoprecipitated using streptavidin 657

Alexa Fluor 647 (anti-Biotin), subjected to SDS-PAGE, and probed using anti-HA-658

HRP (HA-Ub) antibody. IB, Immunoblotting; IP, immunoprecipitation. 659

660



30

Figure 4. In vivo protein levels of JAV1 regulated by the JUL1-26S proteasome 661

system. (A) The plasmid constructs of 35SPΩ:: FLAG-tagged JAV1 (JAV1-FLAG) 662

and V5-tagged JUL1 or JUL1-ring domain mutant (JUL1RM) (JUL1-V5 or 663

JUL1RM-V5) were co-transformed with 35SP::GFP into Arabidopsis leaf protoplast 664

cells, and the cells were then treated with MG132 during the incubation. (B, C) 665

35SPΩ::JAV1-FLAG and 35SP::GFP were co-transformed into protoplast cells 666

prepared from wild type (WT) and either the jul1 mutant (jul1-1) (B) or transgenic 667

plants overexpressing JUL1 (JUL1-OX1) (C), with (+) or without (-) MG132 668

treatment. Total proteins extracted from the cells were subjected to SDS-PAGE and 669

immunoblotted with FLAG-HRP and GFP antibodies. The means and standard 670

errors from three independent experiments (Expt. 1, 2 and 3) are presented in the 671

right panels. Means indicated by different small letters are significantly different, 672

based on a one-way ANOVA with post-hoc Tukey’s HSD (P < 0.05). 673

674

Figure 5. In vivo protein levels of JAV1 in Arabidopsis cells overexpressing JAV1 675

and Arabidopsis leaves. JAV1 protein levels in Arabidopsis T87 cells 676

overexpressing JAV1 with or without JUL1 (A) and leaves of Arabidopsis wild-677

type (WT) and jul1 mutant plants (B) are shown. Total proteins extracted from 678

mature leaves were subjected to SDS-PAGE and immunoblotted with an anti-JAV1 679

antibody. For (A), data were normalized by transcript levels of the transgene of the 680

hygromycin phosphotransferase (HPT) gene. The means and standard errors from 681

three independent experiments are presented at the bottom. Means indicated by 682

different small letters are significantly different, based on a t-test (P < 0.05). 683

684

Figure 6. Regulation of transcript levels of JUL1, JAV1, and PDF1.2. Transcript 685

levels of JUL1, JAV1, and PDF1.2 in leaves of Arabidopsis wild-type (WT) and 686

coi1-1 mutant plants in response to methyl jasmonate (MeJA) treatment for 4 h and 687

damage with Spodoptera litura larvae for 24 h. Transcript levels of genes were 688

measured by RT-qPCR and normalized by those of ACT8. Data represent the mean 689

and standard error (n = 5-7). Asterisks indicate significant differences between WT 690

and coi1-1 mutant plants (B), based on a t-test (**: P < 0.01, *: P < 0.05). 691



31

692

Figure 7. The JAV1/JUL1 system is responsible for defense responses but not plant 693

growth or development properties. The phenotype of rosette plants (A) and 694

seedlings with (+) or without (-) methyl jasmonate (MeJA) treatment (B) of wild 695

type (WT), JUL1-overexpressing lines (JUL1-OX1 and LUL1-OX2), and jul1 and 696

jav1 mutants. (C) Transcript levels of JUL1, PDF1.2, VSP2, and PATHOGENESIS-697

-RELATED 1 (PR1) in the leaves of WT, JUL-OX and mutant lines (n = 5-6). (D) 698

Spodoptera litura second-instar larvae were put onto potted plants. The net body 699

weight that larvae gained during 3 days after they had been placed on the plants 700

was determined (n = 11-13). (E) Likewise, the fungus Botrytis cinerea was 701

inoculated onto leaves of potted plants, and areas of induced lesions were 702

determined after 3 days (n = 10). For C, D, and E, data represent the mean and 703

standard error. Data marked with an asterisk are significantly different from those 704

of WT, based on a one-way ANOVA with Holm's sequential Bonferroni post-hoc 705

test (**: P < 0.01, *: P < 0.05). 706

707

708



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