The OsGSK2 Kinase Integrates Brassinosteroid and Jasmonic Acid Signaling … · 2020-06-25 · Plant Cell Advance Publication. Published on June 25, 2020, ... INTRODUCTION. 60. Brassinosteroids

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RESEARCH ARTICLE 1

The OsGSK2 Kinase Integrates Brassinosteroid and Jasmonic 2

Acid Signaling by Interacting with OsJAZ4 3

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Yuqing He1,2,6, Gaojie Hong2,6, Hehong Zhang1, Xiaoxiang Tan1, Lulu Li1, Yaze 5

Kong2, Tian Sang3, Kaili Xie1, Jia Wei2, Junmin Li1, Fei Yan1, Pengcheng Wang3, 6

Hongning Tong4, Chengcai Chu5, Jianping Chen1,2* and Zongtao Sun1* 7

8 1State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and 9

Safety of Agro-products, Key Laboratory of Biotechnology in Plant Protection of 10

Ministry of Agriculture and Zhejiang Province, Institute of Plant Virology, Ningbo 11

University, Ningbo 315211, China. 12 2Institute of Virology and Biotechnology, Zhejiang Academy of Agricultural Sciences, 13

Hangzhou 310021, China. 14 3Shanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular 15

Plant Sciences, Chinese Academy of Sciences, Shanghai 200032, China. 16 4Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 17

100081, China. 18 5Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 19

Beijing 100101, China. 20 6These authors contributed equally to this work. 21

*Corresponding authors. [email protected] (ZS) or [email protected]

(JC) 23

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Short title: OsGSK2 triggers JA signaling via OsJAZ4 25

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One-sentence summary: OsGSK2 directly interacts with and destabilizes OsJAZ4 to 27

activate JA-mediated defense signaling. 28

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The author responsible for distribution of materials integral to the findings presented in 30

this article in accordance with the policy described in the Instructions for Authors 31

(www.plantcell.org) is: Zongtao Sun ([email protected]). 32

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ABSTRACT 34

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The crosstalk between brassinosteroid (BR) and jasmonic acid (JA) signaling 36

is crucial for plant growth and defense responses. However, the detailed 37

interplay between BRs and JA remains obscure. Here, we found that the rice 38

(Oryza sativa) Glycogen synthase kinase 3 (GSK3)-like kinase OsGSK2, a 39

conserved kinase serving as a key suppressor of BR signaling, enhanced 40

antiviral defense and the JA response. We identified a member of the 41

Plant Cell Advance Publication. Published on June 25, 2020, doi:10.1105/tpc.19.00499

©2020 American Society of Plant Biologists. All Rights Reserved

mailto:[email protected]

mailto:[email protected]

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JASMONATE ZIM-domain (JAZ) family, OsJAZ4, as a OsGSK2 substrate and 42

confirmed that OsGSK2 interacted with and phosphorylated OsJAZ4. We 43

demonstrated that OsGSK2 disrupted the OsJAZ4-OsNINJA complex and 44

OsJAZ4-OsJAZ11 dimerization by competitively binding to the ZIM domain, 45

perhaps helping to facilitate the degradation of OsJAZ4 via the 26S 46

proteasome pathway. We also showed that OsJAZ4 negatively modulated JA 47

signaling and antiviral defense and that the BR pathway was involved in 48

modulating the stability of OsJAZ4 protein in an OsCORONATINE 49

INSENSITIVE 1-dependent manner. Collectively, these results suggest that 50

OsGSK2 enhances plant antiviral defenses by activating JA signaling as it 51

directly interacts with, phosphorylates, and destabilizes OsJAZ4. Thus, our 52

findings provide a clear link between BR and JA signaling. 53

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KEY WORDS 55

GSK3-like kinase, OsJAZ, Brassinosteroids, Jasmonic acid, Rice 56

black-streaked dwarf virus 57

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INTRODUCTION 59

Brassinosteroids (BRs), a class of plant-specific steroidal hormones, play vital 60

roles in various developmental and physiological processes of plants (Clouse 61

and Sasse, 1998; Bishop and Koncz, 2002; Kim and Wang, 2010; Clouse, 62

2011). When the receptor-like kinase BRASSINOSTEROID INSENSITIVE1 63

(BRI1) perceives BRs at the plasma membrane, it activates a signal 64

transduction cascade leading to the transcriptional regulation of BR- 65

responsive genes (Kim and Wang, 2010; Wang et al., 2012). Glycogen 66

synthase kinase 3 (GSK3)-like kinases serves as key negative regulators of 67

BR signaling (Li and Nam, 2002). In Arabidopsis thaliana, 68

BRASSINOSTEROID INSENSITIVE2 (BIN2), a GSK3-like kinase, 69

phosphorylates and destabilizes the two homologous transcription factors 70

BRASSINAZOLE RESISTANT1 (BZR1) and BZR2/BES1, thereby blocking BR 71

signaling (He et al., 2002; Li and Nam, 2002; Yin et al., 2002; Kim et al., 2009). 72

In rice, OsGSK2 (also named OsSK22, OsGSK7), the homolog of BIN2, also 73

negatively mediates BR signaling through OsBZR1 (Tong et al., 2012a), 74

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together with other transcription factors such as DWARF AND 75

LOW-TILLERING (OsDLT) (Tong et al., 2012a), LEAF and TILLER ANGLE 76

INCREASED CONTROLLER (OsLIC) (Zhang et al., 2012), OVATE FAMILY 77

PROTEIN 8 (OsOFP8) (Yang et al., 2016), and REDUCED LEAF 78

ANGLE1/SMALL ORGAN SIZE1(RLA1/SMOS1) (Qiao et al., 2017). GSK3-like 79

kinases are involved in multiple developmental and physiological processes 80

(reviewed in (Saidi et al., 2012; Youn and Kim, 2015; Tong and Chu, 2018)) 81

and also mediate the crosstalk between the auxin (Vert et al., 2008; Cho et al., 82

2014), abscisic acid (ABA) (Cai et al., 2014), gibberellin (GA)(Guo et al., 2018), 83

jasmonic acid (JA) (Gan et al., 2015) and BR pathways. 84

JA and its derivatives, such as MeJA and JA-IIe, not only regulate plant 85

growth and development, but also promote plant defense against insect attack 86

and pathogen infection (Wasternack and Feussner, 2018; Chini et al., 2016; 87

Goossens et al., 2016; Howe et al., 2018; Yan et al., 2018). JA and MeJA 88

treatments induce JA-responses through their conversion to JA-Ile 89

(Wasternack and Feussner, 2018). The JA signaling pathway has been well 90

elucidated in recent years (Gfeller et al., 2010; Howe et al., 2018). The key 91

suppressors in JA signaling are JASMONATE-ZIM DOMAIN (JAZ) proteins 92

(Chini et al., 2007; Thines et al., 2007; Pauwels and Goossens, 2011). In the 93

absence of JA-IIe, JAZ proteins, together with NOVEL INTERACTOR OF JAZ 94

(NINJA) and TOPLESS, bind to and inhibit transcription factors that promote 95

the expression of JA-responsive genes (Pauwels et al., 2010). In contrast, 96

when JA is present, high levels of JA-Ile lead to binding of its receptor COI1 to 97

JAZ proteins, resulting in SCFCOI1-dependent ubiquitination and degradation of 98

JAZ proteins through the 26S proteasome, which in turns activates JA 99

signaling (Chini et al., 2007; Thines et al., 2007; Sheard et al., 2010). Thus, 100

fast turn-over of JAZ proteins holds the key to JA signal output (Gfeller et al., 101

2010; Wasternack and Feussner, 2018; Mao et al., 2017; Chen et al., 2019). 102

Rice black-streaked dwarf virus (RBSDV) is a double-stranded RNA virus, 103

belonging to the genus Fijivirus within the family Reoviridae (Shikata and 104

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Kitagawa, 1977; Mertens, 2004). RBSDV is propagatively and persistently 105

transmitted to rice, maize (Zea mays), barley (Hordeum vulgare) and wheat 106

(Triticum sp.) via the small brown planthopper (Laodelphax striatellus, SBPH) 107

(Uyeda et al., 1995; Wei and Li, 2016). RBSDV infection causes acute growth 108

abnormalities (particularly severe dwarfism) in plants, and results in serious 109

yield losses in cereal crops (Shikata and Kitagawa, 1977; Wu et al., 2013). We 110

have previously shown that JA, BR, and ABA signaling play different roles in 111

the defense response against RBSDV infection (He et al., 2017; Xie et al., 112

2018; Zhang et al., 2019). While the BR pathway mediates susceptibility to 113

RBSDV infection, the JA pathway plays a positive role in rice defense against 114

viral infection (He et al., 2017). Recently, several studies have implicated 115

GSK3-like kinases in the plant immune system (Karlova et al., 2006; Piroux et 116

al., 2007; Millslujan et al., 2015; Mei et al., 2018; Qiu et al., 2018), but the 117

detailed mechanism of GSK3-like kinase-mediated antiviral defense remains 118

unclear in rice. 119

In this study, RBSDV inoculation and primary root inhibition assays using 120

Go (transgenic plants overexpressing OsGSK2) (Tong et al., 2012a) and Gi 121

(OsGSK2 RNAi transgenic plants) lines (Tong et al., 2012a) indicated that 122

OsGSK2 acts as a positive regulator in JA signaling and antiviral defense. We 123

demonstrate that OsGSK2 physically interacts with and phosphorylates 124

OsJAZ4. We provide evidence that OsGSK2 destabilized OsJAZ4 via 125

disrupting the OsJAZ4-OsNINJA complex and OsJAZ4-OsJAZ11 dimerization. 126

In addition, our results reveal that OsJAZ4 negatively regulates JA signaling 127

and antiviral defense. Therefore, we propose that OsGSK2 activates JA 128

signaling by facilitating the degradation of OsJAZ4. 129

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RESULTS 131

OsGSK2 positively regulates the JA response and antiviral defenses. 132

Our previous results showed that BR signaling promoted rice susceptibility to 133

RBSDV infection (He et al., 2017; Zhang et al., 2019). Here, we found the 134

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levels of OsGSK2 protein in RBSDV-infected plants at 30 day post infection 135

(dpi) were higher than those in virus-free rice plants (Supplemental Figure 1A), 136

suggesting a role for OsGSK2 in rice during RBSDV infection. When infested 137

with RBSDV-carrying SBPH, infected Go plants displayed less severe 138

symptoms and less stunting whereas infected Gi plants had more severe 139

symptoms compared with RBSDV-infected wild-type Oryza sativa spp japonica 140

cultivar Zhonghua 11 (Zh11) plants (Figure 1A). About 75% of the control 141

wild-type Zh11 plants became infected while the Go plants (overexpressing 142

OsGSK2) had fewer infected plants (about 43%) and the Gi (OsGSK2 RNAi) 143

plants had more (about 93%) at 30 dpi (Figure 1B). Consistent with the 144

symptoms, RBSDV coat protein (CP) RNA and protein levels were much lower 145

in infected Go plants, but dramatically more in Gi plants than that in Zh11 146

plants (Figure 1C and Supplemental Figure 1B). Evaluation of SBPH 147

resistance, as previously described (He et al., 2017), showed that Go, Gi and 148

Zh11 plants were similarly susceptible to SBPH (Supplemental Figure 1C). 149

Thus, RBSDV resistance conferred by OsGSK2 is independent of SBPH 150

resistance. 151

Specifically blocking GSK3-like kinase activity can attenuate JA signaling 152

in rice, implying its potential role as a link between BR and JA signaling (Gan et 153

al., 2015). Thus, we tested whether OsGSK2 enhanced plant resistance to 154

RBSDV by activating JA signaling. The transcript levels of JA biosynthetic and 155

signaling genes were greatly elevated in Go plants, but lower in Gi plants 156

compared with those in wild-type Zh11 plants (Figure 1D). 157

Quantification of hormone contents revealed that the production of JA was 158

consistently induced in the Go plants but suppressed in the Gi plants. 159

Moreover, significant upregulation of JA-IIe was observed in Go plants relative 160

to that in Zh11 plants (Figure 1E and Supplemental Figure 2), suggesting 161

activation of the JA pathway by OsGSK2. In addition, the inhibitory effect of 162

methyl jasmonate (MeJA) on root growth was significantly enhanced in Go 163

plants but suppressed in Gi plants in comparison with the wild-type Zh11 164

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plants (Figures 1F and G), indicating that overexpression of OsGSK2 165

enhanced rice root sensitivity to JA signaling. These data together suggest a 166

direct involvement of OsGSK2 in regulating both the JA pathway and RBSDV 167

resistance. 168

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OsGSK2 interacts with and phosphorylates OsJAZ4. 170

To explore the role of OsGSK2 in JA signaling in detail, we performed a yeast 171

two-hybrid (Y2H) screen assay using OsGSK2 bait and a normalized rice 172

cDNA prey library. Interestingly, one of the interactors obtained was OsJAZ4, a 173

JASMONATE ZIM-domain (JAZ) family protein (Figure 2A). Further Y2H 174

experiments showed that OsGSK2 interacted with another rice OsJAZ4 175

homolog, OsJAZ3 (Figure 2A and Supplemental Figure 3A). OsJAZ4 also 176

interacted with other OsGSK2 rice homologs, OsGSK3, OsGSK5 and 177

OsGSK7 (Figure 2B and Supplemental Figure 3B). Pull-down assays 178

demonstrated that GST-OsGSK2 could directly interact with His-OsJAZ4 in 179

vitro (Figure 2C and Supplemental Figure 4). 180

Subcellular localization analysis revealed that OsGSK2-GFP and 181

OsJAZ4-mCherry co-localized in the cytoplasm and nucleus of Nicotiana 182

benthamiana epidermal cells when these fusion proteins were co-expressed 183

under strong CaMV 35S promoter (Supplemental Figure 5). Bimolecular 184

fluorescence complementation (BiFC) experiments showed that co-expression 185

of either cYFP-OsGSK2 /OsJAZ4-nYFP or cYFP-OsJAZ4/OsGSK2-nYFP but 186

not cYFP-OsGSK2/OsJAZ11-nYFP or cYFP-OsJAZ11/OsGSK2-nYFP 187

resulted in strong fluorescence signals (Figure 2D and Supplemental Figure 6). 188

To further verify these results, we performed in vivo co-immunoprecipitation 189

(Co-IP) assays using N. benthamiana leaves transformed with HA-OsGSK2 190

and OsJAZ4-myc/OsJAZ11-myc, and found that HA-OsGSK2 and 191

OsJAZ4-myc specifically co-precipitated with one another, but not with 192

OsJAZ11-myc (Figure 2E). Together, these results demonstrated that OsGSK2 193

interacts with OsJAZ4 both in vitro and in vivo. 194

As a kinase, OsGSK2 can phosphorylate most of the proteins with which it 195

interacts (Youn and Kim, 2015). To investigate whether OsJAZ4 is 196

phosphorylated in vivo, we overexpressed OsJAZ4-MYC in stably transformed 197

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wild-type Zh11 plants. We then used anti-myc beads to immunoprecipitate 198

OsJAZ4-myc protein from OsJAZ4-MYC plants, and treated the IP product 199

with calf intestinal alkaline phosphatase (CIP). To avoid protein degradation, 200

we added MG132 and a cocktail of proteinase inhibitors to the protein extracts. 201

The phosphorylated OsJAZ4-myc (OsJAZ4-myc-P) was detected in phos-tag 202

SDS-PAGE using an anti-myc antibody, and it disappeared after CIP treatment 203

(Figure 3A), indicating that the band corresponded to phosphorylated 204

OsJAZ4-myc. 205

To investigate the potential phosphorylation sites of OsJAZ4, the 206

recombinant His-OsJAZ4 was incubated with the recombinant GST-GSK2 in 207

an in vitro kinase assay buffer, separated by SDS-PAGE electrophoresis, and 208

then subjected to liquid chromatography-tandem mass spectrometry analysis. 209

As a result, eight potential phosphorylation sites were identified: Ser-100, 210

Ser-133, Ser-134, Ser-147, Ser-148, Ser-252, Ser-267 and Ser-364, (Figure 211

3B, Supplemental Figures 7 and 8). We then constructed mutated forms of 212

OsJAZ4 with alterations in these sites (S100A, S133A, S134A, S147A, S148A, 213

S252A, S267A and S364A) and named it OsJAZ4Δ8. Y2H assays showed that 214

the physical interaction of OsJAZ4-OsGSK2 was not impaired among these 215

site mutants (Supplemental Figure 9). When transiently co-expressed with 216

HA-OsGSK2, but not with HA-empty vector, or HA-OsGSK2K92R, kinase-dead 217

mutant Of OsGSK2 (Sun et al., 2018), in N. benthamiana leaves, two bands of 218

OsJAZ4-myc were detected from myc-beads immunoprecipitated products, 219

and the upper band could be eluted by CIP treatment (Figure 3C), showing the 220

phosphorylation of OsJAZ4 by occurs OsGSK2 in vivo. Although OsJAZ4Δ8 221

was still phosphorylated by OsGSK2, the phosphorylation level of OsJAZ4Δ8 222

mutants was significantly reduced, indicating that these sites are likely the 223

major ones being phosphorylated by OsGSK2 kinase. These results therefore 224

demonstrate that OsGSK2 directly phosphorylates OsJAZ4. 225

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OsGSK2 promotes the degradation OsJAZ4. 227

GSK3-like kinases usually regulate both the activity and stability of their 228

substrates by direct phosphorylation (Saidi et al., 2012; Youn and Kim, 2015; 229

Tong and Chu, 2018). We therefore investigated whether OsGSK2 affected the 230

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stability of OsJAZ4. When transiently co-expressed with HA-OsGSK2 in N. 231

benthamiana leaves, the accumulation of OsJAZ4-myc at 48 h was less than 232

that when co-expressed with either HA-empty vector or HA-OsGSK2K92R 233

(Supplemental Figures 10A-D). Similarly, we found that OsJAZ4 degraded 234

much faster in the extracts with GST-OsGSK2 than GST or GST-OsGSK2K92R 235

(Figures 4A, B and Supplemental Figure 11) using a cell-free protein 236

degradation system (Qiao et al., 2017). However, accumulation of the mutant 237

OsJAZ4Δ8-myc was not affected when it was transiently co-expressed with 238

either HA-OsGSK2 or the HA-empty vector (Supplemental Figures 10E and F). 239

Using the cell free degradation system, we found that the degradation rate of 240

the recombinant protein His-OsJAZ4Δ8 was much slower than that of 241

His-OsJAZ4, and similar to the control, which contained 50 μM proteasome 242

inhibitor MG132 (Figures 4C and D). Thus, we concluded that phosphorylation 243

by OsGSK2 probably helped to accelerate OsJAZ4 degradation. 244

To test this hypothesis, N. benthamiana leaves transiently co-expressing 245

HA-OsGSK2 and OsJAZ4-myc were treated with Bikinin, a specific inhibitor of 246

the kinase activity of GSK3-like kinases (Rozhon et al., 2014). As expected, 247

Bikinin treatment inhibited OsGSK2-induced degradation of OsJAZ4, 248

indicating that OsJAZ4 is stable in its unphosphorylated form (supplemental 249

Figures 10G and H). The degradation of OsJAZ4 induced by OsGSK2 was 250

also suppressed by the proteasome inhibitor MG132, which suggested that the 251

26S proteasome pathway may be involved in OsGSK2-mediated OsJAZ4 252

degradation (Supplemental Figures 10I and J). Interestingly, the levels of 253

OsJAZ4 protein were obviously lower in the Go plants, but markedly more in 254

the Gi plants, than those in Zh11 plants (Figures 4E, F and Supplemental 255

Figure 11), indicating a negative role for OsGSK2 in OsJAZ4 accumulation in 256

vivo. These data together suggested that accumulation of OsGSK2 decreased 257

levels of OsJAZ4 protein. 258

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OsGSK2 affects the dimerization of OsJAZ4-OsJAZ11. 260

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We next used a domain deletion assay to define the domains involved in the 261

OsGSK2-OsJAZ4 interaction. Based on the conserved N-terminal domain, 262

C-terminal JA-associated (Jas) domain and the ZIM domain found in all JAZ 263

proteins (Pauwels and Goossens, 2011; Wasternack and Feussner, 2018), we 264

constructed and examined the ability of various deletion mutants of OsJAZ4 265

(Figure 5A, the upper schematic diagrams) to interact with OsGSK2. The 266

results showed that the ZIM domain of OsJZA4 was responsible for binding to 267

OsGSK2 (Figure 5A). Previous reports showed that the conserved ZIM domain 268

is responsible for forming homo- and hetero-dimers among JAZ proteins (Chini 269

et al., 2007; Chini et al., 2009; Chung and Howe, 2009). We therefore 270

conducted a Y2H assay using OsJAZ4 as bait and found that OsJAZ4 271

interacted with OsJAZ9, OsJAZ11 and OsJAZ12 (Figure 5B), consistent with 272

previous reports of dimerization within a subset of OsJAZs (Yamada et al., 273

2012; Wu et al., 2015). 274

We then investigated whether OsGSK2 affected the interaction between 275

OsJAZs and OsJAZ4. In a semi-pull-down assay, GST-OsGSK2 attenuated 276

the interaction between OsJAZ4-myc and His-OsJZA11 (Figure 5C). When 277

transiently co-expressed with OsJAZ11-myc in N. benthamiana leaves, the 278

level of OsJAZ4-myc increased, suggesting that dimerization may contribute to 279

the stability of JAZ proteins (Figure 5D). Although the amount of OsJAZ11-myc 280

was not affected by HA-OsGSK2 (Supplemental Figure 12A and B), the 281

amounts of both OsJAZ11-myc and OsJAZ4-myc were lower in the 282

OsJAZ11-myc/OsJAZ4-myc/HA-OsGSK2 combination than in either 283

OsJAZ11-myc/OsJAZ4-myc/HA or OsJAZ11-myc/OsJAZ4-myc/HA- 284

OsGSK2K92R (Figure 5D, Supplemental Figures 12C-F). Interestingly, the 285

levels of OsJAZ11 protein were obviously less in the Go plants, but markedly 286

more in the Gi plants, than in Zh11 plants (Figure 5E and Supplemental Figure 287

11). These results demonstrated that OsGSK2 could compete with OsJAZ11 288

for binding to OsJAZ4 to dissociate the OsJAZ4-OsJAZ11 complex, resulting in 289

suppression of OsJAZs accumulation. 290

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OsGSK2 and OsNINJA compete for binding to OsJAZ4 292

JAZ proteins are suppressors of JA signaling (Chini et al., 2007; Thines et al., 293

2007). In the domain deletion experiment, we demonstrated that the Jas 294

domain of OsJAZ4 is necessary for binding to OsCOI1b in a coronatine (COR, 295

a JA-IIe analogue)-dependent manner, and that the ZIM domain of OsJAZ4 is 296

responsible for interacting with OsNINJA (Figure 6A). These results are 297

consistent with previous reports (Katsir et al., 2008; Chung and Howe, 2009; 298

Miersch, 2009; Sheard et al., 2010), and show that OsJAZ4 is involved in JA 299

signaling. JAZs and NINJA are the corepressors in JA signing, and dissociation 300

of JAZ-NINJA complexes results in degradation of JAZ proteins (Pauwels et al., 301

2010; Wasternack and Feussner, 2018). Since the ZIM domain was also 302

responsible for the OsGSK2-OsJAZ4 interaction (Figure 5A), we investigated 303

whether OsGSK2 affected the interaction between OsJAZ4 and OsNINJA. An 304

in vitro pull-down assay showed that the interaction of OsJAZ4-myc and 305

His-OsNINJA was impaired by an increased amount of GST-OsGSK2 (Figure 306

6B). This result demonstrated that OsGSK2 and OsNINJA compete for binding 307

to OsJAZ4. 308

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OsJAZ4 suppresses JA signaling and antiviral defense. 310

To further test the function of OsJAZ4 in JA signaling, we generated OsJAZ4 311

overexpression (OsJAZ4-OE) and RNA interference (OsJAZ4-RNAi) 312

transgenic rice plants in the O. sativa L. japonica Nipponbare (NIP) 313

background, and confirmed the expected effects on the expression of OsJAZ4 314

by RT-qPCR (Figure 7A). Notably, the transcripts of JA-responsive genes, 315

except HYDROPEROXIDE LYASE (OsHPL3, encoding a competitor of allene 316

oxide synthase AOS for the same substrate) (Tong et al., 2012b), were 317

significantly suppressed in OsJAZ4-OE lines (#1 and #3) but elevated in 318

OsJAZ4-RNAi lines (#14 and #18), compared with wild-type NIP (Figure 7A). 319

When treated with MeJA, the root length reduced more slowly in OsJAZ4-OE 320

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lines (#1 and #3) but faster in OsJAZ4-RNAi lines (#14 and #18) than in NIP 321

(Figures 7B and C), suggesting that OsJAZ4 serves as a suppressor of JA 322

signaling in rice. 323

The expression of OsJAZ4 in RBSDV-infected leaves at 30 dpi was 324

decreased relative to that in uninfected controls (Supplemental Figures 13A 325

and B), indicating that OsJAZ4 was involved in regulating rice immunity. 326

Therefore, the sensitivity of OsJAZ4 transgenic plants to RBSDV infection was 327

assessed. OsJAZ4-OE lines were more susceptible to RBSDV, while 328

OsJAZ4-RNAi lines were more resistant than the NIP control (Figures 6D-F 329

and Supplemental Figure 13C). The susceptibility to RBSDV infection 330

promoted by OsJAZ4 was independent of any effect on SBPH resistance 331

(Supplemental Figure 14). These results together suggest that OsJAZ4 acts as 332

an important repressor of JA signaling and plays a negative role in rice antiviral 333

defense. 334

335

The effect of the OsGSK2-OsJAZ4 interaction on JA and BR signaling 336

crosstalk 337

To further investigate the role of the OsGSK2-OsJAZ4 interaction in JA and BR 338

signaling, we first treated coi1-13 (OsCOI1 knock-down mutant; Yang et al., 339

2012) and NIP control seedlings with brassinolide (BL), which inactivates and 340

degrades OsGSK2 (Kim et al., 2009), and Bikinin, respectively. Treatment of 341

rice seedlings with either BL or Bikinin significantly increased the accumulation 342

of OsJAZ4 protein (Figure 8A and B). However, the BR- and Bikinin-mediated 343

stabilization of OsJAZ4 was inhibited in coi1-13 mutants (Figure 8C and D), 344

indicating involvement of OsCOI1 in BR signaling-mediated OsJAZ4 stability. 345

The chemical Bikinin can specifically inhibit the activity of GSK3-like 346

kinases including BIN2 and OsGSK2, resulting in a BR signaling defect (De 347

Rybe et al., 2009). Thus, we used Bikinin treatment to analyze the function of 348

OsGSK2 in OsJAZ4-RNAi plants. The root sensitivity of MeJA-treated 349

OsJAZ4-RNAi lines was similar in the presence or absence of Bikinin (Figure 350

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8E). These results indicated that suppression of OsGSK2 did not affect JA 351

sensitivity in OsJAZ4-RNAi plants. Together, these data suggest that OsJAZ4 352

is required for OsGSK2-mediated JA signaling and that it acts downstream of 353

OsGSK2. 354

In addition, the BR sensitivity, as shown by the lamina joint assay, was 355

enhanced in OsJAZ4-OE plants but suppressed in OsJAZ4-RNAi lines 356

compared with that in wild-type NIP controls (Figure 8F and G). Consistent 357

with previous reports that the JA pathway was antagonized by the BR pathway 358

(Ren et al., 2010; Kim et al., 2013; Nahar et al., 2013; Gan et al., 2015; He et 359

al., 2017), our results confirmed that OsJAZ4 could enhance BR responses by 360

inhibiting JA signaling. 361

362

DISCUSSION 363

Plant growth and defense responses are coordinated by the interplay among 364

various phytohormones, such as BRs and JA (Kim and Wang, 2010; 365

Vidhyasekaran, 2015). BRs and JA are growth-promoting and defense-related 366

hormones, respectively, (Vidhyasekaran, 2015) and there is extensive 367

crosstalk between the BR and JA signaling pathways during both plant growth 368

and defense responses (Ren et al., 2010; Gan et al., 2015). In Arabidopsis, BR 369

suppresses the inhibition of root elongation by JA, whereas a defect in BR 370

biosynthesis increases sensitivity to the JA response and reduces the negative 371

effect of BR signaling on JA-inhibitory root growth (Ren et al., 2010). In rice, 372

MeJA inhibits the BR-induced increase in lamina joint inclination (Gan et al., 373

2015). Interactions between BR and JA are also involved in modulating plant 374

immunity (Kim et al., 2013; Nahar et al., 2013; He et al., 2017). The mutant 375

gulliver3-D overproduces BR and has reduced sensitivity to MeJA, and JA 376

interrupts BR signaling by repressing DWF4 expression upon Pseudomonas 377

syringae infection (Kim et al., 2013). In rice roots, BR and JA are antagonistic 378

and physiological BR levels suppress the JA-induced resistance to the 379

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root-knot nematode Meloidogyne graminicola (Nahar et al., 2013). Here, we 380

found that the Go plants overexpressing OsGSK2 with its upregulated JA 381

pathway were more resistant to RBSDV infection while the OsGSK2 RNAi Gi 382

plants with suppressed JA pathway were more susceptible compared with the 383

wild-type Zh11 plants. This indicates that the JA pathway is involved in 384

OsGSK2-mediated rice defense (Figure 1 and Supplemental Figure 1). We 385

then demonstrated that OsGSK2 could interact with and phosphorylate 386

OsJAZ4 protein (Figure 2 and 3). We also showed that OsGSK2 disrupted the 387

OsJAZ4-OsNINJA corepressor and OsJAZ4-OsJAZ11 complex via the ZIM 388

domain and that these dual effects of OsGSK2 on OsJAZ4 helped to promote 389

degradation of OsJAZ4 by the 26S proteasome (Figures 4-6). It cannot be 390

ruled out that altered JA content in OsGSK2 overexpressing and RNAi plants 391

may affect the levels of OsJAZ proteins. We further confirmed that OsJAZ4 392

suppressed JA signaling and antiviral defense (Figure 7). The BR- and 393

Bikinin-treatment assays demonstrated that OsCOI1 was involved in BR 394

signaling-mediated OsJAZ4 stability (Figures 8). In addition, the MeJA 395

hypersensitivity in OsJAZ4-RNAi lines were not enhanced by exogenous 396

Bikinin (Figure 8), suggesting that OsGSK2-mediated JA signaling may 397

depend on OsJAZ4. Although the levels of OsGSK2 protein were not altered in 398

OsJAZ4 mutants (Supplemental Figure 15), the BR sensitivity analysis in 399

OsJAZ4 mutants demonstrated that suppression of JA signaling by OsJAZ4 400

may enhance the BR-induced lamina joint inclination (Figures 8). Hence, these 401

results suggested that OsJAZ4-mediated BR sensitivity might be via the 402

downstream signaling pathway of OsGSK2. Our previous study showed that 403

JA mediated resistance and that BR mediated susceptibility to RBSDV 404

infection (He et al., 2017). OsGSK2 also suppresses the BR pathway by 405

inactivating the transcription function of OsBZR1 in rice (Tong et al., 2012a). 406

Thus, we suggest that OsGSK2 positively modulates rice antiviral defense by 407

coordinating JA and BR signaling. The physical interaction between OsGSK2 408

and OsJAZ4 reveals a direct cross-talk between JA and BR signaling at the 409

14

molecular level. 410

It has been reported that Arabidopsis and rice JAZ proteins form homo- 411

and heterodimers and that the ZIM domain is responsible for this dimerization 412

(Chini et al., 2007; Chini et al., 2009; Chung and Howe, 2009; Ren et al., 2010; 413

Yamada et al., 2012; Chen et al., 2019). Our Y2H data also showed that 414

OsJAZ4 formed heterodimeric complexes with OsJAZ9, OsJAZ11 and 415

OsJAZ12 (Figure 5). In Arabidopsis, strongly JA-insensitive phenotypes 416

conferred by overexpression of JAZ10.4 (an alternatively spliced form of 417

JAZ10 that lacks the Jas domain) were suppressed by mutations in the ZIM 418

domain that block JAZ10.4–JAZ interactions (Chung and Howe, 2009). 419

AtJAZ3ΔJas does not interact with AtMYC2, but over-expression of 420

AtJAZ3ΔJas confers jasmonate-insensitivity to Arabidopsis transgenic plants 421

because its ZIM is able to interact with other JAZ proteins (Chini et al., 2007; 422

Chini et al., 2009). These findings suggest that the dimerization mediated by 423

the ZIM domain suppresses JA signaling and that disruption of JAZ 424

dimerization therefore increases JA signaling output. It has been reported that 425

the ZIM domain is essential for regulating JAZs stabilization (Chini et al., 2007; 426

Mao et al., 2017; Chen et al., 2019). Although the ZIM domain is not itself 427

required for the JAZ-COI1 interaction, the heterodimerization of JAZs via the 428

ZIM domain would affect the spatial structure of the C-terminal of JAZs and 429

would likely therefore affect the JAZs–COI1 interaction. For example, 430

AtJAZ3ΔJas protein could interact with AtJAZ1 and AtJAZ9, and inhibit 431

degradation of AtJAZ1 and AtJAZ9 by interfering with COI1 activity (Chini et al., 432

2007). AtSPL9 and AtHARP1 interact with JAZ proteins via the ZIM domain to 433

inhibit degradation of JAZs by interfering with the COI1–JAZs interaction (Mao 434

et al., 2017; Chen et al., 2019). Here, we demonstrated that OsGSK2 interacts 435

with OsJAZ4 via the ZIM domain and competed with OsJAZ11 for binding to 436

OsJAZ4 (Figure 5), which may disrupt the OsJAZ4-OsJAZ11 heterodimer 437

complex. We also found that the accumulation of OsJAZ11 was suppressed 438

when OsGSK2 promoted degradation of OsJAZ4 in vivo (Figures 4 and 5), 439

15

indicating that OsGSK2 disrupts the stability of the OsJAZ4-OsJAZ11 440

heterodimer. Thus, the reduced accumulation of OsJAZs resulting from the 441

dissociation of OsJAZ4-OsJAZs by OsGSK2 may, at least partially, contribute 442

to the JA sensitivity and antiviral defense induced by OsGSK2. 443

The protein phosphorylation pathway has been reported to be involved in 444

the JA signaling system (Kazan and M.Manners, 2013). MYC2 445

phosphorylation is functionally coupled with its action to regulate 446

JA-responsive gene transcription and JA-mediated immunity (Zhai et al., 2013). 447

Phosphorylation of Jasmonate-associated VQ domain protein 1 (JAV1) 448

disintegrates the JAV1-JAZ8-WRKY51 complex to derepress JA biosynthesis 449

and defend against herbivory (Yan et al., 2018). 450

JAZ proteins serve as a key negative regulator in JA-mediated 451

transcriptional responses (Pauwels et al., 2010; Pauwels and Goossens, 2011). 452

However, there has so far been little research on the phosphorylation of JAZ 453

proteins, and no reports of direct physical interactions between JAZ and any of 454

the kinases involved in JA signaling (Gfeller et al., 2010; Yan et al., 2018; Liu et 455

al., 2019). Here, we first demonstrated that OsJAZ4 physically interacted with 456

and was phosphorylated by OsGSK2 (Figure 2 and 3). We then showed that 457

OsGSK2 can disrupt the OsJAZ4-OsNINJA corepressor complex to destabilize 458

OsJAZ4 via the 26S proteasome in an OsCOI1-dependent manner (Figures 4 459

and 8). In addition, we confirmed that OsJAZ4 suppressed JA signaling (Figure 460

6). Furthermore, overexpression of OsGSK2 resulted in decreased levels of 461

OsJAZ proteins, thus increasing JA signaling by alleviating the repression 462

mediated by JAZ (Figure 1, 4 and 5). This is consistent with previous reports 463

that fast turnover of JAZ proteins holds the key to JA signal output (Gfeller et 464

al., 2010; Wasternack and Feussner, 2018; Mao et al., 2017; Chen et al., 465

2019). Therefore, our results provide insight into the physical interaction 466

between a JAZ family member, OsJAZ4, and a GSK3-like kinase, OsGSK2, 467

involved in JA signaling. 468

In conclusion, we propose a model illustrating how OsGSK2 integrates the 469

16

JA and BR signaling pathways and triggers rice antiviral resistance (Figure 7). 470

As the levels of OsGSK2 increase, the repression of BR signaling and 471

BR-induced susceptibility is enhanced. On the other hand, the increased 472

amount of OsGSK2 binds to OsJAZ4 to dissociate the OsJAZ4-OsNINJA 473

corepressor and the OsJAZ4-OsJAZ11 heterodimer complex. The 474

phosphorylated OsJAZ4 and free OsJAZ11 are degraded by the 26S 475

proteasome in an OsCOI1-dependent manner, which in turn enhances the JA 476

response and JA-mediated antiviral resistance. Our findings reveal an 477

important mechanism for the positive role of OsGSK2 in rice antiviral immunity, 478

and provide insight into the crosstalk mechanism between JA and BR 479

signaling. 480

481

METHODS 482

483

Plant materials and insect vectors 484

Rice (Oryza sativa L. japonica) cultivars Wuyujing No. 3 and NIP were used in 485

this study. Go and Gi plants with the corresponding wild-type Zh11 were 486

previously described (Tong et al., 2012a). The JA-insensitive mutant coi1-13 487

with its wild-type NIP was described before (Yang et al., 2012). OsJAZ4-OE 488

(Line #1 and #3), OsJAZ4-RNAi (line #14 and #18) and OsJAZ4-MYC 489

transgenic plants were constructed in this study (constructs are described 490

below). RBSDV-infected rice plants were collected from fields in Shandong 491

Province, China. Virus-free small brown planthoppers (Laodelphax striatellus, 492

SBPHs) were kept and reared on healthy Wuyujing No. 3 seedlings in glass 493

beakers in a glasshouse at 25 °C under artificial light. Rice plants were grown 494

in a greenhouse at 28-30 °C with 14 h light/10 h darkness, light intensity 600 495

µmol m −2 s −1. Nicotiana benthamiana plants were grown in a growth chamber 496

at 25 °C and with 16 h light/8 h darkness. 497

498

RBSDV transmission experiment 499

17

RBSDV transmission via SBPH was performed as described previously with 500

some modifications (Tong et al., 2011; He et al., 2017). Briefly, SBPH carrying 501

RBSDV were transferred to rice seedlings at the 1.5- to 2.0-leaf stage 502

(approximately three viruliferous insects per seedling) and allowed to feed for 3 503

days, and then the insects were completely removed. The inoculated plants 504

were taken and grown in the greenhouse to develop symptoms. Plants 505

infected with RBSDV exhibited symptoms such as stunting and darkening of 506

leaves at 30 dpi and the presence of RBSDV in each plant was confirmed by 507

RT-PCR using virus-specific primers S10-F and S10-R (Supplemental Table 1). 508

The number of diseased plants was used to calculate the viral incidence (% 509

plants infected). For each independent experiment, at least three biological 510

replicates were used and at least 40 seedlings were used for each replicate. 511

512

Total RNA extraction and RT-qPCR 513

Total RNA from leaves was extracted using the Trizol protocol (Invitrogen, USA) 514

in accordance with the manufacturer’s instructions. First-strand cDNA was 515

synthesized from 1 μg total RNA using a HiScript II Q RT for qPRC (+gDNA 516

viper) kit (Vazyme, China). RT-qPCR was performed on a QuantStudio 6 Flex 517

Real-Time PCR System (Applied Biosystems, Singapore) using a CHamQ 518

SYBR qPCR Master Mix kit (Vazyme, China), following the supplier’s protocol. 519

The RT-qPCR conditions were as follows: 95 °C for 3 min; 40 cycles of 95 °C 520

for 15 s, 60 °C for 15 s, and 72 °C for 20 s. The mRNA expression levels were 521

normalized against the expression of housekeeping gene, OsUBQ5, and the 522

fold change was calculated by the comparative Ct method (2-∆∆Ct method) 523

(Livak and Schmittgen, 2001). At least three biological replicate samples were 524

used. Differences were considered significant at p ≤ 0.05. The primers used in 525

this study are listed in Supplemental Table 1. 526

527

Primary root inhibition assay 528

Germinated seeds were cultured in normal rice culture solutions (Yoshida et al., 529

18

1976) supplemented with different concentration of meJA (TCI) and incubated 530

in a growth chamber at 30 °C with 8 h light followed by 25 °C with 16 h 531

darkness. 3 days later, root lengths of seedlings were measured. For each 532

treatment, at least 10 seedlings for each plant were treated and measured. 533

Two independent experiments were performed. 534

535

Y2H screening and Y2H assays 536

For Y2H screening assay, the coding sequences of OsGSK2 were cloned into 537

the pGBKT7 vector and used as the bait to screen a normalized rice cDNA 538

prey library according to the manufacturer’s instructions. 539

For Y2H assays, the ORFs of OsGSKs, OsCOI1b, OsNINJA and OsJAZs 540

with its mutants were cloned into the pGBKT7 (BD) or pGADT7 (AD) vectors. 541

These constructs or the corresponding empty vectors were co-transformed 542

into the yeast strain AH109 and incubated at 30 °C on SD medium lacking Leu, 543

Trp, then spotted on selective media lacking Ade, His, Leu and Trp. Primers 544

used are provided in Supplemental Table 1. 545

546

Co-localization experiments and BiFC analysis 547

All binary vectors used in these studies were derived from the pCV1300 548

plasmid (Sun et al., 2013). For co-localization experiments, the ORFs of 549

OsGSK2 or OsJAZ4 were cloned into pCV-mCherry-N1 or pCV-GFP-N1, 550

respectively, to obtain pCV:OsJAZ4-mCherry and pCV:OsGSK2-GFP 551

constructs as previously described (Sun et al., 2013). These constructs were 552

co-transiently expressed in N. benthamiana leaves by Agrobacterium 553

tumefaciens infiltration. 554

For BIFC assay, the full-length cDNA sequences of OsGSK2 and OsJAZ4 555

were cloned into the cYFP and nYFP vectors to obtain the OsGSK2-nYFP, 556

OsGSK2-cYFP, OsJAZ4-nYFP and OsJAZ4-cYFP constructs, respectively. 557

The constructs were transformed into Agrobacterium tumefaciens strain 558

GV3101 then transiently co-expressed in N. benthamiana leaves. The 559

19

fluorescence signal for each combination was visualized using a Leica TCS 560

SP5 confocal laser scanning microscope system (Leica Microsystems, 561

Bannockburn, IL, USA) 40-44 h after infiltration. 562

563

Pull-down assay 564

The full-length coding sequence of OsGSK2 was cloned into the GST fusion 565

vector (pGEX-6p-1), that of OsJAZ4 into the His fusion vector (pCOLD-TF) and 566

those of OsJAZ11 and OsNINJA into the His fusion vector (pET-32a). The 567

fusions were then transformed into Escherichia coli BL21 (DE3). To induce 568

protein expression, a final concentration of 1 mM isopropyl 569

β-D-thiogalactopyranoside (IPTG) was added when the optical density (OD)600 570

of the cultured cells was 0.6-0.8. For induction of recombinant protein, the 571

cultures were incubated at 28 °C for 8 hours for GST-OsGSK2, OsNINJA and 572

His-OsJAZ11, and at 16 °C for 16 hours for His-OsJAZ4. For the pull-down 573

assay, GST or GST-OsGSK2 was incubated with GST beads (Beaver, China) 574

at 4 °C for 1 h and then His-OsJAZ4 was added. The incubation continued for 575

2 h, and then the beads were washed thoroughly, resolved by SDS-PAGE, and 576

detected using anti-His antibody (ab18184; abcam;1:4000 dilution). The bait 577

proteins were probed with anti-GST antibody (ab92; abcam; 1:3000 dilution). 578

The primers used are listed in Supplemental Table 1. 579

For competitive pull-down assays, 3 μg His-OsJAZ11 or His-NINJA with 2, 580

6, or 15 μg GST-OsGSK2 or GST, were incubated with immobilized 581

OsJAZ4-myc at 4 °C for 1 hour. Proteins retained on the beads were resolved 582

by SDS-PAGE and detected with anti-His antibody. The loading of 583

OsJAZ4-myc was probed with anti-myc antibody (ab9132; abcam; 1:4000 584

dilution). His-OsJAZ11 and GST-OsGSK2, His-OsNINJA and GST-OsGSK2 585

were stained with Coomassie Brilliant Blue. 586

587

Co-immunoprecipitation assay 588

For the co-IP assay in N. benthamiana leaves, the coding sequences of 589

20

OsGSK2, OsJAZ11 and OsJAZ4 were cloned into pCV-4HA-N1 and 590

pCV-3myc-N1 vectors, respectively, to obtain pCV-HA-OsGSK2, 591

pCV-OsJAZ11-myc and pCV-OsJAZ4-myc constructs as previously described 592

(Sun et al., 2013). Then pCV-HA-OsGSK2 and pCV-OsJAZ11-myc, or 593

pCV-HA-OsGSK2 and pCV-OsJAZ4-myc were transiently co-expressed in N. 594

benthamiana leaves. The leaves were collected and ground in liquid nitrogen, 595

then extracted by PierceT IP lysis buffer (Thermo Scientific, 87788) with 1 mM 596

DTT and 1 × complete protease inhibitor cocktail. 30 μl of anti-HA or anti-myc 597

magnetic beads were added to the protein extraction and then incubated at 598

4 °C for 4h. The precipitated samples were washed thoroughly, resolved by 599

SDS-PAGE, and detected with the corresponding antibodies (anti-HA antibody, 600

2999S, Cell Signaling, 1:3000 dilution). The primers used are listed in 601

Supplemental Table 1. 602

603

Kinase assay 604

For the in vivo kinase assay, the OsJAZ4-myc from pooled T1 OsJAZ4-MYC 605

transgenic plants or N. benthamiana leaves co-expressed with different vector 606

combinations was immunoprecipitated with anti-myc beads, and the IP product 607

was treated with CIP. To avoid protein degradation, MG132 and a cocktail of 608

proteinase inhibitors was added. The IP products were separated by 7.5% 609

Phos-tag (50 μM) SDS–PAGE (Wako, Japan) and analyzed with anti-myc 610

antibody. 611

612

Determination of Phosphorylation Sites of OsJAZ4 by OsGSK2 Kinase 613

His-OsJAZ4 was phosphorylated by GST-OsGSK2 as described (Wang et al., 614

2013). The fusion proteins (kinase : substrate = 1 : 5) were added in 25 μL of 615

reaction buffer [25 mMTris (pH 7.5), 12 mM MgCl2, and 1 mM DTT] with 50 616

mM ATP in a 37 °C water bath for 1 h. The phosphorylated His-OsJAZ4 was 617

separated from the SDS-PAGE gel and subjected to in-solution 618

alkylation/tryptic digestion followed by liquid chromatography/tandem mass 619

21

spectrometry as described (Wang et al., 2013). 620

621

Protein Degradation Assay 622

For the degradation assay in N. benthamiana, individual cultures were 623

adjusted to OD600 = 1, and equal volumes were mixed before leaf infiltration. 624

The infiltrated leaves of at least four plants were collected and pooled at 36 h 625

and again at 48 h after infiltration. 50 μM MG132 or 20 μM Bikinin were 626

infiltrated at 36 h, and 12 h later, proteins were collected. 627

For the cell-free protein degradation assay, seven-day-old wild-type NIP 628

seedlings were harvested and ground to a fine power in liquid nitrogen. Total 629

protein was extracted in degradation buffer (25 mM Tris-HCl, pH 7.5, 10 mM 630

NaCl, 10 mM MgCl2, 5 mM DTT, and 10 mM ATP) (Qiao et al., 2017). Extracts 631

containing equal amounts of recombinant proteins were added to the tubes 632

and incubated at 37 °C for the times indicated. 633

634

Plant hormone treatment 635

For BL or Bikinin treatments, seven-day-old rice seedlings were sprayed with 1 636

μM BL (Sigma) or 20 μM Bikinin (Sigma) dissolved in 0.1% Triton X-100. 637

Leaves were collected for protein extraction at the indicated time points. Three 638

independent experiments were performed. 639

For in vivo lamina joint assays, the micro-drop method was performed as 640

described previously (Hong et al., 2003). The lamina joints of the second leaf 641

of four-day-old seedlings were spotted with 1000 ng of BL in 1 μL ethanol by 642

micro-syringe. The angles between the leaf lamina of the second leaf blade 643

and sheath were measured 3 days after treatment by analyzing digital images 644

using Motic Images Plus 2.0 software (China Group Co., Ltd.). At least twenty 645

plants were used for each treatment. Three independent experiments were 646

performed. 647

648

Vector construction and plant transformation 649

22

To generate OsJAZ4-OE plants, the ORF of OsJAZ4 was amplified using 650

CV-OsJAZ4-F/R primers and cloned into the pCAMBIA1300 vector driven by 651

the 35S promoter. To produce the RNA interference lines, two fragments of 652

OsJAZ4 (nt 960 to 1218) were amplified using the primer pairs 653

RNAi-OsJAZ4-F1/R1 and RNAi-OsJAZ4-F2/R2, and then inversely inserted 654

into pTCK303 vector driven by the UBI promoter. The constructs described 655

above were introduced into Agrobacterium tumefaciens strain EHA105 and 656

transformed into the NIP background. pCV-OsJAZ4-myc vector was used to 657

generate OsJAZ4-MYC transgenic plants in a Zh11 background. The T4 658

generation of OsJAZ4-OE and OsJAZ4-RNAi lines and T1 hemizygous 659

OsJAZ4-myc plants were used. The primers used are listed in Supplemental 660

Table 1. 661

662

JA measurement 663

Seven-day-old total leaves from Go, Gi and ZH11 plants were collected, 664

ground in liquid nitrogen and then used for hormone extraction and analysis as 665

described previously (Fu et al., 2012; He et al., 2017). Three biological 666

replicates were used, each of which consisted of at least fifteen pooled plants. 667

668

Phylogenetic analysis 669

The amino acid sequences of OsJAZs and OsJGSK2s were download from 670

the Rice Genome Annotation Project 671

(http://rice.plantbiology.msu.edu/index.shtml) and aligned in ClustalW 672

(https://myhits. sib.swiss /cgi-bin/clustalw). Phylogenetic analyses were 673

conducted using MEGA version 6, and the tree was generated using the 674

neighbor-joining method (complete deletion and 1,000 bootstrap replications) 675

(Tamura et al., 2013). 676

677

Antibody generation and validation 678

The purified recombinant His-OsJAZ11, GST-OsGSK2 protein and peptide of 679

23

OsJAZ4 (CSSNRDESLSLGQPR) were used as antigens to immunize New 680

Zealand rabbits to produce antiserum. The polyclonal antibodies (anti-OsJAZ4, 681

anti-OsJAZ11 and anti-OsGSK2) from the generated antisera were purified by 682

protein G chromatography (Bio-Rad, Shanghai, China) according to the 683

manufacturer’s protocol. Immunoblotting was performed to detect the purified 684

antibody. anti-OsJAZ4, anti-OsJAZ11 and anti-OsGSK2 were used as primary 685

antibody at 3, 4.5 and 1.5 μg/ml, respectively, and one specific band for each 686

antibody was detected within the total protein fraction of plants tested 687

(Supplemental Figure 11). The protein levels of OsJAZ4 and OsJAZ11 688

decreased upon MeJA treatment (Supplemental Figure 11). 689

690

Statistical analysis 691

Differences were analyzed using ANOVA with Fisher’s least significant 692

difference (LSD) tests. A p-value ≤ 0.05 was considered statistically significant. 693

All analyses were performed using ORIGIN 8 software. Statistical data are 694

provided in Supplemental Data Set 1. 695

696

Accession numbers 697

Sequence data from this article can be found in the rice genome annotation 698

project databases under the following accession numbers: 699

OsJAZ1, Os04g55920; OsJAZ2, Os07g05830; OsJAZ3, Os08g33160; 700





OsGSK1, Os01g14860; OsGSK2, Os05g11730; OsGSK3, Os02g14130; 705

OsGSK4, Os01g19150; OsGSK5, Os03g62500; OsGSK6, Os05g04340; 706

OsGSK7, Os01g10840; OsGSK8, Os06g35530. 707

708

SUPPLEMENTARY DATA 709

24

Supplemental Figure 1. Effect of OsGSK2 on Rice black-streaked dwarf virus 710

(RBSDV) infection. 711

712

Supplemental Figure 2. Levels of endogenous JA-IIe in seven-day-old Zh11, 713

Go and Gi plants. 714

715

Supplemental Figure 3. Phylogenetic analysis of OsGSK and OsJAZ amino 716

acid sequences in rice using the neighbor-joining method. 717

718

Supplemental Figure 4. SDS-PAGE analysis of recombinant GST-OsGSK2 719

and His-OsJAZ4 proteins. 720

721

Supplemental Figure 5. Co-localization of OsGSK2-GFP and 722

OsJAZ4-mCherry in N. benthamiana leaf epidermal cells 723

724

Supplemental Figure 6. RT-qPCR analysis of OsJAZ4, OsJAZ11 and 725

OsGSK2 transcript levels for Figure 2D. 726

727

Supplemental Figure 7. Identification of OsJAZ4 phosphorylation sites by 728

OsGSK2 Kinase using LC-MS/MS. 729

730

Supplemental Figure 8. The 8 potential phosphorylation motifs of OsGSK2 in 731

OsJAZ4. 732

733

Supplemental Figure 9. Effects of potential phosphorylation site mutants of 734

OsJAZ4 on the OsJAZ4-OsGSK2 interaction. 735

736

Supplemental Figure 10. Effects of OsGSK2 on OsJAZ4 accumulation in N. 737

benthamiana leaves. 738

739

25

Supplemental Figure 11. Antibody validation. 740

741

Supplemental Figure 12. Accumulation of OsJAZs-myc in N. benthamiana 742

leaves. 743

744

Supplemental Figure 13. Effect of OsJAZ4 on RBSDV infection. 745

746

Supplemental Figure 14. Survival rates of SBPH on OsJAZ4-OE lines (#1 747

and #3), OsJAZ4-RNAi lines (#14 and #18) and NIP. 748

749

Supplemental Figure 15. OsGSK2 protein levels in wild type Nipponbare 750

(NIP) and OsJAZ4 mutant plants. 751

752

Supplemental Figure 16. Full scan data of the immunoblots in this work. 753

754

Supplemental Table 1. Primers used in this work. 755

756

Supplemental File 1. Multiple sequence alignment for Supplemental Figure 3. 757

758

Supplemental Data Set 1. Data for all statistical analyses performed in this 759

study. 760

761

Acknowledgments 762

We are indebted to Prof. Jianxiang Wu (Zhejiang University) for providing 763

RBSDV-CP antibody, to Prof. Kenji Gomi (Kagawa University) for providing 764

OsJAZ plasmids, to Prof. Zuhua He (Shanghai Institutefor Biological Sciences, 765

Chinese Academy of Sciences, China) for the coi1-13 mutant. We thank Mike 766

Adams for critically reading and improving the manuscript. This work was 767

funded by the National Key Research and Development Plan 768

(2016YFD0200804), the International Science & Technology Cooperation 769

26

Program of China (2015DFA30700), Zhejiang Provincial Natural Science 770

Foundation of China (LQ18C140004), National Natural Science Foundation of 771

China (31800249, 31670291, 31670303). This work was sponsored by 772

K.C.Wong Magna Fund in Ningbo University.773

774

Author contributions 775

Y.H. and Z.S. conceived the project and designed the experiments; Y.H. and 776

G.H. carried out the experiments with assistance from H.Z., L.L., Y.K., and K.X.; 777

all authors analyzed and discussed the results; and Y.H., J.C. and Z.S. wrote 778

the manuscript. 779

27

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32

Figure legends 991

Figure 1. OsGSK2 positively regulates antiviral defense and JA response. 992

(A) Rice black-streaked dwarf virus (RBSDV) symptoms on Zh11, Go and Gi 993

plants. Scale bar, 10 cm. 994

(B) Disease incidence in Zh11, Go and Gi plants following RBSDV inoculation. 995

The numbers of healthy and diseased plants in each treatment was 996

determined by reverse transcription polymerase chain reaction 30 days after 997

inoculation and the number of diseased plants was used to calculate the viral 998

incidence (% plants infected). Each treatment used at least 40 seedlings, and 999

at least three biological replicates were performed. Different letters at the top 1000

of columns indicate significant difference between transgenic and control 1001

plants at p ≤ 0.05 by Fisher's LSD tests. 1002

(C) Expression levels of the RBSDV Coat protein (CP) gene as measured by 1003

RT-qPCR at 30 dpi. Data are relative expression levels of CP in Go, Gi plants 1004

compared with that in the wild-type Zh11 plants. OsUBQ5 was used as the 1005

internal reference gene. Error bars indicate the SD of three biological 1006

replicates. *indicates significant difference between transgenic and control 1007

plants at p ≤ 0.05 by Fisher's LSD test. 1008

(D) Expression analysis of JA-responsive genes by RT-qPCR. Seven-day-old 1009

seedlings were collected for total RNA extraction. OsUBQ5 was used as the 1010

internal reference gene. Values are means ± SE of three biological replicates. * 1011

indicates significant difference between transgenic and the control plants at p ≤ 1012

0.05 by Fisher's LSD test. 1013

(E) Levels of endogenous JA in seven-day-old Zh11, Go and Gi plants. The 1014

limit of quantification to JA was 1 ng/ml. Values are means ± SD of three 1015

biological replicates. Different letters at the top of columns indicate significant 1016

difference between transgenic and control plants at p ≤ 0.05 by Fisher's LSD 1017

tests. 1018

(F and G) Images (F) and quantification of root length (G) of Zh11, Go and Gi 1019

after MeJA treatment. The root lengths of three-day-old seedlings grown in 1020

33

normal rice culture solutions supplemented with indicated concentrations of 1021

MeJA were measured. Data shown are the means from at least 10 seedlings 1022

for each indicated plant. Error bars represent SD. Different letters at the top of 1023

columns indicate significant difference between transgenic and control plants 1024

at p ≤ 0.05 by Fisher's LSD tests. Scale bar, 2 cm. 1025

1026

Figure 2. OsGSK2 interacts with OsJAZ4 in vitro and in vivo. 1027

(A) Yeast two-hybrid assay showing the interaction between OsGSK2 and1028

OsJAZ1-15 proteins. Interactions were examined with SD base without Ade, 1029

His, Leu and Trp. 1030

(B) Yeast two-hybrid assay showing the interaction between OsJAZ4 and1031

OsGSK1-8 proteins. Transformed yeast cells were grown on SD-Ade-His 1032

-Leu-Trp medium.1033

(C) Pull-down assay confirming that OsGSK2 interacts with OsJAZ4 in vitro.1034

Immobilized GST and GST-OsGSK2 were used to pull down His-OsJAZ4, and 1035

immunoprecipitated fractions were detected using anti-His antibody. The bait 1036

proteins were probed with anti-GST antibody. 1037

(D) BiFC assay showing the interaction between OsGSK2 and OsJAZ4 in N.1038

benthamiana leaves. OsJAZ11 was used as negative control. Scale bar, 20 1039

µm. 1040

(E) Co-IP assays showing the interaction between OsGSK2 and OsJAZ4 in1041

vivo. The proteins were extracted from N. benthamiana leaves and 1042

immunoprecipitated by anti-myc and anti-HA magnetic beads, respectively. 1043

The coimmunoprecipitated proteins were probed by either anti-Myc or anti-HA 1044

antibody. OsJAZ11-myc was used as negative control. 1045

1046

Figure 3. OsGSK2 phosphorylates OsJAZ4. 1047

(A) Immunoprecipitated OsJAZ4-myc protein from OsJAZ4-MYC plants was1048

treated with calf intestinal alkaline phosphatase (CIP) or water. OsJAZ4-myc 1049

protein was separated in a phos-tag SDS-PAGE gel and detected by anti-myc 1050

34

anti-body (top and middle panel). The slowly migrating band (red arrow) of 1051

OsJAZ4-myc in the phos-tag gel with short exposure time (Short exp.) or long 1052

exposure time (Long exp.) represents the phosphorylated form of OsJAZ4 1053

(OsJAZ4-myc-P). As loading control (bottom panel), equal amounts of the 1054

immunoprecipitated OsJAZ4-myc protein were separated in a normal 1055

SDS-PAGE gel followed by immunoblot analysis. 1056

(B) Potential phosphorylation sites in OsJAZ4.1057

(C) Immunoprecipitated OsJAZ4-myc and OsJAZ4Δ8-myc protein in N.1058

benthamiana leaves transiently co-expressed with HA-empty vector, 1059

HA-OsGSK2 or HA-OsGSK2K92R. The protein immunoprecipitated by anti-myc 1060

beads or CIP-treated OsJAZ4-myc protein from each combination were 1061

separated in a phos-tag SDS-PAGE gel and detected by anti-myc antibody 1062

(top panel). The slowly migrating band (red arrow) of OsJAZ4-myc in the 1063

phos-tag gel with short exposure time (Short exp.) or long exposure time (Long 1064

exp.) is the phosphorylated form of OsJAZ4 (OsJAZ4-myc-P). As loading 1065

controls, OsGSK2 (bottom panel) and different amounts of the 1066

immunoprecipitated OsJAZ4-myc protein (middle panel) were separated in a 1067

normal SDS-PAGE gel followed by immunoblot analysis. 1068

1069

Figure 4. OsGSK2 promotes OsJAZ4 degradation. 1070

(A) Time course of OsJAZ4 degradation in wild-type Nipponbare (NIP) protein1071

extracts treated with GST, GST-OsGSK2 or GST-OsGSK2K92R. Equal amounts 1072

of plant crude extracts were added to equal amounts of the recombinant 1073

proteins in the in vitro cell-free degradation assays. The Coomassie Brilliant 1074

Blue-stained Rubisco large subunit (Rbc L) was used as a loading control. 1075

(B) Quantification analysis of (A). The relative levels of OsJAZ4 in wild-type1076

NIP plant protein extracts at 0 h were defined as “1.” Data are means ± SE (n = 1077

3). 1078

(C) Time course of degradation of His-OsJAZ4 and His-OsJAZ4Δ8 in the1079

wild-type NIP protein extracts with or without MG132. Equal amounts of the 1080

35

recombinant proteins were incubated with equal amounts of plant crude 1081

extracts in the in vitro cell-free degradation assays. 1082

(D) Quantification analysis of (C). The relative levels of His-OsJAZ4 or 1083

His-OsJAZ4Δ8 incubated with wild-type NIP plant protein extracts at 0 h were 1084

defined as “1.” Data are means ± SE (n = 3). 1085

(E) The protein levels of OsJAZ4 in Go, Gi and Zh11 leaves. The OsJAZ4 1086

protein was detected with anti-OsJAZ4 antibody and RbcL was used as a 1087

loading control. Two independent pools of leaves are shown. 1088

(F) Quantification analysis of (E). The relative level of OsJAZ4 in wild-type 1089

ZH11 was set as “1”. Data are means ± SE (n = 3). 1090

1091

Figure 5. OsGSK2 affects OsJAZ4-OsJAZ11 interaction. 1092

(A) Y2H assay shows that the ZIM domain of OsJAZ4 is responsible for 1093

binding to OsGSK2. Schematic diagrams show the truncated versions of 1094

OsJAZ4. Interactions were examined with SD base without Ade, His, Leu and 1095

Trp. 1096

(B) Yeast two-hybrid assay shows the interaction between OsJAZ4 and 1097

OsJAZ1-15 proteins. Transformed yeast cells were grown on SD-Ade -His 1098

-Trp-Leu medium. 1099

(C) In vitro interaction between OsJAZ4-myc and His-JAZ11 is weakened by 1100

GST-OsGSK2. His-OsJAZ11 protein combined with GST-OsGSK2 was 1101

incubated with immobilized OsJAZ4-myc. The immunoprecipitated fractions 1102

were detected by anti-His antibody. The gradient indicates increasing amount 1103

of GST-OsGSK2. OsJAZ4-myc input was probed with anti-myc antibody, and 1104

the loading of His-OsJAZ11 and GST-OsGSK2 is shown in the lower panel by 1105

Coomassie Brilliant Blue (CBB) Staining. 1106

(D) HA-OsGSK2 affects accumulation of OsJAZ4-myc and OsJAZ11-myc. The 1107

respective vectors were co-transiently expressed in the N. benthamiana leaves. 1108

The infiltrated leaves were collected at 48 h after infiltration, and at least four 1109

36

plants were pooled. The Coomassie Brilliant Blue-stained Rubisco large 1110

subunit (Rbc L) was used as a loading control. 1111

(E) The protein levels of OsJAZ11 in Go, Gi and Zh11 plants. The OsJAZ11 1112

protein was detected with anti-OsJAZ11 antibody and Rbc L was used as a 1113


1115

Figure 6. Effect of OsGSK2 on the OsJAZ4-OsNINJA interaction. 1116

(A) OsNINJA and OsCOI1b interact with OsJAZ4. OsNINJA and OsCOI1b 1117

were fused to the GAL4 DNA-binding domain (BD) while OsJAZ4 and its 1118

mutants were fused to the GAL4 activation domain (AD), respectively. 1119

Interactions were examined using SD base without Ade, His, Leu and Trp. For 1120

the interactions between OsJAZ4 and OsCOI1b, 25 μM coronatine (COR) was 1121

added. 1122

(B) OsGSK2 competes with OsNINJA for binding to OsJAZ4. In vitro 1123

interaction between OsJAZ4-myc and His-OsNINJA is weakened by 1124

GST-OsGSK2. His-OsNINJA protein combined with GST-OsGSK2 was 1125

incubated with immobilized OsJAZ4-myc. The immunoprecipitated fractions 1126

were detected by anti-His antibody. The gradient indicates increasing amounts 1127

of GST-OsGSK2. OsJAZ4-myc input was probed with anti-myc antibody, and 1128

the loading of His-OsNINJA and GST-OsGSK2 is shown in the lower panel by 1129

Coomassie Brilliant Blue (CBB) Staining. 1130

1131

Figure 7. OsJAZ4 negatively modulates JA signaling and rice immunity. 1132

(A) JA-responsive gene expression in indicated transgenic plants. RT-qPCR 1133

analysis of the mRNA levels of JA-responsive genes in the wild-type 1134

Nipponbare (NIP), OsJAZ4-OE lines (#1 and #3) and OsJAZ4-RNAi lines (#14 1135

and #18). OsUBQ5 was used as the internal reference gene. Values are 1136

means ± SE of three biological replicates. * indicates significant difference at p 1137

≤ 0.05 (n = 3) by Fisher's LSD tests. 1138

37

(B and C) Images (B) and quantification of root length (C) in indicated plants 1139

following MeJA treatment. The root lengths of three-day-old seedlings grown in 1140

normal rice culture solutions supplemented with different concentrations of 1141

MeJA were measured. Data shown are the means from at least 15 seedlings 1142

for each plant type. Error bars represent SD. Different letters at the top of 1143

columns indicate significant difference at p ≤ 0.05 by Fisher's LSD tests. Scale 1144

bar, 2 cm. 1145

(D) Disease incidence. The numbers of healthy and diseased plants in each1146

treatment was determined by RT-PCR 30 days after inoculation and the 1147

number of the diseased plants was used to calculate the viral incidence (% 1148

plants infected). Each treatment used at least 40 seedlings, and at least three 1149

biological replicates were performed. Different letters at the top of columns 1150

indicate significant difference between transgenic and control plants at p ≤ 1151

0.05 by Fisher's LSD tests. 1152

(E) The relative expression levels of RBSDV Coat Protein (CP) gene1153

measured by RT-qPCR at 30 dpi. Data represent relative expression levels of 1154

the CP gene in the mutant compared with that in the wild-type NIP plants. 1155

OsUBQ5 was used as the internal reference gene. Error bars represent SD. * 1156

indicates significant difference at p ≤ 0.05 (n ≥ 3) by Fisher's LSD tests. 1157

(F) Viral symptoms in OsJAZ4-OE lines (#1 and #3) and OsJAZ4-RNAi lines1158

(#14 and #18). Scale bar, 10 cm. 1159

1160

Figure 8. Effect of OsGSK2-OsJAZ4 interaction on JA- and BR- pathway 1161

crosstalk. 1162

(A and B) OsJAZ4 accumulation increases in response to BL (brassinolide, A) 1163

or Bikinin (B) treatment. The leaves of seven-day-old wild-type Nipponbare 1164

(NIP) seedlings were treated with 1 μM BL or 20 μM Bikinin, and protein was 1165

extracted from the treated leaves 0, 3, 6 or 12 h later. The Coomassie Brilliant 1166

Blue-stained Rubisco large subunit (Rbc L) was used as a loading control. Two 1167

independent pools of leaves are shown. 1168

38

(C and D) The accumulation of OsJAZ4 induced by BL and Bikinin was 1169

inhibited in coi1-13 mutants. Leaves of seven-day-old wild-type NIP and 1170

coi1-13 seedlings were treated with 1 μM BL (C) or 20 μM Bikinin (D), and the 1171

treated leaves were used for protein extraction at different times after 1172

treatment, and detected by Anti-OsJAZ4 antibody. Rbc L was used as a 1173


(E) Effect of Bikinin on MeJA hypersensitivity in OsJAZ4-RNAi plants.1175

Germinated seeds were grown in normal rice culture solutions containing 0 or 1176

1 μM MeJA, with or without 200 μM Bikinin for 5 days and the root length was 1177

then measured. Relative root elongation is expressed as a percentage of root 1178

elongation in solutions with (right section) or without (left section) 200 μM 1179

Bikinin. Error bars represent SE (n≥20). Different letters at the top of columns 1180

indicate significant difference at p ≤ 0.05 by Fisher's LSD tests. 1181

(F) BL sensitivity test of the wild type NIP, OsJAZ4-OE lines (#1 and #3) and1182

OsJAZ4-RNAi lines (#14 and #18) by lamina joint assay. The plus and minus 1183

symbols indicate with/without BL (100 ng). 1184

(G) Quantification of the data shown in (E). Data shown are the means from at1185

least 15 seedlings for each indicated plant. Error bars represent SE. Different 1186

letters at the top of columns indicates significant difference at p ≤ 0.05 by 1187

Fisher's LSD tests. 1188

1189

Figure 9. Model of OsGSK2-mediated plant defense signaling in rice. 1190

OsGSK2 binds to OsJAZ4 to disrupt OsJAZ4-OsNINJA corepressor and 1191

OsJAZ4-OsJAZ11 dimerization, which promotes the degradation of 1192

phosphorylated OsJAZ4 and free OsJAZ11 by the 26S proteasome in an 1193

OsCOI1-dependent manner. The increased amount of OsGSK2 elevates the 1194

JA response but suppresses the BR response, thereby enhancing rice antiviral 1195

defense. Lines ending with arrows show activation, a solid line ending with a 1196

perpendicular line indicates suppression or an antagonistic interaction. 1197

1198

Figure 1. OsGSK2 positively regulates antiviral defense and JA response.

(A) Rice black-streaked dwarf virus (RBSDV) symptoms on Zh11, Go and Gi plants. Scale bar,

10 cm.

(B) Disease incidence in Zh11, Go and Gi plants following RBSDV inoculation. The numbers of

healthy and diseased plants in each treatment was determined by reverse transcription

polymerase chain reaction 30 days after inoculation and the number of diseased plants was

used to calculate the viral incidence (% plants infected). Each treatment used at least 40

seedlings, and at least three biological replicates were performed. Different letters at the top of

columns indicate significant difference between transgenic and control plants at p ≤ 0.05 by

Fisher's LSD tests.

(C) Expression levels of the RBSDV Coat Protein (CP) gene as measured by RT-qPCR at 30

dpi. Data are relative expression levels of CP in Go, Gi plants compared with that in the

wild-type Zh11 plants. OsUBQ5 was used as the internal reference gene. Error bars indicate

the SD of three biological replicates. *indicates significant difference between transgenic and

control plants at p ≤ 0.05 by Fisher's LSD test.

(D) Expression analysis of JA-responsive genes by RT-qPCR. Seven-day-old seedlings were

collected for total RNA extraction. OsUBQ5 was used as the internal reference gene. Values

are means ± SD of three biological replicates. * indicates significant difference between

transgenic and the control plants at p ≤ 0.05 by Fisher's LSD test.

(E) Levels of endogenous JA in seven-day-old Zh11, Go and Gi plants. The limit of

quantification to JA was 1 ng/ml. Values are means ± SD of three biological replicates.

Different letters at the top of columns indicate significant difference between transgenic and

control plants at p ≤ 0.05 by Fisher's LSD tests.

(F and G) Images (F) and quantification of root length (G) of Zh11, Go and Gi after MeJA

treatment. The root lengths of three-day-old seedlings grown in normal rice culture solutions

supplemented with indicated concentrations of MeJA were measured. Data shown are the

means from at least 10 seedlings for each indicated plant. Error bars represent SD. Different

letters at the top of columns indicate significant difference between transgenic and control

plants at p ≤ 0.05 by Fisher's LSD tests. Scale bar, 2 cm.

Figure 2. OsGSK2 interacts with OsJAZ4 in vitro and in vivo.

(A) Yeast two-hybrid assay showing the interaction between OsGSK2 and OsJAZ1-15 proteins.

Interactions were examined with SD base without Ade, His, Leu and Trp.

(B) Yeast two-hybrid assay showing the interaction between OsJAZ4 and OsGSK1-8 proteins.

Transformed yeast cells were grown on SD-Ade-His -Leu-Trp medium.

(C) Pull-down assay confirming that OsGSK2 interacts with OsJAZ4 in vitro. Immobilized GST

and GST-OsGSK2 were used to pull down His-OsJAZ4, and immunoprecipitated fractions

were detected using anti-His antibody. The bait proteins were probed with anti-GST antibody.

(D) BiFC assay showing the interaction between OsGSK2 and OsJAZ4 in N. benthamiana

leaves. OsJAZ11 was used as negative control. Scale bar, 20 µm.

(E) Co-IP assays showing the interaction between OsGSK2 and OsJAZ4 in vivo. The proteins

were extracted from N. benthamiana leaves and immunoprecipitated by anti-myc and anti-HA

magnetic beads, respectively. The coimmunoprecipitated proteins were probed by either

anti-Myc or anti-HA antibody. OsJAZ11-myc was used as negative control.

Figure 3. OsGSK2 phosphorylates OsJAZ4.

(A) Immunoprecipitated OsJAZ4-myc protein from OsJAZ4-MYC plants was treated with calf

intestinal alkaline phosphatase (CIP) or water. OsJAZ4-myc protein was separated in a

phos-tag SDS-PAGE gel and detected by anti-myc anti-body (top and middle panel). The

slowly migrating band (red arrow) of OsJAZ4-myc in the phos-tag gel with short exposure time

(Short exp.) or long exposure time (Long exp.) represents the phosphorylated form of OsJAZ4

(OsJAZ4-myc-P). As loading control (bottom panel), equal amounts of the immunoprecipitated

OsJAZ4-myc protein were separated in a normal SDS-PAGE gel followed by immunoblot

analysis.

(B) Potential phosphorylation sites in OsJAZ4.

(C) Immunoprecipitated OsJAZ4-myc and OsJAZ4Δ8-myc protein in N. benthamiana leaves

transiently co-expressed with HA-empty vector, HA-OsGSK2 or HA-OsGSK2K92R

. The protein

immunoprecipitated by anti-myc beads or CIP-treated OsJAZ4-myc protein from each

combination were separated in a phos-tag SDS-PAGE gel and detected by anti-myc antibody

(top panel). The slowly migrating band (red arrow) of OsJAZ4-myc in the phos-tag gel with

short exposure time (Short exp.) or long exposure time (Long exp.) is the phosphorylated form

of OsJAZ4 (OsJAZ4-myc-P). As loading controls, OsGSK2 (bottom panel) and different

amounts of the immunoprecipitated OsJAZ4-myc protein (middle panel) were separated in a

normal SDS-PAGE gel followed by immunoblot analysis.

Figure 4. OsGSK2 promotes OsJAZ4 degradation.

(A) Time course of OsJAZ4 degradation in wild-type Nipponbare (NIP) protein extracts treated

with GST, GST-OsGSK2 or GST-OsGSK2K92R

. Equal amounts of plant crude extracts were

added to equal amounts of the recombinant proteins in the in vitro cell-free degradation assays.

The Coomassie Brilliant Blue-stained Rubisco large subunit (Rbc L) was used as a loading

control.

(B) Quantification analysis of (A). The relative levels of OsJAZ4 in wild-type NIP plant protein

extracts at 0 h were defined as “1.” Data are means ± SE (n = 3).

(C) Time course of degradation of His-OsJAZ4 and His-OsJAZ4Δ8 in the wild-type NIP protein

extracts with or without MG132. Equal amounts of the recombinant proteins were incubated

with equal amounts of plant crude extracts in the in vitro cell-free degradation assays.

(D) Quantification analysis of (C). The relative levels of His-OsJAZ4 or His-OsJAZ4Δ8

incubated with wild-type NIP plant protein extracts at 0 h were defined as “1.” Data are means

± SE (n = 3).

(E) The protein levels of OsJAZ4 in Go, Gi and Zh11 leaves. The OsJAZ4 protein was

detected with anti-OsJAZ4 antibody and RbcL was used as a loading control. Two

independent pools of leaves are shown.

(F) Quantification analysis of (E). The relative level of OsJAZ4 in wild-type ZH11 was set as “1”.

Data are means ± SE (n = 3).

Figure 5. OsGSK2 affects OsJAZ4-OsJAZ11 interaction.

(A) Y2H assay shows that the ZIM domain of OsJAZ4 is responsible for binding to OsGSK2.

Schematic diagrams show the truncated versions of OsJAZ4. Interactions were examined with

SD base without Ade, His, Leu and Trp.

(B) Yeast two-hybrid assay shows the interaction between OsJAZ4 and OsJAZ1-15 proteins.

Transformed yeast cells were grown on SD-Ade -His -Trp-Leu medium.

(C) In vitro interaction between OsJAZ4-myc and His-JAZ11 is weakened by GST-OsGSK2.

His-OsJAZ11 protein combined with GST-OsGSK2 was incubated with immobilized

OsJAZ4-myc. The immunoprecipitated fractions were detected by anti-His antibody. The

gradient indicates increasing amount of GST-OsGSK2. OsJAZ4-myc input was probed with

anti-myc antibody, and the loading of His-OsJAZ11 and GST-OsGSK2 is shown in the lower

panel by Coomassie Brilliant Blue (CBB) Staining.

(D) HA-OsGSK2 affects accumulation of OsJAZ4-myc and OsJAZ11-myc. The respective

vectors were co-transiently expressed in the N. benthamiana leaves. The infiltrated leaves

were collected at 48 h after infiltration, and at least four plants were pooled. The Coomassie

Brilliant Blue-stained Rubisco large subunit (Rbc L) was used as a loading control.

(E) The protein levels of OsJAZ11 in Go, Gi and Zh11 plants. The OsJAZ11 protein was

detected with anti-OsJAZ11 antibody and Rbc L was used as a loading control. Two

independent pools of leaves are shown.

Figure 6. Effect of OsGSK2 on the OsJAZ4-OsNINJA interaction.

(A) OsNINJA and OsCOI1b interact with OsJAZ4. OsNINJA and OsCOI1b were fused to the

GAL4 DNA-binding domain (BD) while OsJAZ4 and its mutants were fused to the GAL4

activation domain (AD), respectively. Interactions were examined using SD base without Ade,

His, Leu and Trp. For the interactions between OsJAZ4 and OsCOI1b, 25 μM coronatine

(COR) was added.

(B) OsGSK2 competes with OsNINJA for binding to OsJAZ4. In vitro interaction between

OsJAZ4-myc and His-OsNINJA is weakened by GST-OsGSK2. His-OsNINJA protein

combined with GST-OsGSK2 was incubated with immobilized OsJAZ4-myc. The

immunoprecipitated fractions were detected by anti-His antibody. The gradient indicates

increasing amounts of GST-OsGSK2. OsJAZ4-myc input was probed with anti-myc antibody,

and the loading of His-OsNINJA and GST-OsGSK2 is shown in the lower panel by Coomassie

Brilliant Blue (CBB) Staining.

Figure 7. OsJAZ4 negatively modulates JA signaling and rice immunity.

(A) JA-responsive gene expression in indicated transgenic plants. RT-qPCR analysis of the

mRNA levels of JA-responsive genes in the wild-type Nipponbare (NIP), OsJAZ4-OE lines (#1

and #3) and OsJAZ4-RNAi lines (#14 and #18). OsUBQ5 was used as the internal reference

gene. Values are means ± SE of three biological replicates. * indicates significant difference at

p ≤ 0.05 (n = 3) by Fisher's LSD tests.

(B and C) Images (B) and quantification of root length (C) in indicated plants following MeJA

treatment. The root lengths of three-day-old seedlings grown in normal rice culture solutions

supplemented with different concentrations of MeJA were measured. Data shown are the

means from at least 15 seedlings for each plant type. Error bars represent SD. Different letters

at the top of columns indicate significant difference at p ≤ 0.05 by Fisher's LSD tests. Scale bar,

2 cm.

(D) Disease incidence. The numbers of healthy and diseased plants in each treatment was

determined by RT-PCR 30 days after inoculation and the number of the diseased plants was

used to calculate the viral incidence (% plants infected). Each treatment used at least 40

seedlings, and at least three biological replicates were performed. Different letters at the top of

columns indicate significant difference between transgenic and control plants at p ≤ 0.05 by

Fisher's LSD tests.

(E) The relative expression levels of RBSDV Coat Protein (CP) gene measured by RT-qPCR

at 30 dpi. Data represent relative expression levels of the CP gene in the mutant compared

with that in the wild-type NIP plants. OsUBQ5 was used as the internal reference gene. Error

bars represent SD. * indicates significant difference at p ≤ 0.05 (n ≥ 3) by Fisher's LSD tests.

(F) Viral symptoms in OsJAZ4-OE lines (#1 and #3) and OsJAZ4-RNAi lines (#14 and #18).

Scale bar, 10 cm.

Figure 8. Effect of OsGSK2-OsJAZ4 interaction on JA- and BR- pathway crosstalk.

(A and B) OsJAZ4 accumulation increases in response to BL (brassinolide, A) or Bikinin (B)

treatment. The leaves of seven-day-old wild-type Nipponbare (NIP) seedlings were treated

with 1 μM BL or 20 μM Bikinin, and protein was extracted from the treated leaves 0, 3, 6 or 12

h later. The Coomassie Brilliant Blue-stained Rubisco large subunit (Rbc L) was used as a

loading control. Two independent pools of leaves are shown.

(C and D) The accumulation of OsJAZ4 induced by BL and Bikinin was inhibited in coi1-13

mutants. Leaves of seven-day-old wild-type NIP and coi1-13 seedlings were treated with 1 μM

BL (C) or 20 μM Bikinin (D), and the treated leaves were used for protein extraction at different

times after treatment, and detected by Anti-OsJAZ4 antibody. Rbc L was used as a loading

control. Two independent pools of leaves are shown.

(E) Effect of Bikinin on MeJA hypersensitivity in OsJAZ4-RNAi plants. Germinated seeds were

grown in normal rice culture solutions containing 0 or 1 μM MeJA, with or without 200 μM

Bikinin for 5 days and the root length was then measured. Relative root elongation is

expressed as a percentage of root elongation in solutions with (right section) or without (left

section) 200 μM Bikinin. Error bars represent SD (n≥20). Different letters at the top of columns

indicate significant difference at p ≤ 0.05 by Fisher's LSD tests.

(F) BL sensitivity test of the wild type NIP, OsJAZ4-OE lines (#1 and #3) and OsJAZ4-RNAi

lines (#14 and #18) by lamina joint assay. The plus and minus symbols indicate with/without

BL (100 ng).

(G) Quantification of the data shown in (E). Data shown are the means from at least 15

seedlings for each indicated plant. Error bars represent SE. Different letters at the top of

columns indicates significant difference at p ≤ 0.05 by Fisher's LSD tests.

Figure 9. Model of OsGSK2-mediated plant defense signaling in rice.

OsGSK2 binds to OsJAZ4 to disrupt OsJAZ4-OsNINJA corepressor and OsJAZ4-OsJAZ11

dimerization, which promotes the degradation of phosphorylated OsJAZ4 and free OsJAZ11

by the 26S proteasome in an OsCOI1-dependent manner. The increased amount of OsGSK2

elevates the JA response but suppresses the BR response, thereby enhancing rice antiviral

defense. Lines ending with arrows show activation, a solid line ending with a perpendicular line

indicates suppression or an antagonistic interaction.

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https://www.ncbi.nlm.nih.gov/pubmed?term=Bishop%2C G%2E%2C and Koncz%2C C%2E %282002%29%2E Brassinosteroids and plant steroid hormone signaling%2E Plant Cell&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Bishop%2C J%2E%2C Jones%2C H%2EE%2E%2C O%27Sullivan%2C D%2EM%2E%2C and Potts%2C S%2EG%2E %282017%29%2E Elevated temperature drives a shift from selfing to outcrossing in the insect%2Dpollinated legume%2C faba bean %28Vicia faba%29%2E Journal of Experimental Botony&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Cai%2C Z%2E%2C Liu%2C J%2E%2C Wang%2C H%2E%2C Yang%2C C%2E%2C Chen%2C Y%2E%2C Li%2C Y%2E%2C Pan%2C S%2E%2C Dong%2C R%2E%2C Tang%2C G%2E%2C and Barajaslopez%2C J%2ED%2ED%2E %282014%29%2E GSK3%2Dlike kinases positively modulate abscisic acid signaling through phosphorylating subgroup III SnRK2s in Arabidopsis%2E Proceedings of the National Academy of Sciences of the United States of America&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Chen%2C C%2EY%2E%2C Liu%2C Y%2EQ%2E%2C Song%2C W%2EM%2E%2C Chen%2C D%2EY%2E%2C Chen%2C F%2EY%2E%2C Chen%2C X%2EY%2E%2C Chen%2C Z%2EW%2E%2C Ge%2C S%2EX%2E%2C Wang%2C C%2EZ%2E%2C Zhan%2C S%2E%2C Chen%2C X%2EY%2E%2C and Mao%2C Y%2EB%2E %282019%29%2E An effector from cotton bollworm oral secretion impairs host plant defense signaling%2E Proceedings of the National Academy of Sciences of the United States of America&dopt=abstract

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DOI 10.1105/tpc.19.00499; originally published online June 25, 2020;Plant Cell

Zongtao SunJia Wei, Junmin Li, Fei Yan, Pengcheng Wang, Hongning Tong, Chengcai Chu, Jianping Chen and

Yuqing He, GaoJie Hong, Hehong Zhang, Xiaoxiang Tan, Lulu Li, Yaze Kong, Tian Sang, kaili Xie,OsJAZ4

The OsGSK2 Kinase Integrates Brassinosteroid and Jasmonic Acid Signaling by Interacting with

This information is current as of September 20, 2020

Supplemental Data /content/suppl/2020/07/02/tpc.19.00499.DC1.html

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The OsGSK2 Kinase Integrates Brassinosteroid and Jasmonic Acid Signaling … · 2020-06-25 · Plant Cell Advance Publication. Published on June 25, 2020, ... INTRODUCTION. 60. Brassinosteroids