The Chemical Characteristic and Distribution of Brassinosteroids in Plants
The OsGSK2 Kinase Integrates Brassinosteroid and Jasmonic Acid Signaling … · 2020-06-25 ·...
Transcript of The OsGSK2 Kinase Integrates Brassinosteroid and Jasmonic Acid Signaling … · 2020-06-25 ·...
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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
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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
OsJAZ4, Os09g23660; OsJAZ5, Os04g32480; OsJAZ6, Os03g28940; 701
OsJAZ7, Os07g42370; OsJAZ8, Os09g26780; OsJAZ9, Os03g08310; 702
OsJAZ10, Os03g08330; OsJAZ11, Os03g08320; OsJAZ12, Os10g25290; 703
OsJAZ13, Os10g25230; OsJAZ14, Os10g25250; OsJAZ15, Os03g27900; 704
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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990
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
loading control. Two independent pools of leaves are shown. 1114
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
loading control. Two independent pools of leaves are shown. 1174
(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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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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