The Type III Effector AvrBs2 in Xanthomonas oryzae pv. oryzicola Suppresses Rice Immunity and Promotes Disease Development.

Page 1 of 83 Li et al. MPMI

1

The Type III Effector AvrBs2 in Xanthomonas oryzae pv. oryzicola Suppresses

2

Rice Immunity and Promotes Disease Development

Molecular Plant-Microbe Interactions "First Look" paper • http://dx.doi.org/10.1094/MPMI-10-14-0314-R • posted 02/17/2015 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ.

3 4

Shuai Li, Yanping Wang, Shanzhi Wang, Anfei Fang, Jiyang Wang, Lijuan Liu, Kang

5

Zhang, Yuling Mao and Wenxian Sun*

6 7

Department of Plant Pathology, China Agricultural University, Beijing 100193, China;

8

Key Laboratory of Plant Pathology, Ministry of Agriculture, China Agricultural

9

University, Beijing 100193, China

10 11

*

12

Department of Plant Pathology

13

China Agricultural University

14

2 West Yuanmingyuan Rd., Haidian District

15

Beijing 100193, China

16

Telephone: +86 10 6273 3532;

17

Fax: +86 10 6273 3532;

18

E-mail: [email protected]

Correspondence: Wenxian Sun

19 20 21

Running title: AvrBs2 in Xoc suppresses rice immunity

22

1



23 24

Xanthomonas oryzae pv. oryzicola (Xoc), the causal agent of bacterial leaf streak,

25

is one of the most important bacterial pathogens in rice. However, little is known

26

about the function(s) of individual type III effectors in Xoc virulence and

27

pathogenicity. Here, we examined the effect of the mutations of 23 putative

28

non-transcription activator-like effector genes on Xoc virulence. The avrBs2

29

knock-out mutant was significantly attenuated in virulence to rice. In contrast, the

30

xopAA deletion caused enhanced virulence to a certain rice cultivar. It was also

31

demonstrated that six putative effectors including XopN, XopX, XopA, XopY, XopF1

32

and AvrBs2 caused the hypersensitive response on non-host Nicotiana benthamiana

33

leaves. Virulence function of AvrBs2 was further confirmed by transgenic technology.

34

Pathogen-associated molecular pattern-triggered immune responses including the

35

generation of reactive oxygen species and expression of pathogenesis-related genes

36

were strongly suppressed in the AvrBs2-expressing transgenic rice lines. Although not

37

inhibiting flg22-induced activation of mitogen-activated protein kinases, heterologous

38

expression of AvrBs2 greatly promotes disease progression in rice caused by two

39

important bacterial pathogens X. oryzae pvs. oryzae and oryzicola. Collectively, these

40

results indicate that AvrBs2 is an essential virulence factor that contributes to Xoc

41

virulence through inhibiting defense responses and promoting bacterial multiplication

42

in monocot rice.

43 44

2

Page 3 of 83


Li et al. MPMI

45

Plants possess multiple layers of preformed and induced defenses to protect

46

themselves from pathogen attack (Jones and Dangl 2006). During plant-pathogen

47

co-evolution,

48

pathogen-associated molecular patterns (PAMPs) such as bacterial flagellin and

49

fungal chitin and thus trigger the first layer of defense responses, called

50

PAMP-triggered immunity (PTI). PTI includes a series of defense signal transduction

51

cascades that lead to callose deposition, burst of reactive oxygen species (ROS),

52

induced expression of pathogenesis-related (PR) genes, phytoalexin accumulation and

53

the deposition of phenolic compounds (Chisholm et al. 2006; Jones and Dangl 2006).

54

Many Gram-negative phytopathogenic bacteria, such as Pseudomonas and

55

Xanthomonas, translocate a large number of type III effectors (T3Es) into the host

56

cells through the type III secretion system (T3SS) (Kay and Bonas 2009). The

57

secreted T3Es often target essential components in the PTI signaling pathway, such as

58

PRRs, mitogen-activated protein kinases (MAPKs) and transcription factors to

59

interfere with or suppress the plant immune system (Boller and He 2009; Dou and

60

Zhou 2012; Xin and He 2013). Accordingly, plants have evolved to specifically

61

recognize certain T3Es by the intracellular cognate resistance (R) proteins to initiate

62

the second layer of defenses, referred to as effector-triggered immunity (ETI). As

63

compared with PTI, ETI is more robust and effective and is often characteristic of the

64

hypersensitive response (HR) at the invasion site that inhibits pathogen multiplication

65

(Chisholm et al. 2006; Jones and Dangl 2006).

66

pattern

recognition

receptors

(PRRs)

in

plants

recognize

As a major group of virulence factors, the type III effectors and their secretion

3

Page 4 of 83


Li et al. MPMI

67

apparatus encoded by the hypersensitive reaction and pathogenicity (hrp) gene cluster

68

are essential for pathogenesis and virulence of phytopathogenic bacteria (Alfano and

69

Collmer 2004; Kay and Bonas 2009). In Xanthomonas, HrpX is a key hrp regulatory

70

factor and regulates expression of some T3E genes through binding to the PIP

71

(plant-inducible promoter) boxes (Furutani et al. 2006; Li et al. 2011). The PIP-box is

72

a conserved cis-element consisting of the consensus sequence TTCGB-N15-TTCGB

73

(‘B’ represents any base except adenine) that is located at 30-32 bases upstream of the

74

start codon of the effector gene (Furutani et al. 2006; Tsuge et al. 2006). The T3Es

75

usually carry a secretion signal at their N-terminus, which often contains 50 specific

76

amino acid residues (Alfano and Collmer 2004; Cunnac et al. 2004).

77

The T3Es in xanthomonads, also called Xanthomonas outer proteins (Xops), are

78

categorized into up to 39 protein families based on sequence similarity, reflecting

79

genetic diversity of the virulence factor inventory in the pathogens (White et al. 2009).

80

A group of T3Es unique to Xanthomonas are called transcription activator-like (TAL)

81

effectors (Scholze and Boch 2011) and others are grouped into non-TAL effectors

82

(Furutani et al. 2009; White et al. 2009). Various biological functions of T3Es in

83

phytopathogenic bacteria, especially in Pseudomonas, have been recently revealed in

84

succession (Xin and He 2013). Some effectors, such as AvrPtoB, function as E3

85

ubiquitin ligases and promote the degradation of immunity-related proteins in a

86

proteasome-dependent manner (Rosebrock et al. 2007); Some effectors, such as the

87

YopJ/AvrRxv family, target the proteasomal subunit to inhibit the proteasome activity

88

(Üstün et al. 2013); Some effectors, such as HopAO1, alter the phosphorylation state

4

Page 5 of 83


Li et al. MPMI

89

of host proteins (Li et al. 2013; Underwood et al. 2007); Other effectors, especially for

90

TAL effectors including the AvrBs3/PthA family, act as transcription factors that

91

regulate expression of susceptibility genes in hosts and promote bacterial spreading

92

and multiplication (Boch and Bonas 2010; Pereira et al. 2014).

93

In recent years, intensive research has been performed to study function(s) of the

94

Xop proteins. Besides TAL effectors, some non-TAL effector genes including xopR,

95

xopN and xopZ have been demonstrated to be required for full virulence in X. oryzae

96

pv. oryzae (Xoo) (Akimoto-Tomiyama et al. 2012; Sinha et al. 2013; Song and Yang

97

2010). The X. campestris pv. vesicatoria (Xcv) effector XopX contributes to Xcv

98

virulence in host pepper and tomato plants (Metz et al. 2005). Several host targets of

99

the Xops have been identified recently. XopAA and XopY in Xoo interact with

100

OsSERK1/2 and OsRLCK185 to inhibit rice immunity, respectively (Yamaguchi et al.

101

2013a; Yamaguchi et al. 2013b). The XopN homologs target a zinc finger

102

domain-containing protein OsVOZ2 and a putative thiamine synthase OsXNP in rice

103

(Cheong et al. 2013), and the atypical receptor-like kinase TARLK1 in tomato to

104

promote pathogenicity, respectively (Kim et al. 2009). In Xcv, XopD functions as a

105

SUMO protease and interacts with the transcription factor SlERF4 to inhibit ethylene

106

production and to promote pathogen growth in tomato (Kim et al. 2008; Kim et al.

107

2013). It has been recently shown that XopQ in Xcv suppresses ETI by targeting the

108

tomato 14-3-3 isoform SlTFT4, an important component of ETI (Teper et al. 2014).

109

The effector AvrAC secreted by X. campestris pv. campestris (Xcc) functions as a

110

uridylyl transferase that transfers uridine 5´-monophosphate to and masks conserved

5

Page 6 of 83


Li et al. MPMI

111

phosphorylation sites in the activation loop of BIK1 and RIPK, and thus preventing

112

activation of these kinases and suppressing defense signaling in host (Feng et al.

113

2012). Most recently, XopD in Xcc has been demonstrated to target DELLA proteins

114

in Arabidopsis to trigger disease tolerance and increase bacterial survival (Tan et al.

115

2014).

116

Bacterial leaf streak (BLS), caused by X. oryzae pv. oryzicola (Xoc), is currently

117

one of the most serious bacterial diseases in rice and causes significant yield loss in

118

rice-growing regions of South Asia and South China (Niño-Liu et al. 2006). Due to

119

similar biochemical characteristics and disease symptoms on rice leaves, the disease

120

was previously considered to be bacterial blight. Subsequent studies revealed

121

significant differences in infection style and pathogenicity between Xoo and Xoc

122

(Bogdanove et al. 2011; Vauterin et al. 1995). Xoc enters and colonizes the

123

intercellular spaces between mesophyll cells in rice leaves through stomata or wounds.

124

In contrast, Xoo infects rice leaves through vascular tissues and causes a systemic

125

disease (Niño-Liu et al. 2006). Therefore, molecular mechanisms underlying

126

virulence and pathogenicity might be significantly different between Xoo and Xoc.

127

The inventories of effector proteins in many Xanthomonas species have been

128

predicted

129

(http://www.xanthomonas.org/) (Bogdanove et al. 2011). As mentioned above,

130

functional studies on Xops focus on only a few Xanthomonas species including Xoo,

131

Xcc and Xcv. As compared with ever-increasing reports on Xoo effectors, little is

132

known about the function(s) of Xoc effector proteins, particularly for non-TAL

from

publicly

available

genome

6

sequences

of

these

species



133

effectors.

134

AvrBs2, the first reported non-TAL effector in Xcv (Kearney and Staskawicz 1990),

135

is highly conserved in xanthomonads (Swords et al. 1996). AvrBs2 was characterized

136

as an avirulence protein that is recognized by the R protein Bs2 in pepper (Gassmann

137

et al. 2000; Tai et al. 1999). Computational and biochemical evidence showed that

138

AvrBs2 contains a glycerophosphodiesterase catalytic domain that is essential for its

139

virulence function but not for activation of Bs2-mediated disease resistance (Zhao et

140

al. 2011). Functional studies on AvrBs2 have been focused on the dicot plants. In

141

contrast, no experiment has been performed to determine whether avrBs2 in X. oryzae

142

pv. oryzicola is required for bacterial virulence to the monocot rice so far.

143

In this study, we performed mutational analyses of 23 putative Xops in the Xoc

144

strain RS105, revealing that the effector gene avrBs2 is required for full virulence of

145

Xoc. It was also demonstrated that multiple effectors including XopN, XopA, XopX,

146

XopF1, XopY and AvrBs2 triggered HR in non-host N. benthamiana to different

147

degrees. Ectopic expression of AvrBs2 in transgenic rice plants inhibited defense

148

responses including flg22- and chitin-triggered ROS burst and PR gene expression,

149

and importantly, promoted disease progression after infection by rice bacterial

150

pathogens.

151 152

RESULTS

153 154

Construction of gene-deletion mutants for 23 putative non-TAL effector genes in

7



155

X. oryzae pv. oryzicola

156

The effector genes in Xoc have been predicted based on the available genome

157

sequence of the Xoc strain BLS256 (Bogdanove et al. 2011). Among them, at least 26

158

genes were predicted to encode non-TAL effectors. The degree of conservation of

159

these effectors was predicted and compared among Xanthomonas species

160

(Supplementary Fig. S1), and the location of these genes in the genome and the

161

sequences of the PIP-boxes were also identified (Supplementary Table S1). The

162

majority of non-TAL effectors are highly conserved although some effector genes

163

have presence/absence polymorphisms in Xanthomonas species (Bogdanove et al.

164

2011). A total of 11 non-TAL effector families are widely distributed in genome

165

sequence-available Xanthomonas species and thus considered as core effector proteins

166

in xanthomonads (Supplementary Fig. S1). A few effector genes including xopU,

167

xopW, xopY and xopAB are present exclusively in Xanthomonas oryzae pathvars; The

168

xopT and xopAF genes only exist in Xoo and Xoc, respectively; The xopO and xopAJ

169

genes exist in both Xoc and Xcv. Among the 26 non-TAL effectors, the only XopAF is

170

unique to Xoc.

171

To initially understand the roles of putative non-TAL effector genes in Xoc

172

virulence, marker-free deletion mutants of the 23 effector genes were constructed via

173

homologous recombination as described in Materials and Methods. The xopA and

174

hpaA were excluded in mutational analyses since they most likely encode a Harpin

175

and a T3S control protein, respectively, which might not be T3Es; and xopAJ, also

176

called as avrRxo1, was not selected because of failure to make the avrRxo1 knock-out

8

Page 9 of 83


Li et al. MPMI

177

mutant in previous study (Zhao et al. 2004). The mutant strains were used for

178

subsequent functional studies after being confirmed through Southern blot analyses

179

(Supplementary Fig. S2 and S3).

180 181

The mutations of avrBs2 and xopAA cause altered Xoc virulence to rice

182

To determine whether the putative effector genes are required for full virulence

183

of Xoc to rice, all constructed marker-free gene-deletion mutants were pressure

184

inoculated into rice leaves for virulence assays. Virulence of each mutant strain was

185

evaluated by measuring the length of disease lesions at 2 weeks after inoculation as

186

compared with the wild-type strain. The results demonstrated that all gene-deletion

187

mutants except ∆avrBs2 and ∆xopAA exhibited no or little alteration in Xoc virulence

188

under experimental conditions used in the study (Fig. 1A). Disease lesions in the

189

∆avrBs2-inoculated leaves were much shorter than those caused by the wild-type

190

strain (Fig. 1A and Supplementary Fig. S4A). The complementation strain with the

191

plasmid-borne full-length avrBs2 gene largely restored virulence of the ∆avrBs2

192

mutant towards the wild-type level. Enumeration of bacteria extracted from the

193

inoculated rice leaves clearly showed that the in planta population size of ∆avrBs2

194

was much smaller than that of the wild-type and complementation strains during

195

infection (Fig. 1B). Taken together, the data indicate that AvrBs2 is one of the major

196

virulence factors and plays an essential role in virulence and multiplication of Xoc in

197

rice. Interestingly, the ∆xopAA mutant caused significantly longer disease lesions on

198

the leaves of rice cv. Jingang 30 than the wild-type strain (Fig. 1A and Supplementary

9

Page 10 of 83


Li et al. MPMI

199

Fig. S4B). The population size of ∆xopAA was also larger than that of the wild-type

200

strain in the inoculated rice leaves since 8 d after inoculation (Fig. 1C). Introduction

201

of the plasmid-borne xopAA gene reduced virulence of the ∆xopAA mutant to the

202

wild-type level (Fig. 1B, 1C). Virulence of the xopAA mutant to rice was further

203

tested using the other two rice cultivars Nipponbare and Jinhe 2. The results showed

204

that the xopAA mutant and wild-type strains caused almost equal length of disease

205

lesions on these cultivars (Supplementary Fig S5A and S5B). The data suggest that

206

XopAA in Xoc triggers defense responses in a certain rice genetic background.

207 208

The hypersensitive response triggered by putative Xoc effectors on non-host

209

Nicotiana benthamiana leaves

210

X. oryzae pv. oryzicola causes non-host hypersensitive cell death in N.

211

benthamiana. In order to screen which effector candidates have cell death eliciting

212

activities in N. benthamiana, 22 putative effectors except XopAB, XopZ1, XopAA

213

and XopO were transiently expressed in N. benthamina by A. tumefaciens-mediated

214

expression system (Supplementary Table S1). The cell death symptoms were

215

monitored within 3 days after treatment with dexamethasone (DEX), which induced

216

expression of these effector genes. The infiltrated leaf area expressing XopN became

217

necrotic at 1 d after DEX treatment. Expression of XopX and XopA caused similar

218

HR symptoms at ~2 d, while XopF1, XopY and AvrBs2 induced cell death

219

phenotypes at ~3 d. XopN and XopX caused HR on all of 20 inoculated leaves, while

220

XopA, XopY, XopF1 and AvrBs2 induced HR at relatively lower degrees (Fig. 2A).

10


221

Other tested putative effectors did not trigger cell death symptoms in N. benthamiana,

222

although expression of those proteins was all confirmed by Western blotting (Fig. 2B).


223 224

Flg22-induced immunity is suppressed by transient expression of AvrBs2 in

225

Arabidopsis protoplasts

226

The expression of NONHOST1 (NHO1), an essential gene for nonhost resistance,

227

is induced by flg22 in Arabidopsis. It has been well demonstrated that flg22-induced

228

NHO1 expression was inhibited by different type III effectors in Arabidopsis

229

protoplasts (Li et al. 2005; Lu et al. 2001). Transient expression assays in Arabidopsis

230

protoplasts were used herein to investigate if AvrBs2 can suppress flg22-induced

231

NHO1 expression. Protoplasts isolated from the NHO1-luciferase (NHO1-LUC)

232

transgenic Arabidopsis plants were transfected with an empty vector and the hopAI1

233

construct as negative and positive controls, respectively. As shown in Fig. 3A, LUC

234

expression driven by the NHO1 promoter was greatly induced by flg22 in protoplasts

235

transfected with empty vector. Transfection of the avrBs2 construct nearly abolished

236

flg22-induced LUC expression, which was compared to transfection of hopAI1. These

237

data suggest that transient expression of AvrBs2 in protoplasts strongly suppresses

238

flg22-induced expression of defense genes.

239

In order to determine subcellular localization of AvrBs2 in host cells, GFP was

240

fused in frame with the avrBs2 coding sequence at its C terminus in the pUC19

241

plasmid with the 35S promoter. The recombinant AvrBs2-GFP protein was transiently

242

expressed in rice protoplasts. Green fluorescence of AvrBs2-GFP was detected across

11

Page 12 of 83


Li et al. MPMI

243

the entire cell, which is similar to subcellular localization of AvrBs2-GFP in N.

244

benthamiana (Fig. 3B and Supplementary Fig. S6). Transient expression of

245

AvrBs2-GFP in N. benthamiana caused HR symptoms, indicating that the

246

AvrBs2-GFP fusion protein is functional (data not shown).

247 248

Ectopic expression of AvrBs2 suppresses PTI signaling in rice cell cultures

249

To investigate if AvrBs2 suppresses plant immunity in rice, the transgenic rice

250

cell culture with heterologous expression of AvrBs2-FLAG driven by the

251

DEX-inducible promoter was generated in the genetic background of rice cv.

252

Nipponbare. Induced expression of avrBs2-FLAG in the transgenic rice cells was

253

confirmed by quantitative real-time RT-PCR (data not shown) and immunoblotting

254

(Fig. 4A). As virulence factors, the effectors secreted by plant pathogens often

255

suppress defense responses including PAMP-triggered ROS burst, PR gene

256

expression and MAPK activation (Boller and He 2009; Dou and Zhou 2012). Previous

257

studies revealed that rice senses the PAMPs flg22 and chitin through OsFLS2 (Takai

258

et al. 2008) and through synergetic action of CEBiP and CERK1 (Shimizu et al. 2010),

259

respectively. Here, we measured ROS accumulation in the avrBs2 transgenic rice cell

260

culture after DEX treatment followed by flg22 and chitin stimulation using a highly

261

sensitive ROS detection method. ROS bursts were clearly detected in the transgenic

262

cell cultures with mock treatment in response to flg22 and chitin (Fig. 4B and 4C). In

263

contrast, ROS accumulation induced by flg22 was reduced up to ~60% and

264

chitin-induced ROS was inhibited even more dramatically in the avrBs2 transgenic

12

Page 13 of 83


Li et al. MPMI

265

cell cultures after DEX treatment (Fig. 4B and 4C). Furthermore, the defense marker

266

genes OsPBZ1 and OsPAL1 were demonstrated to be up-regulated by flg22 and chitin

267

in the transgenic rice cell cultures with mock treatment. Remarkably, induced

268

expression of the two defense genes was almost completely blocked in the transgenic

269

rice cells after DEX-induced expression of AvrBs2 (Fig. 4D and 4E). These data

270

indicate that AvrBs2 contributes to Xoc virulence by suppressing PAMP-triggered

271

defense responses in rice.

272 273

Ectopic expression of AvrBs2 suppresses PTI signaling and promotes disease

274

progression in rice plants

275

To further confirm virulence function(s) of Xoc AvrBs2 in suppressing plant

276

immunity in rice, stably transformed transgenic rice plant lines with DEX-inducible

277

expression of AvrBs2-FLAG were created. Among 8 independent transgenic lines, the

278

two T2 homozygous lines A4 and A5 were selected for subsequent functional studies.

279

The transgenic A5 line with a low leaked expression had a lower expression level of

280

AvrBs2 than the A4 line after DEX treatment (Fig. 5A). In consistent with the results

281

in transgenic cell cultures, we showed that flg22 and chitin induced expression of

282

OsPBZ1 and OsPAL1 in the mock-treated transgenic plant leaves and the induction

283

was dramatically suppressed by DEX-induced expression of AvrBs2 (Fig. 5B and 5C).

284

In contrast, MAPK activation induced by flg22 or chitin was not significantly altered

285

by AvrBs2 expressed in the transgenic plants after DEX treatment (Supplementary

286

Fig. S7).

13

Page 14 of 83


Li et al. MPMI

287

It is important to investigate whether AvrBs2 expression in the transgenic plants

288

promotes disease development during the infection of bacterial pathogens. Bacterial

289

disease susceptibility in the avrBs2 transgenic plants was first evaluated after Xoc

290

inoculation. Disease symptoms observed on the inoculated leaves showed that disease

291

lesions in the transgenic rice lines A4 and A5 were significantly longer after DEX

292

spraying as compared with those under mock treatment. The lesion length on the A4

293

transgenic line was longer than that on the A5 line, which is in consistent with the

294

higher AvrBs2 expression level in the A4 line after DEX induction (Fig. 5A, 5D and

295

Supplementary Fig. S8A). Bacterial growth curve assays demonstrated that the

296

population size of Xoc in the transgenic lines upon DEX treatment was also

297

significantly higher than that in the wild-type plant at 12 d after pathogen inoculation

298

(Fig. 5E). Meanwhile, these transgenic plants, when treated with DEX, also exhibited

299

more severe disease symptoms with longer disease lesions than the wild-type plants

300

after X. oryzae pv. oryzae inoculation (Fig. 5F and Supplementary Fig. S8B).

301

Collectively, these results indicate that AvrBs2 expression in the transgenic rice plants

302

disrupts plant basal defenses during the compatible interaction and thus promotes

303

bacterial multiplication and disease symptom development.

304 305

DISCUSSION

306

Rice, one of the staple food crops, is subject to infection of various pathogenic

307

microbes. Xoc causes bacterial leaf streak (BLS), which is currently one of the most

308

serious bacterial diseases in rice and has a great threat to commercial production (Xue

14

Page 15 of 83


Li et al. MPMI

309

et al. 2014). Breeding for disease resistance in rice is considered as the most effective,

310

environmentally friendly approach to controlling the disease. However, no

311

endogenous BLS resistance gene has been identified in rice so far, although a bunch

312

of resistance genes are available for bacterial blight (Niño-Liu et al. 2006). The lack

313

of BLS resistant germplasm in rice might be partially explained by the finding that R

314

gene-mediated resistance is suppressed by the T3Es secreted by Xoc (Makino et al.

315

2006). Therefore, it is important to study the effect of individual effectors on Xoc

316

virulence for better understanding of molecular mechanisms underlying Xoc virulence

317

and pathogenicity. In this study, we investigated the contribution of each non-TAL

318

effector gene to Xoc virulence in a genome-wide analysis. Among 23 tested

319

gene-deletion mutants of the Xoc RS105 strain, the avrBs2 mutation is the only one

320

that leads to reduced virulence of Xoc to rice while the deletion of xopAA enhanced

321

Xoc virulence exclusively to rice cv. Jingang 30. It is notable that the mutational study

322

did not include the previously identified effector genes hrpE3 and Xrp5 that have been

323

demonstrated to be required for full virulence of Xoc (Cui et al. 2013; Xue et al. 2014).

324

Meanwhile, we revealed that six putative effectors caused the hypersensitive response

325

in the non-host N. benthamiana plants through Agrobacterium-mediated transient

326

gene expression.

327

Most of the mutations of individual non-TAL effector genes investigated here did

328

not alter Xoc virulence, which is consistent with previous reports on plant bacterial

329

pathogens. No non-TAL effectors but one TAL effector were identified to be required

330

for Xoc virulence through a large-scale screening of Tn5-tagged Xoc mutant library.

15

Page 16 of 83


Li et al. MPMI

331

In contrast, the study showed that many hrp genes are essential for its virulence (Guo

332

et al. 2012). Virulence assays of the deletion mutants of 18 individual Xop genes in

333

Xoo revealed that XopZ is the only effector that is required for full virulence of Xoo in

334

the susceptible host plant (Song and Yang 2010). Similarly, only xopN mutation out

335

of the tested 7 effector genes in Xcv has a detectable phenotypic effect on the

336

pathogenicity (Roden et al. 2004). In addition, the mutants with disruption of eight

337

Xcc Xop genes individually or collectively did not exhibit any alteration in virulence

338

as compared with the wild-type strain on susceptible host plants (Castaneda et al.

339

2005). Experimental studies on other phytopathogenic bacteria also revealed that

340

individual effector genes are often dispensable in pathogenesis of Pseudomonas

341

syringae and Ralstonia solanacearum (Cunnac et al. 2004; Kvitko et al. 2009).

342

Function redundancy of multiple effectors in one pathogenic organism is speculated to

343

be a major factor that prevents detecting obvious phenotypic effect of individual

344

effector mutations on host responses to pathogen infection. The hypothesis is

345

supported by the findings that the same host targets have been identified for different

346

sets of effectors in plant bacterial pathogens (Xin and He 2013). For instance, it was

347

demonstrated that both of the effectors HopZ1a and HopX1 in P. syringae with

348

different biochemical functions target to and promote degradation of JAZ proteins and

349

contribute to bacterial virulence (Gimenez-Ibanez et al. 2014; Jiang et al. 2013). On

350

the other hand, we cannot rule out another explanation. The effect on Xoc virulence of

351

individual gene deletions might not be detected under experimental conditions used in

352

this study. Some effectors have been shown to suppress pre-invasive immunity, such

16

Page 17 of 83


Li et al. MPMI

353

as stomatal closure (Lozano-Duran et al. 2014). However, the pressure infiltration

354

method used here for evaluating Xoc virulence most likely bypasses the first layer of

355

rice immunity, such as stomata defenses, which might be triggered before or during

356

the natural entry of the pathogen into host extracellular spaces (Zeng et al. 2011).

357

Many effector proteins secreted by plant pathogens are known to have multiple

358

activities, e.g. functioning as a virulence factor in the susceptible host plants, acting as

359

an avirulence protein in the presence of its cognate resistance protein, or triggering

360

HR in the nonhost plants (Gohre and Robatzek 2008). Here, we investigated the HR

361

eliciting activity of most of Xoc putative effectors in the nonhost N. benthamiana

362

plants using a heterogeneous expression system and revealed that at least six putative

363

effectors including XopN, XopX, XopA, XopY, XopF1 and AvrBs2 caused the

364

nonhost HR (Fig. 2). Among them, XopN, XopX, XopY and AvrBs2 have been well

365

demonstrated to suppress plant immunity in other Xanthomonas species (Kim et al.

366

2009; Metz et al. 2005; Tai et al. 1999; Yamaguchi et al. 2013b). However, the

367

functions of these effectors in Xoc virulence need to be further elucidated.

368

Interestingly, the xopAA deletion mutant was found to have an enhanced virulence

369

to rice cv. Jingang 30, suggesting that XopAA secreted by Xoc induces immune

370

responses in rice during infection (Fig. 1). Similar phenomenon has been reported for

371

the xopQ mutant of Xoc, which exhibited an elevated virulence to rice cv. IR28 (Pei et

372

al. 2010). We also demonstrated that the xopAA mutant was not more virulent than the

373

wild-type strain to other rice cultivars (Supplementary Fig. S5). In addition, XopAA is

374

relatively less conserved and missing in Xcc and X. axonopodis pv. citri as compared

17

Page 18 of 83


Li et al. MPMI

375

with core effectors in xanthomonads (Supplementary Fig. S1). Therefore, XopAA is

376

less likely recognized as a conserved signature given that PAMPs and PTI signaling

377

are usually highly conserved within species (Thomma et al. 2011). Presumably,

378

XopAA is recognized as an avirulence protein and the R-Avr protein interaction is

379

partially suppressed by virulence factors since no incompatible rice-Xoc interaction

380

has been observed so far. Otherwise, XopAA might target negative regulators of rice

381

defense signaling to initiate disease tolerance, which has been recently reported for

382

XopD in Xcc (Tan et al. 2014). To identify the host target(s) of the effector XopAA

383

will help to explain the phenomenon.

384

On the other hand, bacterial effector proteins often suppress PTI and thereby

385

enhance pathogenesis in their host plants during the compatible interaction (Gohre

386

and Robatzek 2008). Virulence assays revealed that avrBs2, among the 23

387

investigated effector genes, is the only one which mutation caused a significant

388

attenuation effect on Xoc virulence. The finding is interesting since a previous

389

mutagenesis study demonstrated that the mutation of avrBs2 did not significantly alter

390

Xoo virulence (Song and Yang 2010) and the avrBs2 genes in Xoo and Xoc share 97%

391

coding sequence identity. Similarly, it was previously reported that the rpfG mutants

392

of Xcc and Xoc have different virulence-associated phenotypes (Zhang et al. 2013).

393

Distinct infection processes might cause different molecular mechanisms underlying

394

virulence and pathogenicity among Xanthomonas species. In addition, genetic

395

redundancy in the effector inventory of Xoo and diverse genetic background of rice

396

cultivars could also explain this phenomenon. It was reported that the same Xoo strain

18

Page 19 of 83


Li et al. MPMI

397

might cause different phenotypes on different rice cultivars (Sinha et al. 2013).

398

Subsequently, virulence function(s) of AvrBs2 were further confirmed using transient

399

expression and transgenic technology. Ectopic expression of pathogen effector genes

400

in host plants has been widely used to elucidate virulence functions of bacterial and

401

fungal effectors (Hauck et al. 2003; Park et al. 2012). NHO1 in Arabidopsis is

402

essential for preventing the in planta growth of nonhost Pseudomonas bacteria (Lu et

403

al. 2001). It is well known that flg22 induces the expression of NHO1 (Li et al. 2005).

404

We demonstrated that AvrBs2 transiently expressed in Arabidopsis protoplasts

405

suppressed flg22-induced NHO1 expression (Fig. 3A). Furthermore, induced

406

expression of AvrBs2 in transgenic cell cultures was shown to dramatically suppress

407

flg22-induced and chitin-induced immune responses, such as ROS burst and PR gene

408

expression (Fig. 4). Third, the AvrBs2-expressing transgenic rice plants displayed a

409

nearly complete suppression of defense gene expression triggered by the PAMPs

410

flg22 and chitin. Most importantly, heterologous expression of AvrBs2 in the

411

transgenic plants facilitated bacterial colonization and multiplication in planta and

412

accelerated disease progression (Fig. 5). The phenomenon that the AvrBs2-expressing

413

transgenic rice is more susceptible to Xoc than the wild-type plant is likely due to high

414

expression level of AvrBs2 after DEX induction or expression timing of the effector

415

before inoculation. A similar finding has been reported in avrPiz-t transgenic rice

416

plants that were more susceptible to fungal blast than the wild-type plant (Park et al.

417

2012). In contrast, AvrBs2 expression in the transgenic plants did not inhibit flg22- or

418

chitin-triggered MAPK activation (Supplementary Fig. S7), suggesting that AvrBs2

19

Page 20 of 83


Li et al. MPMI

419

blocks immune signaling transduction downstream of MAPK in rice or inhibits other

420

branches of defense signaling independent on MAPK cascades. Taken together, our

421

data indicate that AvrBs2 and the identified HrpE3 and Xrp5 effectors are essential

422

virulence factors, which contribute to full virulence of Xoc.

423

In summary, the study advanced our understanding of Xop-mediated virulence in

424

bacterial leaf streak. AvrBs2 has been demonstrated to be required for full virulence

425

of Xcv on the dicot plants including pepper and tomato through inhibiting host

426

immunity (Zhao et al. 2011). Our results showed that AvrBs2 is also an essential

427

virulence factor in Xoc, which suppresses immune responses in the monocot rice.

428

These data suggest that AvrBs2 in Xcv and Xoc not only share high identity in protein

429

sequence (Supplementary Fig. S1), but also have a high function similarity. AvrBs2

430

might target defense signaling components shared by the dicot and monocot plants.

431

Given the high structural and functional conservation of AvrBs2, it is interesting to

432

investigate whether the resistance gene Bs2 in pepper confers disease resistance

433

across the dicot and monocot plant classes. The finding may provide insight into

434

strategies to exploit alien disease resistance genes in rice breeding for the

435

broad-spectrum and durable resistance to X. oryzae.

436 437

MATERIALS AND METHODS

438 439 440

Plant materials, bacterial strains and culture conditions Oryza sativa L. ssp. japonica cvs. Jingang 30 and Nipponbare (NPB) plants were

20

Page 21 of 83


Li et al. MPMI

441

grown in greenhouse. Bacterial strains and plasmids used in this study were listed in

442

Supplementary Table S2. A rifampicin-resistant Xoc strain RS105 was used as the

443

wild-type. The Xoc strains were grown in NB medium (3 g/L beef extract, 1 g/L yeast

444

extract, 5 g/L tryptone, 10 g/L sucrose) at 28°C. Antibiotics were used at the

445

following concentrations: ampicillin, 100 µg/mL; kanamycin, 50 µg/mL; rifampin, 25

446

µg/mL. All the experiments were repeated at least three times with similar results

447

unless noted.

448 449

Construction of marker-free gene-deletion mutants in Xoc through homologous

450

recombination

451

The predicted effector genes in Xoc and genome sequences were available from

452

the Xanthomonas Resource database (http://www.xanthomonas.org/) and NCBI

453

(http://www.ncbi.nlm.nih.gov/)

454

gene-deletion mutant strains of Xoc was performed as described previously (Zhang et

455

al. 2013). Briefly, genomic DNA was isolated from the Xoc wild-type strain RS105

456

using a genomic DNA isolation kit (New Industry Company, Beijing, China)

457

according to the manufacturer’s instructions. Two DNA fragments (~1 kb), upstream

458

and downstream close to the start and stop codons of each effector gene, respectively,

459

were amplified from Xoc genome using the primer sets listed in Supplementary Table

460

S3. PCR products were gel-purified and added together into a fusion PCR reaction to

461

amplify DNA fragment carrying upstream and downstream regions of each effector

462

gene but lacking the open reading frame (ORF). The resultant PCR products were

(Bogdanove

21

et

al.

2011).

Construction

of

Page 22 of 83


Li et al. MPMI

463

cloned into pUFR80 that carries a sacB gene as the counter-selective marker

464

(Castaneda et al. 2005; Ried and Collmer 1987). The constructed plasmids were

465

conjugated into Xoc RS105 by tri-parental mating. The conjugants were selected on

466

NA solid medium plates with kanamycin selection. After culturing in NB medium

467

without sucrose, the conjugants were plated onto sucrose (5%)-containing NA plates

468

to

469

sucrose-insensitive Xoc colonies were selected using colony PCR. The mutants were

470

subsequently subject to confirmation via Southern blot analyses (Supplementary Fig.

471

S2 and S3).

screen

sucrose-insensitive

clones.

The

gene-deletion

genotypes

of

472 473

Construction of complementation strains for Xoc gene-deletion mutants

474

For complementation, the full-length effector genes containing upstream

475

promoter regions (~800 bp to ~1kb) were amplified by PCR using gene-specific

476

primer sets (Supplementary Table S3). The amplified products were sub-cloned into

477

the wide host-range vector pVSP61 (Loper and Lindow 1987). After being confirmed

478

by sequencing, the plasmid constructs were transferred into the respective Xoc mutant

479

strains by tri-parental conjugation. Single conjugants of Xoc were selected on the

480

kanamycin-containing NA plates.

481 482

Southern blot analysis

483

Southern blot analysis was performed as described previously (Cannon et al.

484

1979). Briefly, genomic DNA was isolated from the wild-type and mutant Xoc strains

22

Page 23 of 83


Li et al. MPMI

485

as described above and then digested with appropriate restriction enzymes overnight.

486

After separated on agarose gel, digested DNA was blotted onto Hybond-N nylon

487

membrane (GE Healthcare). The probes were PCR amplified and isotope (32P)

488

-labeled using the Random Primer DNA Labeling Kit (Takara). The primer sets for

489

amplifying probes were listed in Supplementary Table S3.

490 491

Transient expression of the effector proteins in N. benthamiana

492

The coding sequence of each putative effector gene of Xoc without the stop codon

493

was amplified and cloned into the vector pUC19-35S-FLAG-RBS (Li et al. 2005)

494

after digestion with Xho I and Nar I for avrBs2, Xho I and Csp45 I for other effector

495

genes except xopAA, xopAB, xopO and xopZ1 (Supplementary Table S3). The four

496

effector genes were not constructed for transient expression because no suitable

497

restriction sites are available for these genes. The ORF of each effector gene with

498

3×FLAG

499

pUC19-35S-FLAG-RBS construct with Xho I and Spe I and re-ligated into the

500

DEX-inducible expression vector pTA7001, respectively (Aoyama and Chua 1997).

501

These constructed plasmids were transformed into the Agrobacterium strain C58C1

502

through the freeze-thaw method (Deblaere et al. 1985). Overnight-cultured

503

Agrobacterium strains were collected and re-suspended in 10 mM MgCl2 with 150

504

µM acetosyringone (OD600, ~0.3). After incubation for 2 h or more, Agrobacterium

505

cultures were infiltrated into the leaves of 6-week-old N. benthamiana plants with

506

needleless syringes. These leaves were sprayed with 30 µM DEX at 24 h after

coding

sequence

was

cleaved

23

from

the

corresponding


507

infiltration. The cell death phenotypes on the leaves were observed and photographed

508

at 3 d after DEX spraying.


509 510

Subcellular localization and protoplast transfection assay

511

For subcellular localization, the coding sequence of GFP was amplified from

512

pGWB5 (Nakagawa et al. 2007) and ligated into the pUC19 plasmid after digestion

513

with Sph I and Hind III. The 35S promoter fragment was cleaved from

514

pUC19-35S-FLAG-RBS by EcoR I and Sac I and then re-ligated into pUC19-GFP.

515

The ORF of avrBs2 was amplified using the primer set avrBs2-BamH I-F/avrBs2-Sal

516

I-R (Supplementary Table S3) and then cloned into pUC19-35S-GFP after digestion

517

with BamH I and Sal I. The construct was confirmed by sequencing.

518

The transfection assay in protoplasts was carried out as described previously (Li

519

et al. 2005). Briefly, protoplasts were isolated from 10-day-old etiolated seedlings of

520

Oryza sativa cv. Nipponbare or the leaves of 4-week-old NHO1-LUC transgenic

521

Arabidopsis plants. The protoplasts (~2.5×106 protoplasts/ml) were transfected with

522

10 µg plasmid DNA by polyethylene glycol-mediated transformation. After washing

523

with W5 solution (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, and 2 mM MES-KOH,

524

pH 5.7), the protoplasts were re-suspended in W5 solution and treated with 1 µM

525

flg22 for 12 h under low light. The luciferase activity was then evaluated by

526

measuring luminescence intensity at 10 min after adding 50 µM luciferin to the

527

transfected Arabidopsis protoplasts. GFP fluorescence on the transfected rice

528

protoplasts was observed using confocal microscopy (Nikon Ti2000).

24


529


530

Development of the avrBs2 transgenic plants and cell cultures

531

The pTA7001-avrBs2 construct was introduced into rice calli through

532

Agrobacterium-mediated transformation as described previously (Ozawa 2009).

533

Briefly, the sterilized dehusked seeds (cv. NPB) were plated onto NBi medium (N6

534

macro elements, B5 microelements, B5 vitamin, 27.8 mg/L FeSO4·7H2O, 37.3 mg/L

535

Na2-EDTA, 500 mg/L proline and glutamic acid, 300 mg/L casein hydrolysate, 2

536

mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 100 mg/L inositol, and 30 g/L sucrose)

537

for callus induction. The vigorous calli were picked and incubated with

538

Agrobacterium cultures for 2 min and then transferred onto NBco medium (NBi

539

medium supplied with 100 µM acetosyringone, pH 5.5) for 3 days. The calli were

540

subsequently cultured onto NBs medium (NBi medium supplied with 500 mg/L

541

cephamycin and 30 mg/L hygromycin) after washing with sterile water thoroughly.

542

The resistant calli that emerged after 3~4 weeks were transferred onto NBr medium

543

(NBi medium supplied with 0.5 mg/L α-naphthalene acetic acid, 3 mg/L

544

6-benzylaminopurine, 500 mg/L cephamycin and 30 mg/L hygromycin) for shooting.

545

The regenerated shoots were transferred onto 1/2 × Murashige and Skoog medium

546

containing 100 mg/L inositol for rooting. The independent transgenic plants were

547

transferred to the greenhouse for further growth.

548

Rice cell suspension cultures were initiated from the wild-type and avrBs2

549

transgenic calli as described previously (Ozawa and Komamine 1989). Briefly, the

550

compact calli with vigorous growth were inoculated in 100 mL of liquid N6 medium

25

Page 26 of 83


Li et al. MPMI

551

supplemented with 5 mg/mL 2,4-D and 1 mg/mL kinetin to establish cell suspension

552

cultures. Hygromycin (30 mg/L) was added for culturing the avrBs2 transgenic calli.

553

The suspension cultures were incubated at 26°C on a rotary shaker at 140 rpm.

554 555

Oxidative burst assays

556

ROS burst in rice cell cultures was detected as described previously with minor

557

modifications (Lu et al. 2014; Pérez and Rubio 2006). Briefly, equal volume of calli

558

was incubated in fresh culture medium for 24 h at 26°C. DEX was then added into

559

cell cultures to the final concentration of 10 µM to induce AvrBs2 expression. Ethanol

560

(0.03%) was added into cultured cells as mock control. After incubation for another

561

24 h, cultured cells were washed three times and then incubated in 3 ml of fresh media

562

in a 20 ml vial for 3h at 26°C. The cells were then treated with 1 µM flg22 and 20 µL

563

of 100mg/mL chitin. The supernatant (10 µl) was collected at different time points

564

after treatments and mixed with 1 ml of Co(II)-luminol reagent. Chemiluminescence

565

was recorded immediately by the Infinite F200 (Tecan) with the count time set as 1 s.

566 567

Protein extraction and immunoblotting

568

The avrBs2 transgenic calli treated with DEX or mock (0.03% ethanol) were

569

collected and ground in the centrifuge tubes with small stainless steel balls using a

570

milling apparatus (Retsch, Germany) for total protein extraction. The sample loading

571

buffer (50 mM Tris-HCl, pH6.8, 2% SDS, 6% glycerol, 0.1 M dithiothreitol, 0.01%

572

bromophenol blue) was added directly into the tubes. The samples were boiled for 20

26

Page 27 of 83


Li et al. MPMI

573

min and the supernatants were collected for gel separation. For detecting transient

574

expression of the effectors in N. benthamiana, total proteins were extracted from the

575

ground powder of the infiltrated leaves using the extraction buffer (50 mM Tris-HCl,

576

pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% protease inhibitor

577

cocktail [Merck]).

578

The extracted proteins were separated in a 12% polyacrylamide gel and

579

electrophoretically transferred onto Immobilon-P membrane (Millipore) as described

580

previously (Sun et al. 2012). Non-specific binding sites on the membranes were

581

blocked overnight in blocking solution consisting of 5% (w/v) skimmed milk in

582

TBS-T (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.05% Tween 20) at 4°C. The

583

membranes were then incubated with an anti-FLAG monoclonal antibody (Sigma) at

584

a dilution of 1: 5, 000 in blocking solution for 1 h at room temperature. Following

585

three 10-min washes in TBS-T, the membranes were incubated for 1 h at room

586

temperature in TBS-T containing HRP-conjugated goat anti-mouse IgG secondary

587

antibody (1: 5, 000 dilution) (CWBio, China). After three washes in TBS-T, the

588

membranes were incubated with the eECL Western Blot chemiluminescent substrate

589

(CWBio, China) for 5 min, and then exposed to X-ray films. MAPK activation was

590

detected as described (Schwessinger et al. 2011). Briefly, the immunoblots were

591

blocked in 5% (w/v) BSA (MP Biomedicals, USA) in TBS-T for 1 h at 4ºC.

592

Phospho-serine/threonine sites were detected using anti-Phospho-p44/42 MAPK

593

antibody (1 : 2, 000 dilution) (Cell Signaling Technology, USA), followed by

594

HRP-conjugated anti-rabbit IgG secondary antibody (CWBio, China) at a dilution of

27


595

1 : 5, 000.

596


597

RNA isolation and quantitative real-time RT-PCR

598

The transgenic plant leaves and cell cultures were treated with flg22 and chitin

599

for 6 h after the treatment with DEX to induce AvrBs2 expression. Total RNA was

600

isolated from the treated avrBs2 transgenic plants and cells using an Ultrapure RNA

601

isolation kit according to the manufacturer’s instructions (CWBio, China).

602

Complementary DNA (cDNA) was synthesized by reverse transcriptase Superscript

603

III (Invitrogen) using total RNA as template. The transcript levels of defense genes

604

were quantified through quantitative real time RT-PCR (qRT-PCR) with SYBR®

605

premix Ex TaqTM (Takara) using an ABI PRISM® 7000 Sequence Detection System

606

(Applied Biosystems). The expression level of OsActin1 (Os03g0718100) was used as

607

an internal reference. The primers used to track the expression of OsActin1, OsPBZ1

608

(Os12g0555500) and OsPAL1 (Os02g0627100) were listed in the Supplementary

609

Table S3.

610 611

Virulence assays of Xoc and Xoo on rice

612

Virulence of different Xoc gene-deletion mutant strains was determined on rice

613

cv. Jingang 30 by pressure inoculation (Wang et al. 2007). The avrBs2 transgenic

614

plants were sprayed with 30 µM DEX or 0.1 % ethanol as mock control 1 d before

615

bacterial inoculation. For bacterial leaf streak, Xoc strains were cultured in NB

616

medium overnight and diluted with sterile double-distilled water to an OD600 of 0.3

28

Page 29 of 83


Li et al. MPMI

617

and then pressure infiltrated into 6-week-old rice leaves with needleless syringes. For

618

rice bacterial blight, the Xoo PXO99A (OD600, ~ 0.8) was inoculated into rice leaves

619

by the leaf-clipping method (Kauffman et al. 1973). Disease lesions on the inoculated

620

leaves were measured at 14 d after inoculation. At least 10 inoculated leaves were

621

scored for each tested strain.

622

For establishing bacterial growth curves, leaf sections around inoculation sites

623

were harvested at four time points (0, 4, 8, 12 d after inoculation) and sliced into small

624

pieces, incubated in 5 ml sterile water including rifampicin (25 µg/ml) with shaking

625

for 1 h, and then filtered through two layers of sterilization gauze. The filtrates were

626

diluted and then plated onto NA agar plates with rifampicin. Colonies on the plates

627

were counted after 3 d of incubation at 28°C.

628 629

ACKNOWLEDGMENTS

630

We thank Jianmin Zhou at the Institute of Genetics and Developmental Biology

631

of the Chinese Academy of Sciences for providing the pUC19-35S-FLAG-RBS

632

plasmid vector and NHO1-LUC transgenic Arabidopsis plants. The work is supported

633

by the National High Technology Research and Development program of China

634

2012AA100703, the transgenic crop project 2012ZX08009003-003, the 973 program

635

2011CB100700, the NSFC grant 31272007 and the 111 project B13006 to W. S.

636 637

Author Contributions

638

Li, S., Wang, Y., Wang, S. and Sun, W. conceived and designed the experiments; Li,

29

Page 30 of 83


Li et al. MPMI

639

S., Wang, Y., Wang, S., Fang, A., Wang, J., Liu, L. and Mao, Y. performed the

640

experiments; Li, S., Wang, Y., Wang, S., Fang, A., Wang, J., Liu, L., Mao, Y. and

641

Zhang,

642

reagents/materials/analysis tools; Li, S., Wang, Y., Wang, S. and Sun, W. wrote the

643

manuscript. All authors contributed to final approval of the version to be published.

K.

analyzed

the

data;

Zhang,

K.

and

Sun,

W.

contributed

644 645

LITERATURE CITED

646

Akimoto-Tomiyama, C., Furutani, A., Tsuge, S., Washington, E. J., Nishizawa, Y.,

647

Minami, E., and Ochiai, H. 2012. XopR, a type III effector secreted by

648

Xanthomonas oryzae pv. oryzae, suppresses microbe-associated molecular

649

pattern-triggered immunity in Arabidopsis thaliana. Mol. Plant-Microbe Interact.

650

25:505-514.

651

Alfano, J. R., and Collmer, A. 2004. Type III secretion system effector proteins:

652

double agents in bacterial disease and plant defense. Annu. Rev. Phytopathol.

653

42:385-414.

654 655 656 657

Aoyama, T., and Chua, N. H. 1997. A glucocorticoid-mediated transcriptional induction system in transgenic plants. Plant J. 11:605-612. Boch, J., and Bonas, U. 2010. Xanthomonas AvrBs3 family-type III effectors: discovery and function. Annu. Rev. Phytopathol. 48:419-436.

658

Bogdanove, A. J., Koebnik, R., Lu, H., Furutani, A., Angiuoli, S. V., Patil, P. B., Van

659

Sluys, M., Ryan, R. P., Meyer, D. F., Han, S. Aparna, G., Rajaram, M., Delcher,

660

A. L., Phillippy, A. M., Puiu, D., Schatz, M. C., Shumway, M., Sommer, D. D.,

30

Page 31 of 83


Li et al. MPMI

661

Trapnell, C., Benahmed, F., Dimitrov, G., Madupu, R., Radune, D., Sullivan, S.,

662

Jha, G., Ishihara, H., Lee, S.W., Pandey, A., Sharma, V., Sriariyanun, M., Szurek,

663

B., Vera-Cruz, C. M., Dorman, K. S., Ronald, P. C., Verdier, V., Dow, J. M.,

664

Sonti, R. V., Tsuge, S., Brendel, V. P., Rabinowicz, P. D., Leach, J. E., White, F.

665

F., and Salzberg, S. L. 2011. Two new complete genome sequences offer insight

666

into host and tissue specificity of plant pathogenic Xanthomonas spp. J. Bacteriol.

667

193:5450-5464.

668

Boller, T., and He, S. Y. 2009. Innate immunity in plants: an arms race between

669

pattern recognition receptors in plants and effectors in microbial pathogens.

670

Science 324:742-744.

671

Cannon, F. C., Riedel, G. E., and Ausubel, F. M. 1979. Overlapping sequences of

672

Klebsiella pneumoniae nifDNA cloned and characterised. Mol. Gen. Genet.

673

174:59-66.

674

Castaneda, A., Reddy, J. D., El-Yacoubi, B., and Gabriel, D. W. 2005. Mutagenesis of

675

all eight avr genes in Xanthomonas campestris pv. campestris had no detected

676

effect on pathogenicity, but one avr gene affected race specificity. Mol.

677

Plant-Microbe Interact. 18:1306-1317.

678

Cheong, H., Kim, C., Jeon, J., Lee, B., Moon, J. S., and Hwang, I. 2013.

679

Xanthomonas oryzae pv. oryzae type III effector XopN targets OsVOZ2 and a

680

putative thiamine synthase as a virulence factor in rice. PLoS One 8:e73346.

681

Chisholm, S. T., Coaker, G., Day, B., and Staskawicz, B. J. 2006. Host-microbe

682

interactions: shaping the evolution of the plant immune response. Cell

31



683

124:803-814.

684

Cui, Y., Zou, L., Zou, H., Li, Y., Zakria, M., and Chen, G. 2013. HrpE3 is a type III

685

effector protein required for full virulence of Xanthomonas oryzae pv. oryzicola

686

in rice. Mol. Plant Pathol. 14: 678-692.

687

Cunnac, S., Occhialini, A., Barberis, P., Boucher, C., and Genin, S. 2004. Inventory

688

and functional analysis of the large Hrp regulon in Ralstonia solanacearum:

689

identification of novel effector proteins translocated to plant host cells through

690

the type III secretion system. Mol. Microbiol. 53:115-128.

691

Deblaere, R., Bytebier, B., De Greve, H., Deboeck, F., Schell, J., Van Montagu, M.,

692

and Leemans, J. 1985. Efficient octopine Ti plasmid-derived vectors for

693

Agrobacterium-mediated

694

13:4777-4788.

695 696

gene

transfer to

plants.

Nucleic

Acids

Res.

Dou, D., and Zhou, J. 2012. Phytopathogen effectors subverting host immunity: different foes, similar battleground. Cell Host Microbe 12:484-495.

697

Feng, F., Yang, F., Rong, W., Wu, X., Zhang, J., Chen, S., He, C., and Zhou, J. M.

698

2012. A Xanthomonas uridine 5'-monophosphate transferase inhibits plant

699

immune kinases. Nature 485:114-118.

700

Furutani, A., Nakayama, T., Ochiai, H., Kaku, H., Kubo, Y., and Tsuge, S. 2006.

701

Identification of novel HrpXo regulons preceded by two cis-acting elements, a

702

plant-inducible promoter box and a -10 box-like sequence, from the genome

703

database of Xanthomonas oryzae pv. oryzae. FEMS (Fed. Eur. Microbiol. Soc.)

704

Microbiol. Lett. 259:133-141.

32

Page 33 of 83


Li et al. MPMI

705

Furutani, A., Takaoka, M., Sanada, H., Noguchi, Y., Oku, T., Tsuno, K., Ochiai, H.,

706

and Tsuge, S. 2009. Identification of novel type III secretion effectors in

707

Xanthomonas oryzae pv. oryzae. Mol. Plant-Microbe Interact. 22:96-106.

708

Gassmann, W., Dahlbeck, D., Chesnokova, O., Minsavage, G. V., Jones, J. B., and

709

Staskawicz, B. J. 2000. Molecular evolution of virulence in natural field strains

710

of Xanthomonas campestris pv. vesicatoria. J. Bacteriol. 182:7053-7059.

711

Gimenez-Ibanez, S., Boter, M., Fernandez-Barbero, G., Chini, A., Rathjen, J. P., and

712

Solano, R. 2014. The bacterial effector HopX1 targets JAZ transcriptional

713

repressors to activate jasmonate signaling and promote infection in Arabidopsis.

714

PLoS Biol. 12:e1001792.

715 716

Gohre, V., and Robatzek, S. 2008. Breaking the barriers: microbial effector molecules subvert plant immunity. Annu. Rev. Phytopathol. 46:189-215.

717

Guo, W., Cui, Y., Li, Y., Che, Y., Yuan, L., Zou, L., Zou, H., and Chen, G. 2012.

718

Identification of seven Xanthomonas oryzae pv. oryzicola genes potentially

719

involved in pathogenesis in rice. Microbiology 158: 505-518.

720

Hauck, P., Thilmony, R., and He, S. Y. 2003. A Pseudomonas syringae type III

721

effector suppresses cell wall-based extracellular defense in susceptible

722

Arabidopsis plants. Proc. Natl. Acad. Sci. U.S.A. 100:8577-8582.

723

Jiang, S., Yao, J., Ma, K. W., Zhou, H., Song, J., He, S. Y., and Ma, W. 2013.

724

Bacterial effector activates jasmonate signaling by directly targeting JAZ

725

transcriptional repressors. PLoS Pathog. 9:e1003715.

726

Jones, J. D., and Dangl, J. L. 2006. The plant immune system. Nature 444:323-329.

33

Page 34 of 83


Li et al. MPMI

727

Kang, L., Li, J., Zhao, T., Xiao, F., Tang, X., Thilmony, R., He, S., and Zhou, J. 2003.

728

Interplay of the Arabidopsis nonhost resistance gene NHO1 with bacterial

729

virulence. Proc. Natl. Acad. Sci. U.S.A. 100:3519-3524.

730

Kauffman, H. E., Reddy, A. P. K., and Hsieh, S. P. Y. 1973. An improved technique

731

for evaluation of resistance of rice varieties to Xanthomonas oryzae. Plant Dis.

732

57:537-541.

733 734

Kay, S., and Bonas, U. 2009. How Xanthomonas type III effectors manipulate the host plant. Curr. Opin. Microbiol. 12:37-43.

735

Kearney, B., and Staskawicz, B. J. 1990. Widespread distribution and fitness

736

contribution of Xanthomonas campestris avirulence gene avrBs2. Nature

737

346:385-386.

738

Kim, J., Li, X., Roden, J. A., Taylor, K. W., Aakre, C. D., Su, B., Lalonde, S., Kirik,

739

A., Chen, Y., and Baranage, G. 2009. Xanthomonas T3S effector XopN

740

suppresses PAMP-triggered immunity and interacts with a tomato atypical

741

receptor-like kinase and TFT1. Plant Cell 21:1305-1323.

742

Kim, J., Stork, W., and Mudgett, M. B. 2013. Xanthomonas type III effector XopD

743

desumoylates tomato transcription factor SlERF4 to suppress ethylene responses

744

and promote pathogen growth. Cell Host Microbe 13:143-154.

745

Kim, J., Taylor, K. W., Hotson, A., Keegan, M., Schmelz, E. A., and Mudgett, M. B.

746

2008. XopD SUMO protease affects host transcription, promotes pathogen

747

growth, and delays symptom development in Xanthomonas-infected tomato

748

leaves. Plant Cell 20:1915-1929.

34

Page 35 of 83


Li et al. MPMI

749

Koebnik, R., Krüger, A., Thieme, F., Urban, A., and Bonas, U. 2006. Specific binding

750

of the Xanthomonas campestris pv. vesicatoria AraC-type transcriptional

751

activator HrpX to plant-inducible promoter boxes. J. Bacteriol. 188:7652-7660.

752

Kvitko, B. H., Park, D. H., Velasquez, A. C., Wei, C. F., Russell, A. B., Martin, G. B.,

753

Schneider, D. J., and Collmer, A. 2009. Deletions in the repertoire of

754

Pseudomonas syringae pv. tomato DC3000 type III secretion effector genes

755

reveal functional overlap among effectors. PLoS Pathog. 5:e1000388.

756

Li, W., Yadeta, K. A., Elmore, J. M., and Coaker, G. 2013. The Pseudomonas

757

syringae effector HopQ1 promotes bacterial virulence and interacts with tomato

758

14-3-3 proteins in a phosphorylation-dependent manner. Plant Physiol.

759

161:2062-2074.

760

Li, X., Lin, H., Zhang, W., Zou, Y., Zhang, J., Tang, X., and Zhou, J. 2005. Flagellin

761

induces innate immunity in nonhost interactions that is suppressed by

762

Pseudomonas

763

102:12990-12995.

syringae

effectors.

Proc.

Natl.

Acad.

Sci.

U.S.A.

764

Li, Y., Zou, H., Che, Y., Cui, Y., Guo, W., Zou, L., Chatterjee, S., Biddle, E. M.,

765

Yang, C., and Chen, G. 2011. A novel regulatory role of HrpD6 in regulating

766

hrp-hrc-hpa genes in Xanthomonas oryzae pv. oryzicola. Mol. Plant-Microbe

767

Interact. 24:1086-1101.

768

Loper, J. E., and Lindow, S. E. 1987. Lack of evidence for the in situ fluorescent

769

pigment production by Pseudomonas syringae pv. syringae on bean leaf surfaces.

770

Phytopathology 77:1449-1454.

35

Page 36 of 83


Li et al. MPMI

771

Lozano-Duran, R., Bourdais, G., He, S. Y., and Robatzek, S. 2014. The bacterial

772

effector HopM1 suppresses PAMP-triggered oxidative burst and stomatal

773

immunity. New Phytol. 202:259-269.

774

Lu, F., Wang, H., Wang, S., Jiang, W., Shan, C., Li, B., Yang, J., Zhang, S., and Sun,

775

W. 2014. Enhancement of innate immune system in monocot rice by transferring

776

the dicotyledonous elongation factor Tu receptor. J. Integr Plant Biol. doi:

777

10.1111/jipb.12306.

778 779

Lu, M., Tang, X., and Zhou, J. M. 2001. Arabidopsis NHO1 is required for general resistance against Pseudomonas bacteria. Plant Cell 13:437-447.

780

Makino, S., Sugio, A., White, F., and Bogdanove, A. J. 2006. Inhibition of resistance

781

gene-mediated defense in rice by Xanthomonas oryzae pv. oryzicola. Mol.

782


783

Metz, M., Dahlbeck, D., Morales, C. Q., Sady, B. A., Clark, E. T., and Staskawicz, B.

784

J. 2005. The conserved Xanthomonas campestris pv. vesicatoria effector protein

785

XopX is a virulence factor and suppresses host defense in Nicotiana

786

benthamiana. Plant J. 41:801-814.

787

Nakagawa, T., Kurose, T., Hino, T., Tanaka, K., Kawamukai, M., Niwa, Y., Toyooka,

788

K., Matsuoka, K., Jinbo, T., and Kimura, T. 2007. Development of series of

789

gateway binary vectors, pGWBs, for realizing efficient construction of fusion

790

genes for plant transformation. J. Biosci. Bioeng. 104:34-41.

791

Niño-Liu, D. O., Ronald, P. C., and Bogdanove, A. J. 2006. Xanthomonas oryzae

792

pathovars: Model pathogens of a model crop. Mol. Plant Pathol. 7:303-324.

36


793


794

Ozawa, K. 2009. Establishment of a high efficiency Agrobacterium-mediated transformation system of rice (Oryza sativa L.). Plant Sci. 176:522-527.

795

Ozawa, K., and Komamine, A. 1989. Establishment of a system of high-frequency

796

embryogenesis from long-term cell suspension cultures of rice (Oryza sativa L.).

797

Theor. Appl. Genet. 77:205-211.

798

Park, C. H., Chen, S., Shirsekar, G., Zhou, B., Khang, C. H., Songkumarn, P., Afzal,

799

A. J., Ning, Y., Wang, R., Bellizzi, M., Valent, B., and Wang, G. L. 2012.

800

The Magnaporthe oryzae effector AvrPiz-t targets the RING E3 ubiquitin ligase

801

APIP6 to suppress pathogen-associated molecular pattern-triggered immunity in

802

rice. Plant Cell 24: 4748-4762.

803

Pei, J. G., Zou, L. F., Zou, H. S. and Chen, G.Y. 2010. Functional analysis of

804

xopQ1Xoc gene from Xanthomonas oryzae pv. oryzicola in pathogenesis on rice.

805

Sci. Agric. Sin. 17:3538-3546

806

Pereira, A. L., Carazzolle, M. F., Abe, V. Y., de Oliveira, M. L., Domingues, M. N.,

807

Silva, J. C., Cernadas, R. A., and Benedetti, C. E. 2014. Identification of putative

808

TAL effector targets of the citrus canker pathogens shows functional

809

convergence underlying disease development and defense response. BMC

810

Genomics 15:157.

811

Pérez, F. J., and Rubio, S. 2006. An improved chemiluminescence method for

812

hydrogen peroxide determination in plant tissues. Plant Growth Regul. 48:89-95.

813

Ried, J. L., and Collmer, A. 1987. An nptI-sacB-sacR cartridge for constructing

814

directed,

unmarked

mutations

in

37

Gram-negative

bacteria

by

marker



815

exchange-eviction mutagenesis. Gene 57:239-246.

816

Roden, J. A., Belt, B., Ross, J. B., Tachibana, T., Vargas, J., and Mudgett, M. B. 2004.

817

A genetic screen to isolate type III effectors translocated into pepper cells during

818

Xanthomonas infection. Proc. Natl. Acad. Sci. U.S.A. 101:16624-16629.

819

Rosebrock, T. R., Zeng, L., Brady, J. J., Abramovitch, R. B., Xiao, F., and Martin, G.

820

B. 2007. A bacterial E3 ubiquitin ligase targets a host protein kinase to disrupt

821

plant immunity. Nature 448:370-374.

822

Schechter, L. M., Roberts, K. A., Jamir, Y., Alfano, J. R., and Collmer, A. 2004.

823

Pseudomonas syringae type III secretion system targeting signals and novel

824

effectors studied with a Cya translocation reporter. J. Bacteriol. 186:543-555.

825

Schechter, L. M., Vencato, M., Jordan, K. L., Schneider, S. E., Schneider, D. J., and

826

Collmer, A. 2006. Multiple approaches to a complete inventory of Pseudomonas

827

syringae pv. tomato DC3000 type III secretion system effector proteins. Mol.

828


829 830

Scholze, H., and Boch, J. 2011. TAL effectors are remote controls for gene activation. Curr. Opin. Microbiol. 14:47-53.

831

Schwessinger, B., Roux, M., Kadota, Y., Ntoukakis, V., Sklenar, J., Jones, A., and

832

Zipfel, C. 2011. Phosphorylation-dependent differential regulation of plant

833

growth, cell death, and innate immunity by the regulatory receptor-like kinase

834

BAK1. PLoS Genet. 7:e1002046.

835

Shimizu, T., Nakano, T., Takamizawa, D., Desaki, Y., Ishii Minami, N., Nishizawa,

836

Y., Minami, E., Okada, K., Yamane, H., Kaku, H., and Shibuya, N. 2010. Two

38

Page 39 of 83


Li et al. MPMI

837

LysM receptor molecules, CEBiP and OsCERK1, cooperatively regulate chitin

838

elicitor signaling in rice. Plant J. 64:204-214.

839

Sinha, D., Gupta, M. K., Patel, H. K., Ranjan, A., and Sonti, R. V. 2013. Cell wall

840

degrading enzyme induced rice innate immune responses are suppressed by the

841

type 3 secretion system effectors XopN, XopQ, XopX and XopZ of

842

Xanthomonas oryzae pv. oryzae. PLoS One 8:e75867.

843

Song, C., and Yang, B. 2010. Mutagenesis of 18 type III effectors reveals virulence

844

function of XopZPXO99 in Xanthomonas oryzae pv. oryzae. Mol. Plant-Microbe

845

Interact. 23:893-902.

846

Sun, W., Cao, Y., Labby, K. J., Bittel, P., Boller, T., and Bent, A. F. 2012. Probing

847

the Arabidopsis flagellin receptor: FLS2-FLS2 association and the contributions

848

of specific domains to signaling function. Plant Cell 24:1096-1113.

849

Swords, K. M., Dahlbeck, D., Kearney, B., Roy, M., and Staskawicz, B. J. 1996.

850

Spontaneous and induced mutations in a single open reading frame alter both

851

virulence and avirulence in Xanthomonas campestris pv. vesicatoria avrBs2. J.

852

Bacteriol. 178:4661-4669.

853

Tai, T. H., Dahlbeck, D., Clark, E. T., Gajiwala, P., Pasion, R., Whalen, M. C., Stall,

854

R. E., and Staskawicz, B. J. 1999. Expression of the Bs2 pepper gene confers

855

resistance to bacterial spot disease in tomato. Proc. Natl. Acad. Sci. U.S.A.

856

96:14153-14158.

857

Takai, R., Isogai, A., Takayama, S., and Che, F. 2008. Analysis of flagellin perception

858

mediated by flg22 receptor OsFLS2 in rice. Mol. Plant-Microbe Interact.

39



859

2:1635-1642.

860

Tampakaki, A. P., Fadouloglou, V. E., Gazi, A. D., Panopoulos, N. J., and Kokkinidis,

861

M. 2004. Conserved features of type III secretion. Cell Microbiol. 6:805-816.

862

Tan, L., Rong, W., Luo, H., Chen, Y., and He, C. 2014. The Xanthomonas campestris

863

effector protein XopDXcc8004 triggers plant disease tolerance by targeting DELLA

864

proteins. New Phytol. 204:595-608.

865

Teper, D., Salomon, D., Sunitha, S., Kim, J. G., Mudgett, M. B., and Sessa, G. 2014.

866

Xanthomonas euvesicatoria type III effector XopQ interacts with tomato and

867

pepper 14-3-3 isoforms to suppress effector-triggered immunity. Plant J.

868

77:297-309.

869 870

Thomma, B. P., Nürnberger, T., and Joosten, M. H. 2011. Of PAMPs and effectors: the blurred PTI-ETI dichotomy. Plant Cell 23:4-15.

871

Tsuge, S., Nakayama, T., Terashima, S., Ochiai, H., Furutani, A., Oku, T., Tsuno, K.,

872

Kubo, Y., and Kaku, H. 2006. Gene involved in transcriptional activation of the

873

hrp regulatory gene hrpG in Xanthomonas oryzae pv. oryzae. J. Bacteriol.

874

188:4158-4162.

875

Underwood, W., Zhang, S., and He, S. Y. 2007. The Pseudomonas syringae type III

876

effector tyrosine phosphatase HopAO1 suppresses innate immunity in

877

Arabidopsis thaliana. Plant J. 52:658-672.

878

Üstün, S., Bartetzko, V., and Börnke, F. 2013. The Xanthomonas campestris type III

879

effector XopJ targets the host cell proteasome to suppress salicylic-acid mediated

880

plant defence. PLoS Pathog. 9:e1003427.

40


881


882

Vauterin, L., Hoste, B., Kersters, K., and Swings, J. 1995. Reclassification of Xanthomonas. Int. J. Syst. Bacteriol. 45:472-489.

883

Wang, L., Makino, S., Subedee, A., and Bogdanove, A. J. 2007. Novel candidate

884

virulence factors in rice pathogen Xanthomonas oryzae pv. oryzicola as revealed

885

by mutational analysis. Appl. Environ. Microbiol. 73:8023-8027.

886 887

White, F. F., Potnis, N., Jones, J. B., and Koebnik, R. 2009. The type III effectors of Xanthomonas. Mol. Plant Pathol. 10:749-766.

888

Xin, X. F., and He, S. Y. 2013. Pseudomonas syringae pv. tomato DC3000: a model

889

pathogen for probing disease susceptibility and hormone signaling in plants.

890

Annu. Rev. Phytopathol. 51:473-498.

891

Xue, X., Zou, L., Ma, W., Liu, Z., and Chen, G. 2014. Identification of 17

892

HrpX-regulated proteins including two novel type III effectors, XOC_3956 and

893

XOC_1550, in Xanthomonas oryzae pv. oryzicola. PLoS One 9: e93205.

894

Yamaguchi, K., Nakamura, Y., Ishikawa, K., Yoshimura, Y., Tsuge, S., and Kawasaki,

895

T. 2013a. Suppression of rice immunity by Xanthomonas oryzae type III effector

896

Xoo2875. Biosci. Biotechnol. Biochem. 77:796-801.

897

Yamaguchi, K., Yamada, K., Ishikawa, K., Yoshimura, S., Hayashi, N., Uchihashi, K.,

898

Ishihama, N., Kishi-Kaboshi, M., Takahashi, A., Tsuge, S., Ochiai, H., Tada, Y.,

899

Shimamoto, K., Yoshioka, H., and Kawasaki, T. 2013b. A receptor-like

900

cytoplasmic kinase targeted by a plant pathogen effector is directly

901

phosphorylated by the chitin receptor and mediates rice immunity. Cell Host

902

Microbe 13:347-357.

41

Page 42 of 83


Li et al. MPMI

903

Zeng, W., Brutus, A., Kremer, J. M., Withers, J. C., Gao, X., Jones, A. D., and He, S.

904

Y. 2011. A genetic screen reveals Arabidopsis stomatal and/or apoplastic

905

defenses against Pseudomonas syringae pv. tomato DC3000. PLoS Pathog.

906

7:e1002291.

907

Zhang, Y., Wei, C., Jiang, W., Wang, L., Li, C., Wang, Y., Dow, J. M., and Sun, W.

908

2013. The HD-GYP domain protein RpfG of Xanthomonas oryzae pv. oryzicola

909

regulates synthesis of extracellular polysaccharides that contribute to biofilm

910

formation and virulence on rice. PLoS One 8:e59428.

911

Zhao, B., Ardales, E. Y., Raymundo, A., Bai, J., Trick, H. N., Leach, J. E., and

912

Hulbert, S. H. 2004. The avrRxo1 gene from the rice pathogen Xanthomonas

913

oryzae pv. oryzicola confers a nonhost defense reaction on maize with resistance

914

gene Rxo1 Mol. Plant-Microbe Interact. 17: 771–779.

915

Zhao, B., Dahlbeck, D., Krasileva, K. V., Fong, R. W., and Staskawicz, B. J. 2011.

916

Computational and biochemical analysis of the Xanthomonas effector AvrBs2

917

and its role in the modulation of Xanthomonas type three effector delivery. PLoS

918

Pathog. 7:e1002408.

919 920

FIGURE LEGEND

921

Fig. 1. The effects of mutation of each putative effector gene on virulence of X.

922

oryzae pv. oryzicola to rice. A, Mutation of avrBs2 caused a significant reduction in

923

virulence while the ∆xopAA mutant exhibited an enhanced virulence on rice cv.

924

Jingang 30 in repeatable tests. Virulence of the ∆avrBs2 and ∆xopAA mutants was

42

Page 43 of 83


Li et al. MPMI

925

successfully complemented with plasmid-borne full-length avrBs2 and xopAA genes,

926

respectively. Virulence of each mutant was evaluated by the length of disease lesions

927

after bacteria were inoculated into rice leaves by pressure infiltration as compared

928

with that caused by the wild-type (WT) strain. The length of disease lesions was

929

measured at 14 d after inoculation for 10 to 15 leaves per strain. The gene-deletion

930

mutants were constructed for 23 out of 26 putative effector genes that were all present

931

in the Xoc RS105 strain. B, In planta bacterial population of Xoc RS105, ∆avrBs2 and

932

∆avrBs2(avrBs2) at the indicated time points after inoculation. C, In planta bacterial

933

population of Xoc RS105, ∆xopAA and ∆xopAA(xopAA) at the indicated time points

934

after inoculation. Bars are means ± standard error (SE). Different letters a to d

935

indicate statistically significant difference (P < 0.05). cfu, colony-forming units; dpi,

936

days post-inoculation; ∆avrBs2(avrBs2) and ∆xopAA(xopAA), the complementation

937

strains of ∆avrBs2 and ∆xopAA, respectively.

938 939

Fig. 2. Cell death symptoms on N. benthamiana leaves caused by transient expression

940

of Xoc putative effectors. A, Six proteins out of the 22 tested putative effectors

941

including XopN, XopX, XopA, XopY, XopF1 and AvrBs2 caused the hypersensitive

942

response in N. benthamiana to different degrees. XopQ that did not induce HR was

943

shown as a negative control. Agrobacterium cultures transformed with different

944

effector constructs were infiltrated into N. benthamiana leaves followed by DEX

945

spraying. Representative photos were taken at 3 d after DEX treatment. Numbers in

946

parentheses, for instance 18/20, indicate that 18 out of 20 inoculated leaves exhibited

43

Page 44 of 83


Li et al. MPMI

947

cell death symptoms. CK, empty vector. B, Transient expression of 22 tested putative

948

effectors in N. benthamiana was confirmed via Western blot analyses. The samples

949

for protein extraction were collected before cell death symptoms were visible. The

950

effectors with the FLAG tag were detected by immunoblotting with an anti-FLAG

951

antibody. The kD stands for kilodaltons.

952 953

Fig. 3. Transient expression of AvrBs2 inhibits flg22-induced NHO1-LUC expression

954

in Arabidopsis protoplasts. A, The relative luciferase activity in transfected

955

protoplasts. Protoplasts were transfected either with the avrBs2 gene construct or

956

empty vector (EV). The relative luciferase activity was measured 12 h after flg22

957

treatment. EV- indicates EV-transfected protoplasts treated with dH2O, which was

958

used as a control for basal NHO1-LUC expression. EV+ represents EV-transfected

959

protoplasts treated with 1 µM flg22. Each data point (mean ± SE) consists of ten

960

replicates. The experiments were repeated at least three times with similar results. **

961

indicates P value < 0.01. B, Subcellular localization of AvrBs2-GFP transiently

962

expressed in rice protoplasts. The empty vector pUC19-35S-GFP was used as a

963

control.

964 965

Fig. 4. Heterologous expression of AvrBs2 inhibits pathogen-associated molecular

966

pattern-induced ROS burst and defense gene expression in transgenic rice culture. A,

967

Expression of AvrBs2 in transgenic rice cell culture induced by DEX treatment. The

968

AvrBs2-FLAG fusion was detected by Western blotting with an anti-FLAG antibody.

44

Page 45 of 83


Li et al. MPMI

969

NPB, Nipponbare; DEX, dexamethasone; -, without DEX treatment and +, with DEX

970

treatment; WB, Western blot; Ponceau S staining shows the equal loading of the total

971

proteins. B and C, ROS burst induced by flg22 (B) and chitin (C) was significantly

972

suppressed in the avrBs2 transgenic cell culture after DEX treatment. ROS generation

973

was detected by a luminol-chemiluminescence assay. D and E, Up-regulation of the

974

defense marker genes OsPBZ1 (D) and OsPAL1 (E) induced by flg22 and chitin was

975

almost completely blocked in the avrBs2 transgenic cell culture after DEX treatment.

976

The transgenic cell culture was treated with 10 µM DEX or 0.03% ethanol as mock

977

control for 24 h followed by the treatment of 1 µM flg22 or chitin. ** indicates P

978

value < 0.01. Means ± SE are shown.

979 980

Fig. 5. Heterologous expression of AvrBs2 suppresses rice immunity and promotes

981

disease progression caused by bacterial pathogens in transgenic rice plants. A,

982

Induced expression of AvrBs2 in the T2 transgenic homozygous lines A4 and A5 after

983

DEX treatment. The AvrBs2-FLAG fusion was detected by Western blotting with an

984

anti-FLAG antibody. WT, the wild-type rice; DEX, dexamethasone; -, without DEX

985

treatment and +, with DEX treatment; WB, Western blot; Ponceau S staining shows

986

the equal loading of the total proteins. B and C, Up-regulation of the defense marker

987

genes OsPBZ1 (B) and OsPAL1 (C) induced by flg22 and chitin was almost

988

completely inhibited in the avrBs2 transgenic A4 and A5 lines upon DEX treatment.

989

The rice leaves were sprayed with 30 µM DEX or 0.1% ethanol as mock control

990

followed by incubation with flg22 or chitin for 6 h. D, The length of disease lesions

45

Page 46 of 83


Li et al. MPMI

991

caused by the Xoc strain RS105 in the wild-type Nipponbare and avrBs2 transgenic

992

lines A4 and A5. E, In planta bacterial population of RS105 in the wild-type and

993

avrBs2 transgenic lines A4 and A5 at the indicated time points after inoculation. F,

994

The length of disease lesions caused by the Xoo strain PXO99A in the wild-type and

995

avrBs2 transgenic lines A4 and A5. The wild-type and avrBs2 transgenic lines A4 and

996

A5 were inoculated by Xoc and Xoo at 24 h after spraying with 30 µM DEX and 0.1%

997

ethanol as mock control. * and ** indicate P values < 0.05 and 0.01, respectively.

998

Means ± SE are shown.

999 1000

Fig. S1. The degree of conservation of non-TAL effectors among important

1001

Xanthomonas pathogens. The color shading from yellow to red indicates increasing

1002

conservation of non-TAL effectors among different Xanthomonas species and strains

1003

including X. oryzae pv. oryzicola BLS256, X. oryzae pv. oryzae MAFF311018,

1004

KACC10331, PXO99A, X. axonopodis pv. citri 306, X. campestris pv. campestris

1005

B100, 8004, ATCC33913 and X. campestris pv. vesicatoria 85-10. Conservation of

1006

non-TAL effectors was determined by the presence frequency in these Xanthomonas

1007

species and strains, and by homology comparison as well. Blue dots indicate the

1008

presence frequency of non-TAL effectors in these Xanthomonas species and strains.

1009

Eleven

1010

sequence-available Xanthomonas strains and considered as core effectors in

1011

xanthomonads.

non-TAL

effector

proteins are

1012

46

present

in

all

currently

genome

Page 47 of 83


Li et al. MPMI

1013

Fig. S2 and S3. The marker-free gene-deletion mutants of 23 Xoc putative effector

1014

genes were verified by Southern blot analyses. Genomic DNA digested by the

1015

specified restriction enzymes was separated, blotted onto membranes and then probed

1016

with the isotope-labeled probes that were PCR amplified using the respective primer

1017

sets for each effector gene listed in Supplementary Table S3. WT indicates the

1018

digested genomic DNA isolated from the wild-type strain. One to four independent

1019

candidate mutant strains for each gene were selected for confirmation. The false

1020

gene-deletion mutants indicated by ◊ were discarded.

1021 1022

Fig. S4. Disease symptoms on the leaves of rice cv. Jingang 30 caused by different X.

1023

oryzae pv. oryzicola strains. A, Disease lesions on rice leaves caused by the wild-type,

1024

∆avrBs2 and ∆avrBs2(avrBs2) complementation strains. B, Disease lesions on rice

1025

leaves caused by the wild-type, ∆xopAA and ∆xopAA (xopAA) complementation

1026

strains. Disease symptoms on rice leaves were photographed at 14 d after pressure

1027

inoculation. WT, wild-type.

1028 1029

Fig. S5. Virulence of the wild-type, ∆xopAA and ∆xopAA (xopAA) complementation

1030

strains of Xoc on rice cultivars Nipponbare (A) and Jinhe 2 (B). WT, wild-type;

1031

∆xopAA (xopAA), the complementation strain with the plasmid-borne full-length

1032

xopAA gene. The length of disease lesions was scored at 14 d after pressure

1033

inoculation.

1034

47

Page 48 of 83


Li et al. MPMI

1035

Fig. S6. Subcellular localization of AvrBs2-GFP transiently expressed in N.

1036

benthamiana. The empty vector pGWB5-GFP was used as a control. AvrBs2-GFP

1037

was localized throughout the cells, observed under confocal microscopy.

1038 1039

Fig. S7. Pathogen-associated molecular pattern-induced activation of MAP kinases

1040

was not significantly altered by AvrBs2 expression in transgenic rice plants.

1041

A, flg22-induced activation of MAP kinases in the avrBs2 transgenic rice line A4

1042

before and after DEX treatment at the indicated time points. B, Chitin-induced

1043

activation of MAP kinases in the avrBs2 transgenic rice line A4 before and after DEX

1044

treatment at the indicated time points. The leaves of the A4 line were treated with

1045

1µm flg22 (A) or chitin (B) at 24 h after spraying with 30 µM DEX and 0.1% ethanol

1046

as mock control. Upper panel: Activation of MAP kinases was analyzed by

1047

immunoblotting with an anti-pMAPK antibody. Middle panel: Dex-induced

1048

expression of AvrBs2-FLAG in the transgenic plants was confirmed by Western blot

1049

analyses with an anti-FLAG antibody; Lower panel: Ponceau S staining shows the

1050

equal loading of the total proteins. DEX, dexamethasone; WB, Western blot. 0, 15, 30,

1051

and 60 indicate the treatment time of flg22 or chitin.

1052 1053

Fig. S8. Disease symptoms in the wild-type Nipponbare and avrBs2 transgenic rice

1054

plant lines after inoculation with bacterial pathogens X. oryzae pv. oryzae and X.

1055

oryzae pv. oryzicola. A, Disease lesions formed on the leaves of the wild-type and

1056

avrBs2 transgenic lines A4 and A5 with mock or DEX treatment after pressure

48

Page 49 of 83


Li et al. MPMI

1057

infiltration with the Xoc strain RS105. B, Disease lesions formed on the leaves of the

1058

wild-type and avrBs2 transgenic lines A4 and A5 with mock or DEX treatment after

1059

leaf clipping inoculation with the Xoo strain PXO99A. Disease symptoms on rice

1060

leaves were photographed at 14 d after inoculation.

49


Page 50 of 83

175x209mm (200 x 200 DPI)


Page 51 of 83

87x173mm (300 x 300 DPI)


Page 52 of 83

87x148mm (200 x 200 DPI)


Page 53 of 83

158x207mm (200 x 200 DPI)


Page 54 of 83

165x239mm (200 x 200 DPI)


Page 55 of 83

52x146mm (150 x 150 DPI)


Page 56 of 83

178x228mm (150 x 150 DPI)


Page 57 of 83

177x229mm (200 x 200 DPI)


Page 58 of 83

151x110mm (150 x 150 DPI)


Page 59 of 83

157x80mm (150 x 150 DPI)


Page 60 of 83

91x65mm (150 x 150 DPI)


Page 61 of 83

84x102mm (200 x 200 DPI)


Page 62 of 83

115x241mm (150 x 150 DPI)

Page 63 of 83


Table S1. Putative effector genes in Xanthomonas oryzae pv. oryzicola used in this study

Gene ID

Effector class

CDS location

PIP-box

XOC_0146

AvrBs2

148238…150382

TTCGC-N15-TTCGC

XOC_4450

HpaA

4582359…4583186

None

XOC_4432

XopA(Hpa1)

4567885…4568298

TTCGC-N15-TTCGC

XOC_2511

XopAA

2546510…2548597

None

XOC_1662a

XopAB

compl:1638176…1638757

None

XOC_4459

XopAE(HapF)

4592189…4594129

TTGCG-N15-TTTCG

XOC_0445

XopAF

445159…446019

None

XOC_4048

XopAJ(AvrRxo1)

compl:4182525…4183790

None

XOC_3934

XopAK

4069815…4070603

None

XOC_1264

XopC2

compl:1239507...1241156

None

XOC_4455

XopF1

compl:4586033…4587964

TTCGT-N15-TTCGC

XOC_0821

XopI

819545…820897

None

XOC_3274

XopK

compl:3352471…3355185

TTCGT-N15-TTCGT

XOC_3279

XopL

3357895…3359853

TTCGC-N15-TTCGC

XOC_0350

XopN

compl:343459...345621

TTCGG-N15-TTCTT

XOC_1098a

XopO

1082029…1082664

TTCCT-N15-TTCAT

XOC_1262

XopP1

1233914…1236055

TTCGT-N15-TTCGC

XOC_1263

XopP2

1236172...1238304

None

XOC_0099

XopQ

compl:97236…98630

TTCGT-N15-TTCAC

XOC_4603

XopR

compl:4743500...4744813

TTCGG-N15-TTCGC


Page 64 of 83

XOC_2513

XopU

2550471...2553551

TTCGC-N15-TTCGG

XOC_0649

XopV

compl:649036...650031

TTCGC-N15-TTCTG

XOC_0487a

XopW

487457...488068

TTGTT-N15-TTGCC

XOC_0618

XopX

compl:611633…613903

TTCTG-N15-TTCGC

XOC_3486a

XopY

3589017…3589847

TTCGC-N15-TTCGC

XOC_2415

XopZ1

compl:2438455...2442621

TTCTC-N15-TTCGC

Page 65 of 83

Table S2. Bacterial strains and plasmids used in this study Strains/plasmids

Characteristics

References or source


E. coli DH5a

High efficiency transformation

pRK600

Helper strain in tri-parental mating

X. oryzae pv. oryzicola RS105

Wild-type, RifR

△avrBs2

In frame deletion of XOC_0146, RifR

This study

△xopC2


This study

△xopQ


This study

△xopU


This study

△xopV


This study

△xopW

In frame deletion of XOC_0487a, RifR

This study

△xopX


This study

△xopY


This study

△xopAE


This study

△xopF1


This study

△xopP1


This study

△xopR


This study

△xopI


This study

△xopL


This study

△xopN


This study


Page 66 of 83

△xopK


This study

△xopAF


This study

△xopZ1


This study

△xopAA


This study

△xopP2


This study

△xopAK


This study

△xopO


This study

△xopAB


This study

A transient expression vector containing

(Li et al. 2005)

Plasmids pUC19-35S-FLAG-RBS

the cauliflower mosaic virsu 35S promoter, 3×FLAG, and a Rubisco Small Subunit terminator,AmpR pUC19-35S-FLAG-RBS-avrBs2

This study

pUC19-35S-FLAG-RBS-xopC2

This study

pUC19-35S-FLAG-RBS-xopQ

This study

pUC19-35S-FLAG-RBS-xopU

This study

pUC19-35S-FLAG-RBS-xopV

This study

pUC19-35S-FLAG-RBS-xopW

This study

pUC19-35S-FLAG-RBS-xopX

This study

pUC19-35S-FLAG-RBS-xopY

This study

pUC19-35S-FLAG-RBS-xopAE

This study


Page 67 of 83

pUC19-35S-FLAG-RBS-xopF1

This study

pUC19-35S-FLAG-RBS-xopP1

This study

pUC19-35S-FLAG-RBS-xopP2

This study

pUC19-35S-FLAG-RBS-xopR

This study

pUC19-35S-FLAG-RBS-xopI

This study

pUC19-35S-FLAG-RBS-xopL

This study

pUC19-35S-FLAG-RBS-xopN

This study

pUC19-35S-FLAG-RBS-xopK

This study

pUC19-35S-FLAG-RBS-xopAF

This study

pUC19-35S-FLAG-RBS-xopAK

This study

pUC19-35S-FLAG-RBS-xopAJ

This study

pUC19-35S-FLAG-RBS-xopA

This study

pUC19-35S-FLAG-RBS-hpaA

This study

pUC19-35S-GFP

Transient expression vector in protoplast

pUC19-35S-GFP-avrBs2 pTA7001

This study This study

DEX-induced expression vector,KmR

(Aoyama and Chua 1997)

pTA7001-avrBs2

This study

pTA7001-xopC2

This study

pTA7001-xopQ

This study

pTA7001-xopU

This study

pTA7001-xopV

This study


Page 68 of 83

pTA7001-xopW

This study

pTA7001-xopX

This study

pTA7001-xopY

This study

pTA7001-xopAE

This study

pTA7001-xopF1

This study

pTA7001-xopP1

This study

pTA7001-xopP2

This study

pTA7001-xopR

This study

pTA7001-xopI

This study

pTA7001-xopL

This study

pTA7001-xopN

This study

pTA7001-xopK

This study

pTA7001-xopAF

This study

pTA7001-xopAK

This study

pTA7001-xopAJ

This study

pTA7001-xopA

This study

pTA7001-hpaA

This study

pTA7001-GFP

This study

pTA7001-GFP-avrBs2

This study

pUFR80

pUFR80-avrBs2-del

Suicide vector for homologous

(Ried and Collmer

recombination, KmR

1987)

Vector for avrBs2 gene deletion

This study


Page 69 of 83

pUFR80-xopC2-del

Vector for xopC2 gene deletion

This study

pUFR80-xopQ-del

Vector for xopQ gene deletion

This study

pUFR80-xopU-del

Vector for xopU gene deletion

This study

pUFR80-xopV-del

Vector for xopV gene deletion

This study

pUFR80-xopW-del

Vector for xopW gene deletion

This study

pUFR80-xopX-del

Vector for xopX gene deletion

This study

pUFR80-xopY-del

Vector for xopY gene deletion

This study

pUFR80-xopAE-del

Vector for xopAE gene deletion

This study

pUFR80-xopF1-del

Vector for xopF1 gene deletion

This study

pUFR80-xopP1-del

Vector for xopP1 gene deletion

This study

pUFR80-xopR-del

Vector for xopR gene deletion

This study

pUFR80-xopI-del

Vector for xopI gene deletion

This study

pUFR80-xopL-del

Vector for xopL gene deletion

This study

pUFR80-xopN-del

Vector for xopN gene deletion

This study

pUFR80-xopK-del

Vector for xopK gene deletion

This study

pUFR80-xopAF-del

Vector for xopAF gene deletion

This study

pUFR80-xopZ1-del

Vector for xopZ1 gene deletion

This study

pUFR80-xopAA-del

Vector for xopAA gene deletion

This study

pUFR80-xopP2-del

Vector for xopP2 gene deletion

This study

pUFR80-xopAK-del

Vector for xopAK gene deletion

This study

pUFR80-xopO-del

Vector for xopO gene deletion

This study

pUFR80-xopAB-del

Vector for xopAB gene deletion

This study


Page 70 of 83

pVSP61 Expression vector, KmR (Loper and Lindow

1987)

pVSP61-avrBs2 This study

pVSP61-xopAA This study

Page 71 of 83


Table S3. The designed primers used in this study Gene

Primer name

DNA sequence

avrBs2

avrBs2-XhoI-F

AACTCGAGATGCGTATAGGTCCTCCGCAACC

avrBs2-NarI-R

TTGGCGCCCTCCGGCTCGGTCTGGTTGGCC

avrBs2-del-BamHI-F

AGGATCCGCCATTGTCGCTGGCAG

avrBs2-del-R

ATAGGCGTTTCCGGCGGCACGGTTGCGGAGG ACCTATACG

avrBs2-del-F

CGTATAGGTCCTCCGCAACCGTGCCGCCGGA AACGCCTAT

avrBs2-del-SalI-R

TTTGTCGACGTGGTCACCCTGTCGCTGCT

avrBs2-HA-SalI-R

GTCGACTACGCATAGTCAGGAACATCGTATG GGTACTCCGGCTCGGTCTGGTTGGC

xopC2

avrBs2-BamHI-F

AAGGATCCATGCGTATAGGTCCTCCGCAACC

avrBs2-SalI-R

AAAGTCGACCTCCGGCTCGGTCTGGTTGGCC

avrBs2-probe-F

TACGCGCTACCGGACCTGCAC

avrBs2-probe-R

AGCTTGCCTTGCAGTGCCTG

avrBs2-qRT-F

GAAGGGCTATCGTAATCTGGAG

avrBs2-qRT-R

CGCTGAAATCGTGCATCAAC

xopC2-XhoI-F

AACTCGAGATGGGAGGCAGCGATGTGGGCAT

xopC2-Csp45I-R

TTTTCGAATTTCCGGGCTTTCTCAACCACG

xopC2-del-EcoRI-F

AAGAATTCACGACGGCTCCGATGC

xopC2-del-F

TGCGTGCCAACACCTGATTCCATACCGTGCT

Page 72 of 83

CCAGT xopC2-del-R

ACTGGAGCACGGTATGGAATCAGGTGTTGGC


ACGCA

xopQ

xopC2-del-SalI-R

TTTGTCGACCCACTTCGCTGGATCACCC

xopC2-probe-F

GCACGCCACAAGGTCATCAG

xopC2-probe-R

GCAAAGCACACCCATCTGCC

xopQ-XhoI-F

AACTCGAGCATGCAGCCCACCGCAATCCGC

xopQ-Csp45I-R

AATTCGAAGCGCGCATGTTCCCCCTCGTC

xopQ-del-SalI-F

AAAGTCGACCAGGCAATCAGCGCAG

xopQ-del-R

TTCCGGACGCACAACCTCGGATTGCGGTGGGCT GCAT

xopQ-del-F

ATGCAGCCCACCGCAATCCGAGGTTGTGCGTCC GGAA

xopU

xopQ-del-HindIII-R

TTAAGCTTCCCATACATCTCCCACCCG

xopQ-probe-F

CCGGCATATCGTTCGGCGCC

xopQ-probe-R

GCCCAGGGTGACGACGACGT

xopU-XhoI-F

AACTCGAGCATGAATGAGGTGGCAGCCCA

xopU-Csp45I-R

TTTTCGAATGGCGCGCGCCGACGCTGCCT

xopU-del-BamHI-F

TGGATCCTGCAGAAGGTGGATCAGG

xopU-del-R

GGAACACCTGCTGCATCGTCCGCGATGACCTGG

xopU-del-F

CCAGGTCATCGCGGACGATGCAGCAGGTGTTCC

xopU-del-XhoI-R

AACTCGAGAAAGGCAAACGCCCGGAG

Page 73 of 83


xopV

xopU-probe-F

CTTCCATCCAGCTTCGCAAGT

xopU-probe-R

GAATTCAACATGGGCGGGAAG

xopV-XhoI-F

AACTCGAGCTGCCTCGCATGCGCCGAA

xopV-Csp45I-R

TTTTCGAATTCACCGTGAGGGTCAGAATGC

xopV-del-BamHI-F

TGGATCCTTGTGCAGCAGGGTA

xopV-del-R

GCGTGTACCTGGATCATTCATGCGTGTACTCCT GGG

xopV-del-F

CCCAGGAGTACACGCATGAATGATCCAGGTAC ACGC

xopW

xopV-del-HindIII-R

TTAAGCTTCCAGATTGCTGTGTTC

xopV-probe-F

CGGCACATTGAGAAACAACAC

xopV-probe-R

TTCGGATAGCTTGGCTGTTTC

xopW-XhoI-F

TTCTCGAGTGATGAAACCGACCCACATC

xopW-Csp45I-R

AATTCGAAACTGCCGCTACTGGAGGCGCCG

xopW-del-BamHI-F

AAGGATCCCGACGAGCCCACGATTCTCA

xopW-del-R

TCTTGATCCGCATCAGCTTCCGATGTGGGTCGG TTTCATC

xopW-del-F

GATGAAACCGACCCACATCGGAAGCTGATGCG GATCAAGA

xopW-del-SalI-R

TTTGTCGACGATGCTGACCTGCACGCCT

xopW-HA-SalI-R

GTCGACTACGCATAGTCAGGAACATCGTATGG GTAACTGCCGCTACTGGAGGCGCC

Page 74 of 83


xopX

xopW-probe-F

ATTGAGGAGTACGGGCGCAA

xopW-probe-R

CATCGCCGCGTCCGAATTGA

xopX-XhoI-F

AACTCGAGATGGCAGAGGCATCCTCCAACG

xopX-Csp45I-R

AATTCGAAATGCAGCGTCGAATGACGGCC

xopX-del-EcoRI-F

TGAATTCCACCGAGCCCTGCTGATT

xopX-del-R

GGCGTCTGGAGCGAGGTGCTGTTTCGGTCGAT GGTGCCGG

xopX-del-F

CCGGCACCATCGACCGAAACAGCACCTCGCTC CAGACGCC

xopX-del-HindIII-R

TTAAGCTTGATGATCACCACCGAAGCCATGG

xopX-HA-HindIII-R

AAGCTTACGCATAGTCAGGAACATCGTATGGG TAATGCAGCGTCGAATGACGGCC

xopY

xopX-probe-F

CGAGAAGAGCCCGTTGGTAA

xopX-probe-R

GCGTGATGGCTGCGTTCAAA

xopY-XhoI-F

AACTCGAGTATGAAGCGCTATGGGGATCA

xopY-Csp45I-R

AATTCGAAGGAAGACCGACTGCCAGTGGGC

xopY-del-EcoRI-F

TGAATTCGTCCAGCAGAACTTCGG

xopY-del-R

CAGTGGGCACGGTTGTGATTGCGCATAACCACT CCATGAG

xopY-del-F

CTCATGGAGTGGTTATGCGCAATCACAACCGTG CCCACTG

xopY-del-SalI-R

AAAGTCGACCACGTGATCGCCTGG

Page 75 of 83


xopAE

xopY-probe-F

CTGGAAGCCGAATACCACCTG

xopY-probe-R

GCAATCCAAGGTGGTTCCGTC

xopAE-XhoI-F

AACTCGAGATGCATCCGTTGAGTCAGCCGG

xopAE-Csp45I-R

AATTCGAACCATTCCCGGAGCAGTACATGC

xopAE-del-XhoI-F

AACTCGAGACGTGCGGCAGAATCTGCGC

xopAE-del-R

TCCATGTTGAGCGTCTTGACGGCTGACTCAACG GATGCAA

xopAE-del-F

TTGCATCCGTTGAGTCAGCCGTCAAGACGCTCA ACATGGA

xopAE-del-HindIII-R

TTAAGCTTGTCCGTACACAGCCAAAGGTCC

xopAE-HA-HindIII-R

AAGCTTACGCATAGTCAGGAACATCGTATGGGT AATGGATGCCCCATTCCCGGAGC

xopF1

xopAE-probe-F

GCGTGGAGATTGCGTTGCG

xopAE-probe-R

CCGGTATGGAGCAGCATCAA

xopF1-XhoI-F

AACTCGAGATGGGCTTGCCCTCTTCCAGCG

xopF1-Csp45I-R

TTTTCGAATGCTCGCCCGCTTTGCCACTG

xopF1-del-BamHI-F

TGGATCCTCTGGTAGAGCAG

xopF1-del-R

GGTACGGCTCTAGACGTTTGAAATCTGCGTGTT CTTCC

xopF1-del-F

GGAAGAACACGCAGATTTCAAACGTCTAGAGC CGTACC

xopF1-del-HindIII-R

TTAAGCTTGCCGCAGCGTCAATCAT

Page 76 of 83


xopP1

xopF1-probe-F

GGCGAAGGCGAAACCATTCT

xopF1-probe-R

CGACCGTAGACATTGGTAGGC

xopP1-XhoI-F

AACTCGAGATGAATATCAAAAAACGCATTCCGG

xopP1-Csp45I-R

TTTTCGAACTGCCCACCAGCGCCAGCCG

xopP1-del-XhoI-F

AACTCGAGCGTTGCGCCGATGATTA

xopP1-del-R

GCAGCAGACGCTCACTCGATGGAAAGCCCGCC GAGTT

xopP1-del-F

AACTCGGCGGGCTTTCCATCGAGTGAGCGTCTG CTGC

xopP1-del-HindIII-R

TTAAGCTTGGCCGATGATGATGCGGG

xopP1-HA-HindIII-R

AAGCTTACGCATAGTCAGGAACATCGTATGGGT ATCGCTGCCCACCAGCGCCAG

xopR

xopP1-probe-F

GTCATCCGTCATCCGTCATT

xopP1-probe-R

CGATCATCTTCTGGCATGCG

xopR-XhoI-F

AACTCGAGATGTCGCCGACGTCGTCCTCGC

xopR-Csp45I-R

TTTTCGAATCGGTAACCGTTCTCCATTGAG

xopR-del-BamHI-F

TGGATCCTGTTGGCAATCGTGTGTA

xopR-del-R

CTCGCGGTGTACTAGGGTCGTGCGCATCACTTA CTCC

xopR-del-F

GGAGTAAGTGATGCGCACGACCCTAGTACACC GCGAG

xopR-del-HindIII-R

TTAAGCTTTCGATTTCACCCGTGCGC

Page 77 of 83


xopI

xopR-probe-F

TGAAGACCCTACGCCTACAGA

xopR-probe-R

AGGTGGGAGATGCCAACGAAT

xopI-XhoI-F

AACTCGAGATGGCGGAAGACACCAGGGA

xopI-Csp45I-R

TTTTCGAACGTGTCCATATACCTGCGCGAC

xopI-del-BamHI-F

AGGATCCAACACGTCGCTTGGCTTG

xopI-del-R

AGGTCAGCGGCATGGTTCGGTTGATCGGCATA TCG

xopI-del-F

CGATATGCCGATCAACCGAACCATGCCGCTGA CCT

xopL

xopI-del-SalI-R

AAAGTCGACCAACAGGCCAACCACCA

xopI-probe-F

TAGCATGGAGGCGCTGGACG

xopI-probe-R

ATGGAATCCGCGCCGCAAGC

xopL-XhoI-F

AACTCGAGATGTCGCAATGGCAACAACACTA

xopL-Csp45I-R

TTTTCGAAGCGCGAGGGTTCCGAGGTTGTT

xopL-del-EcoRI-F

TGAATTCGGGCCGATCAAGAACAAA

xopL-del-R

CTTCTGCGCCTTCCAATTGCATTGGACGCACTC TGCT

xopL-del-F

AGCAGAGTGCGTCCAATGCAATTGGAAGGCGC AGAAG

xopL-del-SalI-R

AAAGTCGACGCTGCCGAAGTGGAA

xopL-probe-F

GGCAGTTGCCGTCTCCAACG

xopL-probe-R

TCTGCGTCGTAGTGTTGTTGC

Page 78 of 83


xopN

xopN-XhoI-F

AACTCGAGAAATGTGATGAAACCTGCTGC

xopN-Csp45I-R

TTTTCGAACGGCGGCAGTGCCCGATCCTCC

xopN-del-SalI-F

AAAGTCGACCACGTGGATCCGGAGCTC

xopN-del-R

GCCATGCCTCCTGCCAGGGCGAATGGCTGGGC GATGCG

xopN-del-F

CGCATCGCCCAGCCATTCGCCCTGGCAGGAGG CATGGC

xopN-del-HindIII-R

TTAAGCTTCAAGGTGGATCTGGCCGACG

xopN-HA-HindIII-R

AAGCTTACGCATAGTCAGGAACATCGTATGGG TACGCCGGCGGCAGTGCCCGAT

xopK

xopN-probe-F

CGCACCACGCACGCAGATG

xopN-probe-R

GCTCAATCCCTGTGCCCTGTA

xopK-XhoI-F

AACTCGAGATGAACGTGCTGCAGAAGCGG

xopK-Csp45I-R

TTTTCGAAGGTCGTGGACGCATCAGCTGC

xopK-del-EcoRI-F

TGAATTCTGGGTGTGCTCTGCAACAGC

xopK-del-R

TCCGCCTGCGCGCTGAGCTTCGGTAACTCGGGC GTTGGCA

xopK-del-F

TGCCAACGCCCGAGTTACCGAAGCTCAGCGCG CAGGCGGA

xopK-del-HindIII-R

TTAAGCTTCCCGCTGCCGATGCGCAC

xopK-HA-HindIII-R

AAGCTTACGCATAGTCAGGAACATCGTATGGGT AGGTCGTGGACGCATCAGCTGC

Page 79 of 83


xopAF

xopK-probe-F

TGTAACGCTCCTGGAACCTGC

xopK-probe-R

GGCCGGTGGATGCGCTGATT

xopAF-XhoI-F

AACTCGAGAAGCAAGCGTCCGATGCCG

xopAF-Csp45I-R

TATTCGAAATCGCGGAGCTGCTCCGTAGC

xopAF-del-EcoRI-F

AGAATTCTGTTTGATGCGCGCGTT

xopAF-del-R

TTCACAGCGGGTCAATCGCGTGAGTCATTCATT GCCCAGT

xopAF-del-F

ACTGGGCAATGAATGACTCACGCGATTGACCCG CTGTGAA

xopAJ

xopA

hpaA

xopZ1

xopAF-del-HindIII-R

TAAAGCTTAGGCCATAAGGAAGCGAACG

xopAF-probe-F

AATCCCGCCAACGTCGATGT

xopAF-probe-R

CCGTGATGACCGGCGTATTA

xopAJ-XhoI-F

AACTCGAGCAATAGAATGTACGTGATAGA

xopAJ-Csp45I-R

AATTCGAAAATTAGCTCGCTGTGAGCAGCT

xopA-XhoI-F

AACTCGAGCCACGATGAATTCTTTGAACAC

xopA-Csp45I-R

TTTTCGAACTGCATCGATCCGCTGTCGTTC

hpaA-XhoI-F

TTCTCGAGTTCCCATGATCCGTCGCATCT

hpaA-Csp45I-R

TTTTCGAATGGGCGAACCTCCTGAGCCGC

xopZ1-XhoI-F

AACTCGAGATTCCACCATGCCCCGCATTCC

xopZ1-SpeI-R

AAACTAGTAACGCTACGGGACTGGCTCGTA

xopZ1-del-BamHI-F

TGGATCCTTCCAGCACATGCACGCGG

xopZ1-del-R

GCTGCCGGCCAAGTGCAATTCCTGGAAGGCAAG

Page 80 of 83

CGGCAAT xopZ1-del-F

ATTGCCGCTTGCCTTCCAGGAATTGCACTTGGCC


GGCAGC xopZ1-del-HindIII-R

TTAAGCTTCAGCGGTTGGAAGCGCAAC

xopZ1-HA-HindIII-R

AAGCTTACGCATAGTCAGGAACATCGTATGGGTA CGGGACTGGCTCGTAGGGAATC

xopAA

xopZ1-probe-F

ATTCGGCGTCGGCGTAGTGC

xopZ1-probe-R

CCACTACTCATGCCCAGTCGCTCT

xopAA-KpnI-F

ATGGTACCTCGTAAGGACTCCATCATGCA

xopAA-ClaI-R

TTATCGATTTCCGACTGATGCGCCGGATGC

xopAA-del-BglII-F

TTTAGATCTGCGCGTCAATGCCTTCGTCG

xopAA-del-R

CGACGGTCCCGGCGATGAAGCGTTAGGGCTATGG CTTGCG

xopAA-del-F

CGCAAGCCATAGCCCTAACGCTTCATCGCCGGGA CCGTCG

xopAA-del-HindIII-R

TTAAGCTTGACCGTGAACAACTGTTCGC

xopAA-HA-HindIII-R

AAGCTTACGCATAGTCAGGAACATCGTATGGGTA TTCCGACTGATGCGCCGGATGC

xopP2

xopAA-probe-F

TGGCGGCTGGCAAGAATACC

xopAA-probe-R

ACCGACAGGTTGCCCAGGAAGG

xopP2-XhoI-F

AACTCGAGGCACGCATGCGCACCTCTGAT

xopP2-Csp45I-R

ATTTCGAATTGTTGCCCGCCAGCGCCAGCC

Page 81 of 83

xopP2-del-BamHI-F

AGGATCCAAGCCATCCGACTGCAGC

xopP2-del-R

ACGGCGCGTCGTGGTTATTGCGTTGCGCTCGTCAA


TGACC xopP2-del-F

GGTCATTGACGAGCGCAACGCAATAACCACGACG CGCCGT

xopP2-del-SalI-R

AAAGTCGACCACGCCAGCCCAATGG

xopP2-HA-SalI-R

GTCGACTACGCATAGTCAGGAACATCGTATGGGT ATTGTTGCCCGCCAGCGCCAGC

xopAK

xopP2-probe-F

GCAAACCGTGCGTGGAGTGCT

xopP2-probe-R

TCCCGCACGGAGCTTGCTTT

xopAK-XhoI-F

AACTCGAGATGAAGCCGGCTTGCATCAGACC

xopAK-ClaI-R

TTATCGATCCACGACTTGTAGTAATAGATGCC

xopAK-del-EcoRI-F

AGAATTCCAACCATAGGTTCGGCCGG

xopAK-del-R

TTGATGGCATTCTCCTCGCCGGCTTCATGGAGTCA GAAC

xopAK-del-F

GTTCTGACTCCATGAAGCCGGCGAGGAGAATGCC ATCAA

xopO

xopAK-del-BamHI-R

TGGATCCCTACATGAGCACATTCG

xopAK-probe-F

GATGGCCGATCACGCTACTA

xopAK-probe-R

AATCCATCTTGCGCGAGATT

xopO-KpnI-F

AAGGTACCATGATCAACACTTCCGTCAAGG

xopO-Csp45I-R

AATTCGAACCTGTTGATCCGACGACTTTCCT

Page 82 of 83

xopO-del-BamHI-F

TGGATCCGGTCCCAGGCTTC

xopO-del-R

GAACGCAAATCAGCATTCGCTTGCCGTGACTGAC


GTT xopO-del-F

AACGTCAGTCACGGCAAGCGAATGCTGATTTGCG TTC

xopAB

xopO-del-SalI-R

TTTGTCGACTCGCCGTGCGCAATTTCC

xopO-probe-F

CCATGCAGCCTGGTCCGTCT

xopO-probe-R

TTCCTTCAACAGCCGTGGCG

xopAB-del-BamHI-F

TGGATCCTGGAAATGCTGCGGCAG

xopAB-del-R

AACTTGTGGAGTTCCATCGCACATGCCGTGGCAC

xopAB-del-F

GTGCCACGGCATGTGCGATGGAACTCCACAAGTT

xopAB-del-HindIII-R

TAAAGCTTACGGGTTGTTGCGCAC

xopAB-probe-F

GCAAGCGGCGAGAAGCATCT

xopAB-probe-R

ATCGATGGCTGACGCTGTGGT

OsActin1 OsActin1-qRT-F

OsPBZ1

OsPAL1

gfp

TCCATCTTGGCATCTCTCAG

OsActin1-qRT-R

GTACCCGCATCAGGCATCTG

OsPBZ1-qRT-F

GACATCGTGGATGGCTACTATGG

OsPBZ1-qRT-R

TCACTCACTCT AGGTGGGATATAC

OsPAL1-qRT-F

CTCGAGTGCCTCAAGGAGTG

OsPAL1-qRT-R

GCCTCCACACTCCACTGTTA

gfp-SphI-F

TTAGCATGCATGGTGAGCAAGGGCGAGGAG

gfp-HindIII-R

TTTAAGCTTGCCGCTTTACTTGTACAGCTCGTC


Page 83 of 83

gfp-BstBI-F AATTCGAAATGGTGAGCAAGGGCGAGGAG

gfp-SpeI-R AAACTAGTGCCGCTTTACTTGTACAGCTCGTC

Identification of 17 HrpX-regulated proteins including two novel type III effectors, XOC_3956 and XOC_1550, in Xanthomonas oryzae pv. oryzicola.

Simultaneous Detection of Xanthomonas oryzae pv. oryzae and X. oryzae pv. oryzicola in Rice Seed Using a Padlock Probe-Based Assay.

Complete sequence and detailed analysis of the first indigenous plasmid from Xanthomonas oryzae pv. oryzicola.

XocR, a LuxR solo required for virulence in Xanthomonas oryzae pv. oryzicola.

Effector Diversification Contributes to Xanthomonas oryzae pv. oryzae Phenotypic Adaptation in a Semi-Isolated Environment.

DgcA, a diguanylate cyclase from Xanthomonas oryzae pv. oryzae regulates bacterial pathogenicity on rice.

A systematic analysis of the role of GGDEF-EAL domain proteins in virulence and motility in Xanthomonas oryzae pv. oryzicola.

Genome subtraction for the identification of potential antimicrobial targets in Xanthomonas oryzae pv. oryzae PXO99A pathogenic to rice.

The broad bacterial blight resistance of rice line CBB23 is triggered by a novel transcription activator-like (TAL) effector of Xanthomonas oryzae pv. oryzae.

Effector Mimics and Integrated Decoys, the Never-Ending Arms Race between Rice and Xanthomonas oryzae.

Sensitive detection of Xanthomonas oryzae Pathovars oryzae and oryzicola by loop-mediated isothermal amplification.

Two thiadiazole compounds promote rice defence against Xanthomonas oryzae pv. oryzae by suppressing the bacterium's production of extracellular polysaccharides.

Mutation of the rice XA21 predicted nuclear localization sequence does not affect resistance to Xanthomonas oryzae pv. oryzae.

Transcriptome-Based Identification of Differently Expressed Genes from Xanthomonas oryzae pv. oryzae Strains Exhibiting Different Virulence in Rice Varieties.

Targeted promoter editing for rice resistance to Xanthomonas oryzae pv. oryzae reveals differential activities for SWEET14-inducing TAL effectors.

Retracted: Genetic Analysis and Molecular Identification of Virulence in Xanthomonas oryzae pv. oryzae Isolates.

Crystallization and preliminary X-ray crystallographic analysis of the XoGroEL chaperonin from Xanthomonas oryzae pv. oryzae.

Xanthomonas euvesicatoria type III effector XopQ interacts with tomato and pepper 14-3-3 isoforms to suppress effector-triggered immunity.

PilZ Domain Proteins of the Plant Pathogen Xanthomonas oryzae pv. oryzae Function Differentially in Virulence.

Deciphering the Role of Tyrosine Sulfation in Xanthomonas oryzae pv. oryzae Using Shotgun Proteomic Analysis.

Expression of colSR Genes Increased in the rpf Mutants of Xanthomonas oryzae pv. oryzae KACC10859.

Time-resolved pathogenic gene expression analysis of the plant pathogen Xanthomonas oryzae pv. oryzae.

Suppression of Xo1-Mediated Disease Resistance in Rice by a Truncated, Non-DNA-Binding TAL Effector of Xanthomonas oryzae.

The crystal structure of type III effector protein XopQ from Xanthomonas oryzae complexed with adenosine diphosphate ribose.