Host-induced gene silencing of the MAPKK gene PsFUZ7 ...

1

Plant Physiology 1

Host-induced gene silencing of the MAPKK gene PsFUZ7 2

confers stable resistance to wheat stripe rust 3

Xiaoguo Zhu1, Tuo Qi1, Qian Yang1, Fuxin He1, Chenglong Tan1, Wei Ma1, 4

Ralf Thomas Voegele2, Zhensheng Kang*1, and Jun Guo*1 5

1 State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant 6

Protection, Northwest A&F University, Yangling 712100, Shaanxi, China. 7

2 Department of Phytopathology, Institute of Phytomedicine, Faculty of 8

Agricultural Sciences, University of Hohenheim, 70599 Stuttgart, Germany 9

* Authors for correspondence: 10

Jun Guo: Northwest A&F University, E-mail: [email protected]; 11

Tel/fax: 0086-29-87082439. 12

Zhensheng Kang: Northwest A&F University, E-mail: 13

[email protected]; Tel/fax: 0086-29-87080061 14

Running title: HIGS of PsFUZ7 confers stable resistance to Pst. 15

One sentence summary: Transgenic wheat expressing a double-stranded RNA 16

targeting MAPKK gene PsFUZ7 from Puccinia striiformis f. sp. tritici exhibits strong 17

resistance to stripe rust. 18

List of authors’ contributions 19

J.G. and Z.S.K designed the experiments; X.G.Z., T.Q., Q.Y., F.X.H., C.L.T and 20

Plant Physiology Preview. Published on October 25, 2017, as DOI:10.1104/pp.17.01223

Copyright 2017 by the American Society of Plant Biologists

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2

W.M. performed the experiments; X.G.Z., J.G., R.T.V, and Z.S.K analyzed the 21

data and wrote the paper. All authors discussed the results and commented on 22

the manuscript. 23

Funding information 24

This study was supported by the National Natural Science Foundation of China 25

(No. 31620103913), the National Basic Research Program of China (No. 26

2013CB127700), the 111 Project from the Ministry of Education of China (No. 27

B07049) and Natural Science Basic Research Plan in Shaanxi Province of 28

China (2017JM3007). 29



3

ABSTRACT 30

RNA interference (RNAi) is a powerful genetic tool to accelerate research in 31

plant biotechnology and to control biotic stresses by manipulating target gene 32

expression. However, the potential of RNAi in wheat to efficiently and durably 33

control the devastating stripe rust fungus Puccinia striiformis f. sp. tritici (Pst), 34

remained largely under explored, so far. To address this issue, we generated 35

transgenic wheat lines expressing double-stranded RNA targeting PsFUZ7 36

transcripts of Pst. We analyzed expression of PsFUZ7 and related genes, and 37

resistance traits of the transgenic wheat lines. We show that PsFUZ7 is an 38

important pathogenicity factor which regulates infection and development of 39

Pst. A PsFUZ7 RNAi construct stably expressed in two independent transgenic 40

wheat lines confers strong resistance to Pst. Pst hyphal development is 41

strongly restricted, and necrosis of plant cells in resistance responses was 42

significantly induced. We conclude that trafficking of RNA molecules from 43

wheat plants to Pst may lead to a complex molecular dialogue between wheat 44

and the rust pathogen. Moreover, we confirm the RNAi-based crop protection 45

approaches can be used as a novel control strategy against rust pathogens in 46

wheat. 47



4

INTRODUCTION 48

Global wheat yields are estimated to be reduced by 3% to over 90% per 49

year because of the obligate biotrophic pathogen P. striiformis f. sp. tritici (Pst), 50

jeopardizing global food security (Wellings, 2011; Chen, 2014). It is evident 51

that Pst constitutes a significant threat to wheat production worldwide. 52

Currently, approaches to manage this disease rely on cultivar resistance 53

coupled with fungicide application (Chen, 2014). However, driven by a greater 54

need for wheat production (Singh et al., 2011), the necessity for environmental 55

protection (Ishii, 2006), the constant evolution of virulence in rust fungi (Chen 56

et al., 2009), and the loss of natural resistance in wheat cultivars (Mcintosh et 57

al., 1995), innovative alternative approaches to control rust disease are 58

urgently required. To date, several technologies have been used to transiently 59

silence Pst genes to restrict pathogen development (Panwar et al., 2013; Fu et 60

al., 2014). However, the pathogen is capable of overcoming this transient 61

resistance barrier, and hence, strategies conferring durable resistance to Pst 62

must be sought. 63

A powerful genetic tool, RNA interference (RNAi), a conserved eukaryotic 64

mechanism that performs a crucial role in gene regulation, has been used to 65

enhance crop resistance by silencing critical genes (Bartel, 2004; Baulcombe, 66

2004). A key conserved trait of RNAi is the cleavage of precursor double 67

stranded RNA (dsRNA) into short 21-24 nucleotide small interfering RNAs 68

(siRNAs) by a ribonuclease called DICER, or Dicer-like (DCL) (Fagard et al., 69

2000). siRNAs are then incorporated into the RNA-induced silencing complex 70

(RISC) containing an Argonaute (AGO) protein (Fagard et al., 2000). 71

Subsequently, specific degradation of the target mRNA sharing sequence 72

similarity with the inducing dsRNA takes place (Ghildiyal and Zamore, 2009; 73

Liu, 2010). Numerous reports have demonstrated the efficiency of RNAi to 74

improve control of bacteria, viruses, fungi, insects, nematodes, and parasitic 75

weeds (Saurabh et al., 2014). Insects feeding on transgenic plants carrying 76

RNAi constructs against genes of the pest were severely constrained in their 77



5

development (Huang et al., 2006; Baum et al., 2007; Mao et al., 2007). In 78

genetically engineered RNAi crop plants, defense against fungi was 79

substantially enhanced (Nowara et al., 2010; Koch et al., 2013; Ghag et al., 80

2014). Host-Induced Gene Silencing (HIGS) of the Fusarium cytochrome P450 81

lanosterol C-14α-demethylase (CYP51) gene, which is essential for ergosterol 82

biosynthesis, confers resistance of barley to Fusarium species (Koch et al., 83

2013). During interaction of the host with the pathogen Blumeria graminis, 84

siRNA molecules are exchanged and restrict fungal development in plants 85

carrying RNAi constructs targeting fungal transcripts (Nowara et al., 2010). 86

Mitogen-activated protein kinase (MAPK) cascades regulate a variety of 87

cellular processes in response to extracellular and intracellular stimuli (Van 88

Drogen and Peter, 2002). In our study, the MAPK kinase gene PsFUZ7, which 89

was shown to play an important role in Pst virulence by regulating hyphal 90

morphology and development, was selected as target for RNAi. Our results 91

indicate that the expression of RNAi constructs in transgenic wheat plants 92

confers strong and durable resistance to Pst, along with a severe restriction of 93

Pst development. This efficient inhibition of disease development suggests 94

that HIGS is a powerful strategy to engineer transgenic wheat resistant against 95

the obligate biotrophic pathogen Pst and has potential as an alternative 96

approach to conventional breeding, or chemical treatment for the development 97

of environmentally friendly and durable resistance in wheat and other food 98

crops. 99

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6

RESULTS 102

Three MAPK Cascade Genes are Highly Induced during Pst 103

Differentiation 104

During our search for potential genes that regulate the development of Pst, 105

we identified and cloned five candidate genes from the virulent Pst strain 106

CYR32. These genes were found to be orthologs of Ustilago maydis MAPK 107

signaling pathway-related genes (Supplemental Table S1). Transcript profiles 108

assayed by quantitative real-time PCR (qRT-PCR) show that PsKPP4, 109

PsFUZ7, PsKPP6, and PsCRK1 are all induced at early differentiation stages, 110

whereas PsKPP2 is significantly down-regulated during this phase (Fig. 1). 111

Transcript levels of PsKPP4, PsFUZ7, and PsKPP6 are increased more than 112

30-fold during the very early stage of colonization of wheat by urediospores 113

(12 h), and the time of primary haustorium formation (18 h), the stage 114

indicating successful colonization of the host. PsKPP4 and PsFUZ7 are 115

induced more than 20-fold during secondary hyphae formation (48-72 h), the 116

stage essential for hyphal expansion. These results suggest that PsKPP4, 117

PsFUZ7, and PsKPP6 participate in early Pst development. Therefore, these 118

genes were chosen as target genes for subsequent virus-induced gene 119

silencing (VIGS) experiments. 120

Transient Silencing of PsFUZ7 Significantly Inhibits Growth of Pst 121

VIGS mediated by the barley stripe mosaic virus (BSMV) has been 122

established in barley and wheat (Holzberg et al., 2002; Scofield et al., 2005). 123

To confirm the effect of silencing PsKPP4, PsFUZ7, and PsKPP6 during the 124

interaction between wheat and Pst strain CYR32, two ~250-bp silencing 125

sequences of each gene were derived from the 5’- and the 3’-prime end of the 126

gene to generate different BSMV constructs (Supplemental Table S2), 127

respectively. At 14 days post inoculation (dpi) with Pst, generation of uredia is 128

suppressed in plants inoculated with BSMV-silencing sequences, and those 129

carrying BSMV:PsFUZ7-as constructs show the highest inhibition of uredia 130



7

formation (Fig. 2A). qRT-PCR analysis of total RNA isolated from silenced 131

leaves sampled at 2 and 7 dpi, revealed effective reductions in transcript levels 132

of the fungal target genes (Fig. 2B). Values are expressed relative to the 133

endogenous Pst reference gene EF-1 (PsEF-1), with the empty vector 134

(BSMV:γ) set to 1 (Yin et al., 2011). To demonstrate the specificity of VIGS 135

against PsKPP4, PsFUZ7 and PsKPP6, the expression of their closest 136

homologs at 2 dpi was also examined (Supplemental Fig. S1 and 137

Supplemental Table S3). 138

Microscopic analyses revealed that initial haustorium formation and 139

elongation of secondary hyphae are both reduced in BMSV:PsFUZ7-1as and 140



8

BMSV:PsFUZ7-2as treated plants (Fig. 3). PsFUZ7-2as-silenced plants exhibit 141

a more pronounced inhibition of haustorium formation and mycelial extension 142

with decreased hyphal length and reduced size of infection areas than other 143

silencing constructs. As a result, fewer uredia were produced in plants carrying 144

BMSV:PsFUZ7-2as (Fig. 2C). This result indicates that PsFUZ7 plays an 145

important role in mycelial growth and development which eventually leads to a 146

significant inhibition of uredia generation and virulence of Pst. 147

The Function of PsFUZ7 is Conserved among Phytopathogenic Fungi 148

PsFUZ7 contains the typical domain structure of MAPKKs, including 12 149



9

catalytic kinase subdomains (Hamal et al., 1999), two putative phosphorylation 150

sites, an activation loop (S/TXXXS/T) as the putative target of an upstream 151

MAPKKK, and a DEJL motif (K/R-K/R-K/R-X (1-5)-L/I-X-L/I) at the N-terminus, 152

which is known to function as a MAPK docking site (Supplemental Fig. S2) 153

(Chen et al., 2012). Sequence alignments indicate that PsFUZ7 shares 70 %, 154

82 %, and 84 % sequence identity with orthologs from Magnaporthe oryzae, P. 155

triticina, and P. graminis f. sp. tritici, respectively. Consistent with this high 156

similarity, PsFUZ7 partially complements the Magnaporthe oryzae mst7 157

mutant in appressorium formation and plant infection (Supplemental Fig. S3). 158

Overexpression of PsFUZ7 in fission yeast results in morphological changes 159



10

and an increased sensitivity to environmental stresses (Supplemental Fig. S4). 160

In accordance with these in vivo data, in vitro assays revealed that stripe rust 161

urediospores treated with the kinase inhibitor U0126 germinate at a lower rate 162

and produce a higher frequency of abnormally differentiated and clearly 163

distorted germ tubes or spherical structures along the mycelial apex at 3 h and 164

6 h post-treatment (Supplemental Fig. S5). Overall, these results strengthen 165

the view that PsFUZ7 may have important roles in pathogenesis. 166

RNAi Constructs in Transgenic Wheat Plants Detected by Southern Blot 167

and PCR 168

To test whether the stable expression of PsFUZ7 silencing constructs can 169

confer resistance to Pst, the selected, most effective RNAi construct, a 759-bp 170

cassette containing an inverted repeat derived from PsFUZ7-2as was 171

introduced into vector pAHC25, and transformed into Triticum aestivum L. cv. 172

Xinong1376 (XN1376) by particle bombardment (Supplemental Fig. S6A). The 173

PsFUZ7-RNAi construct in transgenic wheat plants was detected by PCR 174

(Supplemental Fig. S6B), and the stable integration of RNAi constructs into the 175

wheat genome was verified by Southern blot with specific probes 176

(Supplemental Fig. S6C). No consecutive 21-24 nucleotide (nt) sequences 177

were found in wheat and/or other plant or fungal species for PsFUZ7 178

(Supplemental Table S4). To identify potential off-target sites, BLASTN was 179

performed using the target sequence. Among all putative off-targets examined, 180

12 sequences were detected in wheat and Pst at one or more locus with a 1-3 181

bp mismatch, respectively (Supplemental Fig. S7 and S8; Supplemental Table 182

S2). As shown in Supplemental Fig. S7 and S8, no significant down-regulation 183

was found in the potential putative off-target genes. Transgenic wheat lines 184

that contained RNAi constructs, displayed normal morphology, and set viable 185

seeds were assayed for resistance against Pst. 186

Two T3 Transgenic Wheat Lines Confer Strong Resistance to Pst 187



11

Two independent transgenic lines, L65 and L91, which were highly effective 188

in restricting the spread of Pst, were selected in T3 generations and examined 189

at 16 dpi (Fig. 4A). To verify whether this phenotype is caused by the 190

expression and processing of siRNA, Northern blot analysis was performed 191

with RNA extracted from 14-day-old seedlings of the T3 transgenic lines, using 192

the same specific probes as those used for Southern blot analyses. As shown 193

in Fig. 4B, a single band with the expected size of ~21 nt is present in 194

transgenic lines L65 and L91, while no signal can be detected in control plants, 195

documenting the presence of PsFUZ7 siRNAs in the transgenic lines. To 196

further confirm that the expression of siRNA in wheat is able to silence 197

PsFUZ7, qRT-PCR was performed to analyze transcript abundance of 198

PsFUZ7 in Pst-infected transgenic lines at 3, 10, 16 dpi. PsFUZ7 transcripts in 199

L65 were reduced by 42.2 %, 48.3 %, and 49.3 % compared to control lines at 200

3, 10, 16 dpi, respectively. Similarly, PsFUZ7 transcripts were reduced by 201

41.7 %, 69.9 % and 74.8 % in L91 (Fig. 4C). According to the McNeal’s 202

uniform scale system (McNeal et al., 1971) the host response class of 203

transgenic lines L65 and L91 ranged from 1 to 3, indicating high or medium 204

resistance to Pst. By contrast, the response of control lines was in the 6 to 7 205

class, which along with abundant sporulation and little necrotic/chlorotic stripes 206

indicates susceptibility to Pst (Fig. 4D). Biomass analyses showed that both 207

transgenic lines exhibit significant reductions in fungal biomass (P < 0.01) of 208

68-71 % and 50-57 %, respectively, at 16 dpi with Pst (Fig. 4E). In addition, 209

compared with control plants, transcript levels of some MAPK signal 210

pathway-related genes (Fig. 5A), are reduced in Pst-infected transgenic lines 211

L65 and L91, while some plant defense-related genes are up-regulated (Fig. 212

5B). These results demonstrate that transgenic wheat lines carrying RNAi 213

constructs can produce and process siRNA molecules which efficiently 214

down-regulate PsFUZ7 in Pst, and also affect the expression of some related 215

genes in Pst and wheat. 216

217



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Pst Development and Growth is Severely Suppressed in Transgenic 218

Wheat Lines Carrying PsFUZ7 RNAi Constructs 219

To investigate Pst development in transgenic wheat lines L65 and L91, 220

fungal structures were stained with Wheat Germ Agglutinin (WGA) for 221

microscopic observation. Pst in transgenic lines L65 and L91 exhibits poorly 222

developed hyphae with minimal haustorium formation, while a widespread 223

hyphal network with numerous haustoria in mesophyll cells is observed in 224

control plants at 10 dpi with CYR32 (Fig. 6A-C). Notably, large areas of 225

hypersensitive cell death are induced in transgenic lines L65 and L91 (Fig. 6A 226

and D). The observed restriction of fungal development within 227



13

siRNA-producing host tissue is consistent with the measured reduction of 228

fungal biomass and uredia formation. 229

To further visualize mycelial growth in Pst-infected wheat tissue, 230

transmission electron microscopy was used to examine fungal morphology 231

and wheat cells (Fig. 6E). Disassembly of the nuclear envelope and shrinkage 232

of protoplasts are observed in Pst cells during colonization of the tissue of 233

transgenic plants L65 and L91 (Fig. 6E). In addition, plant plasma membranes 234

were ruptured where they contacted Pst hyphae. By contrast, both fungal and 235

host cells developed normally in control lines (Fig. 6E). These results indicate 236

that RNAi molecules stably expressed in transgenic wheat plants are able to 237



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confer genetically stable resistance to rust fungi by targeting fungal PsFUZ7 238

transcripts resulting in the suppression of Pst growth. 239

240

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15

DISCUSSION 242

Currently, the most effective strategy to control stripe rust disease is the 243

application of fungicides. However, fungicide residues on food products remain 244

a threat to human health (Singh et al., 2015). Traditional plant breeding to 245

improve crop traits is another effective strategy but is time-consuming and 246

labor intensive (Saurabh et al., 2014). During the last two decades, research 247

efforts have focused on strategies to convert to biotechnological approaches 248

for crop improvement. The emergence of RNAi, which can be employed to 249

manipulate gene expression to improve quality traits in crops, offers potential 250

promise (Baulcombe, 2004; Saurabh et al., 2014). However, it remained 251

unclear whether Pst has a functional silencing system that can be induced in 252

transgenic wheat carrying RNAi constructs. Our approach was to determine 253

whether the expression of RNAi molecules derived from the MAPK kinase 254

gene PsFUZ7 in transgenic wheat could confer genetically stable resistance to 255

rust pathogens of wheat. PsFUZ7 was selected as the target gene for silencing 256

because it is expressed at high levels during penetration and parasitic stages 257

of the Pst-wheat interaction and showed the most positive phenotype in virus 258

induced transient silencing assays compared with two other kinases (Fig. 1). In 259

the fungal kingdom, MAPK kinases are evolutionarily conserved proteins that 260

function as key signal transduction components regulating a series of cellular 261

processes (Hamel et al., 2012). In the functional screening of candidate genes 262

for the generation of efficient RNAi sequences, supplementary assays 263

confirmed that the PsFUZ7 is functionally conserved with MAPKKs from other 264

fungi and participates in development and morphogenesis critical for virulence 265

of Pst. Off-target effects, resulting in the knockdown of other transcripts with 266

limited similarity to siRNA, often occur during the application of RNAi in plants 267

and humans (Birmingham et al., 2006; De, 2014). In our study, no off-target 268

effects were detected, indicating that the PsFUZ7 fragment selected to 269

generate RNAi constructs could be an ideal target for control of Pst via RNAi 270

(Supplemental Table S3). 271



16

The present study is devoted to illustrate an efficient alternative approach to 272

conventional breeding and fungicide treatment for fungal disease control. As 273

expected, transgenic lines carrying PsFUZ7 RNAi constructs exhibit strong 274

and genetically stable resistance to Pst in T3 generations (Fig. 4A). Generally, 275

the T3 generation is considered the initial true transgenic line in hexaploid 276

wheat (Cheng et al., 2015a). Histological observations revealed differential 277

hyphal growth in transgenic lines carrying PsFUZ7 RNAi constructs compared 278

to the control lines (Fig. 6). Previous studies demonstrated the efficient 279

transport of siRNA molecules from transgenic plant cells to the colonizing 280

pathogen (Nowara et al., 2010; Koch et al., 2013), and that the transported 281

siRNAs interfere with the target gene, affecting fungal growth through an 282

amplification of RNAi molecules and resulting in a resistance response 283

(Panwar et al., 2013; Cheng et al., 2015a). The suppression of Pst growth in 284

our study correlates with the production of siRNAs corresponding to the 285

targeted PsFUZ7 sequences (Fig. 4B), as well as a significant reduction of 286

PsFUZ7 transcripts and fungal biomass (Fig. 4C and E). 287

To explore the basis for the induction of necrosis in plant cells, a schematic 288

presentation for potential processes triggered by silencing PsFUZ7 via the 289

expression of siRNA in transgenic wheat is presented (Fig. 7). In the plant 290

immunity system, two main phases, pathogen-associated molecular patterns 291

(PAMPs) triggered immunity (PTI) and effector-triggered immunity (ETI) are 292

well known (Jones and Dangl, 2006; Boller and Felix, 2009; Tsuda et al., 293

2010). Cell death induced in transgenic wheat lines is in accordance with a 294

hypersensitive response (HR) that is expressed in pathogen specific ETI 295

(Jones and Dangl, 2006; Thomma et al., 2011). In U. maydis, the kinase CRK1 296

downstream of the MAPK cascade negatively regulates transcription and 297

secretion of some effector proteins (Bielska et al., 2014). In our study, we 298

verified the severe down-regulation of PsCRK1 transcripts (Fig. 5) and the 299

interaction between PsFUZ7 and PsCRK1 via the yeast-two hybrid assay 300

(Supplemental Fig. S9). Considering these results, we propose that the 301



17

expression of the siRNA target PsFUZ7 influences effectors in Pst and triggers 302

ETI in transgenic XN1376 wheat (Fig. 7). In addition, many features associated 303

with fungal pathogenesis are dependent on signaling through MAPK 304

cascades, including the biosynthesis, export, and secretion of different factors 305

(Hamel et al., 2012). HIGS of PsFUZ7 may impact biosynthesis or secretion of 306

Pst components that may function as PAMPs in PTI (Chisholm et al., 2006). 307

Our study has confirmed that the expression of siRNA derived from a Pst 308

pathogenicity gene in wheat is an effective strategy to control rust disease. 309

This approach provides a huge reservoir of novel resources to enhance 310

resistance to abiotic and biotic stress by the production of transgenic crops 311



18

that are environmentally friendly. 312

313

MATERIALS AND METHODS 314

Fungi, Plants and Culture Conditions 315

Chinese Yellow Rust isolate 32 (CYR32), which is a predominant race in 316

China (Wan et al., 2007), was used throughout this study. Triticum aestivum L. 317

cv. Suwon11 (Su11) that is susceptible to CYR32 was used in qRT-PCR and 318

VIGS assays. Plant cultivation and inoculation with Pst were performed as 319

described previously (Kang et al., 2002). XN1376, susceptible to CYR32 at the 320

seedling stage, was grown in an experimental field of Northwest A&F 321

University (Yangling, China) and used for transgenic wheat cultivars. 322

VIGS and RNAi Vector Construction 323

Two PsFUZ7 fragments were used in BSMV-induced gene silencing 324

experiments (Supplemental Table S2). PsFUZ7-1as contains 218 bp of the 325

coding sequence from the 5’ region, whereas PsFUZ7-2as encompasses 326

300-bp coding sequence in the 3’ UTR. The two fragments were cloned into 327

BSMV as previously described (Holzberg et al., 2002). The hpRNA cassette 328

contains the Zea mays alcohol dehydrogenase 1 (adh1) gene as intron flanked 329

by the 300-bp fragment of PsFUZ7 in sense and anti-sense orientation. The 330

expression cassette treated with SmaI and SacI was cloned into the binary 331

vector pAHC25 using the same restriction sites (Christensen et al., 1992; Vasil 332

et al., 1993). The bar gene in the T-DNA of the vector as selection gene is 333

driven by the maize ubiquitin promoter, and the hpRNA cassette is also under 334

control of the Ubi promoter and terminated by the NOS terminator. The 335

resulting vectors were pAHC25-hpPsFUZ7 for silencing PsFUZ7, and the 336

empty vector pAHC25 containing only the bar gene without a silencing 337

cassette. 338

Total DNA, RNA Extraction, PCR and qRT-PCR 339



19

Genomic DNA was extracted by the CTAB method (Porebski et al., 1997). 340

RNA was isolated with TRIZOL following the manufacturer’s instructions 341

(Invitrogen, Carlsbad, CA, USA) and transcribed into cDNA also following the 342

manufacturer’s directions (Promega, Madison, WI, USA). In each transgenic 343

generation, genomic DNA from leaves of transgenic wheat and control plants 344

were identified by PCR with two pairs of specific primers (TFUZ-F/R and 345

Bar-F/R, Supplemental Table S2) to detect the presence of the 346

sense-intron-antisense cassette in transgenic plants and the bar gene. To 347

measure fungal biomass, relative quantification of the single-copy target genes 348

in PsEF-1 and TaEF-1 (elongation factor-1) was carried out (Panwar et al., 349

2013). Total genomic DNA of the wheat cultivar XN1376 or the Pst isolate 350

CYR32 was used to prepare standard curves derived from at least six serial 351

dilutions for each. The correlation coefficients for the analysis of the dilution 352

curves were above 0.99. The relative quantities of the PCR products of 353

PsEF-1 and TaEF-1 in mixed/infected samples were calculated using the 354

gene-specific standard curves to quantify the Pst and wheat genomic DNA, 355

respectively. 356

To measure the transcript levels of PsFUZ7 by qRT-PCR, urediospores and 357

in vivo germ tubes of CYR32, plant tissue of Su11 infected with CYR32 at 358

6,12,18, 24, 36, 48, 72, 120, 168, 216 hours post inoculation (hpi) and 359

urediospores (US) were sampled. To analyze VIGS efficiency, qRT-PCR was 360

carried out 48 and 168 hpi with the CYR32 isolate. For transgenic efficiency 361

assay, total transgenic RNA for quantitative real-time PCR was extracted from 362

the second leaves of wheat at 3 dpi, 10 dpi, and 16 dpi with the CYR32 isolate. 363

All qRT-PCR was performed in a 20-μL reaction mixture containing LightCycler 364

SYBR Green I Master Mix (Roche, Basel, Switzerland), 10 pmol each of the 365

forward and reverse gene-specific primers (Supplemental Table S2), and 2 μl 366

of diluted cDNA (1:5) that was reverse transcribed. PCR was run in a 367

LightCycler 480II (Roche) under the conditions previously described (Cheng et 368

al., 2015b). Each sample was analyzed in three biological replications, and 369



20

each PCR analysis included three technical repeats. The data were 370

normalized to the PsEF-1 expression level. 371

372

BSMV-mediated Gene Silencing 373

Capped in vitro transcripts were prepared from linearized plasmids 374

containing the tripartite BSMV genome (Petty et al., 1990) using the RiboMAX 375

TM Large-Scale RNA Production System-T7 and the Ribom7G Cap Analog 376

(Promega), according to the manufacturers’ instructions. PsFUZ7-1as and 377

PsFUZ7-2as were used to inoculate wheat seedlings, while BSMV:TaPDS 378

(TaPDS: wheat phytoene desaturase gene) and BSMV:γ were used as 379

controls for the BSMV infection. Mock plants were inoculated with 1xFES 380

buffer as the negative control. Wheat seedlings with three leaves were used 381

for inoculation with BSMV, and BSMV-infected wheat plants were kept in a 382

growth chamber at 23 ± 2°C. The fourth leaves were further inoculated with 383

fresh CYR32 urediospores at 10 d after virus inoculation, and the plants were 384

then maintained at 16 ± 2°C (Wang et al., 2007). The phenotypes of the fourth 385

leaves were recorded and photographed at 14 days after inoculation with Pst. 386

The number of uredia was counting a 1 cm2 area at 14 dpi with Pst from at 387

least five randomly treated plants. 388

Cytological Observation of Fungal Growth and Host Responses 389

Leaf segments (1.5 cm long) were cut from inoculated leaves, fixed and 390

decolorized in ethanol/trichloromethane (3:1 v/v) containing 0.15% (w/v) 391

trichloroacetic acid for 3-5 days, and then treated as previously described 392

(Cheng et al., 2015b). To obtain high quality images of Pst infection structures 393

in wheat leaves, wheat germ agglutinin conjugated to Alexa-488 (Invitrogen) 394

was used as described (Ayliffe et al., 2011). Stained tissue was examined 395

under blue light excitation (excitation wavelength 450-480 nm, emission 396

wavelength 515 nm). Necrotic areas were observed via the auto-fl Stained t of 397

attacked mesophyll cells. Infection sites (30-50) from three leaves were 398



21

examined to record the number of haustorial mother cells, haustoria, hyphal 399

branches, hyphal length and infection areas of hyphae or necrotic area of host 400

cells in infected wheat per infection unit. The experiments were conducted in a 401

completely randomized block design with three replications. The presence of a 402

substomatal vesicle was defined as an established infection unit. Hyphal 403

length of Pst was measured from the substomatal vesicle to the apex of the 404

longest infection hypha. All microscopic observations were conducted with an 405

Olympus BX-51 microscope (Olympus, Tokyo, Japan). 406

To observe the ultrastructure of the fungus, wheat leaves bearing uredia 407

were cut into 0.5x0.5 cm pieces, immersed in 4% glutaraldehyde in 0.2 M 408

phosphate buffer, pH 6.8, and fixed at 4 °C overnight (Zhan et al., 2014). Fixed 409

leaf samples were washed four times with phosphate buffer for 15 min each. 410

Thereafter, samples were successively dehydrated for 30 min each in 30, 50, 411

70, 80 and 90% ethanol, and finally three times in 100% ethanol. The 412

dehydrated samples were treated with isoamyl acetate twice for 20 min each. 413

After drying in a CO2 vacuum, the samples were sputter-coated with gold in an 414

E-1045 (Hitachi, Tokyo, Japan) and then examined with an S-4800 SEM 415

(Hitachi). 416

Plant Transformation 417

Immature embryos were isolated from spikes of XN1376 at 13-14 day post 418

anthesis in Yangling, Shaanxi. The isolated wheat embryos were cultured on 419

SD2 medium in darks for 7-10 days for calli induction (Li et al., 2008). Then the 420

calli were moved to SD2 medium added with 0.2 mol/l mannitol and 0.2 mol/l 421

sorbitol. After 4-6 hours, the calli were bombarded with 1-μm gold particles 422

coated with 1.5 μg of recombinant pAHC25 DNA using a PDS-1000 He biolistic 423

gun (BioRad, Hercules, CA, USA) at the pressure of 1,100 psi (Vasil et al., 424

1993; Li et al., 2008). The bombarded calli were transferred onto osmotic 425

pressure medium described above for 16-18 h. Regeneration and selection 426

were carried out in the corresponding medium in the presence of 3-5 mg/l 427



22

bialaphos for the next few weeks, and the surviving plantlets with strong roots 428

and shoots were planted in a greenhouse in pots filled with soil (Cheng et al., 429

2015a). 430

431

Northern Blot 432

About 30 μg of total RNA was subjected to electrophoresis on a denaturing 433

19% polyacrylamide gel, transferred to Nytran Super Charge Nylon 434

Membranes (Schleicher und Schuell MicroScience GmbH, Dassel, Germany) 435

and crosslinked using a Stratagene UV Crosslinker. The membranes were 436

prehybridized with PerfectHyb TM (Sigma-Aldrich, St. Louis, MO, USA), and 437

hybridized with the P32-labeled DNA probes overnight in PerfectHyb buffer. 438

The membranes were autoradiographed on X-OMAT BT film (Carestream 439

Health, Rochester, NY, USA) after rinsing with washing buffer. U6 was used as 440

a loading control. The probe sequences are listed in Supplemental Table S2. 441

442

FIGURE LEGENDS 443

Figure 1. Transcript profiles of five MAPK cascade genes at different 444

Pst infection stages. Wheat leaves (Su11) were inoculated with fresh 445

urediospores (CYR32) and kept in the dark and under high humidity for 24 h. 446

Inoculated leaves were sampled at different time points according to the 447

differentiation stage of Pst. Gene expression levels were normalized to the 448

expression level of PsEF-1. Results are expressed as means ± standard errors 449

of three biological replicates. US: urediospores; 6 - 264 h: 6 - 264 hpi with 450

CYR32. PsKPP4: MAP kinase kinase kinase gene; PsFUZ7: MAP kinase 451

kinase gene; PsKPP2, PsKPP6 and PsCRK1: MAP kinase genes. 452

Figure 2. Functional assessment of PsKPP4, PsFUZ7 and PsKPP6 in 453

the wheat-Pst interaction by virus-induced gene silencing. A, Phenotypes 454

of fourth leaves inoculated with CYR32 at 14 dpi. Plants were pre-inoculated 455



23

with FES buffer (mock), BSMV:TaPDS, BSMV:γ, BSMV:PsKPP4-1as, 456

BSMV:PsKPP4-2as, BSMV:PsFUZ7-1as, BSMV:PsFUZ7-2as, 457

BSMV:PsKPP6-1as, or BSMV:PsKPP6-2as, respectively. B, Relative 458

transcript levels of PsKPP4, PsFUZ7 and PsKPP6 in knockdown plants 459

inoculated with CYR32 at 2 and 7 dpi. Wheat leaves inoculated with BSMV:γ 460

and sampled after inoculation with CYR32 were used as controls. Data were 461

normalized to the transcript level of PsEF-1. Asterisks indicate P < 0.05, 462

double asterisks indicate P < 0.01. C, Quantification of uredial density in 463

silenced plants 14 dpi with CYR32. Differences were assessed using Student’s 464

t-tests. Values represent the means ± standard errors of three independent 465

samples. Asterisks indicate P < 0.05, double asterisks indicate P < 0.01. 466

Figure 3. Epifluorescence observation of fungal growth in wheat 467

inoculated with BSMV and infected with CYR32. Leaves inoculated with 468

CYR32 were sampled at 48 and 120 hpi and examined under an 469

epifluorescence microscope after staining with wheat germ agglutinin 470

conjugated to Alexa-488 (Invitrogen, Carlsbad, CA, USA). Treatments include 471

A, BSMV:γ; B, BSMV:PsFUZ7-1as and C, BSMV:PsFUZ7-2as inoculated with 472

CYR32 at 48 hpi (Bars = 10 μm); D, BSMV:γ; E, BSMV:PsFUZ7-1as and F, 473

BSMV:PsFUZ7-2as inoculated with CYR32 at 120 hpi (Bars = 50 μm). SV, 474

substomatal vesicle; HMC, haustorial mother cell; IH, infection hypha; H, 475

haustorium. G, Average number of haustoria (H), hyphal branches (HB) and 476

haustorial mother cells (HMC) in wheat inoculated with BSMV constructs and 477

infected with CYR32 at 48 hpi. H, Average length of infection hyphae (IH) 478

measured from their origin at the substomatal vesicle to the tip of the hypha in 479

wheat inoculated with BSMV constructs and infected with CYR32 at 48 hpi. I, 480

Infection area per infection site in wheat inoculated with BSMV and infected 481

with CYR32 at 120 hpi. Differences in G-I were assessed using Student’s 482


samples. Asterisks indicate P < 0.05, double asterisks indicate P < 0.01. 484



24

Figure 4. Transgenic wheat lines L65 and L91 producing fungal 485

gene-derived siRNAs induce silencing of the target mRNA and confer 486

resistance to Pst infection. A, Phenotypes of transgenic lines L65, L91, and 487

control (CK) in T2 and T3 generations. The second leaves of seedlings were 488

inoculated with urediospores of CYR32 and photographed at 14 dpi with Pst in 489

each generation. CK, transgenic lines carrying empty vector; L65 and L91, 490

transgenic lines carrying RNAi constructs. B, Northern blot analysis of siRNA 491

isolated from T3 transgenic wheat lines detected with specific probe derived 492

from the PsFUZ7 fragment. U6 small nuclear RNA as a loading control. C, 493

Relative expression of PsFUZ7 at 3, 10, and 16 dpi with CYR32 of T3 494

transgenic wheat lines L65, L91, and control (CK). Data were normalized to 495

PsEF-1, and the CK-10 d control was set to 1. D, Host response and infection 496

types in T3 transgenic wheat lines L65, L91, and control (CK) assessed at 16 497

and 25 dpi with CYR32. E, Fungal biomass measured using real-time PCR of 498

total DNA extracted from wheat leaves infected with CYR32 at 14 dpi. Ratio of 499

total fungal DNA to total wheat DNA was assessed using the wheat gene 500

TaEF-1 and the Pst gene PsEF-1. Differences were assessed using Student’s 501


samples. Double asterisks indicate P < 0.01. 503

Figure 5. Transcript profiles of genes involved in the MAPK pathway in 504

Pst and defense-related genes in transgenic wheat plants L65 and L91. 505

(A) Transcript abundance of MAPK-pathway related genes in Pst decreases 506

except for PsKPP2. Wheat leaves were sampled at 3 and 10 dpi with Pst. Data 507

were normalized to the PsEF-1 expression level, and the CK-10d control was 508

set to 1. PsKPP4, MAP kinase kinase kinase; PsFUZ7, MAP kinase kinase; 509

PsKPP2, PsKPP6 and PsCRK1, MAP kinase. (B) Transcript abundance of 510

pathogenesis-related proteins or defense-related genes increase in transgenic 511

wheat plants L65 and L91 at 3 and 10 dpi. TaPR1, pathogenesis-related 512

protein 1; TaPR2, β-1,3-glucanase; TaPAL, phenylalanine ammonia lyase; 513



25

TaAPX, ascorbate peroxidase; TaNOX, NADPH oxidase; TaSOD, superoxide 514

dismutase; TaPOD, peroxidase; TaCAT2, catalase 2; TaCAT1, catalase 1. The 515

data were normalized to the TaEF-1 expression level, and the CK-10d control 516

was set to 1. Values represent the means ± standard errors of three 517

independent samples. 518

Figure 6. Microscopic visualization of the in host-induced gene 519

silencing effect targeting PsFUZ7 on colonization of wheat leaf tissue by 520

Pst. A, Cytological observation of rust interaction with wheat by 521

epifluorescence microscopy. Leaves inoculated with CYR32 were sampled at 522

10 dpi. SV, substomatal vesicle; IH, infection hypha; HMC, haustorial mother 523

cell; H, haustorium; NHC, necrotic host cell. Bars = 50 μm. B, Infection area 524

per infection site is reduced in the transgenic lines L65 and L 91 infected with 525

CYR32 compared with CK at 10 dpi. C, The formation of haustoria in lines L65 526

and L91 is inhibited after inoculation with CYR32 compared with CK at 10 dpi. 527

D, The necrotic area of plants from lines L65 and L91 is increased compared 528

with CK at 10 dpi with CYR32. E, Cytological observations of Pst CYR32 and 529

wheat by transmission electron microscopy at 10 dpi. HMC, haustorial mother 530

cell; H, haustorium; RN, rust nucleus; Ch, chloroplast; Cy, cytoplasm; IH, 531

infection hypha; HC, host cell; NHC, necrotic host cell; Org, organelle. Left 532

bars = 2 μm, Right bars = 500 nm. Differences in D-E were assessed using 533

Student’s t-tests. Values represent the means ± standard errors of three 534

independent samples. Double asterisks indicate P < 0.01. 535

Figure 7. Schematic presentation of possible molecular dialogues 536

between transgenic lines carrying RNAi constructs and colonizing Pst. 537

Fungal dsRNA, produced inside transgenic wheat cells, is cleaved by the plant 538

silencing machinery using endonuclease-type DICER enzymes into small 539

silencing molecules (siRNAs). These siRNAs are trapped by a complex of 540

proteins, and transported to the paramural space. After passing the haustorial 541

cell wall, the silencing molecules trigger RNAi of their mRNA targets, and may 542



26

act as primers leading to the activation of systemic silencing signals, thus 543

inducing the immunity system of transgenic wheat by mechanisms including 544

ETI and PTI. 545

546

SUPPLEMENTAL DATA 547

Supplemental Figure S1. Relative transcript levels of (A) PsKPP4, (B) 548

PsFUZ7, and (C) PsKPP6 homologs in respective knockdown plants 549

inoculated with CYR32 at 2 dpi. 550

Supplemental Figure S2. Multiple sequence alignment of FUZ7 orthologs. 551

Supplemental Figure S3. Complementation of the mst7 mutant of 552

Magnaporthe oryzae with PsFUZ7. 553

Supplemental Figure S4. Overexpression assay of PsFUZ7 in the fission 554

yeast SP-Q01. 555

Supplemental Figure S5. Effect of the immuno-suppressive inhibitor U0126 556

on germination of Pst. 557

Supplemental Figure S6. Structure of the pAHC25-PsFUZ7 RNAi construct 558

and molecular identification in transgenic plants. 559

Supplemental Figure S7. Transcript abundance of putative off-target in Pst 560

after L65, L91 and CK inoculated with CYR32 at 10dpi. 561

Supplemental Figure S8. Transcript abundance of putative off-target in wheat 562

after L65, L91 and CK inoculated with CYR32 at 10dpi. 563

Supplemental Figure S9. Y2H assay using MAPK cascade genes. 564

Supplemental Table S1. MAPK orthologs identified in Pst. 565



27

Supplemental Table S2. Primers used in this study. 566

Supplemental Table S3. Homologs of PsKPP4, PsFUZ7 and PsKPP6 in Pst. 567

Supplemental Table S4. Prediction of PsFUZ7 off-target transcripts. 568

569

570

ACKNOWLEDGMENTS 571

The authors thank Professor Jiankang Zhu from Shanghai Center for Plant 572

Stress Biology, SIBS, CAS, China for providing technical supports on 573

Sourthern blot and Northern blot, and Professor Larry Dunkle from the 574

USDA-Agricultural Research Service at Purdue University, USA for critical 575

reading the manuscript. 576



28

577

578



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http://www.ncbi.nlm.nih.gov/pubmed?term=Ayliffe M%2C Devilla R%2C Mago R%2C White R%2C Talbot M%2C Pryor A%2C Leung H %282011%29 Nonhost resistance of rice to rust pathogens%2E Mol Plant Microbe Interact 24%3A 1143%2D1155&dopt=abstract

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http://scholar.google.com/scholar?as_q=Nonhost resistance of rice to rust pathogens&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Nonhost resistance of rice to rust pathogens&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Ayliffe&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Bartel DP %282004%29 MicroRNAs%3A genomics%2C biogenesis%2C mechanism%2C and function%2E Cell 116%3A 281%2D297&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116: 281-297

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Bartel&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=MicroRNAs: genomics, biogenesis, mechanism, and function&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=MicroRNAs: genomics, biogenesis, mechanism, and function&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Bartel&as_ylo=2004&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Baulcombe D %282004%29 RNA silencing in plants%2E Nature 431%3A 356%2D363&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Baulcombe D (2004) RNA silencing in plants. Nature 431: 356-363

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Baulcombe&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=RNA silencing in plants&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=RNA silencing in plants&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Baulcombe&as_ylo=2004&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Baum JA%2C Bogaert T%2C Clinton W%2C Heck GR%2C Feldmann P%2C Ilagan O%2C Johnson S%2C Plaetinck G%2C Munyikwa T%2C Pleau M %282007%29 Control of coleopteran insect pests through RNA interference%2E Nat Biotechnol 25%3A 1322%2D1326&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Baum JA, Bogaert T, Clinton W, Heck GR, Feldmann P, Ilagan O, Johnson S, Plaetinck G, Munyikwa T, Pleau M (2007) Control of coleopteran insect pests through RNA interference. Nat Biotechnol 25: 1322-1326

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Baum&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Control of coleopteran insect pests through RNA interference&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Control of coleopteran insect pests through RNA interference&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Baum&as_ylo=2007&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Bielska E%2C Higuchi Y%2C Schuster M%2C Steinberg N%2C Kilaru S%2C Talbot NJ%2C Steinberg G %282014%29 Long%2Ddistance endosome trafficking drives fungal effector production during plant infection%2E Nat Commun 5%3A 5097&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Bielska E, Higuchi Y, Schuster M, Steinberg N, Kilaru S, Talbot NJ, Steinberg G (2014) Long-distance endosome trafficking drives fungal effector production during plant infection. Nat Commun 5: 5097

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Bielska&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Long-distance endosome trafficking drives fungal effector production during plant infection.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Long-distance endosome trafficking drives fungal effector production during plant infection.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Bielska&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Birmingham A%2C Anderson EM%2C Reynolds A%2C Ilsleytyree D%2C Leake D%2C Fedorov Y%2C Baskerville S%2C Maksimova E%2C Robinson K%2C Karpilow J %282006%29 3%27 UTR seed matches%2C but not overall identity%2C are associated with RNAi off%2Dtargets%2E Nat Methods 3%3A 199%2D204&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Birmingham A, Anderson EM, Reynolds A, Ilsleytyree D, Leake D, Fedorov Y, Baskerville S, Maksimova E, Robinson K, Karpilow J (2006) 3' UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nat Methods 3: 199-204

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Birmingham&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=3' UTR seed matches, but not overall identity, are associated with RNAi off-targets&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

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http://www.ncbi.nlm.nih.gov/pubmed?term=Boller T%2C Felix G %282009%29 A renaissance of elicitors%3A perception of Microbe%2DAssociated Molecular Patterns and danger signals by Pattern%2DRecognition Receptors%2E Plant Biol 60%3A 379%2D406&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Boller T, Felix G (2009) A renaissance of elicitors: perception of Microbe-Associated Molecular Patterns and danger signals by Pattern-Recognition Receptors. Plant Biol 60: 379-406

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Boller&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=A renaissance of elicitors: perception of Microbe-Associated Molecular Patterns and danger signals by Pattern-Recognition Receptors&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=A renaissance of elicitors: perception of Microbe-Associated Molecular Patterns and danger signals by Pattern-Recognition Receptors&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Boller&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Chen T%2C Zhu H%2C Ke D%2C Cai K%2C Wang C%2C Gou H%2C Hong Z%2C Zhang Z %282012%29 A MAP kinase kinase interacts with SymRK and regulates nodule organogenesis in Lotus japonicus%2E Plant Cell 24%3A 823%2D838&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Chen T, Zhu H, Ke D, Cai K, Wang C, Gou H, Hong Z, Zhang Z (2012) A MAP kinase kinase interacts with SymRK and regulates nodule organogenesis in Lotus japonicus. Plant Cell 24: 823-838

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Chen&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=A MAP kinase kinase interacts with SymRK and regulates nodule organogenesis in Lotus japonicus&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=A MAP kinase kinase interacts with SymRK and regulates nodule organogenesis in Lotus japonicus&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Chen&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Chen WQ%2C Wu LR%2C Liu TG%2C Xu SC%2C Jin SL%2C Peng YL%2C Wang BT %282009%29 Race dynamics%2C diversity%2C and virulence evolution in Puccinia striiformis f%2E sp%2E tritici%2C the causal agent of wheat stripe rust in China from 2003 to 2007%2E Plant Dis 93%3A 1093%2D1101&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Chen WQ, Wu LR, Liu TG, Xu SC, Jin SL, Peng YL, Wang BT (2009) Race dynamics, diversity, and virulence evolution in Puccinia striiformis f. sp. tritici, the causal agent of wheat stripe rust in China from 2003 to 2007. Plant Dis 93: 1093-1101


http://scholar.google.com/scholar?as_q=Race dynamics, diversity, and virulence evolution in Puccinia striiformis f&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Race dynamics, diversity, and virulence evolution in Puccinia striiformis f&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Chen&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Chen XM %282014%29 Integration of cultivar resistance and fungicide application for control of wheat stripe rust%2E Can J Plant Pathol 36%3A 311%2D326&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Chen XM (2014) Integration of cultivar resistance and fungicide application for control of wheat stripe rust. Can J Plant Pathol 36: 311-326


http://scholar.google.com/scholar?as_q=Integration of cultivar resistance and fungicide application for control of wheat stripe rust&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Integration of cultivar resistance and fungicide application for control of wheat stripe rust&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Chen&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Cheng W%2C Song XS%2C Li HP%2C Cao LH%2C Sun K%2C Qiu XL%2C Xu YB%2C Yang P%2C Huang T%2C Zhang JB %282015a%29 Host%2Dinduced gene silencing of an essential chitin synthase gene confers durable resistance to Fusarium head blight and seedling blight in wheat%2E Plant Biotechnol J 13%3A 1335%2D1345&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Cheng W, Song XS, Li HP, Cao LH, Sun K, Qiu XL, Xu YB, Yang P, Huang T, Zhang JB (2015a) Host-induced gene silencing of an essential chitin synthase gene confers durable resistance to Fusarium head blight and seedling blight in wheat. Plant Biotechnol J 13: 1335-1345

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Ghag SB, Shekhawat UK, Ganapathi TR (2014) Host-induced post-transcriptional hairpin RNA-mediated gene silencing of vital fungalgenes confers efficient resistance against Fusarium wilt in banana. Plant Biotechnol J 12: 541-553


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Hamel LP, Nicole MC, Duplessis S, Ellis BE (2012) Mitogen-activated protein kinase signaling in plant-interacting fungi: distinctmessages from conserved messengers. Plant Cell 24: 1327-1351


Holzberg S, Brosio P, Gross C, Pogue GP (2002) Barley stripe mosaic virus-induced gene silencing in a monocot plant. Plant J 30: 315-327


Huang G, Allen R, Davis EL, Baum TJ, Hussey RS (2006) Engineering broad root-knot resistance in transgenic plants by RNAi silencingof a conserved and essential root-knot nematode parasitism gene. Proc Natl Acad Sci USA 103: 14302-14306


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Host-induced gene silencing of the MAPKK gene PsFUZ7 ...

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