1 running head: an orphan gene enhances wheat disease ... · in mock-treated wheat tissues, the 194...

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Running head: An orphan gene enhances wheat disease resistance 1

Corresponding Author: 2

Pr. Fiona Doohan 3

UCD School of Biology and Environmental Science, College of Science and UCD 4

Earth Institute, University College Dublin, Belfield, Dublin 4, Ireland. 5

phone: 0035317162248 6

email: [email protected] 7

Research Area: Signaling and Response 8

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Plant Physiology Preview. Published on October 27, 2015, as DOI:10.1104/pp.15.01056

Copyright 2015 by the American Society of Plant Biologists

www.plantphysiol.orgon March 10, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.

http://www.plantphysiol.org

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Title 26

TaFROG encodes a Pooideae orphan protein that interacts with SnRK1 and 27

enhances resistance to the mycotoxigenic fungus Fusarium graminearum. 28

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Alexandre Perochon, Jia Jianguang, Amal Kahla, Chanemougasoundharam 30

Arunachalam, Steven R. Scofield, Sarah Bowden, Emma Wallington, Fiona M. 31

Doohan. 32

UCD Earth Institute and School of Biology and Environmental Science, College of 33

Science, University College Dublin, Belfield, Dublin 4, Ireland (A.P., J.J., A.K., C.A., 34

F.D.); USDA-ARS, Crop Production and Pest Control Research Unit and Purdue 35

University, Department of Agronomy, 915 West Street, West Lafayette, IN 47907-36

2054, USA (S.S.); NIAB, Huntingdon Road, Cambridge, CB3 0LE, UK (S.B., E.W.). 37

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One sentence summary: A Pooideae orphan protein that enhances plant 39

resistance against a toxigenic fungus physically interacts with a key signalling 40

protein. 41

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Funding information: This work was funded by Science Foundation Ireland project 53

10/IN.1/B3028 and 14/1A/2508, and by the NIAB Trust. 54

Corresponding author email: [email protected] 55

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Abstract 78

All genomes encode taxonomically restricted ‘orphan’ genes and the vast majority 79

are of unknown function. There is growing evidence that such genes play an 80

important role in environmental adaptation of taxa. We report the functional 81

characterization of an orphan gene (designated TaFROG) as a component of 82

resistance to the globally important wheat (Triticum aestivum) disease, Fusarium 83

head blight (FHB). TaFROG is taxonomically restricted to the grass subfamily 84

Pooideae. Gene expression studies showed that it is a component of the early wheat 85

response to the mycotoxin deoxynivalenol (DON), which is a virulence factor 86

produced by the causal fungal agent of FHB, Fusarium graminearum. The temporal 87

induction of TaFROG by F. graminearum in wheat spikelets correlated with the 88

activation of the defence Pathogenesis-Related-1 gene (TaPR1). But unlike TaPR1, 89

TaFROG induction by F. graminearum was toxin-dependent, as determined via 90

comparative analysis of the effects of wild type fungus and a DON-minus mutant 91

derivative. Using virus-induced gene silencing and overexpressing transgenic wheat 92

lines, we present evidence that TaFROG contributes to host resistance to both DON 93

and F. graminearum. TaFROG is an intrinsically disordered protein and it localized to 94

the nucleus. TaSnRK1α, a wheat alpha subunit of the Snf1-related kinase 1 95

(SnRK1), was identified as a TaFROG-interacting protein based on a yeast two-96

hybrid study. In planta bi-molecular fluorescence complementation assays confirmed 97

the interaction. Thus we conclude that TaFROG encodes a new SnRK1-interacting 98

protein and it enhances biotic stress resistance. 99

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Introduction 108

It is estimated that wheat yields must at least double by 2050 to meet growing 109

future demands. We lose billions of dollars worth of wheat grain annually due to 110

diseases caused by fungi (Dean et al., 2012). Some fungal diseases also threaten 111

health because the causal organisms contaminate grain with mycotoxins that are 112

harmful to humans and animals (Arunachalam and Doohan, 2013). Breeding for 113

disease resistance is the most environmentally sustainable method of disease 114

control. Resistance to disease can be quantitative non-specific, or qualitative, 115

pathogen race-specific. We must expand our portfolio of wheat resistance genes 116

selected for within breeding programs if we want to deploy sustainable resistance to 117

a broad spectrum of pathogens. This will be most easily achieved if we obtain better 118

knowledge of the disease resistance mechanisms and the transcriptional patterns 119

important to disease resistance within wheat (Bischof et al., 2011). 120

Many of the plant genes activated in response to pathogens are of unknown 121

function. This includes taxonomically restricted orphan genes that lack homologs in 122

other plant families (Arendsee et al., 2014). A role for orphan genes in lineage-123

specific adaptations is supported by a growing number of examples from a spectrum 124

of diverse organisms, including plants (Tautz and Domazet-Loso, 2011; Arendsee et 125

al., 2014). Orphans were enriched in differentially expressed gene sets from 126

Arabidopsis thaliana and rice following treatments with hormones, abiotic or biotic 127

stressors (Guo et al., 2007; Donoghue et al., 2011). Arabidopsis mutant screens 128

identified several plant orphan genes that alter abiotic stress resistance (Luhua et al., 129

2013). Examples detailing the functional role of orphan genes in plant disease 130

responses are rare. The Brassicaceae-specific EWR1 orphan gene provides 131

resistance to vascular wilt pathogens (Yadeta et al., 2014), while the rice orphan 132

gene OsDR10 has a negative effect on plant resistance to bacterial blight disease 133

(Xiao et al., 2009). 134

Here we characterize a wheat orphan gene that enhances resistance to 135

Fusarium head blight (FHB) disease, and thus was designated TaFROG (Triticum 136

aestivum Fusarium Resistance Orphan Gene). This gene was originally identified as 137

being responsive to a mycotoxic virulence factor, deoxynivalenol (DON), which is 138

produced by the causal fungal agent of FHB, Fusarium graminearum. This fungus 139



6

produces DON to facilitate disease spread within wheat head tissue (Bai et al., 140

2002). The disease results in yield loss and contamination of grain with DON that is 141

harmful to human and animal health (Rocha et al., 2005). Gene expression studies 142

evaluated the effect of DON synthesis on the responsiveness of this gene to F. 143

graminearum. Gene silencing and overexpression studies assessed the contribution 144

of this gene to DON tolerance and FHB resistance. Microscopic analysis of 145

transgenic plants was used to determine the subcellular location of TaFROG. A 146

yeast two-hybrid screen against a wheat cDNA library identified TaSnRK1α as a 147

TaFROG-interacting protein and the interaction was confirmed in planta using 148

bimolecular fluorescence complementation. On the basis of this study we have 149

identified a wheat orphan gene that enhances disease resistance and determined 150

that it interacts with a central stress regulator TaSnRK1α. 151

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RESULTS 154

TaFROG is an orphan gene, taxonomically-restricted to the Pooideae 155

Previous studies within our laboratory identified a novel wheat transcript, 156

responsive to F. graminearum and DON. Using a RACE-PCR, we cloned the full-157

length open reading frame of this gene (TaFROG) from bread wheat cv. CM82036. 158

The deduced coding sequence shares 100, 93 and 85% homology with sequences 159

on chromosomes 4A, 4D and 4B, respectively, of the recently published wheat cv. 160

Chinese Spring genome (IWGSC Consortium, 2014). TaFROG homologs were 161

found within the Triticum genus (E<10-83). They were restricted to the Pooideae 162

subfamily of Poaceae (E<10-4; Fig. 1A). TaFROG gene variants were not identified 163

outside this subfamily, either in transcript databases or in any other sequenced 164

monocotyledon genome. TaFROG shared no significant homology with any 165

characterized gene or protein. The protein did not contain any characterized domain. 166

Thus we conclude that TaFROG is a novel ‘orphan’ gene: orphan genes are strictly 167

defined as having a narrow phylogenetic distribution and not encoding previously-168

identified protein domains (Khalturin et al., 2009). 169

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FROG represents a new family of intrinsically disordered nuclear proteins 171

The FROG family are small basic proteins of between 121-142 amino acids 172

(Supplemental Fig. S1A) and represent new intrinsically disordered proteins (IDPs) 173

with a highly negative folding index profile determined using the FoldIndex prediction 174

tool (Prilusky et al., 2005) (Supplemental Fig. S1B). Between 62 to 72% of protein 175

residues are predicted to be disordered and these lie within four conserved regions 176

(Supplemental Fig. S1A). FROG encodes two conserved motifs: a Nuclear Export 177

Signal (NES) and a Nuclear Localization Sequence (NLS), as shown in 178

Supplemental Fig. S1A, suggesting FROG can localize in the nucleus. We fused 179

wheat FROG (TaFROG, cloned from chromosome 4A of cv. CM82036) to yellow 180

fluorescent protein (YFP) and transiently or stably expressed TaFROG-YFP in wheat 181

leaves or A. thaliana plants, respectively. In wheat epidermal cells, confocal 182

microscopy analyses showed that TaFROG-YFP was restricted to the nucleus within 183

distinct nuclear bodies (Fig. 1B), unlike coexpressed CFP, which was found in both 184

the cytosol and the nucleus. In stable transgenic A. thaliana lines, light sheet 185



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fluorescence microscopy detected TaFROG-YFP in bodies localized within the 186

DAPI-stained nuclei (Fig. 1C). The integrity of A. thaliana TaFROG-YFP fusion was 187

confirmed via western blot analysis (Supplemental Fig. S2). 188

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TaFROG is a mycotoxin-responsive orphan gene 190

The tissue-specificity and DON-responsiveness of TaFROG (chromosome 4A 191

homeolog) were analysed using semi-quantitative RT-PCR (sqRT-PCR) and 192

quantitative real-time PCR (qPCR) analyses. In mock-treated wheat tissues, the 193

basal expression of TaFROG was below or near detectable limits, in contrast to the 194

high level of gene expression in DON-treated tissues (Fig. 2A and Supplemental Fig. 195

S3A). Thus, TaFROG transcription is induced by the toxigenic F. graminearum 196

virulence factor DON. In order to determine if the gene was activated by F. 197

graminearum, the level of expression was measured by qPCR in wheat head 198

spikelets at 1-5 days post-fungal inoculation (dpi). The result showed that the fungus 199

induces TaFROG as early as 1 dpi, induction peaking at 2 dpi (Fig. 2B). We also 200

studied FgTri5 transcription in the same tissue, this being a key gene in the fungal 201

DON biosynthetic pathway and its expression positively correlates with the 202

production of DON (Gardiner et al., 2009). In F. graminearum-infected wheat 203

spikelets, qPCR showed that the peak FgTri5 expression coincided with the peak in 204

wheat TaFROG expression (2 dpi), both the fungal and wheat gene presenting the 205



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same temporal expression profile (Fig. 2B). Based on the fact that TaFROG was 206

responsive to both DON and its’ producer, we hypothesized that toxin biosynthesis 207

might be a requirement for the fungal activation of this gene. Comparative analysis of 208

the response of TaFROG to both wild type fungus and its’ less virulent non-DON-209

producing mutant derivative (GZT40) enabled us to test this hypothesis. Both the 210

mutant and the wild type elicited a similar defence response, as represented by the 211

defence marker gene TaPR1 (Fig. 2B), previously described to be induced as an 212

early response to F. graminearum infection (Pritsch et al., 2000). But, unlike wild 213

type strain GZ3639, mutant GZT40 did not induce TaFROG expression in wheat 214

spikelets (Fig. 2B). Thus we conclude that TaFROG is a component of the early host 215



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response to F. graminearum, elicited in response to DON. The role of TaFROG in 216

the mycotoxin response is not restricted to the chromosome 4A homeolog. The 217

chromosome 4B, 4D homeologs and the barley TaFROG homolog share the same 218

DON-dependent expression pattern in tissues treated with DON or heads infected 219

with F. graminearum (Supplemental Fig. S3, A, B, C and D) (Boddu et al., 2007; 220

Gardiner et al., 2010). 221

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TaFROG contributed to DON tolerance 223

Virus-Induced Gene Silencing (VIGS) was used to determine if reducing 224

TaFROG transcript levels altered the phenotypic response to DON in a toxin-225

resistant wheat genotype. qRT-PCR confirmed that VIGS worked efficiently. DON 226

treatment of central head spikelets induced TaFROG, but in gene-silenced plants 227

(BSMV:FROG plants) the DON induction of TaFROG was reduced by 77%, as 228

compared to the effect of DON on plants treated with the mock virus (BSMV:00) (Fig. 229

3A). In the absence of DON, minimal TaFROG expression was observed, both in 230

BSMV:FROG and BSMV:00-treated plants (Fig. 3A). At a phenotypic level, 231

assessment of heads at 14 days post-toxin treatment showed that BSMV:FROG 232

plants were more sensitive to DON-induced damage than the BSMV:00 plants (Fig. 233

3B). Silencing of TaFROG resulted in a 2.2-fold increase in the number of DON-234

damaged spikelets (in BSMV:FROG versus BSMV:00 plants). Note that we obtained 235

similar results when we conducted an independent experiment using a different 236

BSMV construct targeting a larger fragment of the TaFROG gene (Supplemental Fig. 237

S4 and S5). 238



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The results of the VIGS experiments suggested a direct role for TaFROG in 239

DON tolerance. To confirm this, we generated transgenic wheat (cv. Fielder) lines 240

overexpressing TaFROG and tested their phenotypic response to DON and F. 241

graminearum. Transgenic lines OE-1, OE-2 and OE-3 were selected for F. 242

graminearum studies on the basis that they respectively exhibited a 218, 445 and 243

80-fold increase in TaFROG gene expression, as compared to wild type plants 244

(Supplemental Fig. S6). We evaluated the phenotypic effect of DON on spikelets 21 245

days post-toxin treatment. Fig. 3C illustrates that DON-induced damage of spikelets 246

was significantly reduced for the three overexpressor lines, as compared to the wild 247

type plants, thus confirming the role of TaFROG in DON tolerance. 248

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TaFROG enhances wheat leaf resistance to F. graminearum 250

DON facilitates disease spread in wheat heads (Bai et al., 2002) and, based 251

on its contribution to DON tolerance, we hypothesised that TaFROG might enhance 252

FHB resistance. Using a detached leaf assay, we evaluated the spread and 253

sporulation of F. graminearum (strain GZ3639) on transgenic lines OE-1, OE-2 and 254

OE-3, relative to wild type plants. Amending the fungal inoculum with DON (75 µM) 255

was previously reported to increase the reproducibility of results within detached leaf 256

assays (Chen et al., 2006). Thus we did two experiments; one using F. graminearum 257

plus DON as the treatment (Supplemental Fig. S7) and the other using only the 258



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fungus (Fig. 4). Similar results were obtained for both experiments, disease and 259

sporulation being reduced in the overexpressor as compared to wild type plants. 260

Thus, the overexpression of TaFROG enhanced wheat resistance to colonisation by 261

F. graminearum. 262

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Overexpression of TaFROG retards the spread of FHB disease symptoms 264

We evaluated the effect of TaFROG overexpression on the spread of FHB 265

symptoms from inoculated central spikelets of wheat heads. Results showed that 266



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wild type cv. Fielder had an average of 5.1 diseased spikelets at 21 dpi. All three 267

transgenic lines exhibited less disease spread; significant reductions of 23 and 17% 268

were respectively observed for OE-1 and OE-2, relative to wild type plants (Fig. 5A 269

and Supplemental Fig. S8A). The reduction observed for OE-3 at 21 days post-270

treatment was not significant, however, like OE1, the disease progression for line 271

OE3 (evaluated as AUDPC calculated using disease scores from 7, 14 and 21 dpi) 272

was significantly lower than on wild type plants (WT) (Fig. 5B). 273

The enhanced resistance of the transgenic lines relative to wild type was 274

confirmed under higher disease pressure where whole heads were sprayed with the 275

pathogen. All three transgenic lines showed less disease symptoms at 10 dpi, as 276

depicted in Supplemental Fig. S8B. By 21 dpi, 51 and 49% of the spikelets on OE-1 277

and OE-2 exhibited disease symptoms as compared to 57% on the WT plants (Fig. 278

5C). No reduction in disease was observed for OE-3 at this time point, but like OE-1 279

and OE-2, the disease progression for line OE3 (evaluated as AUDPC) was 280

significantly lower than on wild type plants (WT) (25-15% lower, as depicted in Fig. 281



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5D). Overall, the results of both point and spray inoculation experiments 282

demonstrated that overexpression of TaFROG provided quantitative resistance to 283

FHB. 284

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TaFROG, a new SnRK1-interacting protein 286

TaFROG is an IDP; the unstructured properties of IDPs allow them to bind 287

multiple protein partners and form an important component of the cellular signalling 288

machinery (Wright and Dyson, 2015). A yeast two-hybrid screen was conducted to 289

determine if TaFROG could interact with components of the plant cell signalling 290

machinery. Using TaFROG as bait, we screened a cDNA expression library 291

generated from DON-treated wheat tissue and thus identified putative interacting 292

proteins, including NAC transcription factors, a histone-binding protein and the 293

kinase TaSnRK1α. Among the putative interacting enzyme, TaSnRK1α was selected 294

for further analysis. TaSnRK1α is a wheat alpha subunit of a sucrose non-fermenting 295

1 (SNF1)-related kinase 1, a central regulator of energy and stress signalling in 296

plants (Halford and Hey, 2009). The protein-protein interaction in yeast was 297

confirmed using a GAL4-based yeast two-hybrid system (Fig. 6A; Supplemental Fig. 298

S9A for serial dilution results; Supplemental Fig. S9B for vector swapping results). 299

On the contrary, no growth was observed when we combined TaFROG with another 300

SnRK, TaSnRK2.8, or with the empty vectors. Protein expression from constructs 301

was confirmed by western blot analysis (Supplemental Fig. S9C). Thus we confirmed 302

that TaFROG is able to interact with TaSnRK1α in yeast. 303

We further investigated the interaction between TaFROG and TaSNRK1α in 304

planta using the Bimolecular Fluorescence Complementation (BiFC) system (Gehl et 305

al., 2009). TaFROG and TaSNRK1α genes were fused at their N terminal with either 306

the N or C terminal part of YFP. The resulting constructs were transiently co-307

expressed in Nicotiana benthamiana leaves. We observed a strong YFP signal, 308

predominantly in punctate cytoplasmic bodies, when YFPN-TaFROG was combined 309

with YFPC-TaSNRK1α (Fig. 6B). A smaller number of larger cytoplasmic bodies with 310

an irregular shape were also detected, potentially corresponding to protein 311

aggregates. Similar result were obtained with different combinations of TaSNRK1α 312

and TaFROG (TaFROG-YFPC/TaSnRK1α-YFPN or YFPC-TaFROG/TaSnRK1α-313



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YFPN), the only difference being that we occasionally detected fluorescent punctate 314

bodies in the nucleus of cells (see YFPC-TaFROG/TaSnRK1α-YFPN, Supplemental 315

Fig. S10B). The ability of TaFROG to homodimerize was also evaluated as part of 316

the BiFC experiment. The combination of YFPN-TaFROG/YFPC-TaFROG gave a 317

strong YFP signal, restricted within nuclear bodies (Fig. 6B). As a negative control, 318

the combination YFPN-TaFROG/ YFPC-TaSnRK2.8 gave no clear fluorescence (Fig. 319

6B). Protein expression from constructs was confirmed by western blot analysis 320

(Supplemental Fig. S10C). Collectively these BiFC data indicated that TaFROG can 321

homodimerize in the nucleus and that it can interact with TaSnRK1α in planta, 322

predominantly in cytosolic bodies and potentially (rarely) in nuclear bodies. 323

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DISCUSSION 326

FHB is one of the most devastating diseases of small grain cereals. 327

Resistance to FHB in wheat is quantitative, non-species and non-race specific. Many 328

quantitative trait loci (QTL) are confirmed to contribute to FHB resistance, but the 329

causative genes underpinning them remain to be determined. The overexpression of 330

classical plant defence genes was shown to enhance FHB resistance in wheat; 331

examples include β-1,3-glucanase, the defensin protein α-1-purothionin (Mackintosh 332

et al., 2007) and the key regulator of the salicylic defence signalling pathway, NPR1 333

(Makandar et al., 2006). To our knowledge, TaFROG is the first wheat orphan gene 334

to be functionally characterized and demonstrated to positively enhance biotic stress 335

resistance, specifically FHB resistance. TaFROG and its homeologs map to group 4 336

chromosomes, but with the wheat genome data available at present, there is no 337

evidence that TaFROG co-locates with FHB QTL previously identified on these 338

chromosomes (Buerstmayr et al., 2009). 339

Expression of TaFROG is induced in response to the F. graminearum 340

virulence factor DON. DON inhibits eukaryotic protein synthesis (Arunachalam and 341

Doohan, 2013), leading to cell death (Desmond et al., 2008), but lower 342

concentrations can also inhibit plant programmed cell death (Audenaert et al., 2013). 343

Fusarium species are considered hemibiotrophic, and Audenaert et al. (2013) 344

postulated that low levels of DON could facilitate the initial biotrophism and the 345

subsequent increase in DON production might facilitate the switch to necrotrophism. 346

Wheat may defend itself against the necrotrophic phase by counteracting oxidative 347

stress and inducing cell death (Walter et al., 2010), a theory supported by the 348

positive association between the activity of genes involved in the alleviation of 349

oxidative stress and the inheritance of DON/FHB resistance QTL Fhb1 (Walter et al., 350

2008). QTL Fhb1 is also associated with the detoxification of DON to DON-3-O-351

glucoside (Lemmens et al., 2005). 352

The DON-responsiveness of TaFROG, combined with the effects of gene 353

silencing and overexpression on DON/FHB resistance, led us to deduce that the 354

enhanced FHB resistance afforded by TaFROG is likely a consequence of enhanced 355

DON resistance. Fungal toxin productivity varies from strain to strain and there may 356

be a dose-dependent effect on the TaFROG-associated resistance response. 357



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TaFROG is an IDP and there is growing evidence that these proteins control protein 358

signalling complex assembly in cytosolic and nuclear bodies to regulate cellular 359

signalling pathways (Wright and Dyson, 2015). This information, coupled with the 360

lack of protein domains, led us to speculate that TaFROG is not involved directly in 361

any DON detoxification process, but might be a component of the signalling 362

response to DON, perhaps upstream of DON and FHB resistance responses. The 363

fact that TaFROG is induced as an early response to DON and its’ interaction with 364

the plant cell signalling machinery suggests that this orphan protein might be part of 365

the signalling events established to promote plant resistance to the fungus and the 366

toxin. We confirmed that TaFROG can physically interact with TaSnRK1α, both in 367

yeast and in planta. TaSnRK1α is a wheat alpha subunit of a sucrose non-fermenting 368

1 (SNF1)-related kinase 1, which is analogous to the yeast SNF1 and the 369

mammalian AMP-activated protein kinase (AMPK). SnRK1 proteins are central 370

regulators of plant sugar and stress signalling (Halford and Hey, 2009). It may be 371

that TaFROG is a new component of the SnRK1 signalling pathway. Interestingly, 372

human macrophages show a change in the phosphorylation state of AMPK in 373

response to DON, indicating that the associated signalling pathway is activated by 374

the mycotoxin (Pan et al., 2013). There is growing evidence that the interaction of 375

the SnRK1/SNF1 with novel proteins, including orphan proteins, regulates cellular 376

adaptability. For example, the yeast orphan protein MDF1 physically interacts with 377

SNF1, thereby conferring a selective advantage on yeast grown in rich medium by 378

virtue of their rapid consumption of glucose in early generational stages (Li et al., 379

2014). 380

To date there is no evidence that SnRK1 plays a role in plant defence 381

responses against a fungal toxin that acts as virulence factors. But there are studies 382

supporting the role of SnRK1 in plant defence against viruses, fungus, bacteria and 383

herbivores. For example, tobacco plants overexpressing SnRK1 were more resistant 384

to geminivirus infection (Hao et al., 2003); an SnRK1 null mutant was more 385

susceptible to Magnaporthe oryzae (Kim et al., 2015); SnRK1-silenced plants were 386

impaired in the hypersensitive response induced by the Xanthomonas campestris pv. 387

vesicatoria effector AvrBs1 (Szczesny et al., 2010); GAL83, the β-subunit of SnRK1 388

complex, was shown to have an important role in carbon resource allocation to roots, 389

enhancing tolerance to herbivory (Schwachtje et al., 2006). Thus it is conceivable 390



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that the complex TaSnRK1α/TaFROG plays a role in the wheat defence response 391

against DON and F. graminearum. 392

TaFROG is a nuclear protein, restricted within nuclear bodies. BiFC analysis 393

showed that TaFROG can homodimerize within nuclear bodies and that 394

TaFROG/TaSnRK1α complexes were primarily localized in small cytosolic bodies, 395

(very rarely within nuclear bodies). Although further study is required to validate the 396

subcellular location of the complexes, SnRK1 proteins have been previously 397

reported to localize in both the cytoplasm and in the nucleus, in agreement with the 398

dual functions of SnRK1 as a regulator of cytosolic enzyme activity and gene 399

expression (Halford and Hey, 2009; Cho et al., 2012). Moreover SnRK1 can localize 400

in nuclear bodies when interacting with the DUF581 proteins (Nietzsche et al., 2014) 401

or in cytosolic small fluorescent structures, reflecting both golgi bodies (O'Brien et al., 402

2015) or bodies from unknown origins stimulated from mechanical wounding 403

(Williams et al., 2014). Similarly upon oxidative, metabolic or drug-induced stress, 404

the AMPK localize in cytosolic bodies (Mahboubi et al., 2015). 405

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CONCLUSION 407

In the present study we present the functional characterization of TaFROG as 408

a wheat orphan gene validated to contribute to disease resistance in wheat, 409

providing a new source of resistance genes for crop breeding programmes. Future 410

work will determine the biochemical means by which TaFROG enhances plant 411

disease resistance, including a study to determine if this orphan protein affects the 412

SnRK1 signalling pathway. Because TaFROG is an intrinsically disordered protein 413

localized in cytosolic bodies when interacting with TaSnRK1α, we cautiously 414

speculate that this localization to specific subcellular bodies may affect downstream 415

signalling pathways. Future experiments will determine the nature of these bodies, 416

clarify the subcellular localization of the complex TaFROG/TaSnRK1α using 417

alternative approaches and determine the effect of this interaction on SnRK1 418

signalling. 419

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MATERIALS AND METHODS 421



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Plant and fungal material 422

Arabidopsis thaliana accession Col-0 and Triticum aestivum (wheat) cultivars 423

(cvs.) Fielder and CM82036 were used in this study. Wheat cv. Fielder is susceptible 424

to FHB disease (Badea et al., 2013), while cv. CM82036 is resistant to FHB and 425

DON, a trait that is associated with quantitative trait loci (QTL) located on 426

chromosomes 3B and 5A (Buerstmayr et al., 2003). The wheat cv. CM82036 was 427

used for gene expression and virus induced gene silencing studies, whereas cv. 428

Fielder and its’ transgenic derivatives were used for the disease assessment studies. 429

Wild type F. graminearum strain GZ3639 and its DON-minus mutant derivative 430

GZT40 were used for pathogenicity studies (Proctor et al., 1995; Bai et al., 2002). 431

Asexual conidial inoculum (macroconidia) was produced in Mung bean broth (Bai 432

and Shaner, 1996) and was harvested, washed and adjusted to 106 conidia/ml, all as 433

previously described (Brennan et al., 2005). 434

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Plant material for gene expression studies 436

For root gene expression studies, wheat plants were germinated for 3 days at 437

20°C on moist Whatman No. 1 filter paper (Whatman) and then placed in a new Petri 438

dish on filter paper soaked with 6 ml of either 67.5 µM DON (Santa Cruz) in 0.02% 439

Tween-20 or 0.02% Tween-20 (mock). Dishes were sealed with Parafilm and 440

incubated for 24 hours at 20°C in the dark before harvesting the roots. There were 441

three biological replicates, each including 12 plants (3 Petri dishes) per treatment. 442

For detached leaf gene expression studies, wheat plants were cultivated 443

under contained environment conditions at 20–22°C with a 16 h light/8 h dark 444

photoperiod at 300 μmol m-2 s-1 and 70% relative humidity, as previously described 445

(Ansari et al., 2007). The second leaves (growth stage 10; (Zadoks et al., 1974)) 446

were collected and 8 cm sections were placed in a square petri dish on moist 447

Whatman No. 1 filter paper soaked with 10 ml 0.67 mM benzimidazole (Sigma), 448

adaxial side facing upwards. The ends of the leaf were sandwiched between two 449

slices of 1% agar containing 0.67 mM benzimidazole. Leaves were gently wounded 450

with a glass Pasteur pipette and the wound site was treated with 10 μl of either 451

0.02% Tween-20 (mock) or this solution containing 337.5 µM DON. Dishes were 452

sealed as above and incubated in a growth chamber at 20-22°C with a 16 h light/8 h 453



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dark photoperiod at 200 μmol m-2 s-1 and 70% relative humidity. There were three 454

biological replicates, each including 14 leaves (7 Petri dishes) per treatment. 455

For adult plant DON and FHB studies, plants were cultivated as above under 456

contained environment conditions. At mid-anthesis (growth stage 65; (Zadoks et al., 457

1974)) two central spikelets of the heads were treated; for the DON experiment, they 458

were treated with 15 μl of either 0.02% Tween-20 (mock) or 16.87 mM DON in 459

0.02% Tween-20, while in a separate FHB experiment, the central spikelets were 460

treated with either 20 μl 0.02% Tween-20 (mock) or this solution augmented with 2 x 461

104 conidia of either F. graminearum strain GZ3639 (WT) or its non-DON-producing 462

mutant derivative GZT40. In both the DON and FHB experiment, the treated heads 463

were covered with plastic bags for 2 days to maintain high humidity. Treated 464

spikelets were harvested at various time points post-treatment and plant material 465

were flash-frozen in liquid N2 and stored at -70°C prior to RNA extraction. The DON 466

experiment comprised three biological replicates, while the FHB experiment 467

comprised two biological replicates. In each biological replicate, RNA was extracted 468

form one pooled sample per treatment (representing a pool of 4 heads from 469

individual plants). 470

471

DNA, RNA extraction and cDNA synthesis 472

DNA was extracted using the HP plant DNA mini kit (OMEGA) following the 473

manufacturer’s instructions. Wheat head RNA was extracted as described previously 474

(Ansari et al., 2007). RNA from all other plant tissues was extracted using the 475

RNeasy plant kit (Qiagen), according to the manufacturer’s instructions. DNase 476

treatment of RNA was performed using the TURBO DNA-free TM kit (Ambion Inc., 477

USA), according to the manufacturer’s instructions. The quality, yield and Integrity of 478

the RNA was analysed by measuring the UV absorbance with a NanoDrop 1000 479

(Thermo scientific, USA) and it was visualized following electrophoresis through an 480

agarose gel. The absence of DNA contamination was confirmed by PCR primers 481

targeting the endogenous gene TaGAPDH (primer sets listed in Supplemental Table 482

S1). Reverse transcription of total RNA was performed as described previously 483

(Walter et al., 2008). 484

485



21

Amplification and sequencing of TaFROG 486

The mRNA sequence of TaFROG was obtained by three successive rounds 487

of RACE (Rapid amplification of cDNA ends) using RNA extracted from DON-treated 488

heads of wheat cv. CM82036 and the GeneRacer TM kit (Invitrogen). RNA was 489

desphophorylated, decapped and reverse transcribed using SuperScript™ III RT 490

(Invitrogen), according to manufacturer’s instructions. Gene-specific primers are 491

detailed in Supplemental Table S1. The initial touchdown PCR reaction (50 µl 492

volume final) contained 200 nM forward or reverse gene-specific primers (GSP1 493

primer), 600 nM of either 5’ or 3’ GeneRacer primer, 1X high Fidelity buffer, 2.5 U 494

high fidelity platinum Taq polymerase (Invitrogen), 2 mM MgSO4, 200 µM of each 495

dNTP, 1 µl cDNA. PCR reaction conditions consisted of an initial denaturation step at 496

94°C for 2 min followed by five cycles of at 94°C for 30 s, 72°C for 1 min; five cycles 497

at 94°C for 30 s, 70°C for 1 min; 25 cycles at 94°C for 30 s, final annealing at 68°C 498

for 30 s, one cycle 72°C for 1 min. The two subsequent rounds of nested PCR (50 µl 499

volume) contained 1 μl of a 1:300 dilution of PCR product, 2.5 U LA Taq and 1X 500

TaKaRa LA Taq™ Mg2+ plus buffer (Takara), 200 µM dNTP (Gibco, UK), 200 nM of 501

either the 5’ or 3’ nested GeneRacer primer and 200 nM of either the forward or 502

reverse nested gene-specific primers (GSP2). PCR reaction conditions consisted of 503

an initial denaturation at 94°C for 1 min followed by 25 cycles of 94°C for 30 s, 65°C 504

for 30 s and 68°C for 2 min. The final extension was set at 68°C for 10 min. Amplified 505

fragments were cloned into the TOPO TA Cloning® kit (Invitrogen) and sequenced. 506

507

Bioinformatic analysis 508

Using the sequence obtained via RACE-PCR of TaFROG, the open reading 509

frame (ORF) and the coding sequence (CDS) were deduced using NCBI’s ORF 510

finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Homologs and homeologs were 511

identified via BLASTn query of the nucleotide sequences in the NCBI GenBank and 512

genome databases of: wheat (http://wheat‐urgi.versailles.inra.fr/Seq‐Repository and 513

http://www.cerealsdb.uk.net/cerealgenomics/CerealsDB), barley 514

(http://pgsb.helmholtz-muenchen.de/plant/barley/fpc/searchjsp/index.jsp), 515

Brachypodium (http://pgsb.helmholtz-516

muenchen.de/plant/brachypodium/searchjsp/index.jsp), Triticum urartu and Triticum 517



22

turgidum (http://dubcovskylab.ucdavis.edu/wheat_blast), bamboo 518

(http://www.bamboogdb.org/), rice (http://rice.plantbiology.msu.edu/), Zea mays, 519

Leersia perrieri, Musa acuminata, Setaria italica and Sorghum bicolor 520

(http://plants.ensembl.org/index.html). Prediction of the Nuclear Localization 521

Sequence (NLS) and Nuclear Export Signal (NES) was performed with WolF PSORT 522

(http://psort.hgc.jp/), NLStradamus 523

(http://www.moseslab.csb.utoronto.ca/NLStradamus/) and NetNES 1.1 524

(http://www.cbs.dtu.dk/services/NetNES/). Multiple sequence alignment of TaFROG 525

proteins and homologs was performed using ClustalW 526

(http://www.ebi.ac.uk/Tools/msa/clustalw2/) and Boxshade 527

(http://www.ch.embnet.org/software/BOX_form.html) programs. 528

529

Generation of transgenic wheat and Arabidopsis plants 530

Transgenic wheat cv. Fielder overexpressing TaFROG was generated as 531

follows. The TaFROG CDS was cloned using the gateway cloning strategy (primer 532

sets listed in Supplemental Table S1) and the pDONR207 vector (Invitrogen); the 533

gene was subsequently cloned into binary vector pSc4ActR1R2 (under the control of 534

the rice actin promoter) (McElroy et al., 1990). The recombinant plasmid pEW246-535

TaFROG was then transformed into Agrobacterium tumefaciens strain AGL-1 and 536

co-cultivated with immature wheat embryos at 23 °C in the dark for 2 days (Ishida et 537

al., 2015). Following removal of the embryonic axis, subsequent tissue culture of the 538

plant material was performed essentially as described previously (Risacher et al., 539

2009). DNA isolated from regenerated plantlets was analysed by qPCR to determine 540

the copy number of the nptII selectable marker gene, relative to an internal control 541

(Craze et al., in preparation). T0 transgenic plants carrying T-DNA (1 copy in lines 542

OE-1 and OE-2; 1-2 copies in line OE-3) and overexpressing TaFROG 543

(Supplemental Fig. S6) were propagated to the T3 generation: plants were grown 544

under contained environment conditions at 20–22°C with a 16 h light/8 h dark 545

photoperiod at 300 μmol m-2 s-1 70% relative humidity, as previously described 546

(Ansari et al., 2007). Homozygosity was analysed by testing for the presence-547

absence of the construct and calculating the segregation ratio in each generation. 548



23

For Arabidopsis transformation, the TaFROG coding sequence was fused to 549

YFP (TaFROG-YFP) under the control of the 35S promoter. TaFROG CDS was 550

cloned in pDONR207 (Invitrogen) using a gateway cloning strategy (primer sets 551

listed in Supplemental Table S1) and the gene was subsequently cloned into binary 552

vector pAM-PAT-P35S-YFP (Bernoux et al., 2008). pAM-PAT-P35S-TaFROG-YFP 553

was transformed into Arabidopsis ecotype Col-0 via Agrobacterium-mediated 554

transformation using the floral dipping method (Logemann et al., 2006). Transgenic 555

plants were selected in vitro on ½ MS containing 1% sucrose and BASTA (7.5 µg/ml 556

glufosinate ammonium), pH 5.7. Homozygous lines were propagated to the T2 557

generation at 20–22°C with a 16 h light/8 h dark photoperiod at 200 μmol m-2 s-1 and 558

70% relative humidity. Expression of TaFROG-YFP was confirmed by western blot 559

analysis (Supplemental Fig. S2). 560

561

Virus-induced gene silencing (VIGS) 562

VIGS were performed as described previously (Walter et al., 2015), with some 563

modification. The barley stripe mosaic virus (BSMV)-derived VIGS vectors used in 564

this study consisted of the wild type BSMV ND18 α, β, γ tripartite genome (Holzberg 565

et al., 2002; Scofield et al., 2005). Two different gene fragments were used for VIGS 566

of TaFROG (Supplemental Fig. S4). These were amplified from the CDS of TaFROG 567

(primer sets listed in Supplemental Table S1) via PCR. VIGS target sequences were 568

chosen to preferentially silence TaFROG (chromosome 4A) (100% homology) but 569

could also potentially target the two other homeologs (the two VIGS fragments 570

sharing 92% homology to the variants on chromosome 4B and 4D, based on cv. 571

Chinese spring sequences). PCR amplicons of the silencing fragments were cloned 572

into NotI/PacI -digested γ RNA vector pSL038-1 (Scofield et al., 2005). A BSMV γ 573

RNA construct containing a 185bp fragment of the barley phytoene desaturase 574

(PDS) gene served as a positive control for VIGS and has been previously described 575

(Scofield et al., 2005). 576

Vectors containing the BSMV α and γ genomes (BSMV:00), the γ RNA 577

genome encoding silencing fragments of FROG (FROG or FROG2), or PDS were 578

linearized with MluI. The vector carrying the BSMV β genome was linearized using 579

SpeI. Capped in vitro transcripts were prepared from the linearized plasmids using 580



24

the mMessage mMachine T7 in vitro transcription kit (AM1344, Ambion, USA) 581

following the manufacturer’s protocol. Flag leaves of wheat cv. CM82036 at growth 582

stage 47 (just before the emergence of the first wheat head, (Zadoks et al., 1974)) 583

were rub-inoculated with BSMV constructs following the protocol described by 584

(Scofield et al., 2005) Rub inoculations were done with 1:1:1 ratio mixtures of the in 585

vitro transcripts from BSMV α and β and either γ RNA (BSMV:00), BSMV:PDS, 586

BSMV:FROG or BSMV:FROG2. At mid-anthesis (growth stage 65), two florets of two 587

central spikelets of heads of BSMV-infected tillers were treated with 15 μl of either 588

0.02% Tween-20 (mock) or 16.87 mM DON in 0.02% Tween-20. Treated heads 589

were covered with plastic bags for 2 days to maintain high humidity. One spikelet 590

above the treated spikelets was sampled 24h after DON treatment, flash frozen in 591

liquid N2, and stored at -70° C prior to RNA extraction for gene expression studies. 592

The number of damaged (discoloured and necrotic) spikelets (including treated 593

spikelets) was assessed 14 days after DON treatment. In total there were two VIGS 594

experiments conducted, each including two biological replicates. Experiment 1 and 2 595

respectively used BSMV:FROG and BSMV:FROG2 for gene silencing 596

(Supplemental Fig. S5). Each biological replicate included positive and negative 597

controls. For BSMV:FROG, each biological replicate included 10-18 heads (5-9 598

plants) per treatment combination. For BSMV:FROG2, each biological replicate 599

included 10-15 heads (15 plants) per treatment combination. 600

601

Quantitative real-time and semi-quantitative PCR analysis 602

Reverse transcription of total RNA was performed as described previously 603

(Walter et al., 2008). Quantitative real-time PCR (qPCR) analysis was conducted 604

using the Stratagene Mx3000TM Real-Time PCR. Each reaction contained 1.25 μl of 605

a 1:5 (v/v) dilution of cDNA, 0.2 μM of the each primers and 1X SYBR® Premix Ex 606

Taq™ (Tli RNase H plus, RR420A, Takara) in a total reaction volume of 12.5 μl. 607

PCR conditions were: 1 cycle of 1 min at 95°C; 40 cycles of 5 s at 95°C and 20 s at 608

60°C; and a final cycle of 1 min at 95°C, 30 s at 55°C and 30 s at 95°C for the 609

dissociation curve. All qPCR analysis were conducted in duplicate (two cDNA 610

generated from independent reverse transcriptions). The threshold cycle (Ct) values 611

obtained by qPCR were used to calculate the relative gene expression using the 612



25

formula 2-(Ct target gene – Ct housekeeping gene) as described previously (Livak and 613

Schmittgen, 2001). To measure the fold change, an efficiency corrected calculation 614

model was used using the formula ((Etarget)∆Ct target (control – sample))/(Ehousekeeping)∆Ct 615 housekeeping (control – sample)) (Souaze et al., 1996). 616

Semi-quantitative RT-PCR (sqRT-PCR) was conducted in order to quantify 617

TaFROG transcript levels in different wheat tissues after calibration of the cDNA 618

template with the wheat alpha-tubulin. PCR conditions were: 1 cycle of 5 min at 619

95°C; 30 or 40 cycles of 45 s at 95°C, 45 s at 60°C, 45 s at 72°C; and a final cycle of 620

5 min at 72°C. PCR products were visualized after electrophoresis through a 1.5% 621

agarose gel. 622

623

Primers for qPCR 624

Primer sets used are listed in Supplemental Table S1. TaFROG, TaFROG-B 625

and TaFROG-D primers were designed to be specific to the chromosome 4A, 4B 626

and 4D variants, respectively. Unless otherwise stated, primers used for gene 627

expression studies were specific to the chromosome 4A variant. TaFROG primers 628

used for validation of gene silencing were designed so as to not overlap with the 629

gene fragments used for the VIGS. The wheat alpha-tubulin (GB No.: U76558.1) 630

(Xiang et al., 2011) was used as a housekeeping gene for TaFROG gene expression 631

analysis and verified to be not differentially expressed either in the microarray 632

expression data available in the PLEXdb (http://www.plexdb.org/) or in the conditions 633

used in our analysis. For F. graminearum, the FgActin gene (FGSG locus ID: 634

07335.3) (Brown et al., 2011) was used as a reference to calculate the relative 635

expression of FgTri5 gene (FGSG locus ID: 03537.3). The specificity of all primers 636

used was verified by analysis of the dissociation curve, sequence and 637

electrophoretic profile of PCR products. 638

639

Detached leaf disease assessment study 640

Detached leaf disease experiments were performed as described previously 641

(Browne and Cooke, 2004), with some modifications. An 8 cm section was cut from 642

the second leaf of 3-leaf-stage plants (growth stage 13; (Zadoks et al., 1974)). Leaf 643



26

sections were placed with the adaxial side facing upwards on the surface of a Petri 644

dish (90 mm diameter). The cut ends were placed between a sandwich of 1 % plant 645

agar pH 5.7 (Duchefa) containing 0.5 mM benzimidazole (agar was removed from 646

the centre of the plate to prevent excessive fungal growth at the point of leaf 647

inoculation). The centre of each leaf section was punctured and treated with a 4 μl 648

droplet of 0.02 % Tween-20 (mock) or this solution augmented with either 106 649

conidia/ml of F. graminearum strain GZ3639 or 106 conidia/ml of strain GZ3639 plus 650

75 µM DON. Plates were incubated at 20 °C under a 16 h light/8 h dark cycle. Leaf 651

sections were analysed 4 days post-inoculation. Disease leaf area was estimated 652

using FIJI software of photographed leaf sections (Schindelin et al., 2012). Leaf 653

sections were dipped in water and vigorously vortexed and macroconidia production 654

were counted using a haemocytometer (Hycor Biomedical). There were three 655

biological replicates, each including 6 plates per treatment, and each plate including 656

two leaf sections per wheat genotype. 657

658

FHB assessment and DON studies 659

Wheat cv. Fielder and transgenic lines overexpressing TaFROG were grown 660

and, for the point inoculation experiment, heads were treated as described above 661

(VIGS) with: 10 μl of DON or Tween-20 (mock) (DON experiment); 20 μl of 106 662

conidia/ml of F. graminearum strain GZ3639 or Tween-20 (mock) (FHB experiment). 663

In the case of the spray inoculation experiment, heads were sprayed with a total of 2 664

ml inoculum of 2 x 105 conidia/ml of F. graminearum using a hand-held sprayer. 665

Control plants were sprayed with Tween-20 (mock). Spray inoculated heads were 666

covered with plastic bags for 4 days to promote infection. For the DON experiment, 667

the number of damaged spikelets was assessed as above (VIGS) 21 days after DON 668

treatment. For the FHB experiments (point and spray inoculation), the level of 669

infection was calculated by visually scoring the number of infected spikelets at 7, 14 670

and 21 days post inoculation and data were used to calculate area under the disease 671

progress curve (AUDPC) (Shaner, 1977). For each experiment, there were three 672

biological replicates, each including at least 20 heads (10 plants) per treatment. 673

674

Yeast Two-Hybrid Analysis 675



27

Yeast two-hybrid screening was performed by Hybrigenics Services, S.A.S., 676

Paris, France (http://www.hybrigenics-services.com). Briefly the coding sequence for 677

TaFROG was cloned into pB29 as an N-terminal fusion to LexA. The construct was 678

used as a bait to screen a random-primed wheat cDNA library produced with RNA 679

extracted from DON-treated heads of wheat cv. CM82036 and cloned into pP6. A 680

total of 62 million clones were screened using a mating approach and positive 681

colonies were selected on a medium lacking tryptophan, leucine and histidine. The 682

prey fragments of the positive clones were amplified by PCR and sequenced. 683

Sequences were used to identify the corresponding proteins in the GenBank 684

database (NCBI). For analysis of specific protein-protein interactions, full-length CDS 685

of TaSnRK1α and TaSnRK2.8 (Zhang et al., 2010) were cloned into the vector 686

pDONR207 using the Gateway cloning technology (primer sets listed in 687

Supplemental Table S1) as described previously for TaFROG. They were then 688

recombined into bait and prey vectors derived from pGADT7 and pGBKT7 plasmids 689

(Clontech). Analysis of protein-protein interactions was performed using the Gal4 690

two-hybrid assay as described previously (Perochon et al., 2010). 3-AT was included 691

as a competitive inhibitor of the HIS3 protein in order to increase the stringency and 692

prevent auto-activation. 693

694

Subcellular localization of fluorescent fusion proteins and bimolecular 695

fluorescent complementation 696

Vector pAM-PAT-P35S-CFP and pAM-PAT-P35S-TaFROG-YFP (described 697

above) were used to biolistically transform detached wheat seedling leaves for 698

subcellular localization studies. Transformation was performed with 2-5 μg of total 699

DNA coated onto 1μm gold particles using the Bio-Rad PDS-1000/He biolistic 700

particle delivery system (Bio-Rad) and the manufacturers’ instructions (pressure set 701

at 1100 psi). After bombardment, leaves were placed on moist Whatman filter paper 702

in a petri dish and incubated for 24 hours at 21°C in the dark before analysis with a 703

confocal laser scanning microscope (Olympus fluoview FV1000) equipped with a 704

UPLSAPO 40X objective (numerical aperture, 0.95). CFP and YFP excitation was 705

performed at 405 nm and 515 nm respectively, and emission detected in the 460-706



28

500 nm range for CFP and 530-630 nm range for YFP. The experiment comprised 707

three biological replicates, each including three leaves. 708

TaFROG fused to the YFP was visualized within roots of three independent 709

stable transgenic Arabidopsis plants (described above) using a light sheet 710

fluorescence microscope (Lightsheet Z.1, Carl Zeiss Microscopy GmbH) equipped 711

with a W Plan-Apochromat 20X objective (numerical aperture, 1). Briefly, one-week-712

old Arabidopsis seedling grown on ½MS 0.8% agar medium were collected and 713

stained by manual vacuum infiltration with a 1 µg/ml DAPI (Sigma, D9542) solution in 714

PBS buffer (incubated for 20 min in the solution). As described by (von Wangenheim 715

et al., 2014), seedlings were then embedded within a cylinder of melted 1% agarose 716

type VI (Sigma). DAPI and YFP excitation was performed at 458 and 515 nm 717

respectively, and emission was detected in the 465-514 nm range for DAPI and 525-718

600 nm range for YFP. 719

For the in planta analysis of protein-protein interactions, the gateway cloning 720

strategy was used to subclone TaFROG, TaSnRK1α and TaSnRK2.8 CDS into the 721

bimolecular fluorescence complementation (BiFC) vectors pDEST-GW VYCE, 722

pDEST-VYCE(R)GW, pDEST- GWVYNE, pDEST-VYNE(R)GW (Gehl et al., 2009). This 723

generated constructs wherein proteins were fused at either their N- or C- terminal to 724

the YFP C-terminal (YFPC) or N-terminal fragment (YFPN). Vectors were introduced 725

into A. tumefaciens. A mix of Agrobacterium transformants was prepared: OD 600 = 726

0.5, 0.5 and 0.1 respectively for the YFPC construct, YFPN construct and the P19 727

silencing construct 728

(http://www.plantsci.cam.ac.uk/research/davidbaulcombe/methods/protocols/pbin61-729

p19.doc/view). This mix was syringe-infiltrated into leaf epidermal cells of 4-week-old 730

Nicotiana benthamiana. Epidermal cells were assayed for fluorescence 2-3 days 731

after infiltration. At 30 min prior to observation, cell nuclei were stained by pressure 732

infiltration of the leaves with a DAPI solution at 1 µg/ml. Image analysis were carried 733

using a confocal laser scanning microscope (Olympus fluoview FV1000) equipped 734

with the same objective described above. The DAPI excitation was performed at 405 735

nm and emission detected in the 425-475 nm range. Excitation/emission for the YFP 736

was performed as described above. 737



29

All microscopy image stacks were saved on the computer as TIFF files and 738

further processed using FIJI software. 739

740

Western blot 741

Protein extracts from yeast (Y2H studies), N. benthamiana leaf cells (BiFC 742

studies) and Arabidopsis leaves (localisation studies) were used for western blot 743

analysis. Yeast cells were grown at 28 °C overnight in Trp/Leu drop-out liquid 744

medium and subsequently for 3-5 h in YPD liquid medium. Total protein extracts 745

were prepared from yeast cells according to the yeast protocols handbooks 746

(Clontech). N. benthamiana leaves and Arabidopsis leaves were snap-frozen and 747

ground with beads in a Tissuelyser II (Qiagen). Total plant protein extracts were 748

obtained using Protein Extraction Buffer (Agrisera) and the manufacturer’s 749

instruction. Proteins were electrophoresed using a NuPAGE system and 10% Bis-750

Tris PAGE gels (Life technologies) according to the manufacturers’ instructions. After 751

protein transfer to nitrocellulose membranes using an iBlot gel transfer system (Life 752

technologies), proteins fused to the Gal4 activating domain (AD) or the N terminal of 753

the YFP (YFPN) were detected via their epitope HA using an anti-HA antibody at 754

1/1000 dilution (Roche). In the case of the proteins fused to the Gal4 binding domain 755

(BD) or C terminal part of the YFP (YFPC), an anti-c-Myc antibody at 1/500 dilution 756

(Roche) was used to detect their epitope cMyc. Following electrochemiluminescence 757

assay, emitted signal was imaged with the Fusion-FX (Vilber Lourmat). 758

759

Statistical analysis 760

All the statistical analysis was performed using the SPSS statistic version 20 761

software (SPSS). The normality of the data distribution was evaluated with the 762

Shapiro-Wilk test. In the case of the gene expression, conidial production, DON and 763

Fusarium head blight data sets, individual treatments were compared using either 764

the Mann-Whitney U or Kruskal-Wallis test. The normally-distributed infected leaf 765

area data from detached leaf experiments was assessed by ANOVA incorporating 766

Tukey's HSD test at the 0.05 level of significance. 767

768



30

Accession numbers 769

TaFROG (GB No.: KR611570), TaSnRK1α (GB No.: KR611568) and 770

TaSnRK2.8 (GB No.: KR611569) were cloned from the wheat cultivar (cv. 771

CM82036). TaFROG homeolog sequences were identified within wheat genome 772

contigs (Chromosome 4A: contigs_longerthan_200_7074280, chromosome 4B: 773

contigs_longerthan_200_4893246, chromosome 4C: 774

contigs_longerthan_200_2310089). Homologs from Triticum turgidum and Triticum 775

urartu were identified within contigs UCW_Tt-k25_contig_48942 and UCW_Tu-776

k51_contig_30323, respectively. Homologs from Aegilops tauschii (GB No.: 777

EMT01121), Hordeum vulgare (GB No.: BAJ91423) and Festuca pratensis (GB No.: 778

GO795446) were collected from the NCBI GenBank database. The homolog from 779

Brachypodium distachyon (Bradi4g22656.1) was collected from the MIPS Plants 780

database. 781

782

Supplemental Data 783

The following supplemental materials are available. 784

Figure S1. Characteristics of FROG protein sequences. 785

Figure S2. Immunoblot analysis with GFP antibody using the total proteins extracted 786

from wild type (WT) or TaFROG-YFP Arabidopsis leaves. 787

Figure S3. Expression of TaFROG homeologs and its barley homolog in different 788

tissues and in response to DON or F. graminearum. 789

Figure S4. Schematic representation of the position of the two VIGS gene fragments 790

(FROG and FROG2) and the qPCR target region within the TaFROG gene. 791

Figure S5. Effect of TaFROG silencing on DON tolerance. 792

Figure S6. Molecular characterization of transgenic wheat cv. Fielder 793

overexpressing TaFROG under the control of a rice actin promoter. 794

Figure S7. Effect of TaFROG overexpression on wheat leaf resistance to F. 795

graminearum. 796

Figure S8. Symptoms of FHB on TaFROG overexpression lines. 797



31

Figure S9. Yeast two-hybrid analysis. 798

Figure S10. Results for positive and negative controls included within the 799

Bimolecular Fluorescence Complementation analysis. 800

Supplemental Table S1. Primer sets used in this study. 801

802

ACKNOWLEDGEMENTS 803

The authors thank B. Moran, K. Heinrich-Lynch, B. Fagan, P. Grandjean, M. 804

Craze and R. Bates for technical assistance, H. Buerstmayr (IFA Tulln, Austria) for 805

wheat cultivars, Robert Proctor (USDA, USA) for F. graminearum fungi. C. Power 806

(Carl Zeiss UK Ltd) and E. G. Reynaud (UCD, Ireland) for assistance with light sheet 807

microscopy and C.K. Ng (UCD) for assistance with cell biology. 808

809

Author contributions 810

A.P., J.J., A.K. and C.A. performed the experiments; E.W. and S.B. 811

transformed wheat with TaFROG and determined gene copy number. A.P. and F.D. 812

designed the experiment and wrote the manuscript. S.S. contributed to VIGS 813

experimental design and provided constructs. 814

815

Author competing interests 816

The authors have a patent pending related to this material. 817

818

Figure legends 819

Figure 1. Phylogenetic analysis and subcellular localization of TaFROG. A, 820

Phylogenetic tree showing grass species that contain homologs of TaFROG. BLAST 821

E-value and the percentage of protein sequence identity (% id) calculated using 822

TaFROG from the chromosome 4A are presented. B and C, Microscopic analyses of 823

TaFROG within the nucleus of wheat and Arabidopsis thaliana cells, respectively. 824

Wheat seedling leaves were biolistically co-transformed with vectors harbouring 825

either CFP or TaFROG-YFP. Stable plant roots overexpressing TaFROG-YFP was 826



32

analysed for Arabidopsis. Cells were observed by confocal microscopy for wheat 827

leaves or by light sheet fluorescence microscopy for Arabidopsis roots. YFP and 828

either CFP or DAPI images are shown both separately and as an overlay. Scale bar 829

indicates 10 µm. 830

831

Figure 2. TaFROG transcript levels in wheat tissues after treatment with DON or F. 832

graminearum. A, Tissues at different wheat development stages and flowering 833

spikelets treated with DON. After 24 h, TaFROG gene expression was assessed via 834

sqRT-PCR (number in parenthesis = number of PCR cycles). B, TaFROG, FgTri5 835

(top graph) and TaPR1 (bottom graph) gene expression in wheat heads treated with 836

F. graminearum was assessed via qPCR. The wheat alpha-tubulin and F. 837

graminearum FgActin genes were used as internal reference to calculate the relative 838

expression of FgTri5 and TaFROG genes respectively, using the formula 2-(Ct target 839 gene – Ct housekeeping gene). Wheat spikelets were treated with either wild type F. 840

graminearum GZ3639, its DON-minus mutant derivative GZT40 or mock. The 841

tissues were harvested at various days post-inoculation as indicated. Results 842

represent the mean (two biological replicates and 4 technical replicates per 843

treatment from a pooled biological sample) and error bars indicate ± SEM (n = 8). 844

Asterisks show significant differences between treatments and mock (Kruskal-Wallis 845

test; *, P<0.05; **, P<0.01). 846

847

Figure 3. Effect of TaFROG silencing and overexpression on DON tolerance. A and 848

B, In the VIGS experiment, gene silencing of TaFROG in wheat spikelets was 849

quantified by (A) qPCR analysis and (B) the phenotypic response to DON was 850

assessed at 14 days post toxin treatment. For the qPCR analysis the wheat alpha-851

tubulin gene was used as internal reference to calculate the relative expression of 852

TaFROG gene using the formula 2-(Ct target gene – Ct housekeeping gene). VIGS constructs 853

used were empty vector BSMV:00 or the construct BSMV:FROG that targets the 854

TaFROG gene for silencing. Constructs were applied to the flag leaf and the central 855

flowering spikelets were treated with either 16.87 mM DON or Tween-20 (mock). C, 856

The phenotypic response of TaFROG overexpressor lines (OE-1, OE-2 and OE-3) 857

and control plants (WT) to DON treatment (as above) at 21 days post toxin 858



33

treatment. Asterisks show significant differences between DON-treated (A, B) 859

BSMV:00 and BSMV:FROG constructs or (C) overexpressor lines and the WT 860

(Mann-Whitney U test; *, P<0.05; **, P<0.01; ***, P<0.001). Error bars indicate ± 861

SEM (A, B: n = 20-36; C: n = 60). 862

863

Figure 4. Effect of TaFROG overexpression on wheat leaf resistance to F. 864

graminearum. A, B and C, TaFROG overexpressor lines (OE-1, OE-2 and OE-3) and 865

control plants (WT) were used for phenotypic analysis. A, Representative leaf 866

symptoms, B, diseased leaf area and C, conidia production were determined 4 days 867

post-treatment of detached wheat leaves with F. graminearum. Error bars indicate ± 868

SEM (B, C: n = 36). Asterisks show significant differences compared to the WT ((B) 869

Tukey's HSD test; (C) Mann-Whitney U test; *, P<0.05; **, P<0.01; ***, P<0.001). 870

871

Figure 5. Effect of TaFROG overexpression on the susceptibility of wheat to FHB 872

disease. At mid-anthesis, central flowering spikelets from overexpressor lines (OE-1, 873

OE-2 and OE-3) or control plants (WT) were (A, B) point-inoculated or (C, D) spray-874

inoculated with F. graminearum. A, B, C and D, disease was assessed at different 875

days post inoculation and data presented correspond to, the (A) score or (C) 876

percentage of infected spikelets per head at 21 days, or (B ,D) to the AUDPC. Error 877

bars indicate ± SEM (A, B, C, D: n = 60). Asterisks show significant differences 878

compared to the WT (Mann-Whitney U test; *, P<0.05; **, P<0.01; ***, P<0.001). 879

880

Figure 6. Interaction of TaFROG with TaSnRK1α. A, Yeast two-hybrid assay using 881

the yeast co-transformed with TaSnRK1α and TaFROG cloned in the Gal4 bait/prey 882

vectors. Yeast was grown for five days under selective Trp/Leu/His drop-out medium 883

in the presence of 3-AT (-TLH + 0.1 mM 3-AT) or non- selective Trp/Leu drop-out 884

medium (-TL) conditions. B, In planta protein-protein interaction visualised by the 885

Bimolecular Fluorescence Complementation (BiFC) assay. Confocal microscopy 886

images of representative N. benthamiana epidermal leaf cells expressing proteins 887

fused to N- or C-terminal part of the YFP as indicated. YFP, DAPI fluorescence and 888

Differential Interference Contrast (DIC) images are shown both separated and as an 889



34

overlay. Scale bar indicates 10 µm. In A and B, TaSnRK2.8 was used as a negative 890

control. 891



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Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Arendsee ZW, Li L, Wurtele ES (2014) Coming of age: orphan genes in plants. Trends Plant Sci 19: 698-708Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Arunachalam C, Doohan FM (2013) Trichothecene toxicity in eukaryotes: cellular and molecular mechanisms in plants and animals.Toxicol Lett 217: 149-158


Audenaert K, Vanheule A, Hofte M, Haesaert G (2013) Deoxynivalenol: a major player in the multifaceted response of fusarium toits environment. Toxins (Basel) 6: 1-19


Badea A, Eudes F, Laroche A, Graf R, Doshi K, Amundsen E, Nilsson D, Puchalski B (2013) Antimicrobial peptides expressed inwheat reduce susceptibility to Fusarium head blight and powdery mildew. Canadian Journal of Plant Science 93: 199-208


Bai G-H, Shaner G (1996) Variation in Fusarium graminearum and cultivar resistance to wheat scab. Plant Disease 80: 975-979Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Bai GH, Desjardins AE, Plattner RD (2002) Deoxynivalenol-nonproducing fusarium graminearum causes initial infection, but doesnot cause disease spread in wheat spikes. Mycopathologia 153: 91-98


Bernoux M, Timmers T, Jauneau A, Briere C, de Wit PJ, Marco Y, Deslandes L (2008) RD19, an Arabidopsis cysteine proteaserequired for RRS1-R-mediated resistance, is relocalized to the nucleus by the Ralstonia solanacearum PopP2 effector. Plant Cell20: 2252-2264


Bischof M, Eichmann R, Huckelhoven R (2011) Pathogenesis-associated transcriptional patterns in Triticeae. J Plant Physiol 168:9-19


Boddu J, Cho S, Muehlbauer GJ (2007) Transcriptome analysis of trichothecene-induced gene expression in barley. Mol PlantMicrobe Interact 20: 1364-1375


Brennan JM, Egan D, Cooke BM, Doohan FM (2005) Effect of temperature on head blight of wheat caused by Fusarium culmorumand F. graminearum. Plant Pathology 54: 156-160


Brown NA, Bass C, Baldwin TK, Chen H, Massot F, Carion PW, Urban M, van de Meene AM, Hammond-Kosack KE (2011)Characterisation of the Fusarium graminearum-Wheat Floral Interaction. J Pathog 2011: 626345


Browne RA, Cooke BM (2004) Development and Evaluation of an in vitro Detached Leaf Assay forc Pre-Screening Resistance toFusarium Head Blight in Wheat. European Journal of Plant Pathology 110: 91-102



http://www.ncbi.nlm.nih.gov/pubmed?term=%28Theor Appl Genet%5BTitle%5D%29 AND 114%5BVolume%5D AND 927%5BPagination%5D AND Ansari KI%2C Walter S%2C Brennan JM%2C Lemmens M%2C Kessans S%2C McGahern A%2C Egan D%2C Doohan FM%5BAuthor%5D&dopt=abstract

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http://scholar.google.com/scholar?as_q=QTL mapping and marker-assisted selection for Fusarium head blight resistance in wheat: a review&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Buerstmayr&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

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http://scholar.google.com/scholar?as_q=Molecular mapping of QTLs for Fusarium head blight resistance in spring wheat&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Buerstmayr&as_ylo=2003&as_allsubj=all&hl=en&c2coff=1

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http://scholar.google.com/scholar?as_q=Characterization of Arabidopsis thaliana-Fusarium graminearum interactions and identification of variation in resistance among ecotypes&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Chen&as_ylo=2006&as_allsubj=all&hl=en&c2coff=1

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http://scholar.google.com/scholar?as_q=Regulatory functions of SnRK1 in stress-responsive gene expression and in plant growth and development&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Cho&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

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http://scholar.google.com/scholar?as_q=The Fusarium mycotoxin deoxynivalenol elicits hydrogen peroxide production, programmed cell death and defence responses in wheat&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Desmond&as_ylo=2008&as_allsubj=all&hl=en&c2coff=1

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http://scholar.google.com/scholar?as_q=Evolutionary origins of Brassicaceae specific genes in Arabidopsis thaliana.&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=Evolutionary origins of Brassicaceae specific genes in Arabidopsis thaliana.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Donoghue&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

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http://scholar.google.com/scholar?as_q=Nutrient profiling reveals potent inducers of trichothecene biosynthesis in Fusarium graminearum&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Gardiner&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

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http://scholar.google.com/scholar?as_q=Transcriptome analysis of the barley-deoxynivalenol interaction: evidence for a role of glutathione in deoxynivalenol detoxification&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Gardiner&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

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http://www.ncbi.nlm.nih.gov/pubmed?term=%28A chromosome%2Dbased draft sequence of the hexaploid bread wheat Triticum aestivum genome%2E%5BTitle%5D%29 AND IWGSC The International Wheat Genome Sequencing Consortium%5BAuthor%5D&dopt=abstract

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http://scholar.google.com/scholar?as_q=A chromosome-based draft sequence of the hexaploid bread wheat (Triticum aestivum) genome.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=IWGSC&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28 transformation using immature embryos%2E Methods Mol Biol%5BTitle%5D%29 AND 1223%5BVolume%5D AND 189%5BPagination%5D AND Ishida Y%2C Tsunashima M%2C Hiei Y%2C Komari T%5BAuthor%5D&dopt=abstract

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http://www.ncbi.nlm.nih.gov/pubmed?term=%28Physiol Plant%5BTitle%5D%29 AND 148%5BVolume%5D AND 322%5BPagination%5D AND Luhua S%2C Hegie A%2C Suzuki N%2C Shulaev E%2C Luo X%2C Cenariu D%2C Ma V%2C Kao S%2C Lim J%2C Gunay MB%2C Oosumi T%2C Lee SC%2C Harper J%2C Cushman J%2C Gollery M%2C Girke T%2C Bailey%2DSerres J%2C Stevenson RA%2C Zhu JK%2C Mittler R%5BAuthor%5D&dopt=abstract

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http://scholar.google.com/scholar?as_q=Isolation of an efficient actin promoter for use in rice transformation&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=%28The complex becomes more complex%3A protein%2Dprotein interactions of SnRK1 with DUF581 family proteins provide a framework for cell%2D and stimulus type%2Dspecific SnRK1 signaling in plants%2E%5BTitle%5D%29 AND Nietzsche M%2C Schiessl I%2C Bornke F%5BAuthor%5D&dopt=abstract

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

http://scholar.google.com/scholar?as_q=The complex becomes more complex: protein-protein interactions of SnRK1 with DUF581 family proteins provide a framework for cell- and stimulus type-specific SnRK1 signaling 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=The complex becomes more complex: protein-protein interactions of SnRK1 with DUF581 family proteins provide a framework for cell- and stimulus type-specific SnRK1 signaling in plants.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Nietzsche&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28J Exp Bot%5BTitle%5D%29 AND 66%5BVolume%5D AND 2475%5BPagination%5D AND O%27Brien M%2C Kaplan%2DLevy RN%2C Quon T%2C Sappl PG%2C Smyth DR%5BAuthor%5D&dopt=abstract

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

http://scholar.google.com/scholar?as_q=PETAL LOSS, a trihelix transcription factor that represses growth in Arabidopsis thaliana, binds the energy-sensing SnRK1 kinase AKIN10&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=PETAL LOSS, a trihelix transcription factor that represses growth in Arabidopsis thaliana, binds the energy-sensing SnRK1 kinase AKIN10&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=O'Brien&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Toxicol Appl Pharmacol%5BTitle%5D%29 AND 268%5BVolume%5D AND 201%5BPagination%5D AND Pan X%2C Whitten DA%2C Wu M%2C Chan C%2C Wilkerson CG%2C Pestka JJ%5BAuthor%5D&dopt=abstract

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http://scholar.google.com/scholar?as_q=Global protein phosphorylation dynamics during deoxynivalenol-induced ribotoxic stress response in the macrophage&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Pan&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Biochem Biophys Res Commun%5BTitle%5D%29 AND 398%5BVolume%5D AND 747%5BPagination%5D AND Perochon A%2C Dieterle S%2C Pouzet C%2C Aldon D%2C Galaud JP%2C Ranty B%5BAuthor%5D&dopt=abstract

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http://scholar.google.com/scholar?as_q=Interaction of a plant pseudo-response regulator with a calmodulin-like protein&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=Interaction of a plant pseudo-response regulator with a calmodulin-like protein&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Perochon&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Bioinformatics%5BTitle%5D%29 AND 21%5BVolume%5D AND 3435%5BPagination%5D AND Prilusky J%2C Felder CE%2C Zeev%2DBen%2DMordehai T%2C Rydberg EH%2C Man O%2C Beckmann JS%2C Silman I%2C Sussman JL%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Prilusky J, Felder CE, Zeev-Ben-Mordehai T, Rydberg EH, Man O, Beckmann JS, Silman I, Sussman JL (2005) FoldIndex: a simple tool to predict whether a given protein sequence is intrinsically unfolded. Bioinformatics 21: 3435-3438

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

http://scholar.google.com/scholar?as_q=FoldIndex: a simple tool to predict whether a given protein sequence is intrinsically unfolded&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=FoldIndex: a simple tool to predict whether a given protein sequence is intrinsically unfolded&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Prilusky&as_ylo=2005&as_allsubj=all&hl=en&c2coff=1

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http://scholar.google.com/scholar?as_q=Fungal development and induction of defense response genes during early infection of wheat spikes by Fusarium graminearum&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Pritsch&as_ylo=2000&as_allsubj=all&hl=en&c2coff=1

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http://scholar.google.com/scholar?as_q=Reduced virulence of Gibberella zeae caused by disruption of a trichothecene toxin biosynthetic gene&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Proctor&as_ylo=1995&as_allsubj=all&hl=en&c2coff=1

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http://scholar.google.com/scholar?as_q=Components of the gene network associated with genotype-dependent response of wheat to the Fusarium mycotoxin deoxynivalenol&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Walter&as_ylo=2008&as_allsubj=all&hl=en&c2coff=1

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http://scholar.google.com/scholar?as_q=Regulation of Sucrose non-Fermenting Related Kinase 1 genes in Arabidopsis thaliana.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Williams&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

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http://scholar.google.com/scholar?as_q=Intrinsically disordered proteins in cellular signalling and regulation&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=Intrinsically disordered proteins in cellular signalling and regulation&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Wright&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

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http://scholar.google.com/scholar?as_q=Overexpression of a common wheat gene TaSnRK2.8 enhances tolerance to drought, salt and low temperature in Arabidopsis.&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=Overexpression of a common wheat gene TaSnRK2.8 enhances tolerance to drought, salt and low temperature in Arabidopsis.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhang&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1


1 running head: an orphan gene enhances wheat disease ... · in mock-treated wheat tissues, the 194...

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