2 9 10 11 12 13 14 Plant Biology Division support for JM’s lab came from the ... Sucrose breakdown in root nodules, ... Sucrose synthase is necessary for efficient nitrogen fixation

Running title 9

Nodule-specific sucrose transporter 10

Corresponding author 12

Michael Udvardi 13

Plant Biology Division 14

The Samuel Roberts Noble Foundation 15

Ardmore Oklahoma 73401 16

USA 17

Tel +15802246601 18

Email mudvardinobleorg 19

Primary research area 22

Membranes Transport and Bioenergetics 23

Plant Physiology Preview Published on March 28 2016 as DOI101104pp1501910

MtSWEET11 a Nodule-Specific Sucrose Transporter of Medicago truncatula Root 25

Nodules 26

Igor S Kryvoruchko15 Senjuti Sinharoy16 Ivone Torres-Jerez1 Davide Sosso2 Catalina 28

I Pislariu17 Dian Guan3 Jeremy Murray3 Vagner A Benedito4 Wolf B Frommer2 and 29

Michael K Udvardi1 30 31 1Plant Biology Division 32

USA 35 36 2Department of Plant Biology 37

Carnegie Institution of Science 38

Stanford California 94305 39

USA 40

41 3Department of Cell and Developmental Biology 42

John Innes Centre 43

Norwich NR4 7UH 44

United Kingdom 45 46 4Division of Plant amp Soil Sciences 47

West Virginia University 48

Morgantown West Virginia 26506 49

USA 50 51 5Current address 52

Bioengineering Department 53

Kafkas University 54

Kars 36100 55

Turkey 56

57 6Current address 58

Department of Biotechnology 59

University of Calcutta 60

35 Ballygaunge Circular Road 61

Kolkata 700019 62

India 63

Department of Biology and Chemistry 66

Texas AampM International University 67

Laredo Texas 78041 68

USA 69

70 Both authors contributed equally to this work 71

One sentence summary 73

A nodule-specific sucrose transporter of Medicago truncatula 74

Financial support 76

This work was supported by the Agriculture and Food Research Initiative Competitive Grants 77

Program Grant No 2010-65115-20384 from the USDA National Institute of Food and 78

Agriculture and the National Science Foundation Major Research Instrumentation Program 79

(NSF DBI-0400580 DBI-0722635) Financial support for JMrsquos lab came from the Biotechnology 80

and Biological Sciences Research Council Grants BBG0238321 and BBL0103051 Work in 81

WBFrsquos lab was supported by the Division of Chemical Sciences Geosciences and Biosciences 82

Office of Basic Energy Sciences at the US Department of Energy (DOE DE-FG02-04ER15542) 83

and the National Science Foundation (IOS-1258018) 84

Abstract 86

Optimization of nitrogen fixation by rhizobia in legumes is a key area of research for sustainable 87

agriculture Symbiotic nitrogen fixation (SNF) occurs in specialized organs called nodules and 88

depends on a steady supply of carbon to both plant and bacterial cells Here we report the 89

functional characterization of a nodule-specific sucrose transporter MtSWEET11 from 90

Medicago truncatula MtSWEET11 belongs to a clade of plant SWEET proteins that are capable 91

of transporting sucrose and play critical roles in pathogen susceptibility When expressed in 92

mammalian cells MtSWEET11 transported sucrose but not glucose The MtSWEET11 gene 93

was found to be expressed in infected root hair cells and in the meristem invasion zone and 94

vasculature of nodules Expression of an MtSWEET11-GFP fusion protein in nodules resulted in 95

green fluorescence associated with the plasma membrane of uninfected cells and infection 96

thread and symbiosome membranes of infected cells Two independent Tnt1-insertion sweet11 97

mutants were uncompromised in SNF Therefore although MtSWEET11 appears to be involved 98

in sucrose distribution within nodules it is not crucial for SNF probably because other sucrose 99

transporters can fulfill its role(s) 100

Introduction 102

Legumes can establish nitrogen-fixing symbioses with soil bacteria called rhizobia that provide 103

reduced nitrogen primarily ammonia to the plant for growth In return the bacteria receive 104

reduced carbon from plant photosynthesis along with all other nutrients required for metabolism 105

and growth (Udvardi and Poole 2013) Legume-rhizobia symbioses are a primary entry point for 106

nitrogen (N) into the terrestrial biological N-cycle which makes them key components of natural 107

and agricultural ecosystems (Peoples et al 2009) Over the last three decades considerable 108

progress has been made in our understanding of how various solutes are translocated between 109

symbiotic partners Nevertheless key transporters including those involved in transport of 110

sugars into nodule cells and between cellular compartments remain largely unknown (Udvardi 111

and Poole 2013 Clarke et al 2014 Benedito et al 2010) 112

Sucrose transport from phloem cells to cells in sink organs can occur in two ways apoplasmic 113

flow via plasma membrane (PM)-located transporters and symplasmic flow via plasmodesmal 114

connections (Patrick 1997) Increased frequencies of plasmodesmata have been documented 115

in nodules of legumes (Complainville et al 2003) and the non-legume Datisca glomerata 116

(Schubert et al 2011) In nodule primordia of Medicago sativa plasmodesmata between stele 117

pericycle endodermis and cortex cells were significantly more abundant compared to non-118

inoculated root controls (Complainville et al 2003) In D glomerata root nodules induced by 119

Frankia spp extensive plasmodesmal connections were observed between all cell types of the 120

nodule including mature infected cells (Schubert et al 2011) 121

Sucrose is the primary form of photosynthetic carbon exported via the phloem from source 122

tissues to various sinks including root nodules (Vance et al 1998 Lalonde et al 2004 Ayre 123

2011) Sucrose breakdown in root nodules and more generally in higher plants is mediated by 124

sucrose synthases and invertases (Morell and Copeland 1984 Winter and Huber 2000) 125

Nodule-upregulated genes encoding sucrose synthases and invertases have been isolated and 126

characterized in several legumes and one actinorhizal species (Kuumlster et al 1993 Gordon and 127

James 1997 Gordon et al 1999 Baier et al 2007 Horst et al 2007 Welham et al 2009 128

Schubert et al 2011) Sucrose synthase is necessary for efficient nitrogen fixation in Pisum 129

sativum (Gordon et al 1999) M truncatula (Baier et al 2007) and Lotus japonicus (Horst et 130

al 2007) 131

Sugar uptake studies using nodule cell protoplasts isolated from broad bean revealed that 132

uninfected protoplasts but not those containing rhizobia were able to import sucrose and 133

glucose in a proton symport-dependent manner (Peiter and Schubert 2003) 134

The first nodule-enhanced sugar transporter to be described was LjSUT4 in L japonicus 135

(Flemetakis et al 2003) SUTs are a family of sucroseproton symporters within the Major 136

Facilitator Superfamily (MFS) and LjSUT4 was found to be expressed in vascular bundles inner 137

cortex and infected and uninfected cells of nodules Later it was localized to the tonoplast and 138

was shown to transport a range of sugars including sucrose and maltose (Reinders et al 2008) 139

A proposed function for LjSUT4 is efflux of sugars stored in the vacuole for use in the 140

cytoplasm Another sugar transporter induced in nitrogen-fixing nodules DgSTP1 was 141

characterized in the non-legume D glomerata (Schubert et al 2011) DgSTP1 belongs to the 142

sugar porter (SP) family and has the highest relative uptake rate for glucose It is also capable 143

of transporting galactose xylose and mannose albeit at much lower rates Because of the 144

specific increase of DgSTP1 transcripts in infected nodule cells and an unusually low pH 145

optimum of the protein it has been suggested to fulfill the function of glucose export toward 146

symbiotic bacteria prior to the onset of nitrogen fixation (Schubert et al 2011) 147

A porter family of sugar transporters called SWEET was discovered recently (Chen et al 148

2010 Chen et al 2012) Plant SWEET-genes are up-regulated in pathogenic interactions with 149

bacteria and fungi where they are believed to transport sugars to the microbes (Yang et al 150

2006 Ferrari et al 2007 Antony et al 2010) It has been suggested that members of this 151

family may perform similar functions in mutualistic associations given the fact that the nodule-152

specific gene MtN3 (designated MtSWEET15c in this study Figure 1) discovered almost 20 153

years ago (Gamas et al 1996) was shown to be a member of the SWEET family recently 154

(Chen et al 2010 Eom et al 2015) The SWEET family is subdivided into four clades 155

Members of the SWEET family capable of sucrose transport fall into Clade III and have been 156

localized primarily to the PM (Chen et al 2012 Lin et al 2014) Two Clade III SWEET 157

transporters from rice were shown to be directly regulated by bacterial transcription activator-like 158

(TAL) effectors (Antony et al 2010) They are thought to be involved in sucrose efflux to the 159

apoplasm for consumption by pathogens as mentioned above (Yang et al 2006 Antony et al 160

2010) 161

Here we describe the first functional characterization of a SWEET transporter in legume 162

nodules MtSWEET11 from M truncatula 163

RESULTS 164

MtSWEET11 is a Nodule-Specific Transporter in Clade III of the SWEET family 166

We conducted a systematic search for SWEET genes in the M truncatula genome Using 167

IMGAG v40 of the genome we identified 26 genes with high similarity to known SWEET 168

transporter genes from Arabidopsis (Fig 1) To identify symbioses-related SWEET genes we 169

analyzed the expression patterns of these genes in plants using the Medicago truncatula Gene 170

Expression Atlas (MtGEA v3 Benedito et al 2008) One of the genes Medtr3g098930 171

(Affymetrix probeset Mtr208451S1_at) was found to be expressed in a nodule-specific 172

manner (Supplemental Fig S1) Transcript levels of this gene in nodules six days post 173

inoculation (6dpi) were 228-fold higher than in roots Two different names of the gene have 174

been suggested recently MtSWEET13 (Breakspear et al 2014) and MtSWEET11 (Lin et al 175

2014) In this manuscript we follow the naming system from the latter study Secondary 176

structure prediction for the MtSWEET11 protein indicated 7 putative trans-membrane domains 177

(TMDs Supplemental Fig S2) Phylogenetic analysis of the MtSWEET11 protein (Fig 1) placed 178

it into Clade III of the SWEET family with AtSWEET10 to 15 of Arabidopsis (Chen et al 2012) 179

as well as OsSWEET11 and OsSWEET14 of rice (Yang et al 2006 Antony et al 2010) all of 180

which are capable of sucrose transport (Chen et al 2012) 181

MtSWEET11 is a Sucrose-Specific Transporter 182

To characterize the transport activity of MtSWEET11 we co-expressed the coding sequence 183

(CDS) of MtSWEET11 with the high-sensitivity Foumlrster resonance energy transfer (FRET) 184

sucrose sensor FLIPsuc90micro∆1V (Lager et al 2006) in human embryonic kidney (HEK) 293T 185

cells which have low endogenous sucrose uptake activity (Chen et al 2012) MtSWEET11 186

expression enabled HEK293T cells to accumulate sucrose as evidenced by a sucrose-induced 187

negative FRET ratio change (Fig 2) Under the same test conditions but using a glucose FRET 188

sensor glucose was not imported by MtSWEET11 (Supplemental Fig S3) 189

MtSWEET11 is Expressed in Rhizobia-Infected Root Hair Cells and the Meristem 190

Invasion Zone the Distal Portion of the Vasculature of Nodules 191

MtSWEET11 is upregulated in nodules (Supplemental Fig S1) and in root hairs of WT and skl 192

plants inoculated with S meliloti compared to plants inoculated with non-Nod factor producing 193

control rhizobia (Breakspear et al 2014) This suggests that MtSWEET11 expression is 194

correlated with successful rhizobial infection We tested this hypothesis by using the same 195

system to measure MtSWEET11 expression in root hairs of S meliloti-inoculated nin plants 196

Expression of MtSWEET11 was significantly reduced in root hairs of nin seedlings relative to the 197

WT controls (Fig 3A) To investigate further MtSWEET11 gene expression patterns we 198

measured transcript levels of this gene in roots and in nodules of genotypes A17 and R108 at 199

different stages of development and in different nodule zones using quantitative real-time 200

polymerase chain reaction (qRT-PCR) assays MtSWEET11 transcripts were barely detectable 201

in wild-type R108 roots before inoculation and 2 dpi with rhizobia (Fig 3B) Transcript levels 202

increased significantly by 4 dpi and reached a peak at 6 dpi where the expression was over 203

3000-fold higher than in non-inoculated roots A steady decline in the transcript abundance was 204

observed at 8 10 15 and 21 dpi although levels in nodules at 21 dpi were still about 1000-fold 205

higher than in non-inoculated roots (Fig 3B) To better define the spatial pattern of MtSWEET11 206

expression in nodules qRT-PCR was performed on five manually-dissected developmental 207

zones of mature 28 dpi nodules from ecotype A17 (Fig 3C) MtSWEET11 transcript levels were 208

highest in the nodule meristem followed by the invasion zone and were virtually undetectable in 209

the other three nodule zones (interzone nitrogen fixation zone and senescence zone) To 210

further visualize MtSWEET11 expression in developing nodules wild type R108 roots were 211

transformed with the GUS-reporter gene fused to 1705 bp of the MtSWEET11 promoter and 212

subsequently inoculated with S meliloti strain Sm1021 containing a plasmid with the lacZ-213

reporter gene pMtSWEET11GUS expression was detectable as early as 1 dpi in dividing root 214

cortical cells (Fig 3D) underlying sites of rhizobia infection indicated by Magenta-Gal staining 215

for rhizobial LacZ activity Further strong GUS staining was found in actively dividing nodule 216

primordia at 3 dpi (Fig 3E) At 10 dpi and 21 dpi MtSWEET11 expression was associated 217

predominantly with the meristem the invasion zone and distal part of the vascular bundles (Fig 218

3F-I) Weak GUS signal was detected in the proximal and basal portions of the vascular system 219

of 10 dpi nodules after prolonged (6 h) staining (Fig 3G) No GUS signal was found in the 220

interior of the nitrogen fixation zone (Fig 3F-I) even when nodules were transversely sectioned 221

prior to staining (results not shown) 222

MtSWEET11 Protein is Associated with the Plasma Membrane of Uninfected Nodule Cells 223

and with Infection Thread and Symbiosome Membranes of Infected Cells 224

In order to determine the possible sub-cellular location(s) of MtSWEET11 protein we analyzed 225

the distribution pattern of an MtSWEET11-GFP (Green Fluorescent Protein) fusion protein 226

expressed in plant cells The fusion protein was transiently expressed under the control of the 227

constitutive 35S promoter in tobacco epidermal cells or the maize polyubiquitin promoter 228

(Christensen et al 1992) or the native promoter (17 kb) in Medicago nodules Transformation 229

of tobacco (Nicotiana benthamiana) leaf epidermal cells with p35SMtSWEET11-GFP resulted 230

in a fluorescent signal at the PM but not at any other organelle membranes (Supplemental Figs 231

S5A B) Expression of pUBIMtSWEET11-GFP in Medicago nodules resulted in green 232

fluorescence predominantly at the PM of uninfected cortical cells and at internal structures of 233

infected cells but not in uninfected cells of the same tissue (Supplemental Figs S5C D) Since 234

this contrasted with our gene expression analysis we tested whether the internal cellular 235

location of MtSWEET11-GFP was an artifact of ectopic over-expression from the polyubiquitin 236

promoter For this we used the native MtSWEET11 promoter to drive MtSWEET11-GFP 237

expression in nodules Green fluorescence associated with expression of 238

pMtSWEET11MtSWEET11-GFP in nodules was detected on trans-cellular infection threads 239

(Fig 4F) and symbiosomes in the infected cells with no signal at the PM of either the nodule 240

meristematic cells or cortical cells (Fig 4B D F-H) Surprisingly the protein localization pattern 241

of MtSWEET11-GFP driven by the native MtSWEET11 promoter did not match that of the GUS 242

protein driven by the same promoter (Fig 3F-I) Therefore we generated a control construct in 243

which the MtSWEET11 promoter was fused to GFP alone Green fluorescence resulting from 244

expression of pMtSWEET11GFP was observed only in the invasion zone and vascular bundles 245

of transgenic nodules (Fig 4A C E) consistent with the pMtSWEET11GUS results 246

Symbiotic Phenotype of sweet11 Mutants 247

Two independent sweet11 mutants were isolated from a Tnt1-insertional mutant population of 248

M truncatula using a PCR-based approach (Tadege et al 2008 Cheng et al 2011) In lines 249

NF12718 and NF17758 the Tnt1 transposon is located in exon 3 at positions 160 bp (sweet11-250

1 allele) and 232 bp (sweet11-2 allele) respectively relative to the translational start of 251

MtSWEET11 (Supplemental Fig S6) 252

Homozygous sweet11-1 and sweet11-2 mutants were obtained and phenotyped with respect to 253

nodule primordia nodule number leaf color plant size fresh weight and nitrogenase activity 254

(acetylene reduction assay) No significant differences between mutants and wild type controls 255

were found for these parameters except for biomass which was slightly lower in one mutant 256

but not the other (Supplemental Figs S7-S9) 257

Given the biochemical function of MtSWEET11 in sucrose transport we looked for conditional 258

phenotypes of the sweet11 mutants associated with altered sucrose transport in nodules under 259

conditions where sucrose supply to nodules was limited sweet11 mutants their wild-type-like 260

siblings and genotype R108 controls were grown under low-light conditions with 7 or 20 of 261

normal light intensity No significant difference in leaf color plant size fresh weight or 262

nitrogenase activity was found between the mutants and control plants (Supplemental Fig S9 263

exemplifies the results for line NF12718) In all cases low light reduced plant growth and 264

nitrogen fixation as expected 265

Loss of MtSWEET11 Activity is not Associated with Transcriptional Activation of other 266

Potential Sucrose Transporter Genes 267

In order to rule out the unlikely possibility that Tnt1 was excised from MtSWEET11 transcripts in 268

mutant nodules to produce transcripts that encode functional transporter we performed RT-269

PCR using MtSWEET11 coding sequence-specific primers (Supplemental Table 1) and nodule 270

cDNA from wild-type R108 and the two mutant lines As expected no wild-type MtSWEET11 271

transcript was present in the two mutants (Supplemental Fig S10) 272

The absence of symbiotic defects in the sweet11 mutants pointed to the existence of other 273

nodule sucrose transporters that can compensate for the loss of MtSWEET11 activity in these 274

mutants Several SWEET genes of clade III and other clades were found from MtGEA v3 data 275

to be expressed in nodules including MtSWEET1b (Medtr3g0891251) MtSWEET2b 276

(Medtr2g0731901) MtSWEET3c (Medtr1g0284601) MtSWEET12 (Medtr8g0963201) and 277

MtSWEET15c (Medtr7g4057301) (Supplemental Dataset 3 Benedito et al 2008) Likewise 278

members of the un-related SUT family of sucrose transporter genes and several genes from the 279

sugar porter (SP) family were expressed in nodules including MtSUT1-1 (Medtr1g096910) 280

MtSUT4-1 (Medtr5g067470 Reinders et al 2008) MtSUT4-2 (Medtr3g110880) and MtSTP13 281

(Medtr1g104780) (Supplemental Dataset 3) Some of these genes were induced during nodule 282

development including MtSWEET1b (Medtr3g0891251) MtSWEET15c (Medtr7g4057301) 283

MtSUT4-2 (Medtr3g110880) and MtSTP13 (Medtr1g104780) (Supplemental Dataset 3) We 284

confirmed these result for genes MtSWEET1b (Medtr3g0891251) MtSWEET12 285

(Medtr8g0963201) MtSWEET15c (Medtr7g4057301) MtSUT4-1 (Medtr5g067470) MtSUT4-2 286

(Medtr3g110880) and MtSTP13 (Medtr1g104780) by qRT-PCR (Supplemental Fig S11) 287

However none of these genes were induced in sweet11 mutant nodules in response to loss of 288

MtSWEET11 Analysis of the laser-capture microdissection data for different nodule zones from 289

Roux et al (2014) indicates that Medtr3g090950 and Medtr6g007623 are also expressed in 290

nodules In summary transcripts of several putative sucrose transporter genes are present in 291

nodules that could potentially fulfill the role(s) of MtSWEET11 in its absence although none of 292

the genes tested were expressed at higher levels in the sweet11 mutants than in the wild-type 293

DISCUSSION 294

Previous studies have demonstrated the importance of sucrose metabolism in nodules for 296

symbiotic nitrogen fixation (e g Baier et al 2007 see Vance et al 2008 for a review) Sucrose 297

is delivered to nodules via the phloem (Vance et al 1998) and distributed to other cell types via 298

apoplasmic andor symplasmic routes Uptake of sucrose from the apoplasm requires specific 299

transport proteins in the plasma membrane (see Ayre et al 2011 for a review) Movement of 300

sucrose between compartments of individual cells also requires transporters in the intervening 301

membranes Many putative sucrose transporter genes have been identified in legumes and are 302

expressed in nodules (Wienkoop and Saalbach 2003 Benedito et al 2010 Limpens et al 303

2013 Clarke et al 2015) although none of these have been characterized genetically In this 304

study we characterized a nodule-specific sucrose transporter of M truncatula MtSWEET11 at 305

the molecular biochemical cellular and genetic levels 306

MtSWEET11 belongs to Clade III of the SWEET protein family (Fig 1) a branch of the family 307

that is thought to specialize in sucrose transport (Chen et al 2012 Lin et al 2014 see Eom et 308

al 2015 for a review) Our experiments confirmed the substrate specificity of MtSWEET11 309

which transported sucrose but not glucose when expressed in HEK293T cells (Fig 2 310

Supplemental Fig S3) 311

MtSWEET11 promoter analysis using either GUS or GFP reporter genes expressed in 312

transformed roots and nodules indicated that MtSWEET11 expression is limited to the nodule 313

apex (meristem and infection zone) and to the vascular system (Figs 3F-I 4A C E) This 314

spatial pattern of gene expression was supported by quantitative measurements of 315

MtSWEET11 transcript levels which were found to be highest in the meristem followed by the 316

invasion zone with relatively little expression in other zones of mature nitrogen-fixing nodules 317

(Fig 3C) Our transcript data are consistent with the expression pattern revealed in the 318

RNAseq-based study of Roux et al (2014) where MtSWEET11 was expressed most strongly in 319

the nodule apex including the distal invasion zone and the meristem (Supplemental Dataset 3 320

and Supplemental Dataset 4) Maximal expression of MtSWEET11 was found in young nodules 321

6dpi which declined in older nodules but remained approximately 1000-fold higher in nodules 322

than in roots (Fig 3B) The temporal and spatial patterns of MtSWEET11 gene expression 323

indicate that the encoded transporter plays roles in sucrose transport during the first few days of 324

the symbiosis with rhizobia in infected root hair cells and underlying cortical cells as well as 325

throughout nodule development and during symbiotic nitrogen fixation in mature nodules 326

Localization of the MtSWEET11 protein using an MtSWEET11-GFP fusion protein expressed in 327

genetically-transformed cells indicated a plasma membrane location in uninfected cells and 328

different locations in infected cells namely infection thread (IT) and symbiosome membranes 329

(Fig 4) Differential localization of membrane proteins in infected versus uninfected cells is not 330

unprecedented in plant-microbe interactions and has been observed for the phosphate 331

transporter MtPT4 during arbuscular mycorrhizal symbiosis in Medicago (Pumplin et al 2012) 332

and potato proteins that are rerouted to the plant-pathogen interface in the heterologous 333

expression system Nicotiana benthamiana - Phytophthora infestans interaction (Bozkurt et al 334

2015) Taken together with the gene expression data described above the protein localization 335

data indicate that MtSWEET11 plays different roles in sucrose transport in different cell types In 336

uninfected cells where MtSWEET11 is located in the plasma membrane the transporter 337

probably plays a role in sucrose uptake from the apoplasm Based on our MtSWEET11 338

promoter-GUSGFP results apoplasmic sucrose uptake via MtSWEET11 appears to be 339

important in vascular tissues which are the primary conduit for sucrose entry into nodules 340

Sucrose taken up by cells within or surrounding the vascular tissues may subsequently move to 341

adjacent and distant cells via symplasmic sucrose transport through plasmodesmata which are 342

prevalent in nodules cells (Complainville et al 2003) Nonetheless MtSWEET11 expression in 343

cells of the meristem indicates that these cells are probably also able to take up sucrose from 344

the apoplasm presumably to fuel hyper-active metabolism and cell division 345

MtSWEET11 expression is induced in root hairs shortly after contact with rhizobia and its 346

expression is dependent on the transcription factor NIN (Fig 3A Supplemental Fig S4 347

Breakspear et al 2014) This regulation by NIN could be direct but may also be indirect as nin 348

mutants cannot form infection threads (Marsh et al 2007) Given the apparent location of 349

MtSWEET11 in trans-cellular infection threads of cortical cells it seems reasonable to speculate 350

that MtSWEET11 resides in the IT membrane of infected root hair cells as well although we 351

were unable to observe it there perhaps because of relatively low levels of expression of the 352

gene in those cells In any case the infection thread membrane is contiguous with the plasma 353

membrane so lateral movement of MtSWEET11 from the plasma membrane to the IT 354

membrane is conceivable An IT membrane localization of MtSWEET11 would put it in a 355

strategic location to supply sucrose to rhizobia within infection threads In this context it is 356

salient to note that SWEET transporters in other plant species are induced by pathogens to 357

ensure their sugar supply (Yang et al 2006 Antony et al 2010) Since purified Nod-factors 358

from rhizobia are insufficient to induce full-scale MtSWEET11 expression in root hair cells 359

(Supplemental Fig S4 Breakspear et al 2014) it would be interesting to determine whether 360

other bacterial factors are involved Although dicarboxylic acids are believed to be the primary 361

source of carbon for mature nitrogen-fixing bacteroids in nodules (Udvardi and Poole 2013 362

Benedito et al 2010) it is possible that other C-compounds that are common in root exudates 363

such as sugars and amino acids (Hirsch et al 2013) could be provided to rhizobia in ITs 364

(analogous to the apoplasm) prior to their release into cells within symbiosomes Expression 365

data from Roux et al (2014) based on laser capture microdissection of cells from different 366

nodule zones show expression of agl thu and frc rhizobial genes involved in the uptake andor 367

catabolism of sucrose (Willis et al 1999) trehalose (Jensen et al 2002) and fructose (Lambert 368

et al 2001) in the nodule apex suggesting the importance of sucrose and derived sugars as 369

energy sources for rhizobia contained within the infection thread It would be interesting to 370

determine whether sugar supply to rhizobia is reduced in sweet11 mutants perhaps using a 371

sugar-sensitive reporter gene in rhizobia 372

It came as a surprise to us that MtSWEET11-GFP fusion protein expressed from the 373

MtSWEET11 promoter accumulated in cells of the nitrogen-fixing zone (Fig 4) given that free 374

GFP or GUS expressed from the same promoter accumulated in the meristem and invasion 375

zones and in vascular bundles (Figs 3F-I 4A C E) locations consistent with the transcript data 376

(Fig 3C Supplemental Dataset 3 Supplemental Dataset 4) Given the congruence between the 377

three other types of data we conclude that MtSWEET11-GFP localization in nodules does not 378

accurately reflect MtSWEET11 gene expression and we speculate that intercellular movement 379

of the SWEET-GFP protein may account for its unexpected distribution in nodules There are 380

precedents for membrane proteins accumulating in cells other than those that express the 381

corresponding genes (Kuumlhn et al 1997) 382

Our MtSWEET11-GFP fusion protein experiments point to possible locations for MtSWEET11 383

on the plasma membrane of uninfected cells and the SM of infected cells in the nitrogen-fixing 384

zone of nodules (Fig 4F-H) Sugar uptake data from infected or uninfected cells isolated from 385

broad bean nodules indicated that uninfected cells were competent in sucrose uptake while 386

infected cells were not (Peiter and Schubert 2003) It would be interesting to obtain similar data 387

for cells isolated from sweet11 mutant nodules to determine whether MtSWEET11 contributes 388

to sucrose uptake in uninfected nodule cells 389

What are the implications of the possible SM location of the MtSWEET11 protein As for an IT 390

location a SM location would place MtSWEET11 in a position to transport sucrose towards the 391

rhizobia within symbiosomes Although sugars are unlikely to be primary sources of carbon for 392

nitrogen-fixing bacteroids (Udvardi and Poole 2013) sucrose and glucose have been found in 393

the symbiosome space of soybean nodules at concentrations of 6 and 3 respectively 394

(Tejima et al 2003) Consistent with this the rhizobial aglEFGAK operon encoding the sucrose-395

inducible α-glucoside transport system although mainly expressed in the nodule apex is 396

significantly expressed in the N-fixation zone (Roux et al 2014 Mauchline et al 2006 Jensen 397

et al 2002) It would be interesting to determine whether sucrose levels in the symbiosome 398

space of sweet11 mutants are lower than those of the wild-type Proteomic analysis of SM 399

purified from nodules of L japonicus revealed the presence of a putative sucrose transporter of 400

the SUC family (Wienkoop and Saalbach 2003) Thus if MtSWEET11 is active on the SM of 401

Medicago nodules it may not be the only active sucrose transporter there This leads us to the 402

subject of functional redundancy of sucrose transporters in Medicago nodules 403

Loss of MtSWEET11 function in sweet11 mutants had no significant effect on nodulation or 404

nitrogen fixation under conditions tested in this study (Supplemental Figs S7-9) Although it is 405

possible that subtle conditional phenotypes may be found for the sweet11 mutants in further 406

studies our results indicate that other sucrose transporters are active in Medicago nodules and 407

can perform the role(s) of MtSWEET11 in its absence Several SWEET genes of clade III and 408

other clades are expressed in nodules including MtSWEET1b (Medtr3g0891251) 409

MtSWEET2b (Medtr2g0731901) MtSWEET3c (Medtr1g0284601) MtSWEET12 410

(Medtr8g0963201) and MtSWEET15c (Medtr7g4057301) (Supplemental Dataset 3 Benedito 411

et al 2008) In fact analysis of the entire SWEET family across nodule zones indicates that 412

although MtSWEET11 accounts for 75 of SWEET gene expression in the nodule apex it only 413

accounts for 8 of the total reads in the N-fixation zone (data from Roux et al 2014 414

Supplemental Dataset 4) Likewise members of the un-related SUT family of sucrose 415

transporter genes and several genes from the sugar porter (SP) family are expressed in 416

nodules including MtSUT1-1 (Medtr1g096910) MtSUT4-1 (Medtr5g067470 Reinders et al 417

2008) MtSUT4-2 (Medtr3g110880) and MtSTP13 (Medtr1g104780) (Supplemental Dataset 3) 418

Clearly there are many putative sucrose transporters expressed in nodules presumably a 419

reflection of the importance of sucrose and sucrose transport for legume nodule metabolism 420

MATERIALS AND METHODS 421

Identification of SWEET Family Members in M truncatula 423

SWEET family genes were identified by sequence similarity to 17 A thaliana SWEETs using 424

the BLASTN algorithm at the IMGAG v4 CDS database (httpbioinfo3nobleorgdoblast) The 425

phylogenetic tree was constructed with MEGA 5 software (Tamura et al 2011) MtSWEET11 426

(IMGAG v4 Medtr3g0989301) was identified as nodule-specific based on data in the Medicago 427

truncatula Gene Expression Atlas (Benedito et al 2008) 428

Plant Growth Conditions 429

Seeds of M truncatula ecotype R108 and mutant plants were scarified with concentrated H2SO4 430

for 8 min rinsed five times with sterile water and sterilized for 3 min with a 33 (vv) solution of 431

commercial bleach (6 wv of Cl) containing 01 (vv) Tween 20 Seeds were then rinsed 10 432

times with sterile water and left in the dark in 50 ml Falcon tubes overnight at room temperature 433

(RT) to imbibe After transfer to sterile water-soaked filter paper in a Petri dish seeds were 434

incubated in the dark for two days at 4degC and then for two days at RT Seedlings were 435

transferred to a 12 mixture of vermiculiteturface (washed and double-autoclaved) in plastic 436

cones and placed in a walk-in growth chamber set at 200 μE mndash2 sndash1 for 16 h light dark period 8 437

h 21degC daynight and 40 humidity Up to the day of inoculation with rhizobia plants were 438

supplied only with distilled water Seven days after germination plants were inoculated with a 439

20 to 24 h culture of Sinorhizobium meliloti Sm1021 (OD600 075 to 120 TY medium) re-440

suspended in 2L zero-nitrogen half-strength BampD medium (Broughton and Dilworth 1971) to 441

OD600 = 002 Each plant was supplied with 50 mL of the inoculum to the surface of the soil by 442

the stem Any remaining suspension was added to the tray Subsequently distilled water was 443

added to the tray every 4 days up to the point of harvesting Thus atmospheric nitrogen fixation 444

was the only controlled source of nitrogen for these plants They were not exposed to N-445

containing fertilizers or N-rich soil at any stage before the measurements For seed production 446

plants inoculated with rhizobia were transferred from vermiculiteturface to soil 14 dpi and 447

cultivated in a greenhouse In the light-limitation experiment on the day of inoculation plants 448

were covered with domes made of commercial anti-mosquito net (Charcoal Fiberglass Screen 449

Cloth New York Wire) in order to reduce available light down to 7 and 20 of normal (3 and 2 450

layers of the net respectively) Control plants (100 light) were left uncovered 451

Isolation of Tnt1 Mutant Lines for MtSWEET11 452

Reverse genetic screening of a Tnt1 mutant population of M truncatula (Tadege et al 2008) for 453

insertions in the MtSWEET11 gene was carried out as described previously (Cheng et al 454

2011) Oligonucleotide sequences used as primers for PCR and genotyping of sweet11-1 and 455

sweet11-2 insertion alleles are provided in Supplemental Table S1 To reduce the number of 456

unlinked Tnt1 insertions in sweet11 mutants the mutants were backcrossed twice to the wild-457

type R108 lines as described (Taylor et al 2011) 458

Construction of DsRed-encoding Destination Vectors pUBIcGFP-DR and pRRcGFP for 459

Protein Localization with a C-terminal GFP Tag 460

To enable red fluorescence-based selection of transformed roots we modified commercial 461

vectors pMBb7Fm21GW-UBIL (VIB) and pKGW RedRoot (VIB) A 2515 bp region containing 462

the DsRed cassette of pKGW RedRoot was PCR-amplified with primers DsRED-F-KpnI and 463

DsRED-R-AvrII (Supplemental Table S1) cut with KpnI and AvrII and ligated to adapters 464

converting KpnI and AvrII sites into AflII and SacI sites respectively The adapters were formed 465

from oligonucleotides supplied with 5rsquo-phosphate (AflII_KpnI_SAS and SacI_AvrII_SAS 466

Supplemental Table S1) This DsRed product was introduced into AflIISacI-cut 467

pMBb7Fm21GW-UBIL in place of 1663 bp containing p35S and BAR which were eliminated to 468

make the resulting vector pUBIcGFP-DR shorter A 950 bp region containing the GFP gene 469

and terminator was amplified from pK7FWG2 (VIB) with primers GFP-F-SpeIT35S-R-HindIII 470

(Supplemental Table S1) digested with SpeI and HindIII and ligated into pKGW RedRoot pre-471

cut with the same restriction endonucleases to produce vector pRRcGFP 472

Cloning the CDS and Promoter Region of MtSWEET11 473

Primers for PCR-based cloning of MtSWEET11 CDS from M truncatula ecotype R108 474

(Supplemental Table S1) were designed on the basis of a partial genome sequence assembly 475

performed at the Noble Foundation The predicted R108 MtSWEET11 CDS differed by six base 476

pairs from that of ecotype Jemalong A17 deposited at the International Medicago Genome 477

Annotation Group database (IMGAG v4) This was later confirmed by sequencing of the CDS 478

amplified from R108 cDNA 479

cDNA produced from mRNA of 21 dpi root nodules of M truncatula ecotype R108 served as 480

template for PCR-amplification of the 810 bp MtSWEET11 CDS with primers SW11-CDS-F and 481

SW11-CDS-R (Supplemental Table S1) the latter of which was designed to eliminate the stop 482

codon The PCR product was cloned into pDONR207 (Life Technologies) For substrate 483

transport assays in HEK293T cells the entry clone was recombined with pcDNA32V5-DEST 484

(Life Technologies) For constitutive expression of MtSWEET11 the CDS was cloned into 485

destination vector pUBIcGFP 1705 bp of the promoter region upstream of the translational start 486

codon was amplified from M truncatula ecotype Jemalong A17 (primers SW11-pro-F and 487

SW11-pro-R Supplemental Table S1) cloned into pDONR207 and recombined with 488

destination vector pKGWFS70 (VIB) for GUS assays For subcellular localization of the 489

MtSWEET11 protein the 1705 bp promoter fragment was fused to the MtSWEET11 CDS by 490

overlap extension-PCR (OE-PCR Higuchi et al 1988) Individual elements for the fusion were 491

amplified from corresponding templates (1st stage PCR) with primers SW11-pro-FSW11-fus-R 492

from genomic DNA and SW11-fus-FSW11-CDS-R from the expression clone The products 493

were mixed in 11 molar ratio and used as an overlapping template for the 2nd stage PCR that 494

involved primers SW11-pro-F and SW11-CDS-R alone (see Supplemental Table S1 for primer 495

sequences) The resulting fusion was cloned into pDONR207 and recombined with a destination 496

vector pRRcGFP In both destination vectors the GFP CDS was located 3rsquo to the MtSWEET11 497

CDS All clones were verified by sequencing in both directions 498

Transport Assays with HEK293T Cells 499

Sucrose and glucose uptake assays in HEK293T cells were conducted as described in Chen et 500

al (2012) 501

Root Transformation Histochemical Staining and Light Microscopy 502

Plants with composite transgenic roots were generated using an axenic Agrobacterium 503

rhizogenes ARqua1-mediated procedure described in the Medicago truncatula Handbook 504

(httpwwwnobleorgGlobalmedicagohandbook) without antibiotic selection To enable 505

visualization of rhizobia in roots and nodules S meliloti strain Sm1021 was transformed with 506

the lacZ-reporter plasmid pXLGD4 (Boivin et al 1990) Inoculation with rhizobia was conducted 507

two weeks after transfer of plants to substrate For localization of promoter-GUS activity entire 508

root systems were harvested at different time points and stained with X-Gluc (Gold 509

Biotechnology) as described in Boivin et al (1990) Roots were also stained with Magenta-Gal 510

(Gold Biotechnology) for lacZ-activity as described in Arrighi et al (2008) Whole-nodule 511

images were taken with a Nikon SMZ 1500 fluorescent stereo microscope using a Nikon RS 512

Ri1 digital camera pMtSWEET11GUS fusion experiments were conducted in three biological 513

replicates with approximately 24 plants transformed in each replicate Sections of GUS-stained 514

nodules (50 μm) were obtained with a Vibratome 1000 Plus (Technical Product International) 515

An Olympus BX14 light microscope equipped with an Olympus DP72 digital camera was used 516

to visualize the sections Localization of proteins expressed under the control of the ubiquitin 517

promoter was performed in two biological replicates Native promoter MtSWEET11-GFP and 518

GFP-only localization experiments were performed in four and two biological replicates 519

respectively Each time approximately 24 plants were transformed Five or more nodules with 520

the strongest DsRed signal (transformation marker) were collected under the RFP channel 10 to 521

12 dpi and handmade sections were prepared Images were acquired with a Leica TCS SP2 522

AOBS confocal laser scanning microscope and LCS Lite software (Leica Microsystems) or a 523

PerkinElmer Ultra View Spinning Disc confocal microscope supplied with Velocity 611 software 524

package (PerkinElmer) Digital images were processed with Adobe Photoshop CS4 525

Phenotypic Analysis and Acetylene Reduction Assay 526

Visual assessment of nodules shoots and roots of wild-type and mutant plants was made 10 527

dpi 21 dpi and 42 to 44 dpi Images of representative plants were acquired with a Canon 528

DS126131 EOS 30D digital camera Fresh weight of whole plants was recorded with analytical 529

scales Entire root systems of 42 dpi plants were dissected and placed into sealed test tubes for 530

acetylene reduction assays (ARA) Gas measurements were performed with an Agilent 7890A 531

gas chromatograph (Agilent Technologies) after 18 to 24 h of incubation with 10 (vv) 532

acetylene at 28degC as described previously (Oke and Long 1999) 533

Nodule and Root Hair Collection RNA Extraction and Complementary DNA Synthesis 534

For the nodulation time course (0 to 21 dpi) we used cDNA generated by Sinharoy et al 535

(2013) 0 to 4 dpi samples were collected from the root zone susceptible to nodulation whereas 536

individual nodules were collected for samples of 6 dpi and onwards For gene expression 537

profiling in different nodule zones 28 dpi nodules (Jemalong A17) were dissected into the 538

meristematic zone invasion zone interzone II-III nitrogen fixation and senescence zone 539

Leghemoglobin coloration and overall nodule morphology were used to discriminate between 540

different nodule zones Hand-dissection was performed on unfixed nodules under a dissecting 541

microscope Three independent biological replicates each consisting of multiple samples were 542

prepared for each nodule zone qRT-PCR and hybridization to the GeneChipreg Medicago 543

Genome Array (Affymetrix) were performed on each of these biological replicates Root hairs 544

were removed from 10 day old nin-1 seedlings 5 dpi with S meliloti 1021 and RNA was isolated 545

for microarray analysis as previously described (Breakspear et al 2014) Data were normalized 546

with data from Breakspear et al (2014) skl-1 5 dpi with S meliloti 1021 WT (Jemalong A17) 547

plants 5 dpi with S meliloti 1021 and WT 5 dpi with S meliloti nodΔD1ABC (SL44) Nodules 548

from wild-type and mutant plants were collected 8 dpi (S meliloti Sm1021) immediately frozen 549

in liquid nitrogen and stored at -80degC Total RNA of frozen nodules and nodule zones was 550

extracted with TRIZOL reagent (Life Technologies) as described in Chomczynski and Mackey 551

(1995) Isolated RNA was digested with RNase-free DNase I (Ambion) following manufacturerrsquos 552

recommendations and further column-purified with an RNeasy MinElute CleanUp kit (Qiagen) 553

RNA was quantified using a Nanodrop Spectrophotometer ND-100 (NanoDrop Technologies) 554

and the purity assessed with a Bioanalyzer 2100 (Agilent) Nodule-derived cDNA was 555

synthesized with SuperScript III Reverse Transcriptase (Life Technologies) using oligo(dT)20 or 556

Random Hexamers to prime the reaction as described previously (Kakar et al 2008) cDNA 557

synthesis was performed individually for three biological replicates each corresponding to 17 to 558

20 plants Hybridization to the Medicago Genome Array was conducted following the 559

manufacturerrsquos instructions (Affymetrix) Microarray data was analyzed as previously described 560

(Benedito et al 2008) 561

qRT-PCR Analysis 562

PCR reactions were carried out in an ABI PRISM 7900 HT Sequence Detection System 563

(Applied Biosystems) Reaction mixtures were set up in an optical 384-well plate so that a 5 μL 564

reaction contained 25 μL SYBR Green Power Master Mix reagent (Applied Biosystems) 15 ng 565

cDNA and 200 nM of each gene-specific primer Amplification of templates followed the 566

standard PCR protocol 50degC for 2 min 95degC for 10 min 40 cycles of 95degC for 15 s and 60degC for 567

1 min and SYBR Green fluorescence was measured continuously Melting curves were 568

generated after 40 cycles by heating the sample up to 95degC for 15 s followed by cooling down to 569

60degC for 15 s and heating the samples to 95degC for 15 s Transcript levels were normalized using 570

the geometric mean of three housekeeping genes MtPI4K (Medtr3g091400) MtPTB2 571

(Medtr3g090960) and MtUBC28 (Medtr7g116940) qRT-PCR was performed for three 572

biological replicates and displayed as relative expression values To ensure specificity of 573

primers to each MtSWEET gene subjected to qRT-PCR all primer pairs (14 pairs) were tested 574

on four cDNA libraries (Supplemental Fig S11) which yielded melting curves corresponding to 575

unique products in each case 576

Statistical Analysis 577

Statistical significance of observed differences was determined with a Studentrsquos t-test 578

implemented using the TTEST function in Microsoft Excel with two-tailed distribution and 579

unequal variances 580

Accession Numbers 581

Supplemental Dataset 1 contains IMGAG v40 gene identifiers and CDSs of genes used in this 582

study as well as corresponding NCBI accession numbers Absence of the accession number 583

indicates sequences not present in the database Accession numbers of genes with IMGAG 584

v40 sequences different from NCBI entries are shown in brackets For members of MtSWEET 585

family Transporter Classification Database (TCDB) family numbers are also shown CDS of 586

MtSWEET11 from M truncatula ecotype R108 is available from NCBI under accession number 587

KF998083 588

ACKNOWLEDGEMENTS 589

We would like to thank Xiaofei Cheng and Jiangqi Wen for help with identification of the Tnt1 591

mutants Shulan Zhang for help with experiments Frank Coker Colleen Elles Janie Gallaway 592

and Vicki Barrett for technical assistance in maintaining the M truncatula Tnt1 lines Mark 593

Taylor for backcrossing the mutants Pascal Ratet Kirankumar S Mysore and Million Tadege 594

are acknowledged for construction of the Tnt1 mutant population We would also like to express 595

our gratitude to Maria Harrison Rebecca Dickstein and Carroll Vance for sharing their data and 596

helpful discussions Special thanks are addressed to Nick Krom for help with bioinformatics Jin 597

Nakashima for support with cellular imaging Christopher Town for acquisition of genomic 598

sequences 599

AUTHOR CONTRIBUTIONS 600

IK SS JM VAB WF and MKU contributed to the experimental design IK SS IT-602

J DS CIP DG and JM were involved in the experimental work DS performed transport 603

assays in HEK293T cells CIP provided data on transcriptional profiling of nodule 604

developmental zones JM conducted transcriptional profiling of root hairs under symbiotic 605

conditions IK SS IT-J and DS were responsible for the data analysis IK SS and 606

MKU wrote the manuscript 607

FIGURE LEGENDS 608

Figure 1 Phylogenetic tree of 26 Medicago truncatula and 17 Arabidopsis thaliana SWEET 610

family members The tree was constructed with MEGA 51 using protein sequences from M 611

truncatula (IMGAG v4) and Arabidopsis (Phytozomenet) Tree branch values are bootstrap 612

values (1000 reiterations) The tree demonstrates that MtSWEET11 falls into Clade III of 613

SWEET transporters together with six Arabidopsis homologs (AtSWEET9-15) Color code of 614

Clades I-IV blue I green II red III yellow IV 615

Figure 2 Identification of sucrose as a substrate of MtSWEET11 Sucrose uptake was 616

measured in HEK293T cells expressing MtSWEET11 together with the cytosolic FRET sucrose 617

sensor FLIPsuc90micro∆1V Individual cells were analyzed by quantitative ratio imaging of CFP and 618

Venus emission (acquisition interval 10 s) HEK293TFLIPsuc90micro∆1V cells were perfused with 619

medium followed by a pulse of 10 mM sucrose HEK293T cells transfected with sensor only 620

(ldquocontrolrdquo light blue) served as a negative control Cells containing the sensor and Arabidopsis 621

SWEET12 (ldquoAtSWEET12rdquo dark blue) constituted a positive control Similar to AtSWEET12 622

MtSWEET11 showed clear sucrose influx (ldquoMtSWEET11rdquo red) indicated by the decrease in 623

normalized intensity ratio in response to sucrose application Values represent mean plusmn SE n ge 624

4 repeated with comparable results at least three times 625

Figure 3 Expression of MtSWEET11 is limited to root hairs of infected plants the nodule 626

meristem the invasion zone and the nodule vasculature A MtSWEET11 expression in root 627

hairs 5 dpi with S meliloti 1021 in WT (Jemalong A17) in comparison with skl-1 and nin-1 628

mutants The control (CT) is WT 5 dpi with the non-Nod factor producing S meliloti nodΔD1ABC 629

(SL44) Data for WT and skl-1 from Breakspear et al (2014) B Time course of MtSWEET11 630

expression in nodules (qRT-PCR) C MtSWEET11 expression in manually-dissected nodule 631

zones of a mature nodule (28 dpi) measured using the Medicago Genome Array (Affymetrix) 632

Error bars in A represent SEM values of three biological replicates Error bars in B and C 633

represent SD values of three biological replicates Nodule zones M ndash meristem Inv ndash invasion 634

zone Int ndash interzone Nfix ndash nitrogen fixation zone S ndash senescence zone D-I Staining for GUS 635

activity D Nodule primodium Infection threads (1 dpi) showing rhizobia stained with Magenta-636

Gal (lacZ-reporter) E Nodule primordium (3 dpi) F Young nodule (10 dpi) after one-hour 637

staining for GUS activity G Young nodule (10 dpi) after six-hour staining for GUS activity H 638

Mature nodule (21 dpi) after one-hour staining for GUS activity I Mature nodule (21 dpi) after 639

six-hour staining for GUS activity Bars D and E 100 μm F and G 300 μm H and I 1000 μm 640

Figure 4 MtSWEET11-GFP fusion driven by the native promoter localizes to symbiosomes 641

infection threads and plasma membrane of non-infected nodule cells Images were taken with a 642

confocal laser scanning microscope except for panel H which was acquired with a confocal 643

spinning-disk microscope A C and E GFP driven by the SWEET11 native promoter (control) 644

B D F G and H GFP fused to the C-terminus of MtSWEET11 driven by the native promoter 645

A-D Overlay with bright-field images of transverse sections (A B) and cross-sections (C D) of 646

12 dpi nodules E-H Overlay with red fluorescence channel showing rhizobia (mCherry 647

plasmid) M ndash nodule meristem V ndash nodule vasculature IT ndash infection thread IC ndash infected cell 648

UC ndash uninfected cell CV ndash central vacuole G Fully developed infected cell from nitrogen 649

fixation zone Individual rod-shaped symbiosomes are visible H Symbiosomes released from a 650

dissected cell Bars A-D 100 μm E-F 20 μm G-H 10 μm Note that localization pattern of 651

GFP expressed from the native MtSWEET11 promoter is virtually the same as seen using the 652

corresponding GUS reporter in Figure 3 653

Supplemental Data 654

Supplemental Figure S1 MtSWEET11 is expressed exclusively in root hairs of infected plants 656

and nodules Expression profile of MtSWEET11 corresponding to Affymetrix probeset 657

Mtr208451S1_at is shown for selected microarray samples from the Medicago truncatula 658

Gene Expression Atlas v3 (MtGEA v3 Benedito et al 2008) downloaded from 659

httpmtgeanobleorgv3 The values are means of three biological replicates rsquoRoot Hairs 5 660

dpi Sm1021 controlrsquo refers to inoculation with S meliloti mutant cells carrying nodD1ABC 661

deletion (Nod-factor signalling defective Breakspear et al 2014) 662

Supplemental Figure S2 Predicted protein structure of MtSWEET11 A Seven 663

transmembrane helices (TMH) a short N-terminal loop at the outer side of the membrane (out) 664

and a long C-terminal loop at the inner side of the membrane (in) were predicted with TMHMM 665

software v20 (httpwwwcbsdtudkservicesTMHMM) The graphics were generated using 666

the Geneious software package (Biomatters) B TMHMM posterior probability plots for 667

MtSWEET11 668

Supplemental Figure S3 MtSWEET11 shows no transport of glucose Glucose uptake was 669

measured in HEK293T cells expressing MtSWEET11 together with the cytosolic FRET glucose 670

sensor FLIPglu600mD13V Individual cells were analyzed by quantitative ratio imaging of CFP 671

and Venus emission (acquisition interval 10 s) HEK293TFLIPglu600mD13V cells were 672

perfused with medium followed by a pulse of 25 mM glucose and then a pulse of 5 mM 673

glucose HEK293T cells transfected with sensor only (ldquocontrolrdquo light blue) served as a negative 674

control Cells containing the sensor and Arabidopsis SWEET1 (ldquoAtSWEET1rdquo dark blue) 675

constituted a positive control Similar to the negative control MtSWEET11 showed no influx of 676

glucose (ldquoMtSWEET11rdquo red) indicated by no decrease in normalized intensity ratio in response 677

to glucose application Values represent mean plusmn SE n ge 4 repeated with comparable results at 678

least three times 679

Supplemental Figure S4 MtSWEET11 expression is correlated with successful infection 680

Expression profile of MtSWEET11 corresponding to Affymetrix probeset Mtr208451S1_at is 681

shown for selected microarray samples from the Medicago truncatula Gene Expression Atlas v3 682

(MtGEA v3 Benedito et al 2008) downloaded from httpmtgeanobleorgv3 The values are 683

means of three biological replicates rsquoSm1021 nodD1ABCrsquo refers to inoculation with S meliloti 684

mutant cells carrying a deletion of nodD1ABC (defective for Nod-factor biosynthesis) 685

Supplemental Figure S5 MtSWEET11-GFP fusion expressed ectopically localizes essentially 686

to plasma membrane and symbiosomes but no other membranes Images were acquired with a 687

confocal laser scanning microscope A and B Transient expression in Agrobacterium-infiltrated 688

Nicotiana benthamiana leaves demonstrates strong localization of p35S-driven MtSWEET11-689

GFP fusion to the PM Blue fluorescence indicates a chloroplast adjacent to the PM Epidermal 690

chloroplasts adhere to the PM and thus serve as a marker for discrimination between the PM 691

and the tonoplast In contrast vacuole-associated chloroplasts are surrounded by the tonoplast 692

in epidermal cells Fluorescent signals were visualized 3 days after Agrobacterium infiltration 693

Infiltration and imaging were repeated with comparable results at least three times C and D 694

MtSWEET11-GFP fusion driven by the maize polyubiquitin promoter in M truncatula nodules 695

targets GFP to PM of nodule cortex cells (C) but also to symbiosomes (D) Panel D represents 696

a junction between the nodule cortex and the invasion zone IC ndash infected cell UI ndash uninfected 697

cell Bars A 10 μm B 5 μm C 50 μm D 20 μm 698

Supplemental Figure S6 Exon-intron structure of MtSWEET11 in ecotype R108 and relative 699

positions of Tnt1 insertions Genomic DNA of MtSWEET11 in ecotype R108 is 758 bp shorter 700

than in ecotype Jemalong A17 due to shorter nucleotide sequences of all five introns 701

(Supplemental Dataset 2) Tnt1 insertions in mutant lines NF12718 and NF17758 are located 702

within exon 3 of MtSWEET11 at nucleotide positions 160 and 232 respectively Dark boxes 703

indicate exons Bar = 500 bp 704

Supplemental Figure S7 sweet11 mutants exhibit growth characteristics similar to those of 705

corresponding wild-type controls A Nodule primordia and nodule number during early phases 706

of nodule development Measurements were conducted on 9-13 plants per variant on the whole 707

root system of the plant Error bars show SEM Double asterisk indicates the only significant 708

difference observed in this experiment (line NF17758 p lt 001 Studentrsquos t-test) B Whole-plant 709

images taken 44 dpi Note absence of major differences in plant size and appearance C Fresh 710

weight of wild-type wild-type-like and mutant populations represented in A and B Whole plants 711

were used for measurements of fresh weight Sample sizes R108 n=54 wt-like (NF12718) 712

n=10 mutant (NF12718) n=12 wt-like (NF17758) n=20 mutant (NF17758) n=22 Column 713

heights correspond to mean values Error bars show SEM Asterisks indicate p-value 714

comparison with wt-like (NF12718) lowastlowast

p lt 001 (Studentrsquos t-test) Note that nodule primordia 715

number and fresh weight of mutant plants is moderately affected in one Tnt1 line Wild-type-like 716

and mutant plants derive from separate BC2-F3 progenies of BC2-F2 individuals 717

Supplemental Figure S8 BC2-F3 progenies of heterozygous BC2-F2 sweet11 mutants show 718

no segregation for phenotypic characteristics such as fresh weight and nitrogenase actvity A 719

Fresh weight of wild-type-like (homozygous) and mutant (homozygous) plants 42 dpi Whole 720

plants were used for measurements of fresh weight B Nitrogenase activity (acetylene reduction 721

assay) of the same genotypic groups as shown in A Whole roots were used for these 722

measurements shortly after dissection of shoots Sample sizes wt-like (NF12718) n=11 723

mutant (NF12718) n=13 wt-like (NF17758) n=13 mutant (NF17758) n=18 Column heights 724

correspond to mean values Error bars indicate SEM Wild-type-like and mutant plants derive 725

from BC2-F3 progenies segregating for the Tnt1 insertion 726

Supplemental Figure S9 Light-deprivation does not cause a gene-specific effect on growth 727

and nitrogenase activity of sweet11 mutants A Fresh weight of wild-type wild-type-like and 728

mutant populations (44 dpi) grown at light-deprived conditions (20 of normal light intensity) 729

Whole plants were used for measurements of fresh weight Note absence of statistically 730

significant difference between the mutant and R108 B Nitrogenase activity (acetylene 731

reduction assay) of the same plants as shown in A Whole roots were used for these 732

measurements shortly after dissection of shoots Note absence of statistically significant 733

difference between the mutant and the wild-type-like control indicating that the effect is caused 734

by background insertions Sample sizes R108 n=16 wt-like (NF12718) n=16 mutant 735

(NF12718) n=15 Column heights correspond to mean values Error bars indicate SEM Wild-736

type-like and mutant plants derive from separate BC2-F3 progenies of BC2-F2 individuals 737

Supplemental Figure S10 RT-PCR with MtSWEET11 CDS-specific primers on cDNA 738

preparations from wild-type and the two mutant lines Wild-type size of MtSWEET11 CDS is 813 739

bp Predominant mutant forms correspond to the entire Tnt1 integrated into CDS (6147 bp) and 740

a splicing variant that lacks exon 3 (599 bp) as confirmed by sequencing 741

Supplemental Figure S11 Expression of 14 sucrose transporter genes in different organs 742

measured by qRT-PCR relative to three housekeeping genes N ndash nodule (28 dpi) R ndash root F ndash 743

flower P - seed pod Error bars represent SD of three biological replicates Note that only some 744

of tested genes are expressed in nodules Expression data for MtSWEET9a and MtSWEET15d 745

are not available through the Medicago truncatula Gene Expression Atlas (MtGEA v3 see also 746

Supplemental Dataset 3) 747

Supplemental Table S1 List of primers and sequences used in this study 748

Supplemental Dataset 1 Gene IDs and CDSs used in this study 749

Supplemental Dataset 2 Genomic DNA sequences of MtSWEET11 from ecotypes R108 and 750

Jemalong A17 751

Supplemental Dataset 3 Sugar transporters suscrose synthases and invertases potentially 752

relevant to the nodule function 753

Supplemental Dataset 4 Relative expression of MtSWEET11 vs other MtSWEET family 754

members in different nodule zones 755

REFERENCES 756

Antony G Zhou J Huang S Li T Liu B White F Yang B (2010) Rice xa13 Recessive 758

Resistance to Bacterial Blight Is Defeated by Induction of the Disease Susceptibility Gene Os-759

11n3 Plant Cell 22 3864-3876 760

Arrighi JF Godfroy O de Billy F Saurat O Jauneau A Gough C (2008) The RPG Gene of 761

Medicago truncatula Controls Rhizobium-Directed Polar Growth During Infection Proc Natl 762

Acad Sci U S A 105 9817ndash9822 763

Ayre BG (2011) Membrane-Transport Systems for Sucrose in Relation to Whole-Plant Carbon 764

Partitioning Mol Plant 4 377-394 765

Baier MC Barsch A Kuumlster H Hohnjec N (2007) Antisense Repression of the Medicago 766

truncatula Nodule-Enhanced Sucrose Synthase Leads to a Handicapped Nitrogen Fixation 767

Mirrored by Specific Alterations in the Symbiotic Transcriptome and Metabolome Plant Physiol 768

145 1600-1618 769

Benedito VA Torres-Jerez I Murray JD Andriankaja A Allen S Kakar K Wandrey M 770

Verdier J Zuber H Ott T Moreau S Niebel A Frickey T Weiller G He J Dai X Zhao PX 771

Tang Y Udvardi MK (2008) A Gene Expression Atlas of the Model Legume Medicago 772

truncatula Plant J 55 504-513 773

Benedito VA Li H Dai X Wandrey M He J Kaundal R Torres-Jerez I Gomez SK 774

Harrison MJ Tang Y et al (2010) Genomic Inventory and Transcriptional Analysis of 775

Medicago truncatula Transporters Plant Physiol 152 1716ndash1730 776

Boivin C Camut S Malpica CA Truchet G Rosenberg C (1990) Rhizobium meliloti Genes 777

Encoding Catabolism of Trigonelline Are Induced under Symbiotic Conditions The Plant Cell 778

Online 2 1157-1170 779

Bozkurt TO Belhaj K Dagdas YF Chaparro-Garcia A Wu CH Cano LM Kamoun S (2015) 780

Rerouting of Plant Late Endocytic Trafficking Toward a Pathogen Interface Traffic 16 204-226 781

Breakspear A Liu C Roy S Stacey N Rogers C Trick M Morieri G Mysore KS Wen J 782

Oldroyd GE Downie JA Murray JD (2014) The Root Hair Infectome of Medicago truncatula 783

Uncovers Changes in Cell Cycle Genes and Reveals a Requirement for Auxin Signaling in 784

Rhizobial Infection Plant Cell 26 4680-4701 785

Broughton WJ Dilworth MJ (1971) Control of Leghemoglobin Synthesis in Snake Beans 786

Biochem J 125 1075-1080 787

Chen LQ Hou BH Lalonde S Takanaga H Hartung ML Qu XQ Guo WJ Kim JG 788

Underwood W Chaudhuri B Chermak D Antony G White FF Somerville SC Mudgett 789

MB Frommer WB (2010) Sugar Transporters for Intercellular Exchange and Nutrition of 790

Pathogens Nature 468 527-532 791

Chen LQ Qu X-Q Hou B-H Sosso D Osorio S Fernie AR Frommer WB (2012) Sucrose 792

Efflux Mediated by SWEET Proteins as a Key Step for Phloem Transport Science 335 207-211 793

Cheng X Wen J Tadege M Ratet P Mysore KS (2011) Reverse Genetics in Medicago 794

truncatula Using Tnt1 Insertion Mutants Methods Mol Biol 678 179-190 795

Christensen AH Sharrock RA Quail PH (1992) Maize Polyubiquitin Genes Structure 796

Thermal Perturbation of Expression and Transcript Splicing and Promoter Activity Following 797

Transfer to Protoplasts by Electroporation Plant Mol Biol 18 675-689 798

Clarke VC Loughlin PC Day DA Smith PM (2014) Transport Processes of the Legume 799

Symbiosome Membrane Front Plant Sci 5 699 800

Clarke VC Loughlin PC Gavrin A Chen C Brear EM Day DA Smith PM (2015) Proteomic 801

Analysis of the Soybean Symbiosome Identifies New Symbiotic Proteins Mol Cell Proteomics 802

14 1301-1322 803

Complainville A Brocard L Roberts I Dax E Sever N Sauer N Kondorosi A Wolf S 804

Oparka K Crespi M (2003) Nodule Initiation Involves the Creation of a New Symplasmic Field 805

in Specific Root Cells of Medicago Species Plant Cell 15 2778-2791 806

Eom JS Chen LQ Sosso D Julius BT Lin IW Qu XQ Braun DM Frommer WB (2015) 807

SWEETs Transporters for Intracellular and Intercellular Sugar Translocation Curr Opin Plant 808

Biol 25 53-62 809

Ferrari S Galletti R Denoux C De Lorenzo G Ausubel FM Dewdney J (2007) Resistance 810

to Botrytis cinerea Induced in Arabidopsis by Elicitors is Independent of Salicylic Acid Ethylene 811

or Jasmonate Signaling but Requires PHYTOALEXIN DEFICIENT3 Plant Physiol 144 367ndash812

379 813

Flemetakis E Dimou M Cotzur D Efrose RC Aivalakis G Colebatch G Udvardi M 814

Katinakis P (2003) A Sucrose Transporter LjSUT4 Is up-Regulated During Lotus japonicus 815

Nodule Development J Exp Bot 54 1789-1791 816

Gamas P Niebel FdeC Lescure N Cullimore J (1996) Use of a Subtractive Hybridization 817

Approach to Identify New Medicago truncatula Genes Induced During Root Nodule 818

Development Mol Plant Microbe Interact 9 233ndash242 819

Gordon AJ James CL (1997) Enzymes of Carbohydrate and Amino Acid Metabolism in 820

Developing and Mature Nodules of White Clover J Exp Bot 48 895-903 821

Gordon AJ Minchin FR James CL Komina O (1999) Sucrose Synthase in Legume Nodules 822

Is Essential for Nitrogen Fixation Plant Physiol 120 867-878 823

Higuchi R Krummel B Saiki RK (1988) A General Method of In Vitro Preparation and Specific 824

Mutagenesis of DNA Fragments Study of Protein and DNA Interactions Nucleic Acids Res 16 825

7351ndash7367 826

Hirsch PR Miller AJ Dennis PG (2013) Do Root Exudates Exert More Influence on 827

Rhizosphere Bacterial Community Structure Than Other Rhizodeposits In FJ de Bruijn ed 828

Molecular Microbial Ecology of the Rhizosphere Ed 1 Vol 1 John Wiley amp Sons Inc Hoboken 829

NJ USA pp 229-242 830

Horst I Welham T Kelly S Kaneko T Sato S Tabata S Parniske M Wang TL (2007) Tilling 831

Mutants of Lotus japonicus Reveal That Nitrogen Assimilation and Fixation Can Occur in the 832

Absence of Nodule-Enhanced Sucrose Synthase Plant Physiol 144 806-820 833

Jensen JB Peters NK Bhuvaneswari TV (2002) Redundancy in Periplasmic Binding Protein-834

Dependent Transport Systems for Trehalose Sucrose and Maltose in Sinorhizobium meliloti J 835

Bacteriol 184 2978-2986 836

Kakar K Wandrey M Czechowski T Gaertner T Scheible WR Stitt M Torres-Jerez I Xiao 837

Y Redman JC Wu HC Cheung F Town CD Udvardi MK (2008) A Community Resource for 838

High-Throughput Quantitative RT-PCR Analysis of Transcription Factor Gene Expression in 839

Medicago truncatula Plant Methods 4 1746-4811 840

Kuumlhn C Franceschi VR Schulz A Lemoine R Frommer WB (1997) Macromolecular 841

Trafficking Indicated by Localization and Turnover of Sucrose Transporters in Enucleate Sieve 842

Elements Science 275 1298ndash1300 843

Kuumlster H Fruhling M Perlick AM Puhler A (1993) The Sucrose Synthase Gene Is 844

Predominantly Expressed in the Root Nodule Tissue of Vicia faba Mol Plant Microbe In 6 507-845

514 846

Lager I Looger LL Hilpert M Lalonde S Frommer WB (2006) Conversion of a Putative 847

Agrobacterium Sugar-Binding Protein into a FRET Sensor with High Selectivity for Sucrose J 848

Biol Chem 281 30875-30883 849

Lalonde S Wipf D Frommer WB (2004) Transport Mechanisms for Organic Forms of Carbon 850

and Nitrogen between Source and Sink Annu Rev Plant Biol 55 341-372 851

Lambert A Oslashsterarings M Mandon K Poggi MC Le Rudulier D (2001) Fructose Uptake in 852

Sinorhizobium meliloti is Mediated by a High-Affinity ATP-Binding Cassette Transport System J 853

Bacteriol 183 4709-4717 854

Limpens E Moling S Hooiveld G Pereira PA Bisseling T Becker JD and Kuster H (2013) 855

Cell- and Tissue-Specific Transcriptome Analyses of Medicago truncatula root nodules PloS 856

One 8 e64377 857

Lin IW Sosso D Chen LQ Gase K Kim SG Kessler D Klinkenberg PM Gorder MK Hou 858

BH Qu XQ et al (2014) Nectar Secretion Requires Sucrose Phosphate Synthases and the 859

Sugar Transporter SWEET9 Nature 508 546-549 860

Marsh JF Rakocevic A Mitra RM Brocard L Sun J Eschtruth A Long SR Schultze M 861

Ratet P Oldroyd GED (2007) Medicago truncatula NIN is Essential for Rhizobial-Independent 862

Nodule Organogenesis Induced by Autoactive CalciumCalmodulin-Dependent Protein Kinase 863

Plant Physiol 144 324ndash335 864

Mauchline TH Fowler JE East AK Sartor AL Zaheer R Hosie AH Poole PS Finan TM 865

(2006) Mapping the Sinorhizobium meliloti 1021 Solute-Binding Protein-Dependent 866

Transportome Proc Natl Acad Sci U S A 103 17933-17938 867

Morell M Copeland L (1984) Enzymes of Sucrose Breakdown in Soybean Nodules Alkaline 868

Invertase Plant Physiol 74 1030-1034 869

Oke V Long SR (1999) Bacterial Genes Induced within the Nodule During the Rhizobium-870

Legume Symbiosis Mol Microbiol 32 837-849 871

Patrick JW (1997) Phloem Unloading Sieve Element Unloading and Post-Sieve Element 872

Transport Annu Rev Plant Physiol Plant Mol Biol 48 191-222 873

Peiter E Schubert S (2003) Sugar Uptake and Proton Release by Protoplasts from the 874

Infected Zone of Vicia faba L Nodules Evidence against Apoplastic Sugar Supply of Infected 875

Cells J Exp Bot 54 1691-1700 876

Peoples MB Brockwell J Herridge DF Rochester IJ Alves BJR Urquiaga S Boddey RM 877

Dakora FD Bhattarai S Maskey S et al (2009) The Contributions of Nitrogen-Fixing Crop 878

Legumes to the Productivity of Agricultural Systems Symbiosis 48 1-17 879

Pumplin N Zhang X Noar RD Harrison MJ (2012) Polar Localization of a Symbiosis-Specific 880

Phosphate Transporter is Mediated by a Transient Reorientation of Secretion Proc Natl Acad 881

Sci U S A 109 E665-672 882

Reinders A Sivitz AB Starker CG Gantt JS Ward JM (2008) Functional Analysis of LjSUT4 883

a Vacuolar Sucrose Transporter from Lotus japonicus Plant Mol Biol 68 289-299 884

Roux B Rodde N Jardinaud MF Timmers T Sauviac L Cottret L Carregravere S Sallet E 885

Courcelle E Moreau S et al (2014) An Integrated Analysis of Plant and Bacterial Gene 886

Expression in Symbiotic Root Nodules Using Laser-Capture Microdissection Coupled to RNA 887

Sequencing Plant J 77 817ndash837 888

Schubert M Koteyeva NK Wabnitz PW Santos P Buttner M Sauer N Demchenko K 889

Pawlowski K (2011) Plasmodesmata Distribution and Sugar Partitioning in Nitrogen-Fixing 890

Root Nodules of Datisca glomerata Planta 233 139-152 891

Sinharoy S Torres-Jerez I Bandyopadhyay K Kereszt A Pislariu CI Nakashima J 892

Benedito VA Kondorosi E Udvardi MK (2013) The C2H2 Transcription Factor Regulator of 893

Symbiosome Differentiation Represses Transcription of the Secretory Pathway Gene 894

VAMP721a and Promotes Symbiosome Development in Medicago truncatula Plant Cell 25 895

3584-3601 896

Tadege M Wen J He J Tu H Kwak Y Eschstruth A Cayrel A Endre G Zhao PX 897

Chabaud M Ratet P Mysore KS (2008) Large-Scale Insertional Mutagenesis Using the Tnt1 898

Retrotransposon in the Model Legume Medicago truncatula Plant J 54 335-347 899

Tamura K Peterson D Peterson N Stecher G Nei M Kumar S (2011) MEGA5 Molecular 900

Evolutionary Genetics Analysis Using Maximum Likelihood Evolutionary Distance and 901

Maximum Parsimony Methods Mol Biol Evol 28 2731-2739 902

Taylor M Blaylock L Nakashima J McAbee D Ford J Harrison M Udvardi M (2011) 903

Medicago truncatula Hybridization Supplemental Videos In U Mathesius EP Journet LWA 904

Sumner eds Medicago truncatula Handbook Version July 2011 The Samuel Roberts Noble 905

Foundation Ardmore Oklahoma pp 1ndash5 httpwwwnobleorgMedicagoHandbook 906

Tejima K Arima Y Yokoyama T Sekimoto H (2003) Composition of Amino Acids Organic 907

Acids and Sugars in the Peribacteroid Space of Soybean Root Nodules Soil Sci Plant Nutr 49 908

239-247 909

Udvardi M Poole PS (2013) Transport and Metabolism in Legume-Rhizobia Symbioses In SS 910

Merchant ed Annu Rev Plant Biol Vol 64 Annual Reviews Palo Alto pp 781-805 911

Vance CP Miller SS Driscoll BT Robinson DL Trepp G Gantt JS Samas DA (1998) 912

Nodule Carbon Metabolism Organic Acids for N2 Fixation In C Elmerich A Kondorosi WE 913

Newton eds Biological Nitrogen Fixation for the 21st Century Vol 31 Springer Netherlands pp 914

443-448 915

Vance CP (2008) Carbon and Nitrogen Metabolism in Legume Nodules In M Dilworth E 916

James J Sprent W Newton eds Nitrogen-Fixing Leguminous Symbioses Vol 7 Springer 917

Netherlands pp 293-320 918

Welham T Pike J Horst I Flemetakis E Katinakis P Kaneko T Sato S Tabata S Perry J 919

Parniske M Wang TL (2009) A Cytosolic Invertase Is Required for Normal Growth and Cell 920

Development in the Model Legume Lotus Japonicus J Exp Bot 60 3353-3365 921

Wienkoop S Saalbach G (2003) Proteome Analysis Novel Proteins Identified at the 922

Peribacteroid Membrane from Lotus japonicus Root Nodules Plant Physiol 131 1080-1090 923

Willis LB Walker GC (1999) A Novel Sinorhizobium meliloti Operon Encodes an Alpha-924

Glucosidase and a Periplasmic-Binding-Protein-Dependent Transport System for Alpha-925

Glucosides J Bacteriol 181 4176-4184 926

Winter H Huber SC (2000) Regulation of Sucrose Metabolism in Higher Plants Localization 927

and Regulation of Activity of Key Enzymes Crit Rev Biochem Mol Biol 35 253-289 928

Yang B Sugio A White FF (2006) Os8N3 Is a Host Disease-Susceptibility Gene for Bacterial 929

Blight of Rice Proc Natl Acad Sci U S A 103 10503-10508 930

Parsed CitationsAntony G Zhou J Huang S Li T Liu B White F Yang B (2010) Rice xa13 Recessive Resistance to Bacterial Blight Is Defeated byInduction of the Disease Susceptibility Gene Os-11n3 Plant Cell 22 3864-3876

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

Arrighi JF Godfroy O de Billy F Saurat O Jauneau A Gough C (2008) The RPG Gene of Medicago truncatula Controls Rhizobium-Directed Polar Growth During Infection Proc Natl Acad Sci U S A 105 9817-9822

Ayre BG (2011) Membrane-Transport Systems for Sucrose in Relation to Whole-Plant Carbon Partitioning Mol Plant 4 377-394Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Baier MC Barsch A Kuumlster H Hohnjec N (2007) Antisense Repression of the Medicago truncatula Nodule-Enhanced SucroseSynthase Leads to a Handicapped Nitrogen Fixation Mirrored by Specific Alterations in the Symbiotic Transcriptome andMetabolome Plant Physiol 145 1600-1618

Benedito VA Torres-Jerez I Murray JD Andriankaja A Allen S Kakar K Wandrey M Verdier J Zuber H Ott T Moreau S Niebel AFrickey T Weiller G He J Dai X Zhao PX Tang Y Udvardi MK (2008) A Gene Expression Atlas of the Model Legume Medicagotruncatula Plant J 55 504-513

Benedito VA Li H Dai X Wandrey M He J Kaundal R Torres-Jerez I Gomez SK Harrison MJ Tang Y et al (2010) GenomicInventory and Transcriptional Analysis of Medicago truncatula Transporters Plant Physiol 152 1716-1730

Boivin C Camut S Malpica CA Truchet G Rosenberg C (1990) Rhizobium meliloti Genes Encoding Catabolism of Trigonelline AreInduced under Symbiotic Conditions The Plant Cell Online 2 1157-1170

Bozkurt TO Belhaj K Dagdas YF Chaparro-Garcia A Wu CH Cano LM Kamoun S (2015) Rerouting of Plant Late EndocyticTrafficking Toward a Pathogen Interface Traffic 16 204-226

Breakspear A Liu C Roy S Stacey N Rogers C Trick M Morieri G Mysore KS Wen J Oldroyd GE Downie JA Murray JD (2014)The Root Hair Infectome of Medicago truncatula Uncovers Changes in Cell Cycle Genes and Reveals a Requirement for AuxinSignaling in Rhizobial Infection Plant Cell 26 4680-4701

Broughton WJ Dilworth MJ (1971) Control of Leghemoglobin Synthesis in Snake Beans Biochem J 125 1075-1080Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Chen LQ Hou BH Lalonde S Takanaga H Hartung ML Qu XQ Guo WJ Kim JG Underwood W Chaudhuri B Chermak D AntonyG White FF Somerville SC Mudgett MB Frommer WB (2010) Sugar Transporters for Intercellular Exchange and Nutrition ofPathogens Nature 468 527-532

Chen LQ Qu X-Q Hou B-H Sosso D Osorio S Fernie AR Frommer WB (2012) Sucrose Efflux Mediated by SWEET Proteins as aKey Step for Phloem Transport Science 335 207-211

Cheng X Wen J Tadege M Ratet P Mysore KS (2011) Reverse Genetics in Medicago truncatula Using Tnt1 Insertion MutantsMethods Mol Biol 678 179-190

Pubmed Author and Title wwwplantphysiolorgon May 14 2018 - Published by Downloaded from

CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Christensen AH Sharrock RA Quail PH (1992) Maize Polyubiquitin Genes Structure Thermal Perturbation of Expression andTranscript Splicing and Promoter Activity Following Transfer to Protoplasts by Electroporation Plant Mol Biol 18 675-689

Clarke VC Loughlin PC Day DA Smith PM (2014) Transport Processes of the Legume Symbiosome Membrane Front Plant Sci 5699

Clarke VC Loughlin PC Gavrin A Chen C Brear EM Day DA Smith PM (2015) Proteomic Analysis of the Soybean SymbiosomeIdentifies New Symbiotic Proteins Mol Cell Proteomics 14 1301-1322

Complainville A Brocard L Roberts I Dax E Sever N Sauer N Kondorosi A Wolf S Oparka K Crespi M (2003) Nodule InitiationInvolves the Creation of a New Symplasmic Field in Specific Root Cells of Medicago Species Plant Cell 15 2778-2791

Eom JS Chen LQ Sosso D Julius BT Lin IW Qu XQ Braun DM Frommer WB (2015) SWEETs Transporters for Intracellular andIntercellular Sugar Translocation Curr Opin Plant Biol 25 53-62

Ferrari S Galletti R Denoux C De Lorenzo G Ausubel FM Dewdney J (2007) Resistance to Botrytis cinerea Induced inArabidopsis by Elicitors is Independent of Salicylic Acid Ethylene or Jasmonate Signaling but Requires PHYTOALEXINDEFICIENT3 Plant Physiol 144 367-379

Flemetakis E Dimou M Cotzur D Efrose RC Aivalakis G Colebatch G Udvardi M Katinakis P (2003) A Sucrose TransporterLjSUT4 Is up-Regulated During Lotus japonicus Nodule Development J Exp Bot 54 1789-1791

Gamas P Niebel FdeC Lescure N Cullimore J (1996) Use of a Subtractive Hybridization Approach to Identify New Medicagotruncatula Genes Induced During Root Nodule Development Mol Plant Microbe Interact 9 233-242

Gordon AJ James CL (1997) Enzymes of Carbohydrate and Amino Acid Metabolism in Developing and Mature Nodules of WhiteClover J Exp Bot 48 895-903

Gordon AJ Minchin FR James CL Komina O (1999) Sucrose Synthase in Legume Nodules Is Essential for Nitrogen Fixation PlantPhysiol 120 867-878

Higuchi R Krummel B Saiki RK (1988) A General Method of In Vitro Preparation and Specific Mutagenesis of DNA FragmentsStudy of Protein and DNA Interactions Nucleic Acids Res 16 7351-7367

Hirsch PR Miller AJ Dennis PG (2013) Do Root Exudates Exert More Influence on Rhizosphere Bacterial Community StructureThan Other Rhizodeposits In FJ de Bruijn ed Molecular Microbial Ecology of the Rhizosphere Ed 1 Vol 1 John Wiley amp SonsInc Hoboken NJ USA pp 229-242

Horst I Welham T Kelly S Kaneko T Sato S Tabata S Parniske M Wang TL (2007) Tilling Mutants of Lotus japonicus Reveal ThatNitrogen Assimilation and Fixation Can Occur in the Absence of Nodule-Enhanced Sucrose Synthase Plant Physiol 144 806-820

Jensen JB Peters NK Bhuvaneswari TV (2002) Redundancy in Periplasmic Binding Protein-Dependent Transport Systems forTrehalose Sucrose and Maltose in Sinorhizobium meliloti J Bacteriol 184 2978-2986

Kakar K Wandrey M Czechowski T Gaertner T Scheible WR Stitt M Torres-Jerez I Xiao Y Redman JC Wu HC Cheung F TownCD Udvardi MK (2008) A Community Resource for High-Throughput Quantitative RT-PCR Analysis of Transcription Factor GeneExpression in Medicago truncatula Plant Methods 4 1746-4811

Kuumlhn C Franceschi VR Schulz A Lemoine R Frommer WB (1997) Macromolecular Trafficking Indicated by Localization andTurnover of Sucrose Transporters in Enucleate Sieve Elements Science 275 1298-1300

Kuumlster H Fruhling M Perlick AM Puhler A (1993) The Sucrose Synthase Gene Is Predominantly Expressed in the Root NoduleTissue of Vicia faba Mol Plant Microbe In 6 507-514

Lager I Looger LL Hilpert M Lalonde S Frommer WB (2006) Conversion of a Putative Agrobacterium Sugar-Binding Protein intoa FRET Sensor with High Selectivity for Sucrose J Biol Chem 281 30875-30883

Lalonde S Wipf D Frommer WB (2004) Transport Mechanisms for Organic Forms of Carbon and Nitrogen between Source andSink Annu Rev Plant Biol 55 341-372

Lambert A Oslashsterarings M Mandon K Poggi MC Le Rudulier D (2001) Fructose Uptake in Sinorhizobium meliloti is Mediated by aHigh-Affinity ATP-Binding Cassette Transport System J Bacteriol 183 4709-4717

Limpens E Moling S Hooiveld G Pereira PA Bisseling T Becker JD and Kuster H (2013) Cell- and Tissue-SpecificTranscriptome Analyses of Medicago truncatula root nodules PloS One 8 e64377

Lin IW Sosso D Chen LQ Gase K Kim SG Kessler D Klinkenberg PM Gorder MK Hou BH Qu XQ et al (2014) Nectar SecretionRequires Sucrose Phosphate Synthases and the Sugar Transporter SWEET9 Nature 508 546-549

Marsh JF Rakocevic A Mitra RM Brocard L Sun J Eschtruth A Long SR Schultze M Ratet P Oldroyd GED (2007) Medicagotruncatula NIN is Essential for Rhizobial-Independent Nodule Organogenesis Induced by Autoactive CalciumCalmodulin-Dependent Protein Kinase Plant Physiol 144 324-335

Mauchline TH Fowler JE East AK Sartor AL Zaheer R Hosie AH Poole PS Finan TM (2006) Mapping the Sinorhizobium meliloti1021 Solute-Binding Protein-Dependent Transportome Proc Natl Acad Sci U S A 103 17933-17938

Morell M Copeland L (1984) Enzymes of Sucrose Breakdown in Soybean Nodules Alkaline Invertase Plant Physiol 74 1030-1034Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Oke V Long SR (1999) Bacterial Genes Induced within the Nodule During the Rhizobium-Legume Symbiosis Mol Microbiol 32837-849

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title wwwplantphysiolorgon May 14 2018 - Published by Downloaded from

Patrick JW (1997) Phloem Unloading Sieve Element Unloading and Post-Sieve Element Transport Annu Rev Plant Physiol PlantMol Biol 48 191-222

Peiter E Schubert S (2003) Sugar Uptake and Proton Release by Protoplasts from the Infected Zone of Vicia faba L NodulesEvidence against Apoplastic Sugar Supply of Infected Cells J Exp Bot 54 1691-1700

Peoples MB Brockwell J Herridge DF Rochester IJ Alves BJR Urquiaga S Boddey RM Dakora FD Bhattarai S Maskey S et al(2009) The Contributions of Nitrogen-Fixing Crop Legumes to the Productivity of Agricultural Systems Symbiosis 48 1-17

Pumplin N Zhang X Noar RD Harrison MJ (2012) Polar Localization of a Symbiosis-Specific Phosphate Transporter is Mediated bya Transient Reorientation of Secretion Proc Natl Acad Sci U S A 109 E665-672

Reinders A Sivitz AB Starker CG Gantt JS Ward JM (2008) Functional Analysis of LjSUT4 a Vacuolar Sucrose Transporter fromLotus japonicus Plant Mol Biol 68 289-299

Roux B Rodde N Jardinaud MF Timmers T Sauviac L Cottret L Carregravere S Sallet E Courcelle E Moreau S et al (2014) AnIntegrated Analysis of Plant and Bacterial Gene Expression in Symbiotic Root Nodules Using Laser-Capture MicrodissectionCoupled to RNA Sequencing Plant J 77 817-837

Schubert M Koteyeva NK Wabnitz PW Santos P Buttner M Sauer N Demchenko K Pawlowski K (2011) PlasmodesmataDistribution and Sugar Partitioning in Nitrogen-Fixing Root Nodules of Datisca glomerata Planta 233 139-152

Sinharoy S Torres-Jerez I Bandyopadhyay K Kereszt A Pislariu CI Nakashima J Benedito VA Kondorosi E Udvardi MK (2013)The C2H2 Transcription Factor Regulator of Symbiosome Differentiation Represses Transcription of the Secretory Pathway GeneVAMP721a and Promotes Symbiosome Development in Medicago truncatula Plant Cell 25 3584-3601

Tadege M Wen J He J Tu H Kwak Y Eschstruth A Cayrel A Endre G Zhao PX Chabaud M Ratet P Mysore KS (2008) Large-Scale Insertional Mutagenesis Using the Tnt1 Retrotransposon in the Model Legume Medicago truncatula Plant J 54 335-347

Tamura K Peterson D Peterson N Stecher G Nei M Kumar S (2011) MEGA5 Molecular Evolutionary Genetics Analysis UsingMaximum Likelihood Evolutionary Distance and Maximum Parsimony Methods Mol Biol Evol 28 2731-2739

Taylor M Blaylock L Nakashima J McAbee D Ford J Harrison M Udvardi M (2011) Medicago truncatula HybridizationSupplemental Videos In U Mathesius EP Journet LWA Sumner eds Medicago truncatula Handbook Version July 2011 TheSamuel Roberts Noble Foundation Ardmore Oklahoma pp 1-5 httpwwwnobleorgMedicagoHandbook

Tejima K Arima Y Yokoyama T Sekimoto H (2003) Composition of Amino Acids Organic Acids and Sugars in the PeribacteroidSpace of Soybean Root Nodules Soil Sci Plant Nutr 49 239-247

Udvardi M Poole PS (2013) Transport and Metabolism in Legume-Rhizobia Symbioses In SS Merchant ed Annu Rev Plant BiolVol 64 Annual Reviews Palo Alto pp 781-805

Pubmed Author and TitleCrossRef Author and Title

Google Scholar Author Only Title Only Author and Title

Vance CP Miller SS Driscoll BT Robinson DL Trepp G Gantt JS Samas DA (1998) Nodule Carbon Metabolism Organic Acids forN2 Fixation In C Elmerich A Kondorosi WE Newton eds Biological Nitrogen Fixation for the 21st Century Vol 31 SpringerNetherlands pp 443-448

Vance CP (2008) Carbon and Nitrogen Metabolism in Legume Nodules In M Dilworth E James J Sprent W Newton eds Nitrogen-Fixing Leguminous Symbioses Vol 7 Springer Netherlands pp 293-320

Welham T Pike J Horst I Flemetakis E Katinakis P Kaneko T Sato S Tabata S Perry J Parniske M Wang TL (2009) A CytosolicInvertase Is Required for Normal Growth and Cell Development in the Model Legume Lotus Japonicus J Exp Bot 60 3353-3365

Wienkoop S Saalbach G (2003) Proteome Analysis Novel Proteins Identified at the Peribacteroid Membrane from Lotus japonicusRoot Nodules Plant Physiol 131 1080-1090

Willis LB Walker GC (1999) A Novel Sinorhizobium meliloti Operon Encodes an Alpha-Glucosidase and a Periplasmic-Binding-Protein-Dependent Transport System for Alpha-Glucosides J Bacteriol 181 4176-4184

Winter H Huber SC (2000) Regulation of Sucrose Metabolism in Higher Plants Localization and Regulation of Activity of KeyEnzymes Crit Rev Biochem Mol Biol 35 253-289

Yang B Sugio A White FF (2006) Os8N3 Is a Host Disease-Susceptibility Gene for Bacterial Blight of Rice Proc Natl Acad Sci U SA 103 10503-10508

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

MtSWEET11 a Nodule-Specific Sucrose Transporter of Medicago truncatula Root 25

Nodules 26

Igor S Kryvoruchko15 Senjuti Sinharoy16 Ivone Torres-Jerez1 Davide Sosso2 Catalina 28

I Pislariu17 Dian Guan3 Jeremy Murray3 Vagner A Benedito4 Wolf B Frommer2 and 29

Michael K Udvardi1 30 31 1Plant Biology Division 32

USA 35 36 2Department of Plant Biology 37

Carnegie Institution of Science 38

Stanford California 94305 39

USA 40

41 3Department of Cell and Developmental Biology 42

John Innes Centre 43

Norwich NR4 7UH 44

United Kingdom 45 46 4Division of Plant amp Soil Sciences 47

West Virginia University 48

Morgantown West Virginia 26506 49

USA 50 51 5Current address 52

Bioengineering Department 53

Kafkas University 54

Kars 36100 55

Turkey 56

Kolkata 700019 62

India 63

USA 69

Abstract 86

Introduction 102

al 2007) 131

2010) 161

RESULTS 164

DISCUSSION 294

al (2012) 501

KF998083 588

sequences 599

FIGURE LEGENDS 608

MtSWEET11 668

Jemalong A17 751

REFERENCES 756

11n3 Plant Cell 22 3864-3876 760

145 1600-1618 769

Online 2 1157-1170 779

Biochem J 125 1075-1080 787

14 1301-1322 803

Biol 25 53-62 809

379 813

7351ndash7367 826

NJ USA pp 229-242 830

Bacteriol 184 2978-2986 836

514 846

Biol Chem 281 30875-30883 849

Bacteriol 183 4709-4717 854

One 8 e64377 857

Cells J Exp Bot 54 1691-1700 876

Sci U S A 109 E665-672 882

3584-3601 896

239-247 909

443-448 915

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

Kolkata 700019 62

India 63

USA 69

Abstract 86

Introduction 102

al 2007) 131

2010) 161

RESULTS 164

DISCUSSION 294

al (2012) 501

KF998083 588

sequences 599

FIGURE LEGENDS 608

MtSWEET11 668

Jemalong A17 751

REFERENCES 756

11n3 Plant Cell 22 3864-3876 760

145 1600-1618 769

Online 2 1157-1170 779

Biochem J 125 1075-1080 787

14 1301-1322 803

Biol 25 53-62 809

379 813

7351ndash7367 826

NJ USA pp 229-242 830

Bacteriol 184 2978-2986 836

514 846

Biol Chem 281 30875-30883 849

Bacteriol 183 4709-4717 854

One 8 e64377 857

Cells J Exp Bot 54 1691-1700 876

Sci U S A 109 E665-672 882

3584-3601 896

239-247 909

443-448 915

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

Abstract 86

Introduction 102

al 2007) 131

2010) 161

RESULTS 164

DISCUSSION 294

al (2012) 501

KF998083 588

sequences 599

FIGURE LEGENDS 608

MtSWEET11 668

Jemalong A17 751

REFERENCES 756

11n3 Plant Cell 22 3864-3876 760

145 1600-1618 769

Online 2 1157-1170 779

Biochem J 125 1075-1080 787

14 1301-1322 803

Biol 25 53-62 809

379 813

7351ndash7367 826

NJ USA pp 229-242 830

Bacteriol 184 2978-2986 836

514 846

Biol Chem 281 30875-30883 849

Bacteriol 183 4709-4717 854

One 8 e64377 857

Cells J Exp Bot 54 1691-1700 876

Sci U S A 109 E665-672 882

3584-3601 896

239-247 909

443-448 915

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

Abstract 86

Introduction 102

al 2007) 131

2010) 161

RESULTS 164

DISCUSSION 294

al (2012) 501

KF998083 588

sequences 599

FIGURE LEGENDS 608

MtSWEET11 668

Jemalong A17 751

REFERENCES 756

11n3 Plant Cell 22 3864-3876 760

145 1600-1618 769

Online 2 1157-1170 779

Biochem J 125 1075-1080 787

14 1301-1322 803

Biol 25 53-62 809

379 813

7351ndash7367 826

NJ USA pp 229-242 830

Bacteriol 184 2978-2986 836

514 846

Biol Chem 281 30875-30883 849

Bacteriol 183 4709-4717 854

One 8 e64377 857

Cells J Exp Bot 54 1691-1700 876

Sci U S A 109 E665-672 882

3584-3601 896

239-247 909

443-448 915

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

Introduction 102

al 2007) 131

2010) 161

RESULTS 164

DISCUSSION 294

al (2012) 501

KF998083 588

sequences 599

FIGURE LEGENDS 608

MtSWEET11 668

Jemalong A17 751

REFERENCES 756

11n3 Plant Cell 22 3864-3876 760

145 1600-1618 769

Online 2 1157-1170 779

Biochem J 125 1075-1080 787

14 1301-1322 803

Biol 25 53-62 809

379 813

7351ndash7367 826

NJ USA pp 229-242 830

Bacteriol 184 2978-2986 836

514 846

Biol Chem 281 30875-30883 849

Bacteriol 183 4709-4717 854

One 8 e64377 857

Cells J Exp Bot 54 1691-1700 876

Sci U S A 109 E665-672 882

3584-3601 896

239-247 909

443-448 915

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

2010) 161

RESULTS 164

DISCUSSION 294

al (2012) 501

KF998083 588

sequences 599

FIGURE LEGENDS 608

MtSWEET11 668

Jemalong A17 751

REFERENCES 756

11n3 Plant Cell 22 3864-3876 760

145 1600-1618 769

Online 2 1157-1170 779

Biochem J 125 1075-1080 787

14 1301-1322 803

Biol 25 53-62 809

379 813

7351ndash7367 826

NJ USA pp 229-242 830

Bacteriol 184 2978-2986 836

514 846

Biol Chem 281 30875-30883 849

Bacteriol 183 4709-4717 854

One 8 e64377 857

Cells J Exp Bot 54 1691-1700 876

Sci U S A 109 E665-672 882

3584-3601 896

239-247 909

443-448 915

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

RESULTS 164

DISCUSSION 294

al (2012) 501

KF998083 588

sequences 599

FIGURE LEGENDS 608

MtSWEET11 668

Jemalong A17 751

REFERENCES 756

11n3 Plant Cell 22 3864-3876 760

145 1600-1618 769

Online 2 1157-1170 779

Biochem J 125 1075-1080 787

14 1301-1322 803

Biol 25 53-62 809

379 813

7351ndash7367 826

NJ USA pp 229-242 830

Bacteriol 184 2978-2986 836

514 846

Biol Chem 281 30875-30883 849

Bacteriol 183 4709-4717 854

One 8 e64377 857

Cells J Exp Bot 54 1691-1700 876

Sci U S A 109 E665-672 882

3584-3601 896

239-247 909

443-448 915

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

DISCUSSION 294

al (2012) 501

KF998083 588

sequences 599

FIGURE LEGENDS 608

MtSWEET11 668

Jemalong A17 751

REFERENCES 756

11n3 Plant Cell 22 3864-3876 760

145 1600-1618 769

Online 2 1157-1170 779

Biochem J 125 1075-1080 787

14 1301-1322 803

Biol 25 53-62 809

379 813

7351ndash7367 826

NJ USA pp 229-242 830

Bacteriol 184 2978-2986 836

514 846

Biol Chem 281 30875-30883 849

Bacteriol 183 4709-4717 854

One 8 e64377 857

Cells J Exp Bot 54 1691-1700 876

Sci U S A 109 E665-672 882

3584-3601 896

239-247 909

443-448 915

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

DISCUSSION 294

al (2012) 501

KF998083 588

sequences 599

FIGURE LEGENDS 608

MtSWEET11 668

Jemalong A17 751

REFERENCES 756

11n3 Plant Cell 22 3864-3876 760

145 1600-1618 769

Online 2 1157-1170 779

Biochem J 125 1075-1080 787

14 1301-1322 803

Biol 25 53-62 809

379 813

7351ndash7367 826

NJ USA pp 229-242 830

Bacteriol 184 2978-2986 836

514 846

Biol Chem 281 30875-30883 849

Bacteriol 183 4709-4717 854

One 8 e64377 857

Cells J Exp Bot 54 1691-1700 876

Sci U S A 109 E665-672 882

3584-3601 896

239-247 909

443-448 915

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

DISCUSSION 294

al (2012) 501

KF998083 588

sequences 599

FIGURE LEGENDS 608

MtSWEET11 668

Jemalong A17 751

REFERENCES 756

11n3 Plant Cell 22 3864-3876 760

145 1600-1618 769

Online 2 1157-1170 779

Biochem J 125 1075-1080 787

14 1301-1322 803

Biol 25 53-62 809

379 813

7351ndash7367 826

NJ USA pp 229-242 830

Bacteriol 184 2978-2986 836

514 846

Biol Chem 281 30875-30883 849

Bacteriol 183 4709-4717 854

One 8 e64377 857

Cells J Exp Bot 54 1691-1700 876

Sci U S A 109 E665-672 882

3584-3601 896

239-247 909

443-448 915

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

DISCUSSION 294

al (2012) 501

KF998083 588

sequences 599

FIGURE LEGENDS 608

MtSWEET11 668

Jemalong A17 751

REFERENCES 756

11n3 Plant Cell 22 3864-3876 760

145 1600-1618 769

Online 2 1157-1170 779

Biochem J 125 1075-1080 787

14 1301-1322 803

Biol 25 53-62 809

379 813

7351ndash7367 826

NJ USA pp 229-242 830

Bacteriol 184 2978-2986 836

514 846

Biol Chem 281 30875-30883 849

Bacteriol 183 4709-4717 854

One 8 e64377 857

Cells J Exp Bot 54 1691-1700 876

Sci U S A 109 E665-672 882

3584-3601 896

239-247 909

443-448 915

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

al (2012) 501

KF998083 588

sequences 599

FIGURE LEGENDS 608

MtSWEET11 668

Jemalong A17 751

REFERENCES 756

11n3 Plant Cell 22 3864-3876 760

145 1600-1618 769

Online 2 1157-1170 779

Biochem J 125 1075-1080 787

14 1301-1322 803

Biol 25 53-62 809

379 813

7351ndash7367 826

NJ USA pp 229-242 830

Bacteriol 184 2978-2986 836

514 846

Biol Chem 281 30875-30883 849

Bacteriol 183 4709-4717 854

One 8 e64377 857

Cells J Exp Bot 54 1691-1700 876

Sci U S A 109 E665-672 882

3584-3601 896

239-247 909

443-448 915

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

al (2012) 501

KF998083 588

sequences 599

FIGURE LEGENDS 608

MtSWEET11 668

Jemalong A17 751

REFERENCES 756

11n3 Plant Cell 22 3864-3876 760

145 1600-1618 769

Online 2 1157-1170 779

Biochem J 125 1075-1080 787

14 1301-1322 803

Biol 25 53-62 809

379 813

7351ndash7367 826

NJ USA pp 229-242 830

Bacteriol 184 2978-2986 836

514 846

Biol Chem 281 30875-30883 849

Bacteriol 183 4709-4717 854

One 8 e64377 857

Cells J Exp Bot 54 1691-1700 876

Sci U S A 109 E665-672 882

3584-3601 896

239-247 909

443-448 915

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

al (2012) 501

KF998083 588

sequences 599

FIGURE LEGENDS 608

MtSWEET11 668

Jemalong A17 751

REFERENCES 756

11n3 Plant Cell 22 3864-3876 760

145 1600-1618 769

Online 2 1157-1170 779

Biochem J 125 1075-1080 787

14 1301-1322 803

Biol 25 53-62 809

379 813

7351ndash7367 826

NJ USA pp 229-242 830

Bacteriol 184 2978-2986 836

514 846

Biol Chem 281 30875-30883 849

Bacteriol 183 4709-4717 854

One 8 e64377 857

Cells J Exp Bot 54 1691-1700 876

Sci U S A 109 E665-672 882

3584-3601 896

239-247 909

443-448 915

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

al (2012) 501

KF998083 588

sequences 599

FIGURE LEGENDS 608

MtSWEET11 668

Jemalong A17 751

REFERENCES 756

11n3 Plant Cell 22 3864-3876 760

145 1600-1618 769

Online 2 1157-1170 779

Biochem J 125 1075-1080 787

14 1301-1322 803

Biol 25 53-62 809

379 813

7351ndash7367 826

NJ USA pp 229-242 830

Bacteriol 184 2978-2986 836

514 846

Biol Chem 281 30875-30883 849

Bacteriol 183 4709-4717 854

One 8 e64377 857

Cells J Exp Bot 54 1691-1700 876

Sci U S A 109 E665-672 882

3584-3601 896

239-247 909

443-448 915

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

al (2012) 501

KF998083 588

sequences 599

FIGURE LEGENDS 608

MtSWEET11 668

Jemalong A17 751

REFERENCES 756

11n3 Plant Cell 22 3864-3876 760

145 1600-1618 769

Online 2 1157-1170 779

Biochem J 125 1075-1080 787

14 1301-1322 803

Biol 25 53-62 809

379 813

7351ndash7367 826

NJ USA pp 229-242 830

Bacteriol 184 2978-2986 836

514 846

Biol Chem 281 30875-30883 849

Bacteriol 183 4709-4717 854

One 8 e64377 857

Cells J Exp Bot 54 1691-1700 876

Sci U S A 109 E665-672 882

3584-3601 896

239-247 909

443-448 915

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

al (2012) 501

KF998083 588

sequences 599

FIGURE LEGENDS 608

MtSWEET11 668

Jemalong A17 751

REFERENCES 756

11n3 Plant Cell 22 3864-3876 760

145 1600-1618 769

Online 2 1157-1170 779

Biochem J 125 1075-1080 787

14 1301-1322 803

Biol 25 53-62 809

379 813

7351ndash7367 826

NJ USA pp 229-242 830

Bacteriol 184 2978-2986 836

514 846

Biol Chem 281 30875-30883 849

Bacteriol 183 4709-4717 854

One 8 e64377 857

Cells J Exp Bot 54 1691-1700 876

Sci U S A 109 E665-672 882

3584-3601 896

239-247 909

443-448 915

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

KF998083 588

sequences 599

FIGURE LEGENDS 608

MtSWEET11 668

Jemalong A17 751

REFERENCES 756

11n3 Plant Cell 22 3864-3876 760

145 1600-1618 769

Online 2 1157-1170 779

Biochem J 125 1075-1080 787

14 1301-1322 803

Biol 25 53-62 809

379 813

7351ndash7367 826

NJ USA pp 229-242 830

Bacteriol 184 2978-2986 836

514 846

Biol Chem 281 30875-30883 849

Bacteriol 183 4709-4717 854

One 8 e64377 857

Cells J Exp Bot 54 1691-1700 876

Sci U S A 109 E665-672 882

3584-3601 896

239-247 909

443-448 915

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

KF998083 588

sequences 599

FIGURE LEGENDS 608

MtSWEET11 668

Jemalong A17 751

REFERENCES 756

11n3 Plant Cell 22 3864-3876 760

145 1600-1618 769

Online 2 1157-1170 779

Biochem J 125 1075-1080 787

14 1301-1322 803

Biol 25 53-62 809

379 813

7351ndash7367 826

NJ USA pp 229-242 830

Bacteriol 184 2978-2986 836

514 846

Biol Chem 281 30875-30883 849

Bacteriol 183 4709-4717 854

One 8 e64377 857

Cells J Exp Bot 54 1691-1700 876

Sci U S A 109 E665-672 882

3584-3601 896

239-247 909

443-448 915

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

KF998083 588

sequences 599

FIGURE LEGENDS 608

MtSWEET11 668

Jemalong A17 751

REFERENCES 756

11n3 Plant Cell 22 3864-3876 760

145 1600-1618 769

Online 2 1157-1170 779

Biochem J 125 1075-1080 787

14 1301-1322 803

Biol 25 53-62 809

379 813

7351ndash7367 826

NJ USA pp 229-242 830

Bacteriol 184 2978-2986 836

514 846

Biol Chem 281 30875-30883 849

Bacteriol 183 4709-4717 854

One 8 e64377 857

Cells J Exp Bot 54 1691-1700 876

Sci U S A 109 E665-672 882

3584-3601 896

239-247 909

443-448 915

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

MtSWEET11 668

Jemalong A17 751

REFERENCES 756

11n3 Plant Cell 22 3864-3876 760

145 1600-1618 769

Online 2 1157-1170 779

Biochem J 125 1075-1080 787

14 1301-1322 803

Biol 25 53-62 809

379 813

7351ndash7367 826

NJ USA pp 229-242 830

Bacteriol 184 2978-2986 836

514 846

Biol Chem 281 30875-30883 849

Bacteriol 183 4709-4717 854

One 8 e64377 857

Cells J Exp Bot 54 1691-1700 876

Sci U S A 109 E665-672 882

3584-3601 896

239-247 909

443-448 915

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

MtSWEET11 668

Jemalong A17 751

REFERENCES 756

11n3 Plant Cell 22 3864-3876 760

145 1600-1618 769

Online 2 1157-1170 779

Biochem J 125 1075-1080 787

14 1301-1322 803

Biol 25 53-62 809

379 813

7351ndash7367 826

NJ USA pp 229-242 830

Bacteriol 184 2978-2986 836

514 846

Biol Chem 281 30875-30883 849

Bacteriol 183 4709-4717 854

One 8 e64377 857

Cells J Exp Bot 54 1691-1700 876

Sci U S A 109 E665-672 882

3584-3601 896

239-247 909

443-448 915

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

Jemalong A17 751

REFERENCES 756

11n3 Plant Cell 22 3864-3876 760

145 1600-1618 769

Online 2 1157-1170 779

Biochem J 125 1075-1080 787

14 1301-1322 803

Biol 25 53-62 809

379 813

7351ndash7367 826

NJ USA pp 229-242 830

Bacteriol 184 2978-2986 836

514 846

Biol Chem 281 30875-30883 849

Bacteriol 183 4709-4717 854

One 8 e64377 857

Cells J Exp Bot 54 1691-1700 876

Sci U S A 109 E665-672 882

3584-3601 896

239-247 909

443-448 915

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

Jemalong A17 751

REFERENCES 756

11n3 Plant Cell 22 3864-3876 760

145 1600-1618 769

Online 2 1157-1170 779

Biochem J 125 1075-1080 787

14 1301-1322 803

Biol 25 53-62 809

379 813

7351ndash7367 826

NJ USA pp 229-242 830

Bacteriol 184 2978-2986 836

514 846

Biol Chem 281 30875-30883 849

Bacteriol 183 4709-4717 854

One 8 e64377 857

Cells J Exp Bot 54 1691-1700 876

Sci U S A 109 E665-672 882

3584-3601 896

239-247 909

443-448 915

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

Jemalong A17 751

REFERENCES 756

11n3 Plant Cell 22 3864-3876 760

145 1600-1618 769

Online 2 1157-1170 779

Biochem J 125 1075-1080 787

14 1301-1322 803

Biol 25 53-62 809

379 813

7351ndash7367 826

NJ USA pp 229-242 830

Bacteriol 184 2978-2986 836

514 846

Biol Chem 281 30875-30883 849

Bacteriol 183 4709-4717 854

One 8 e64377 857

Cells J Exp Bot 54 1691-1700 876

Sci U S A 109 E665-672 882

3584-3601 896

239-247 909

443-448 915

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

145 1600-1618 769

Online 2 1157-1170 779

Biochem J 125 1075-1080 787

14 1301-1322 803

Biol 25 53-62 809

379 813

7351ndash7367 826

NJ USA pp 229-242 830

Bacteriol 184 2978-2986 836

514 846

Biol Chem 281 30875-30883 849

Bacteriol 183 4709-4717 854

One 8 e64377 857

Cells J Exp Bot 54 1691-1700 876

Sci U S A 109 E665-672 882

3584-3601 896

239-247 909

443-448 915

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

14 1301-1322 803

Biol 25 53-62 809

379 813

7351ndash7367 826

NJ USA pp 229-242 830

Bacteriol 184 2978-2986 836

514 846

Biol Chem 281 30875-30883 849

Bacteriol 183 4709-4717 854

One 8 e64377 857

Cells J Exp Bot 54 1691-1700 876

Sci U S A 109 E665-672 882

3584-3601 896

239-247 909

443-448 915

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

NJ USA pp 229-242 830

Bacteriol 184 2978-2986 836

514 846

Biol Chem 281 30875-30883 849

Bacteriol 183 4709-4717 854

One 8 e64377 857

Cells J Exp Bot 54 1691-1700 876

Sci U S A 109 E665-672 882

3584-3601 896

239-247 909

443-448 915

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

Cells J Exp Bot 54 1691-1700 876

Sci U S A 109 E665-672 882

3584-3601 896

239-247 909

443-448 915

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

239-247 909

443-448 915

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Parsed Citations

Documents

2 9 10 11 12 13 14 Plant Biology Division support for JM’s lab came from the ... Sucrose breakdown in root nodules, ... Sucrose synthase is necessary for efficient nitrogen fixation