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Calcium pumps and interacting BON1 protein modulate calcium signature stomatal 1

closure and plant immunity 2

Dong-Lei Yang12sect Zhenying Shi23sect Yongmei Bao12sect Jiapei Yan2sect Ziyuan Yang1 Huiyun 3

Yu2 Yun Li1 Mingyue Gou24 Shu Wang2 Baohong Zou12 Dachao Xu1 Zhiqi Ma1 Jitae Kim2 4

Jian Hua21 5

1State Key Laboratory of Crop Genetics and Germplasm Enhancement Nanjing Agricultural 6

University Nanjing 210095 China 7

2 School of Integrative Plant Science Plant Biology Section Cornell University Ithaca NY 8

14853 USA 9

3present address Key Laboratory of Insect Developmental and Evolutionary Biology Institute of 10

Plant Physiology and Ecology Shanghai Institutes for Biological Sciences Chinese Academy of 11

Sciences Shanghai 200032 China 12

4present address Biology Department Brookhaven National Laboratory 50 Bell Avenue Upton 13

NY 11973 USA 14

sectThese authors contribute equally to this work 15

Correspondence should be addressed JH (email jh299cornelledu) or D-LY (email 16

dlyangnjaueducn) 17

18

Author Contributions 19

JH and DLY conceived the project DLY ZS YB JY and JH designed the experiments analyzed 20

the data and made the figures DLY ZS YB JY ZY HY YL MG SW BZ DX ZM and JK 21

conducted experiments JH and DLY wrote the paper 22

23

Plant Physiology Preview Published on July 12 2017 as DOI101104pp1700495

Copyright 2017 by the American Society of Plant Biologists

httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved

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One-sentence summary 24

Calcium pumps ACA10 and ACA8 and their interacting protein BON1 regulate calcium 25

signatures and impact stomatal movement and plant immunity in Arabidopsis 26


3

Abstract 27

Calcium signaling is essential for environmental responses including immune responses 28

Here we provide evidence that the evolutionarily conserved protein BONZAI1 (BON1) functions 29

together with autoinhibited calcium ATPase 10 (ACA10) and ACA8 to regulate calcium signals 30

in Arabidopsis BON1 is a plasma membrane localized protein that negatively regulates the 31

expression of immune receptor genes and positively regulates stomatal closure We found that 32

BON1 interacts with the autoinhibitory domains of ACA10 and ACA8 and the aca10 loss of 33

function (LOF) mutants have an autoimmune phenotype similar to that of the bon1 LOF mutants 34

Genetic evidences indicate that BON1 positively regulates the activities of ACA10 and ACA8 35

Consistent with this idea the steady level of calcium concentration is increased in both aca10 36

and bon1 mutants Most strikingly cytosolic calcium oscillation imposed by external calcium 37

treatment was altered in aca10 aca8 and bon1 mutants in guard cells In addition calcium and 38

pathogen induced stomatal closure was compromised in the aca10 and bon1 mutants Taken 39

together this study indicates that ACA108 and BON1 physically interact on plasma membrane 40

and function in the generation of cytosol calcium signatures that are critical for stomatal 41

movement and impact plant immunity 42

43


4

Introduction 44

Calcium ion (Ca2+) is an important cellular second messenger for diverse developmental 45

processes and environmental responses in both plants and animals (Lewis 2001 Dodd et al 46

2010 Kudla et al 2010) The extreme low calcium concentration in cytosol (100-200 nM) 47

creates a unique environment where calcium concentration can be regulated dynamically (Bush 48

1995) Increases of calcium transient in cytosol are activated in various environmental and 49

developmental processes including root growth stomatal movement pollen growth abiotic 50

stress responses and plant-microbe interaction Calcium spiking with unique magnitude 51

frequency shape and duration in response to environmental and endogenous cues is referred as 52

ldquocalcium signaturerdquo and is thought to encode stimulus-specific information It is shown that in 53

the guard cell where calcium is a key signal for stomatal movement control (Murata et al 2015) 54

calcium oscillation is indeed essential for stomatal closure (Allen et al 2000) 55

The stimulus-specific calcium signature is thought to be generated by coordinated action of 56

various Ca2+ influx and efflux systems including channels pumps and exchangers located at 57

different cellular membranes However the molecular identities of calcium specific influx 58

channels remain controversial (Steinhorst and Kudla 2013) Plasma membrane (PM) localized 59

cyclic nucleotide-gated channels (CNGCs) glutamate receptor like (GLR) and TWO-PORE 60

CHANNEL (TPC) are considered as main channels that release Ca2+ in plant (Dodd et al 2010 61

Kudla et al 2010) Extrusion of Ca2+ from cytosol to external cell and intercellular store 62

compartment is believed to be achieved by Ca2+-ATPases (Ca2+ pumps) and Ca2+H+ antiporters 63

(CAXs) driven by ATP and proton motive force respectively (Geisler et al 2000 Sze et al 64

2000 Shigaki and Hirschi 2006) Ca2+ pumps fall into two groups type IIA or ECA for ER type 65

Ca2+-ATPases and type IIB or ACA for autoinhibited Ca2+-ATPase (Geisler et al 2000) ACAs 66


5

have an N-terminal calmodulin-binding autoinhibitory domain that inhibits the ATPase activity 67

in the C-terminus and they are localized to diverse membrane compartments (Sze et al 2000 68

Boursiac and Harper 2007) Calcium pumps in vacuole ER and nucleus were found to be 69

important for calcium signal generation in response to environmental stimuli The loss of 70

function (LOF) of PCA1 a vacuole localized ACA resulted in sustained rather than transient 71

elevated Ca2+ in cytosol under salt treatment in the moss Physcomitrella patens (Qudeimat et al 72

2008) In tobacco knock-down of NbCA1 an ER localized ACA greatly increased the 73

amplitude and duration of calcium spikes induced by cryptogein (Zhu et al 2010) The LOF of 74

MCA a nucleus localized EAC resulted in greatly reduced nuclear calcium spiking in response 75

to Nod factor in Medicago truncatula (Capoen et al 2011) The involvement of PM localized 76

calcium pumps in calcium signal is not known yet in plants 77

Calcium signaling has recently been genetically connected with plant immunity Distinct 78

calcium signatures are rapidly induced upon pathogen invasion (Lecourieux et al 2006 Ma and 79

Berkowitz 2007) Disruption of putative calcium channels such as PM-localized CNGCs and 80

GLR either enhance or compromise plant immune responses (Clough et al 2000 Ford and 81

Roberts 2014) The PM-localized calcium pumps ACA8 and ACA10 are found to be associated 82

with immune receptor FLS2 and their LOF mutants were susceptible to bacterial pathogens (Frei 83

dit Frey et al 2012) Multiple calcium binding proteins are also involved in plant immunity 84

regulation For instance a calmodulin (CaM)-binding protein MLO negatively regulates 85

resistance to powdery mildew in barley (Kim et al 2002) and a Ca2+- and CaM-binding 86

transcription factor AtSR1CAMTA3 is a negative regulator of salicylic acid (SA)-dependent 87

defense responses in Arabidopsis (Du et al 2009) A CaM gene in Nicotiana benthamiana (N 88

benthamiana) is required for immune responses triggered by silencing of the ER calcium pump 89


6

CA1 (Zhu et al 2010) and overexpression of a pepper CaM gene induces programmed cell 90

death (PCD) and enhances disease resistance (Choi et al 2009) A chloroplast calcium regulated 91

protein CAS also plays an important role in plant immunity (Nomura et al 2012) In addition 92

calcium-dependent protein kinases (CDPK or CPK) are critical regulators of plant immune 93

responses both to pathogen-associated molecular patterns (PAMP) and effectors (Boudsocq and 94

Sheen 2013) Four CDPKs (CPK45611) are found to be critical for transcriptional 95

reprogramming and ROS production in responses to PAMPs (Boudsocq et al 2010) 96

CPK1245611 are shown to be involved in downstream events including transcriptional 97

reprogramming and reactive oxygen species (ROS) production after activation of plant immune 98

receptor NLR (NOD1 like Receptors) genes in response to pathogen effectors (Gao et al 2013) 99

Recently CPK28 is shown to phosphorylate BIK1 a substrate of multiple PAMP receptors and 100

therefore attenuating PAMP signaling (Monaghan et al 2014) 101

One intriguing component involved in calcium signaling and plant immunity is the 102

Arabidopsis BON1 gene BON1 is a member of an evolutionarily conserved copine family found 103

in protozoa plants nematodes and mammals (Creutz et al 1998) The copine proteins have two 104

calcium-dependent phospholipid-binding C2 domains at their amino (N)-terminus and a putative 105

protein-protein interaction VWA (von Willebrand A) or A domain at their carboxyl (C)-terminus 106

(Rizo and Sudhof 1998 Whittaker and Hynes 2002) The BON1 protein resides on the PM 107

mainly through myristoylation of its second residue glycine (Hua et al 2001 Li et al 2010) 108

Mutating residue glycine (G) 2 to alanine (A) abolishes PM localization of BON1 and results in 109

the loss of BON1 activity (Li et al 2010) BON1 has conserved aspartate residues important for 110

calcium binding in the two C2 domains Mutating all these Asp residues in either C2A or C2B 111

domains abolishes BON1 activity in rescuing the bon1-1 defects indicating that calcium binding 112


7

is required for BON1 function (Li et al 2010) 113

BON1 is a negative regulator of plant immune responses and a positive regulator of stomatal 114

closure response In the LOF mutant bon1-1 in Col-0 background a plant immune receptor NLR 115

gene SNC1 (suppressor of NPR1 constitutive 1) is increased at transcript level through initial 116

upregulation followed by SA-mediated amplification (Yang and Hua 2004 Li et al 2007) The 117

bon1-1 mutant thus has constitutive defense (autoimmune) responses and is consequently dwarf 118

However these defects observed is dependent on a Col-0 specific NB-LRR gene SNC1 and the 119

LOF bon1-2 allele in the Wassilewakija (Ws) accession does not exhibit constitutive defense 120

activation because there is no functional SNC1 in Ws (Yang and Hua 2004) In addition to the 121

function in negatively regulating SNC1 BON1 is recently shown to promote stomatal closure in 122

response to stimuli including abscisic acid (ABA) and bacterial pathogen (Gou et al 2015) This 123

function is independent of SNC1 or NB-LRR signaling and the bon1 mutant is defective in 124

stomatal closure when autoimmunity is suppressed by the SNC1 LOF mutation snc1-11 (Gou et 125

al 2015) Therefore BON1 has a more general role in regulating signaling events in addition to 126

NLR gene expression 127

It is intriguing that an evolutionarily conserved calcium binding copine protein can regulate 128

stomatal closure and impact NLR gene expression Here we identified calcium pumps ACA10 129

and ACA8 as interacting proteins of BON1 both physically and genetically The LOF mutants of 130

these genes have altered calcium signature and calcium homeostasis and are defective in 131

stomatal closure and plant immunity Thus we have uncovered a critical role for PM-localized 132

calcium pumps ACA108 in calcium signature generation as well as their regulation by an 133

evolutionarily conserved BON1 protein in Arabidopsis 134

135


8

136


9

Results 137

BON1 and the calcium ATPase ACA10 interact physically 138

To further understand how BON1 functions in immunity we searched for BON1 co-139

expressed genes as they might function together with BON1 Among the top 300 genes with high 140

co-expression values in the ATTED-II database (Obayashi et al 2009) 33 genes were annotated 141

as calcium binding proteins including calcium channels pumps calmodulin and calcium 142

dependent kinases (Supplementary Table 1) Five of them encode calcium efflux pumps 143

including ACA1 ACA2ACA10 ACA11 and ACA1 Previously ACA1 was identified as a 144

putative BON1 interacting protein by BON1 immunoprecipitation followed by Mass 145

Spectrometry (Gou et al 2015) suggesting that BON1 could physically interact with ACA 146

proteins Because ACA1 2 and 11 are localized to plastid envelope ER and vacuole 147

respectively (Huang et al 1993 Harper et al 1998 Lee et al 2007) we chose the PM-148

localized ACA10 for further analysis because BON1 is PM localized This is also due to the fact 149

that the loss of function (LOF) mutant of ACA10 in the Nossen-0 (No-0) accession had a 150

phenotype reminiscent to that of bon1 (see below section) 151

We first analyzed potential physical interaction between BON1 and ACA10 We were not 152

able to clone the full length of ACA10 cDNA without mutations in E coli which was also 153

reported for the full-length cDNAs of ACA9 the close homologue of ACA10 (Schiott et al 154

2004) We therefore used a cDNAgenomic DNA chimera (where the genomic fragment is used 155

after the 22nd exon) for ACA10 for expression analysis When expressed in N benthamiana the 156

ACA10-GFP fusion protein showed signals only on the PM (Supplementary Fig 1) confirming 157

that ACA10 is indeed a PM-localized protein (Bonza et al 2000 Schiott et al 2004) 158

Bimolecular Fluorescence Complementation (BiFC) (Bracha-Drori et al 2004) assays were used 159


10

to determine the interaction of BON1 and ACA10 in N benthamiana Strong fluorescence 160

signals were observed when both the ACA10 fusion with N-terminal of YFP and the BON1 161

fusion with C-terminal of YFP were expressed (Fig 1A supplemental Fig 2) and the signals 162


11

were on the PM where both BON1 and ACA10 are localized (Supplemental Fig 1A) The 163

positive BiFC signal of ACA10 with BON1 is not a non-specific interaction of BON1 with any 164

PM protein as another PM protein OPT3 (oligopeptide transporter 3) did not give a positive 165

signal when co-expressed with BON1 (Fig 1A and supplemental Fig 2) 166

We subsequently used BiFC assay to test which segment of ACA10 (Supplemental Fig 1B) 167

interacts with BON1 and found that BON1 interacts with the N-terminal segment I that includes 168

the autoinhibited domain of the ACA10 (Fig 1B) Four other segments (II to IV) were also 169

analyzed but no positive signals were observed (Fig 1B) Further test will determine their 170

expression levels in order to determine whether or not these segments interact with BON1 in 171

addition to segment I The interaction between BON1 and the segment I of ACA10 was analyzed 172

further by splitndashLUC (luciferase) assay (Fig 1C) Co-expression of the fusion of BON1 and the 173

N-terminal LUC (BON1-NLUC) with the fusion of C-terminal LUC and segment I of ACA10 174

(CLUC-ACA10I) in N benthamiana gave a strong luminescence signal from the LUC activity 175

while the controls without co-expressing the two proteins showed no luminescence (Fig 1C) 176

This interaction was further verified by the yeast two hybrid (Y2H) assay where the C-terminal A 177

domain of BON1 (BON1-A) was found to interact with the segment I of ACA10 (Fig 1D) The 178

full-length BON1 did not exhibit interaction with segment I (Fig 1D) likely because the N-179

terminal part of BON1 targets to the PM and inhibits the fusion protein to go to the nucleus to 180

activate gene expression Because the segment I of ACA10 contains the putative autoinhibitory 181

motif that confers auto-inhibition of many ACA proteins (Bonza et al 2000 Curran et al 2000 182

Geisler et al 2000) BON1 may regulate ACA10 activity by binding to this motif or its nearby 183

sequences 184

185


12

ACA10 mutant in the Nossen-0 (No-0) background has an autoimmune phenotype 186

We found that ACA10 like BON1 is a negative regulator of plant immunity An aca10 187

mutant in No-0 accession named cif1-1 was reported to have a compact inflorescence (George 188

et al 2008) This aca10 mutant allele will be referred as aca10-cif1 to be consistent with other 189

mutant names We suspected that the growth phenotype of aca10-cif1 is at least partially due to 190

constitutive activation of immune responses induced by plant immune receptor NB-LRR genes 191

based on the following observations Under long day growth condition the young leaves of 192

aca10-cif1 display water soaked phenotype that is similar to the autoimmune mutant bon1-1 193

(Fig 2A Yang and Hua 2014) In addition the compact inflorescence phenotype of aca10-cif1 is 194

reminiscent of that of bon1 bon2 bon3 pad4 mutant (Yang et al 2006) Phenotype of water-195

soaked leaf was suppressed by a higher growth temperature of 28degC (Fig 2A) reminiscent of the 196

suppression of NB-LRR mediated plant defense responses by an elevated temperature (Wang and 197

Hua 2009) Furthermore the aca10-cif1 phenotype is only present in No-0 but not in Col-0 or 198

Ws which was due to an accession difference at the CIF2 locus (George et al 2008) The CIF2 199

gene is not yet cloned and it is likely to be a NB-LRR gene as the accession specificity is often 200

the property of such genes (Noel et al 1999 Clark et al 2007) 201

We therefore assayed the growth of virulent pathogen Pseudomonas syringe pv tomato (Pst) 202

DC3000 in aca10-cif1 The bacterial growth was reduced by 10 times in the aca10-cif1 mutant 203

compared to that in the wild-type No-0 plants at 22degC but not 28degC (Fig 2B) This enhanced 204

resistance likely results from a constitutive upregulation of defense responses at 22degC The 205

defense marker gene PR1 which is induced by pathogen in the wild type is upregulated in 206

aca10-cif1 in the absence of pathogen inoculation and this upregulation of PR1 expression was 207

observed at 22degC but not at 28degC (Fig 2C) To further test the hypothesis that growth defect is 208


13

due to activation of NB-LRR mediated defense we introduced in aca10-cif1 mutant the LOF 209

mutation in the PAD4 gene that plays an important role in such a defense (Wiermer et al 2005) 210

The pad4-1 mutant in Col-0 background was crossed to aca10-cif1 in No-0 and F2 plants were 211


14

genotyped at the ACA10 CIF2 and PAD4 loci All plants with genotypes of aca10-cif1 cif2-n 212

(No-0 allele of CIF2) exhibited water-soaked leaf phenotype while all aca10-cif1 cif2-n pad4 213

plants exhibited a wild-type phenotype (Fig 2D) The suppression of cif1 mutant phenotype by 214

pad4 is further confirmed by analyzing progenies from F2 plants with aca10-cif1 cif2-n pad4+ 215

genotype In addition to the growth phenotype pad4 mutation also reduced PR1 expression in 216

aca10-cif1 (Fig 2E) Taken together ACA10 is a negative regulator of disease resistance in a 217

PAD4-dependent manner in the No-0 accession and constitutive immune responses result in 218

growth retardation in aca10-cif1 mutant 219

220

ACA10 and ACA8 are negative regulators of plant immune responses 221

Earlier study reported that ACA8 the closest homolog of ACA10 interacts with a PAMP 222

receptor FLS2 and is a positive regulator of plant immune response (Frei dit Frey et al 2012) It 223

was also reported that using surface inoculation method in which bacteria enter the plants 224

through stomata the aca8 aca10 and aca10aca8 mutants in the Col-0 background were more 225

susceptible to virulent pathogen Pst DC3000 compared to the wild type Col-0 (Frei dit Frey et 226

al 2012) However our characterization of the aca10-cif1 mutant as well as the genetic and 227

physical interaction of ACA10 and BON1 indicate that ACA10 is a negative regulator of immune 228

responses To investigate this discrepancy we analyzed the disease phenotype of aca10 and aca8 229

mutants in the Col-0 background The single LOF mutants of aca10-2 or aca8-2 in Col-0 did not 230

exhibit obvious growth defects in early developmental stage but the double mutant had water-231

soaked leaves and exhibited dwarfism (Fig 3A B) When analyzed for resistance to the virulent 232

pathogen Pst DC3000 the aca10-2 and the aca10-2aca8-2 mutants consistently exhibited 233

enhanced resistance compared to the wild-type Col-0 (Fig 3C D E) Using syringe infiltration 234


15

and dipping methods aca10-2 but not aca8-2 had reduced pathogen growth while aca10 aca8 235

mutant had a further reduction of pathogen growth compared to the wild-type Col-0 (Fig 3C D) 236

We subsequently used the same spray method as described in the prior study (Frei dit Frey et al 237


16

2012) for inoculation and found a smaller but significant reduction of pathogen growth in the 238

aca10-2 and aca10-2aca8-2 mutants (Fig 3E) We could not explain the discrepancy between 239

our result and the prior result by different inoculation methods or plant growth conditions 240

although we cannot exclude the possibility that a difference in an undefined plant growth 241

condition or pathogen preparation caused a difference in disease resistance We conclude that 242

ACA10 and ACA8 are negative regulators of defense responses to bacterial pathogen Pst DC3000 243

from our analysis of their mutants This is supported by the upregulation of a defense response 244

marker gene PR1 in the aca10 and aca10aca8 mutants compared to the wild-type Col-0 (Fig 245

3F) 246

247

Mutants of ACA10 and BON1 have synergistic interactions 248

The physical interaction of BON1 and ACA10 proteins as well as a similar phenotype in 249

immunity indicates that they might function together To further test this hypothesis we 250

constructed double mutant of aca10 and bon1 in the Ws background as neither of the LOF 251

mutants aca10-1 nor bon1-2 in the Ws background exhibited visible developmental defects (Fig 252

4A and Supplemental Fig 3A) The double mutant had smaller curled and water soaked leaves 253

and produced a more compact inflorescence compared to the wild type or the single mutants at 254

22 but not 28degC (Fig 4A and Supplemental Fig 3A-D) Pathogen growth assay demonstrated 255

that the disease resistance was increased in the aca10-1bon1-2 double mutant compared to wild-256

type and single mutant under 22 but not 28degC (Fig 4b) The aca10-1bon1-2 double mutant had a 257

significant increase of PR1 expression compared to wild-type and the single mutants at 22 but 258

not 28degC (Fig 4C) All the defects in the double mutant were suppressed by either pad4 or eds1 259

The two triple mutants aca10-1 bon1-2 eds1-1 and aca10-1 bon1-2 pad4-5 in contrast to aca10-260


17

1 bon1-2 had a wild-type leaf and inflorescence morphology (Fig 4D and Supplemental Fig 261

3E) These triple mutants also had the wild-type level of disease resistance and PR1 upregulation 262

(Fig 4E F) Thus the aca10-1 and bon1-2 has synergistic effect on immune responses in the Ws 263


18

background We hypothesize that NLR genes other than SNC1 are activated in the bon1-2 aca10-264

1 mutant in the Ws background similar to SNC1 activation in the bon1-1 mutant in the Col-0 265

background 266

267

Overexpression of BON1 suppressed the aca10-cif1 but not the aca10aca8 defects 268

The synergistic genetic interaction between BON1 and ACA10 single mutants is not 269

apparently consistent with the hypothesis that BON1 and ACA10 function in the same protein 270

complex andor regulate each other in a signaling pathway However this can be explained if 271

members of the BON1 or the ACA10 family each have similar functions In this case over-272

expression of one family member of the upstream component might be able to compensate for 273

the loss of one but not all family members of the downstream component To test this hypothesis 274

we overexpressed BON1 in aca10-cif1 mutant More than 20 transgenic lines were generated 275

and majority of the lines showed a rescued phenotype with wild-type leaf and inflorescent 276

morphology (Fig 5A and Supplemental Fig 4A) In addition bacterial growth was increased and 277

the PR1 expression was reduced in BON1-OEaca10-cif1 compared to aca10-cif1 (Fig 5B C) 278

In contrast when we overexpressed BON1 in the double mutant aca10aca8 none of the over 279

10 transgenic lines of BON1-OEaca10aca8 exhibited any difference from the aca10aca8 mutant 280

(Fig 5D) To exclude the possibility that the non-rescue was due to lower expression of BON1 in 281

aca10aca8 than in aca10-cif we analyzed the BON1 protein level by protein blot BON1 protein 282

level was in general lower in BON1-OEaca10aca8 than in BON1-OEaca10-cif1 however one 283

BON1-OEaca10-cif1 line exhibiting a rescued phenotype had a lower expression than several 284

BON1-OEaca10aca8 lines with a non-rescued phenotype (Supplemental Fig 4B) To confirm 285

the non-rescue phenotype in aca10aca8 we crossed a high BON1-expression line in aca10-cif1 286


19

(in No-0) with aca10aca8 (in Col-0) and analyzed the F2 progenies The aca10aca8 plants (now 287

in mixed No-0 and Col-0 background) exhibited a similar growth defect irrespective of the 288

presence of the BON1-OE transgene The non-rescuing of aca10aca8 defects by BON1-OE was 289


20

further confirmed in the F3 progenies of several lines of BON1-OEaca10aca8 in mixed 290

background (Fig 5E) Therefore BON1-OE rescues the defects of aca10 single mutant but not 291

the aca10 aca8 double mutant which suggests that BON1 functions upstream of both ACA10 292

and ACA8 293

294

ACA8 and BON1 have physical interaction 295

We subsequently tested the hypothesis that BON1 physically interacts with ACA8 as well 296

as ACA10 In the BiFC assay in N benthamiana co-expression of the full length ACA8 and 297

BON1 exhibited a strong fluorescence signal while co-expression of BON1 and a control PM 298

protein did not (Fig 6A supplemental Fig 2) The interaction between ACA8 and BON1 was 299

verified by a positive signal when the N-terminal segment I of ACA8 and BON1 were co-300

expressed in the split-LUC assay (Fig 6B) Therefore both ACA8 and ACA10 interact with 301

BON1 and the BON1-interacting domains in ACA8 and ACA10 are localized in the same region 302

of the protein 303

304

ACA10 ACA8 and BON1 affect calcium homeostasis and calcium signals 305

Because ACA10 and ACA8 are calcium pumps we hypothesize that the loss of the pump 306

function or its regulation will alter calcium homeostasis To test this hypothesis we monitored 307

calcium homeostasis and signature in plant cells using a FRET reporter Yellow Cameleon (YC) 308

A plant version of this calcium sensor YC36 under the control of the constitutive 35S promoter 309

(Yang et al 2008) was introduced into aca10-2 aca8-2 and bon1-1 mutants Steady levels of 310

calcium were measured in guard cells where YC36 has a strong expression Calcium 311

concentration was measured higher in the aca10 mutants compared to the wild type in both the 312


21

Ws and No-0 backgrounds (Fig 7A) which is consistent with earlier report based on another 313

calcium reporter (Frei dit Frey et al 2012) Interestingly the bon1 mutants also exhibited an 314

increase of calcium at steady status in both Ws and Col-0 backgrounds (Fig 7A) 315


22

Furthermore cytosolic calcium signals generated in response to imposed calcium were 316

altered in the aca10 and bon1 mutants In wild-type Col-0 an external application of 10 mM 317

calcium rapidly induced cytosolic calcium oscillation in guard cells (Fig 7B) as reported earlier 318


23

(Allen et al 2000 Hubbard et al 2012) In aca10-2 aca8-2 and bon1-1 LOF mutants only one 319

calcium spike was observed and no more calcium peaks followed (Fig 7B) Moreover the 320

decreasing of calcium level from the first spike in the three mutants was drastically delayed 321

compared to the wild-type Col-0 (Fig 7B) Therefore the initial influx of calcium ions happened 322

normally but the calcium oscillation pattern was lost This observation indicates an essential role 323

of BON1 ACA10 and ACA8 in cytosolic calcium oscillation and supports the hypothesis that 324

BON1 functions closely with ACA10 and ACA8 in regulating calcium signature 325

326

ACA10 ACA8 and BON1 regulate stomatal movement 327

Calcium signaling is critical in controlling stomatal movement (Kim et al 2010) The altered 328

calcium signature in bon1 aca10 and aca8 guard cells suggests that these mutants might have 329

defects in stomata closure in response to environmental stimuli Indeed the bon1 mutant does 330

not close stomata in response to calcium ABA or pathogen and this defect is not a consequence 331

of autoimmunity as it is independent of SNC1 or PAD4 (Gou et al 2015) (Fig 8A) None of the 332

aca10 aca8 and aca10aca8 mutants in No-0 Ws or Col-0 closed their stomata in response to 10 333

mM calcium either (Fig 8A) Furthermore ACA10 and ACA8 similar to BON1 functions in 334

pathogen induced stomata closure control as well When applied with a coronatine-deficient 335

(CORndash) Pst DC3000 strain which does not induce stomata reopening (Melotto et al 2006) none 336

of the aca10 and aca8 single or double mutants closed their stomata in contrast to the wild type 337

(Fig 8B) We further examined the stomatal movement over a time course in response to Pst 338

DC3000 CORndash as well as Pst DC3000 which causes reclose of stomata in the wild type Col-0 As 339

expected the wild type Col-0 opened stomata at 15 hours and 3 hours in response to both Pst 340

DC3000 CORndash and Pst DC3000 and closeed its stomata at 45 hours when applied with Pst 341


24

DC3000 but not Pst DC3000 CORndash (Fig 8C) In contrast the aca8 aca10 mutant kept its stomata 342

closed at 15 3 and 45 hours when applied with either of the pathogen strains except for a slight 343

opening at 3 hours when applied with Pst DC3000 (Fig 8C) This further supports that the 344


25

aca10 aca8 double mutant is insensitive or less sensitive to pathogens in stomatal closure 345

compared to the wild type Therefore the BON1 and ACA108 regulate stomata closure and 346

plant immune responses in a similar fashion and they might do so through modulating calcium 347

signatures 348

349

350


26

Discussion 351

Calcium signaling is universal and complex Despite rapid progress in deciphering calcium 352

signaling and network in various developmental and environmental responses it remains largely 353

unknown in many processes what calcium transporters are used to generate calcium signals and 354

how these transporters are regulated Here we identified Ca2+-ATPases ACA10 and ACA8 and 355

calcium-binding protein BON1 as important regulators of calcium signature (Fig 9) They are 356

essential to generate calcium oscillation in response to externally applied calcium and without 357

their functions only one single calcium spike is produced instead of multiple repeated spikes 358

This could be due to a failure to produce additional spikes or more likely to terminate the first 359

spike due to the lack of ACA108 function Prior studies also found an effect of ACAs on 360

calcium signatures For instances mutations of tonoplast-localized PCA in moss and ER-361

localized NbCA1 in tobacco caused elevated calcium concentration and increased the amplitude 362

and duration of calcium spikes respectively (Qudeimat et al 2008 Zhu et al 2010) Ca2+-363

ATPases are high affinity but low-capacity transporters whereas Ca2+-exchangers are low affinity 364

high-capacity transporters Based on their biochemistry characteristics it is hypothesized that 365

Ca2+-ATPase is the primarily component that terminates Ca2+ signaling whereas Ca2+-exchanger 366

is the primarily factor that removes Ca2+ following cytosolic Ca2+ elevation (Sze et al 2000) 367

The finding here that the loss of ACA108 function compromises cytosolic calcium oscillation 368

not only establishes a critical role of PM-localized ACAs in calcium signals but also indicates a 369

broader role of ACAs in calcium signature generation than previously thought 370

Environmental signals such as PAMPs trigger opening of calcium permeable channels for 371

calcium influx leading to the cytosolic calcium spike and sometimes multiple spikes forming an 372

oscillation (Thor and Peiter 2014) Our study indicates that calcium pumps ACA10 and ACA8 373


27

are needed for the oscillation but not the first spike It is likely that calcium level in cytosol are 374

reset after the first induction before subsequent spikes can be generated and calcium pumps are 375

needed for this resetting BON1 whose activity is promoted by calcium binding (Li et al 2010) 376


28

potentially modulates calcium oscillation through its binding to and activating ACA10 and 377

ACA8 ACA10 and ACA8 both have an auto-inhibitory domain that overlaps with the 378

calmodulin binding motif and calmodulin binding is thought to activate ACAs (Tidow et al 379

2012) The binding of BON1 to this auto-inhibitory domain of ACA108 may alter the protein 380

conformation of ACA108 or facilitate calmodulin binding of ACA108 leading to the release of 381

auto-inhibition of ACA108 (Fig 9) Because BON1 and calmodulin are both calcium binding 382

proteins the ACA108 pump activity could be controlled by calcium itself thus forming a 383

feedback regulation Future analysis of the interaction among ACA108 BON1 and calmodulin 384

will reveal further the regulatory mechanisms of calcium efflux conferred by ACA108 385

Calcium oscillation is generated in response to a number of stimuli in plants including NOD 386

factor in a variety of legume species (Granqvist et al 2015) In legume calcium oscillation 387

occurs in nucleus in response to NOD factor and an ER- or nuclear envelope- localized calcium 388

ATPase was found to be essential to generate nuclear calcium oscillation (Capoen et al 2011) 389

The finding of a role of PM-localized calcium ATPases in calcium oscillation in cytosol indicate 390

that calcium ATPases have general roles in calcium signature generation in various cellular 391

compartments Further study should determine whether or not ACA108 and BON1 affect 392

calcium signatures in response to other stimuli as well The role of BON1 in calcium signature 393

generation also has implications for the copine protein family where BON1 is a member Some 394

copines in animals were identified as genetic modifiers of channel or receptor mutants (Church 395

and Lambie 2003 Gottschalk et al 2005) and they have also been implicated in receptor 396

mediated signaling from cell culture analysis (Tomsig et al 2004 Heinrich et al 2010) It 397

would be interesting to test if copines have a regulatory role of calcium signals involved in those 398

processes 399


29

This study also finds opposing functions of ACA10 and ACA8 in two layers of plant 400

immunity similarly to BON1 On one hand the aca10 and aca8 mutants are insensitive to 401

calcium and pathogen in stomatal closure response similarly to the bon1 mutants This feature 402

presumably makes these mutant plants more susceptible to pathogen at the invasion stage An 403

earlier report did find a more susceptible phenotype for the aca8 and aca10 mutants (Frei dit 404

Frey et al 2012) However we found in this study that the aca10 and aca10 aca8 mutants have 405

enhanced resistance when bacterial propagation in plants is assayed by either infiltration or 406

surface inoculation methods This discrepancy might have resulted from differences in 407

unidentified plant or pathogen growth conditions because resistance phenotype was observed 408

even when we replicated the experiment reported in Frei dit Frey et al 2012 Nevertheless the 409

enhanced resistance we observed is temperature- and PAD4-dependent indicating of activation 410

of plant immune receptor NLR genes reminiscent of the activation of NLR genes in the bon1 411

mutant Apparently this layer of regulation over-rides the stomatal layer of immunity regulation 412

as both bon1 and aca10 mutants have enhanced resistance measured by pathogen growth 413

How do BON1 and ACA108 regulate two layers of plant immunity Conceivably their 414

positive role in stomatal closure control is directly tied with their roles in calcium signature 415

generating and this might represent a more direct role of these proteins Their roles in controlling 416

pathogen growth likely results from their negative regulation of immune receptor gene 417

expression which could be associated with calcium homeostasis Altered steady state of calcium 418

might mimic signals from sustained pathogen invasion and therefore upregulate or activate NLR 419

genes Indeed disruption of calcium channels and pumps have been shown to affect plant 420

immunity (Clough et al 2000 Zhu et al 2010) For instances two vacuole-localized ACA 421

members ACA4 and ACA11 repress cell death and innate immunity in Arabidopsis (Boursiac et 422


30

al 2010) Although these molecules are located in different parts of the cell including plasma 423

membrane ER or vacuole they could all regulate calcium levels in cytosol and perhaps nucleus 424

as well 425

Although both bon1 and aca10aca8 have autoimmunity their growth phenotypes are not 426

identical This might be due to specific functions these proteins have in addition to their 427

overlapping roles It could also be due to overlapping functions among the BON1 family 428

members or the ACA10 family members BON1 has two other homologs BON2 and BON3 in 429

Arabidopsis while ACA10 has ACA8 and ACA9 as close homologs as well as ACA12 and 430

ACA13 as homologs It is likely that BON1 family members interact with ACA10 family 431

members each pair with a different regulatory strength Therefore a partial loss of the family 432

activity could result in different statuses of calcium homeostasis This variation in calcium steady 433

level might be associated with activation of different NLR genes Identifying such NLR genes 434

activated in various mutants of calcium channels and pumps will lead to a further understanding 435

of the mode of action of calcium signatures as well as their roles in plant immunity 436

437

Materials and Methods 438

Arabidopsis Mutants and Plant Growth 439

The seeds of aca10-cif1 and aca10-1 were kindly provided by R Sharrock (George et al 440

2008) The aca10-2 (GK-044H01) and aca8-2 (GK-688H09) lines were obtained from the 441

Arabidopsis Stock Centre (httparabidopsisinfo) For growth phenotyping the Arabidopsis 442

plants were grown under constant light condition with 100 mmol mndash2 sndash1 and relative humidity at 443

50 to 70 For pathogen growth assays plants were grown under 12 or 16 hour light 444

condition N benthamiana plants were grown in the greenhouse at 24degC for 4 to 6 weeks before 445


31

use for transient expression studies 446

447

Protein Subcellular Localization Assay 448

An ACA10 genomiccDNA hybrid was constructed and cloned into the Gateway entry 449

vector pCR8 TOPO TA vector (Invitrogen) For localization of ACA10 protein the ACA10 gene 450

was transferred from the entry vector to the Hpt-psatn1-ACA10 (GW) vector and transformed 451

into the Agrobacterium tumefaciens (A tumefaciens) strain GV3101 for infiltration into the 452

abaxial surface side of 4- to 6-week-old N benthamiana plant leaves as previously described 453

(Gou et al 2015) Fluorescence of the epidermal cell layer of the lower leaf surface was 454

examined at 2 to 4 days post inoculation (DPI) Images were captured by a Leica TCS SP2 455

Confocal Microscope with excitation wavelength at 488 and 496 nm and emission wavelength 456

between 520 and 535 nm for GFP signals 457

458

BiFC Assay 459

The full-length complementary DNA (cDNA) fragments (without stop codon) of BON1 460

ACA8 OPT3 (Zhai et al 2014) and ACA10 (genomiccDNA hybrid) were amplified using 461

primers in Supplemental Table S2 and cloned into the Gateway entry vectors pCR8 TOPO TA or 462

pENTRD TOPO (Invitrogen) For BiFC experiments BON1 was cloned into pSPYCE-35S GW 463

(Schutze et al 2009) using LR clonase (Invitrogen) to generate BON1cYFP constructs while 464

ACA8 ACA10 and OPT3 were cloned similarly into pSPYNE-35S GW to generate 465

corresponding nYFP constructs respectively A previously described protocol (Schutze et al 466

2009) was followed to observe BiFC signals with minor modification The constructs were 467

transformed into the A tumefaciens strain GV3101 Overnight cell cultures were collected and 468


32

resuspended in 1 mL of AS medium (10 mM MES-KOH pH56 10 mM MgCl2 and 150 microM 469

acetosyringone) to optical density at 600 nm (OD600) to 08 The working suspensions were 470

prepared by mixing appropriate clones containing the BiFC constructs and the gene-silencing 471

inhibitor pBA-HcPro plasmid (Menke et al 2005) at 111 ratio and let them stand for 2 to 4 472

hours The A tumefaciens suspensions were then co-infiltrated into the abaxial surface of 4-473

week-old N benthamiana plant leaves Fluorescence of the epidermal cell layer of the lower leaf 474

surface was examined at 3 to 4 DPI Images were captured by a Leica SP5 confocal microscope 475

with excitation wavelength at 514 nm and emission wavelength between 500 and 550 nm for 476

YFP signals 477

478

Split-Luc Assay 479

The full length cDNA of BON1 was amplified using the oligos listed in Supplemental Table 480

2 The PCR fragment of BON1 was ligated into the pCAMBIA-NLuc vector (Chen et al 2008) 481

digested by BamH1 and SalI using the ClonExpressTM MultiS One Step Cloning Kit (Vazyme) to 482

generate BON1-NLuc The N-terminal parts of ACA8 and ACA10 were each amplified using the 483

oligos listed in Supplemental Table 2 The PCR fragments of ACA8 and ACA10 were ligated 484

into the pCAMBIA-CLuc vector (Chen et al 2008) digested by Kpn1 and SalI using a similar 485

strategy as above to generate ACA8I-CLuc and ACA10I-CLuc These constructs were 486

transformed into A tumefaciens strain GV3101 487

A tumefaciens GV3101 strains containing recombinant constructs were grown in liquid 488

Luria-Bertani medium with rifampicin and kanamycin for 2 days pelleted and resuspended in 489

Murashige and Skoog with 10 mM MES medium containing 200 μM acetosyringone to a final 490

concentration of OD600=06 After 2 hoursrsquo induction the bacterial suspensions were infiltrated 491


33

into young expanded leaves of N benthamiana plants with a needleless syringe After infiltration 492

the plants were covered with dark bag at 23degC for 48 hours The plants were then kept under 493

light for 16 hours sprayed with 1 mM luciferin in 001 Triton X100 solution and kept in dark 494

for 5 min to quench the fluorescence A deep cooling CCD imaging apparatus (DU934P-BV 495

Andor) was used to capture the fluorescence image The camera was cooled to -80degC and all 496

images were taken after 3 min exposure 497

498

Pathogen Growth Assay 499

Pst DC3000 grown on plates with Kingrsquos B medium were washed collected and diluted 500

with 10 mM MgCl2 and 002 Silwet L-77 Syringe infiltration was used for inoculation unless 501

stated otherwise where dipping or spray methods were used For syringe infiltration bacteria 502

were diluted to OD600 of 0002 and syringe-infiltrated on leaves of 3 to 4 week old plants Six 503

inoculated leaf were collected as one sample weighed ground in 1 ml of 10 mM MgCl2 and 504

shaken at room temperature for 1 hour Dipping inoculation was performed as previously 505

described (Gou et al 2015) and bacteria were diluted to OD600=005 for dipping seedlings of 2 506

weeks old Three whole seedlings were collected as one sample Spray inoculation was 507

performed as described previously (Frei dit Frey et al 2012) and the bacteria were diluted to 508

OD600=02 for spray 509

To determine bacterial growth (for all inoculation methods) serial dilutions of the ground 510

tissue solution were spotted on KB medium and the number of cfu (colony forming unit) per 511

fresh weight was calculated Three to four samples were analyzed for each genotype and time 512

point 513


34

514

Yeast Two Hybrid Assay 515

The yeast (Saccharomyces cerevisiae) two-hybrid constructs were made in the pDEST-516

GBKT7 and pDEST-GADT7 Gateway vectors (Rossignol et al 2007) The DNA fragment of 517

the N-terminal segment I of ACA10 was cloned into the pCR8 TOPO TA vector and transferred 518

to the pDEST-GBKT7 vector to obtain the BDACA10-I construct The ADBON1 ADBON1-519

A and BD-BAP1 constructs were previously described (Hua et al 2001) The yeast two-hybrid 520

assay was performed as previously described (Hua et al 2001 Li et al 2010) 521

522

Northern Blotting and Real-time RT-PCR 523

Total RNA was extracted from 3-week old seedling using Trizol reagent (Invitrogen) as 524

instructed RNA of 20 μg was resolved in a 12 gel containing formaldehyde RNA blots were 525

hybridized with gene specific and 32P labeled single strand DNA probe 526

For Real-time PCR SuperScript II Reverse Transcriptase (Invitrogen) was used to 527

synthesize cDNA from isolated RNA Real time RT-PCR was performed using PR1 primers 528

listed in Supplemental Table S2 The ACTIN2 gene was used as internal controls Advanced 529

Universal SYBR Green Supermix (Bio-Rad) was used for Real time RT-PCR 530

531

Western Blotting 532

The vector of 35SBON1-HA used to overexpression BON1 was reported previously (Gou 533

et al 2015) Arabidopsis leaves grown in soil under constant light for 3 weeks were used for 534

protein extraction and western blotting with anti-HA antibody (Sigma Aldrich Catalog H3663) 535

following a previously described method (Gou et al 2015) 536


35

537

Stomatal Closure Assay 538

Stomatal aperture assay was performed as described (Nomura et al 2012) Young rosette 539

leaves from 17- to 25-day-old Arabidopsis plants were detached floated on the stomatal opening 540

buffer containing 10 mM Tris-Mes (pH 615) 5 mM KCl and 50 μM CaCl2 and incubated for 541

25 hours under white light (200-250μmol m-2 s-1) at 22degC Each leaf was then clung onto a cover 542

slip with a medical adhesive (Hollister Libertyville USA) and their mesophyll cells were 543

removed by a razor blade The epidermal strips were then transferred into the stomatal closing 544

buffer containing 10 mM Tris-Mes (pH 615) 5mM KCl and 10mM CaCl2 and incubated for 545

another 2 hours under white light Then the epidermal strips were observed under an inverted 546

microscopy (Model D1 Carl Zeiss) before and after the closing buffer treatment The stomatal 547

aperture was calculated as the ratio of the inner widthouter length of each pair of stomata For 548

each sample more than 50 guard cells were calculated and the experiments were repeated for 4 549

times 550

551

Calcium Level Measurement 552

For calcium oscillation experiment the epidermal strips after the light treatment was 553

acquired as described above Cover slips were then placed in a perfusion chamber which was fit 554

to an inverted microscopy (Model D1 Carl Zeiss Germany) equipped with an emission filter 555

wheel (Lambda XL Novato USA) and a CCD camera (AndorTM Technology Belfast Northern 556

Ireland) Imaging calcium in the guard cells was conducted by monitoring the ratio 557

(535nm442nm) of YC36 using the MetaFluor fluorescence ratio imaging software The 558

epidermal strips were firstly measured for approximately 100 seconds and then the opening 559


36

buffer was changed into the closing buffer by an injector when the same epidermal strips were 560

measured for 700 seconds The interval of image acquisition was 3 seconds For each genotype 561

more than 30 guard cells were measured and the experiments were repeated for 3 times 562

Measurement of steady level of Ca2+ concentration using the YC36 system was performed 563

with ZESS LSM710 confocal laser scanning microscope following the protocol previously 564

described (Krebs et al 2012) 565

566

Acknowledgement 567

We thank Y Wang for the assistance in microscopy Y Tan for technical assistance on 568

YC36 assay R Sharrock for the seeds of cif1-1 and aca10-2 and J Schroeder for the seeds of 569

35SYC36 transgenic plant This work was supported by grants from National Science 570

Foundation of USA (IOSndash0919914 and IOS-1353738) to J Hua the National Key Research and 571

Development Program of China (2016YFD0100600) to D-L Yang Natural Science Foundation 572

of Jiangsu (BK20150659) to D-L Yang National Science Foundation of China (31330061) to B 573

Zou and Jiangsu Collaborative Innovation Center for Modern Crop Production to J Hua and D-574

L Yang We thank China Scholarship Council for fellowships to B Zou H Yu and Y Bao and 575

Shanghai Institute of Plant Physiology and Ecology for a fellowship to Z Shi 576

577

Figure legends 578

Figure 1 Protein-protein interaction between ACA10 and BON1 579

A BiFC assay of ACA10 and BON1 interaction ACA10 fused with N-terminal part of YFP 580


37

(ACA10-NE) and BON1 fused with C-terminal part of YFP (BON1-CE) were transiently 581

expressed in Nicotiana benthamiana (N benthamiana) by Agrobacterium mediated 582

transformation A control plasma membrane protein OPT3 co-expressed with BON1 is shown at 583

the bottom panel Bar=50microm 584

B BiFC assay of interaction between BON1 and each of the five segments of ACA10 I II III 585

IV and V (supplemental Fig 1B) Bar=50microm 586

C Split-LUC assay of interaction of BON1 and the first segment of ACA10 Fusion of segment I 587

of ACA10 with C-terminus LUC (ACA10I-Cluc) was co-expressed with fusion of BON1 with 588

N-terminus LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of 589

ACA10I-Cluc with Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc 590

(lower right) were used as controls 591

D Yeast-two-hybrid assay between the first segment of ACA10 and the A domain of BON1 592

BAP1 was used as a positive control Shown is growth of serial dilutions of yeasts on synthetic 593

medium without leucine or tryptophan (SC-LT) or synthetic medium without leucine tryptophan 594

adenine or histidine (SC-LTAH) AD Activation domain of GAL4 BD DNA-binding domain 595

of GAL4 596

597

Figure 2 The loss of ACA10 in Nossen-0 (No-0) background leads to autoimmune responses 598

A Growth phenotype of aca10-cif1 and wild-type No-0 plants at 22ordmC and 28ordmC 599

B Pst DC3000 bacterial growth in aca10-cif1 and No-0 plants under 22ordmC and 28ordmC ldquordquo 600

indicates significant difference by studentrsquos t-test (P-valuelt0001) 601

C PR1 expression level in aca10-cif1 and No-0 plants at 22ordmC and 28ordmC analyzed by Northern 602

blotting 603


38

D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604

E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605

RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606

t-test (P-valuelt0001) 607

608

Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609

background 610

A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611

B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612

C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613

methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614

statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615

F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616

PCR assay Actin2 is used as a control gene 617

618

Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619

A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620

22ordmC and 28ordmC 621

B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622

C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623

amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624

D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625

mutants grown at 22degC 626


39

E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627

F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628

Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629

2pad4-5 630

Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631

of various genotypes 632

633

Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634

aca10aca8 mutants 635

A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636

22degC 637

B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638

C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639

control gene 640

D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641

22degC 642

E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643

and No-0 background Shown as the second and the last plant respectively are the two parental 644

lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645

two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646

BON1-OE transgene 647

Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648



40

650

Figure 6 Physical interaction of ACA8 and BON1 651

A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652

BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653

benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654

was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655

protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656

images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657

B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658

with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659

LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660

Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661

used as controls 662

663

Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664

mutants 665

A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666

aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667

deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668

guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669

type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670

B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671

aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672


41

genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673

each genotype indicate the number of cells with the representative calcium pattern versus the 674

number of total cells analyzed 675

676

677

Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678

A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679

bon1 in No-0 Ws andor Col-0 backgrounds 680

B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681

and bon1 in the Col-0 background 682

C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683

the wild type and the aca8aca10 plants 684

ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685

valuelt0001 and P-valuelt005 respectively 686

687

Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688

responses 689

Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690

when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691

concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692

extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693

cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694

calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695


42

pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696

Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697

defense response genes 698

699

700

Supplementary tables 701

Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702

molecules 703

Supplementary Table 2 Primers used in this study 704

705

Supplementary Figures 706

Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707

Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708

Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709

EDS1 and PAD4 710

Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711

712

713

714

715

716


43

717

718

719

720


Parsed CitationsAllen GJ Chu SP Schumacher K Shimazaki CT Vafeados D Kemper A Hawke SD Tallman G Tsien RY Harper JF Chory JSchroeder JI (2000) Alteration of stimulus-specific guard cell calcium oscillations and stomatal closing in Arabidopsis det3 mutantScience 289 2338-2342

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

Balagueacute C Lin B Alcon C Flottes G Malmstroumlm S Koumlhler C Neuhaus G Pelletier G Gaymard F Roby D (2003) HLM1 anessential signaling component in the hypersensitive response is a member of the cyclic nucleotide-gated channel ion channelfamily Plant Cell 15 365-379


Bonza MC Morandini P Luoni L Geisler M Palmgren MG De Michelis MI (2000) At-ACA8 encodes a plasma membrane-localizedcalcium-ATPase of Arabidopsis with a calmodulin-binding domain at the N terminus Plant Physiol 123 1495-1506


Boudsocq M Sheen J (2013) CDPKs in immune and stress signaling Trends Plant Sci 18 30-40Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Boudsocq M Willmann MR McCormack M Lee H Shan L He P Bush J Cheng SH Sheen J (2010) Differential innate immunesignalling via Ca2+ sensor protein kinases Nature 464 418-422


Boursiac Y Harper JF (2007) The origin and function of calmodulin regulated Ca2+ pjmps in plants J Bioenerg Biomembr 39 409-414


Boursiac Y Lee SM Romanowsky S Blank R Sladek C Chung WS Harper JF (2010) Disruption of the vacuolar calcium-ATPasesin Arabidopsis results in the activation of a salicylic acid-dependent programmed cell death pathway Plant Physiol 154 1158-1171


Bracha-Drori K Shichrur K Katz A Oliva M Angelovici R Yalovsky S Ohad N (2004) Detection of protein-protein interactions inplants using bimolecular fluorescence complementation Plant Journal 40 419-427


Bush DS (1995) Calcium Regulation in Plant-Cells and Its Role in Signaling Annual Review of Plant Physiology and Plant MolecularBiology 46 95-122


Capoen W Sun J Wysham D Otegui MS Venkateshwaran M Hirsch S Miwa H Downie JA Morris RJ Ane JM Oldroyd GE (2011)Nuclear membranes control symbiotic calcium signaling of legumes Proc Natl Acad Sci U S A 108 14348-14353


Chen H Zou Y Shang Y Lin H Wang Y Cai R Tang X Zhou JM (2008) Firefly luciferase complementation imaging assay forprotein-protein interactions in plants Plant Physiol 146 368-376


Choi HW Lee DH Hwang BK (2009) The Pepper Calmodulin Gene CaCaM1 Is Involved in Reactive Oxygen Species and NitricOxide Generation Required for Cell Death and the Defense Response Molecular Plant-Microbe Interactions 22 1389-1400


Church DL Lambie EJ (2003) The promotion of gonadal cell divisions by the Caenorhabditis elegans TRPM cation channel GON-2is antagonized by GEM-4 copine Genetics 165 563-574

Pubmed Author and TitleCrossRef Author and Title


Google Scholar Author Only Title Only Author and Title

Clark RM Schweikert G Toomajian C Ossowski S Zeller G Shinn P Warthmann N Hu TT Fu G Hinds DA Chen H Frazer KAHuson DH Scholkopf B Nordborg M Ratsch G Ecker JR Weigel D (2007) Common sequence polymorphisms shaping geneticdiversity in Arabidopsis thaliana Science 317 338-342


Clough SJ Fengler KA Yu IC Lippok B Smith RK Jr Bent AF (2000) The Arabidopsis dnd1 defense no death gene encodes amutated cyclic nucleotide-gated ion channel Proc Natl Acad Sci U S A 97 9323-9328


Creutz CE Tomsig JL Snyder SL Gautier MC Skouri F Beisson J Cohen J (1998) The copines a novel class of C2 domain-containing calcium-dependent phospholipid-binding proteins conserved from Paramecium to humans J Biol Chem 273 1393-1402


Curran AC Hwang I Corbin J Martinez S Rayle D Sze H Harper JF (2000) Autoinhibition of a calmodulin-dependent calciumpump involves a structure in the stalk that connects the transmembrane domain to the ATPase catalytic domain J Biol Chem 27530301-30308


Dodd AN Kudla J Sanders D (2010) The language of calcium signaling Annu Rev Plant Biol 61 593-620Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Du L Ali GS Simons KA Hou J Yang T Reddy AS Poovaiah BW (2009) Ca2+calmodulin regulates salicylic-acid-mediated plantimmunity Nature 457 1154-1158


Forde BG Roberts MR (2014) Glutamate receptor-like channels in plants a role as amino acid sensors in plant defenceF1000Prime Rep 6 37


Frei dit Frey N Mbengue M Kwaaitaal M Nitsch L Altenbach D Haweker H Lozano-Duran R Njo MF Beeckman T Huettel BBorst JW Panstruga R Robatzek S (2012) Plasma membrane calcium ATPases are important components of receptor-mediatedsignaling in plant immune responses and development Plant Physiol 159 798-809


Gao X Chen X Lin W Chen S Lu D Niu Y Li L Cheng C McCormack M Sheen J Shan L He P (2013) Bifurcation of ArabidopsisNLR immune signaling via Ca2+-dependent protein kinases PLoS Pathog 9 e1003127


Geisler M Axelsen KB Harper JF Palmgren MG (2000) Molecular aspects of higher plant P-type Ca2+-ATPases Biochim BiophysActa 1465 52-78


George L Romanowsky SM Harper JF Sharrock RA (2008) The ACA10 Ca2+-ATPase regulates adult vegetative development andinflorescence architecture in Arabidopsis Plant Physiol 146 716-728


Gottschalk A Almedom RB Schedletzky T Anderson SD Yates JR 3rd Schafer WR (2005) Identification and characterization ofnovel nicotinic receptor-associated proteins in Caenorhabditis elegans Embo J 24 2566-2578


Gou M Zhang Z Zhang N Huang Q Monaghan J Yang H Shi Z Zipfel C Hua J (2015) Opposing Effects on Two Phases ofDefense Responses from Concerted Actions of HEAT SHOCK COGNATE70 and BONZAI1 in Arabidopsis Plant Physiol 169 2304-2323 httpsplantphysiolorgDownloaded on January 22 2021 - Published by

Copyright (c) 2020 American Society of Plant Biologists All rights reserved


Granqvist E Sun J Op den Camp R Pujic P Hill L Normand P Morris RJ Downie JA Geurts R Oldroyd GE (2015) Bacterial-induced calcium oscillations are common to nitrogen-fixing associations of nodulating legumes and nonlegumes New Phytol 207551-558


Harper JF Hong B Hwang I Guo HQ Stoddard R Huang JF Palmgren MG Sze H (1998) A novel calmodulin-regulated Ca2+-ATPase (ACA2) from Arabidopsis with an N-terminal autoinhibitory domain J Biol Chem 273 1099-1106


Heinrich C Keller C Boulay A Vecchi M Bianchi M Sack R Lienhard S Duss S Hofsteenge J Hynes NE (2010) Copine-IIIinteracts with ErbB2 and promotes tumor cell migration Oncogene 29 1598-1610


Hua J Grisafi P Cheng SH Fink GR (2001) Plant growth homeostasis is controlled by the Arabidopsis BON1 and BAP1 genesGenes Dev 15 2263-2272


Huang LQ Berkelman T Franklin AE Hoffman NE (1993) Characterization of a Gene Encoding a Ca2+-Atpase-Like Protein in thePlastid Envelope Proceedings of the National Academy of Sciences of the United States of America 90 10066-10070


Hubbard KE Siegel RS Valerio G Brandt B Schroeder JI (2012) Abscisic acid and CO2 signalling via calcium sensitivity priming inguard cells new CDPK mutant phenotypes and a method for improved resolution of stomatal stimulus-response analyses Ann Bot109 5-17


Kim MC Panstruga R Elliott C Muller J Devoto A Yoon HW Park HC Cho MJ Schulze-Lefert P (2002) Calmodulin interacts withMLO protein to regulate defence against mildew in barley Nature 416 447-451


Kim TH Bohmer M Hu H Nishimura N Schroeder JI (2010) Guard cell signal transduction network Advances in understandingabscisic acid CO2 and Ca2+ signaling Annu Rev Plant Bio 61561-591


Krebs M Held K Binder A Hashimoto K Den Herder G Parniske M Kudla J Schumacher K (2012) FRET-based geneticallyencoded sensors allow high-resolution live cell imaging of Ca2+ dynamics Plant J 69 181-192


Kudla J Batistic O Hashimoto K (2010) Calcium signals the lead currency of plant information processing Plant Cell 22 541-563Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Lecourieux D Ranjeva R Pugin A (2006) Calcium in plant defence-signalling pathways New Phytol 171 249-269Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Lee SM Kim HS Han HJ Moon BC Kim CY Harper JF Chung WS (2007) Identification of a calmodulin-regulated autoinhibitedCa2+-ATPase (ACA11) that is localized to vacuole membranes in Arabidopsis FEBS Lett 581 3943-3949


Lewis RS (2001) Calcium signaling mechanisms in T lymphocytes Annu Rev Immunol 19 497-521Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title


Li Y Gou M Sun Q Hua J (2010) Requirement of calcium binding myristoylation and protein-protein interaction for the CopineBON1 function in Arabidopsis J Biol Chem 285 29884-29891


Li Y Yang S Yang H Hua J (2007) The TIR-NB-LRR gene SNC1 is regulated at the transcript level by multiple factors Mol PlantMicrobe Interact 20 1449-1456


Ma W Berkowitz GA (2007) The grateful dead calcium and cell death in plant innate immunity Cell Microbiol 9 2571-2585Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Melotto M Underwood W Koczan J Nomura K He SY (2006) Plant stomata function in innate immunity against bacterial invasionCell 126 969-980


Menke FL Kang HG Chen Z Park JM Kumar D Klessig DF (2005) Tobacco transcription factor WRKY1 is phosphorylated by theMAP kinase SIPK and mediates HR-like cell death in tobacco Mol Plant Microbe Interact 18 1027-1034


Monaghan J Matschi S Shorinola O Rovenich H Matei A Segonzac C Malinovsky FG Rathjen JP MacLean D Romeis T ZipfelC (2014) The calcium-dependent protein kinase CPK28 buffers plant immunity and regulates BIK1 turnover Cell Host Microbe 16605-615


Murata Y Mori IC Munemasa S (2015) Diverse stomatal signaling and the signal integration mechanism Annu Rev Plant Biol 66369-392


Noel L Moores TL van Der Biezen EA Parniske M Daniels MJ Parker JE Jones JD (1999) Pronounced intraspecific haplotypedivergence at the RPP5 complex disease resistance locus of Arabidopsis Plant Cell 11 2099-2112


Nomura H Komori T Uemura S Kanda Y Shimotani K Nakai K Furuichi T Takebayashi K Sugimoto T Sano S Suwastika INFukusaki E Yoshioka H Nakahira Y Shiina T (2012) Chloroplast-mediated activation of plant immune signalling in Arabidopsis NatCommun 3 926


Obayashi T Hayashi S Saeki M Ohta H Kinoshita K (2009) ATTED-II provides coexpressed gene networks for ArabidopsisNucleic Acids Res 37 D987-991


Qudeimat E Faltusz AM Wheeler G Lang D Holtorf H Brownlee C Reski R Frank W (2008) A PIIB-type Ca2+-ATPase is essentialfor stress adaptation in Physcomitrella patens Proc Natl Acad Sci U S A 105 19555-19560


Rizo J Sudhof TC (1998) C2-domains structure and function of a universal Ca2+-binding domain J Biol Chem 273 15879-15882Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Rossignol P Collier S Bush M Shaw P Doonan JH (2007) Arabidopsis POT1A interacts with TERT-V(I8) an N-terminal splicingvariant of telomerase J Cell Sci 120 3678-3687


Schiott M Romanowsky SM Baekgaard L Jakobsen MK Palmgren MG Harper JF (2004) A plant plasma membrane Ca2+ pump isrequired for normal pollen tube growth and fertilization Proc Natl Acad Sci U S A 101 9502-9507



Schutze K Harter K Chaban C (2009) Bimolecular fluorescence complementation (BiFC) to study protein-protein interactions inliving plant cells Methods Mol Biol 479 189-202


Shigaki T Hirschi KD (2006) Diverse functions and molecular properties emerging for CAX cationH+ exchangers in plants PlantBiol (Stuttg) 8 419-429


Steinhorst L Kudla J (2013) Calcium and reactive oxygen species rule the waves of signaling Plant Physiol 163 471-485Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Sze H Liang F Hwang I Curran AC Harper JF (2000) Diversity and regulation of plant Ca2+ pumps insights from expression inyeast Annu Rev Plant Physiol Plant Mol Biol 51 433-462


Thor K Peiter E (2014) Cytosolic calcium signals elicited by the pathogen-associated molecular pattern flg22 in stomatal guardcells are of an oscillatory nature New Phytol 204 873-881


Tidow H Poulsen LR Andreeva A Knudsen M Hein KL Wiuf C Palmgren MG Nissen P (2012) A bimodular mechanism of calciumcontrol in eukaryotes Nature 491 468-472


Tomsig JL Sohma H Creutz CE (2004) Calcium-dependent regulation of tumour necrosis factor-alpha receptor signalling bycopine Biochem J 378 1089-1094


Wang Y Hua J (2009) A moderate decrease in temperature induces COR15a expression through the CBF signaling cascade andenhances freezing tolerance Plant J 60 340-349


Whittaker CA Hynes RO (2002) Distribution and evolution of von Willebrandintegrin A domains widely dispersed domains withroles in cell adhesion and elsewhere Mol Biol Cell 13 3369-3387


Wiermer M Feys BJ Parker JE (2005) Plant immunity the EDS1 regulatory node Curr Opin Plant Biol 8 383-389Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Yang S Hua J (2004) A haplotype-specific Resistance gene regulated by BONZAI1 mediates temperature-dependent growthcontrol in Arabidopsis Plant Cell 16 1060-1071


Yang S Yang H Grisafi P Sanchatjate S Fink GR Sun Q Hua J (2006) The BONCPN gene family represses cell death andpromotes cell growth in Arabidopsis Plant J 45 166-179


Yang Y Costa A Leonhardt N Siegel RS Schroeder JI (2008) Isolation of a strong Arabidopsis guard cell promoter and itspotential as a research tool Plant Methods 4 6


Zhai Z Gayomba SR Jung HI Vimalakumari NK Pineros M Craft E Rutzke MA Danku J Lahner B Punshon T Guerinot ML SalthttpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved

DE Kochian LV Vatamaniuk OK (2014) OPT3 Is a Phloem-Specific Iron Transporter That Is Essential for Systemic Iron Signalingand Redistribution of Iron and Cadmium in Arabidopsis Plant Cell 26 2249-2264


Zhu X Caplan J Mamillapalli P Czymmek K Dinesh-Kumar SP (2010) Function of endoplasmic reticulum calcium ATPase in innateimmunity-mediated programmed cell death Embo J 29 1007-1018



Parsed Citations

Reviewer PDF

Parsed Citations

2

One-sentence summary 24

Calcium pumps ACA10 and ACA8 and their interacting protein BON1 regulate calcium 25

signatures and impact stomatal movement and plant immunity in Arabidopsis 26


3

Abstract 27
















43


4

Introduction 44
























5

























6

























7























135


8

136


9

Results 137
























10





11






















sequences 184

185


12

























13





14









220
















15





16









3F) 246

247















17





18



background 266

267





















19





20




and ACA8 293

294









of the protein 303

304










21





22





23








326

















24





25




signatures 348

349

350


26

Discussion 351
























27





28























processes 399


29

























30



as well 425












437










31


447











458

BiFC Assay 459











32









YFP signals 477

478

Split-Luc Assay 479














33







498















point 513


34

514








522









531







35

537













times 550

551










36







566

Acknowledgement 567










577

Figure legends 578




37
















of GAL4 596

597






blotting 603


38





608


background 610








618










39




2pad4-5 630



633




22degC 637



control gene 640


22degC 642









40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

3

Abstract 27
















43


4

Introduction 44
























5

























6

























7























135


8

136


9

Results 137
























10





11






















sequences 184

185


12

























13





14









220
















15





16









3F) 246

247















17





18



background 266

267





















19





20




and ACA8 293

294









of the protein 303

304










21





22





23








326

















24





25




signatures 348

349

350


26

Discussion 351
























27





28























processes 399


29

























30



as well 425












437










31


447











458

BiFC Assay 459











32









YFP signals 477

478

Split-Luc Assay 479














33







498















point 513


34

514








522









531







35

537













times 550

551










36







566

Acknowledgement 567










577

Figure legends 578




37
















of GAL4 596

597






blotting 603


38





608


background 610








618










39




2pad4-5 630



633




22degC 637



control gene 640


22degC 642









40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

4

Introduction 44
























5

























6

























7























135


8

136


9

Results 137
























10





11






















sequences 184

185


12

























13





14









220
















15





16









3F) 246

247















17





18



background 266

267





















19





20




and ACA8 293

294









of the protein 303

304










21





22





23








326

















24





25




signatures 348

349

350


26

Discussion 351
























27





28























processes 399


29

























30



as well 425












437










31


447











458

BiFC Assay 459











32









YFP signals 477

478

Split-Luc Assay 479














33







498















point 513


34

514








522









531







35

537













times 550

551










36







566

Acknowledgement 567










577

Figure legends 578




37
















of GAL4 596

597






blotting 603


38





608


background 610








618










39




2pad4-5 630



633




22degC 637



control gene 640


22degC 642









40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

5

























6

























7























135


8

136


9

Results 137
























10





11






















sequences 184

185


12

























13





14









220
















15





16









3F) 246

247















17





18



background 266

267





















19





20




and ACA8 293

294









of the protein 303

304










21





22





23








326

















24





25




signatures 348

349

350


26

Discussion 351
























27





28























processes 399


29

























30



as well 425












437










31


447











458

BiFC Assay 459











32









YFP signals 477

478

Split-Luc Assay 479














33







498















point 513


34

514








522









531







35

537













times 550

551










36







566

Acknowledgement 567










577

Figure legends 578




37
















of GAL4 596

597






blotting 603


38





608


background 610








618










39




2pad4-5 630



633




22degC 637



control gene 640


22degC 642









40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

6

























7























135


8

136


9

Results 137
























10





11






















sequences 184

185


12

























13





14









220
















15





16









3F) 246

247















17





18



background 266

267





















19





20




and ACA8 293

294









of the protein 303

304










21





22





23








326

















24





25




signatures 348

349

350


26

Discussion 351
























27





28























processes 399


29

























30



as well 425












437










31


447











458

BiFC Assay 459











32









YFP signals 477

478

Split-Luc Assay 479














33







498















point 513


34

514








522









531







35

537













times 550

551










36







566

Acknowledgement 567










577

Figure legends 578




37
















of GAL4 596

597






blotting 603


38





608


background 610








618










39




2pad4-5 630



633




22degC 637



control gene 640


22degC 642









40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

7























135


8

136


9

Results 137
























10





11






















sequences 184

185


12

























13





14









220
















15





16









3F) 246

247















17





18



background 266

267





















19





20




and ACA8 293

294









of the protein 303

304










21





22





23








326

















24





25




signatures 348

349

350


26

Discussion 351
























27





28























processes 399


29

























30



as well 425












437










31


447











458

BiFC Assay 459











32









YFP signals 477

478

Split-Luc Assay 479














33







498















point 513


34

514








522









531







35

537













times 550

551










36







566

Acknowledgement 567










577

Figure legends 578




37
















of GAL4 596

597






blotting 603


38





608


background 610








618










39




2pad4-5 630



633




22degC 637



control gene 640


22degC 642









40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

8

136


9

Results 137
























10





11






















sequences 184

185


12

























13





14









220
















15





16









3F) 246

247















17





18



background 266

267





















19





20




and ACA8 293

294









of the protein 303

304










21





22





23








326

















24





25




signatures 348

349

350


26

Discussion 351
























27





28























processes 399


29

























30



as well 425












437










31


447











458

BiFC Assay 459











32









YFP signals 477

478

Split-Luc Assay 479














33







498















point 513


34

514








522









531







35

537













times 550

551










36







566

Acknowledgement 567










577

Figure legends 578




37
















of GAL4 596

597






blotting 603


38





608


background 610








618










39




2pad4-5 630



633




22degC 637



control gene 640


22degC 642









40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

9

Results 137
























10





11






















sequences 184

185


12

























13





14









220
















15





16









3F) 246

247















17





18



background 266

267





















19





20




and ACA8 293

294









of the protein 303

304










21





22





23








326

















24





25




signatures 348

349

350


26

Discussion 351
























27





28























processes 399


29

























30



as well 425












437










31


447











458

BiFC Assay 459











32









YFP signals 477

478

Split-Luc Assay 479














33







498















point 513


34

514








522









531







35

537













times 550

551










36







566

Acknowledgement 567










577

Figure legends 578




37
















of GAL4 596

597






blotting 603


38





608


background 610








618










39




2pad4-5 630



633




22degC 637



control gene 640


22degC 642









40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

10





11






















sequences 184

185


12

























13





14









220
















15





16









3F) 246

247















17





18



background 266

267





















19





20




and ACA8 293

294









of the protein 303

304










21





22





23








326

















24





25




signatures 348

349

350


26

Discussion 351
























27





28























processes 399


29

























30



as well 425












437










31


447











458

BiFC Assay 459











32









YFP signals 477

478

Split-Luc Assay 479














33







498















point 513


34

514








522









531







35

537













times 550

551










36







566

Acknowledgement 567










577

Figure legends 578




37
















of GAL4 596

597






blotting 603


38





608


background 610








618










39




2pad4-5 630



633




22degC 637



control gene 640


22degC 642









40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

11






















sequences 184

185


12

























13





14









220
















15





16









3F) 246

247















17





18



background 266

267





















19





20




and ACA8 293

294









of the protein 303

304










21





22





23








326

















24





25




signatures 348

349

350


26

Discussion 351
























27





28























processes 399


29

























30



as well 425












437










31


447











458

BiFC Assay 459











32









YFP signals 477

478

Split-Luc Assay 479














33







498















point 513


34

514








522









531







35

537













times 550

551










36







566

Acknowledgement 567










577

Figure legends 578




37
















of GAL4 596

597






blotting 603


38





608


background 610








618










39




2pad4-5 630



633




22degC 637



control gene 640


22degC 642









40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

12

























13





14









220
















15





16









3F) 246

247















17





18



background 266

267





















19





20




and ACA8 293

294









of the protein 303

304










21





22





23








326

















24





25




signatures 348

349

350


26

Discussion 351
























27





28























processes 399


29

























30



as well 425












437










31


447











458

BiFC Assay 459











32









YFP signals 477

478

Split-Luc Assay 479














33







498















point 513


34

514








522









531







35

537













times 550

551










36







566

Acknowledgement 567










577

Figure legends 578




37
















of GAL4 596

597






blotting 603


38





608


background 610








618










39




2pad4-5 630



633




22degC 637



control gene 640


22degC 642









40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

13





14









220
















15





16









3F) 246

247















17





18



background 266

267





















19





20




and ACA8 293

294









of the protein 303

304










21





22





23








326

















24





25




signatures 348

349

350


26

Discussion 351
























27





28























processes 399


29

























30



as well 425












437










31


447











458

BiFC Assay 459











32









YFP signals 477

478

Split-Luc Assay 479














33







498















point 513


34

514








522









531







35

537













times 550

551










36







566

Acknowledgement 567










577

Figure legends 578




37
















of GAL4 596

597






blotting 603


38





608


background 610








618










39




2pad4-5 630



633




22degC 637



control gene 640


22degC 642









40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

14









220
















15





16









3F) 246

247















17





18



background 266

267





















19





20




and ACA8 293

294









of the protein 303

304










21





22





23








326

















24





25




signatures 348

349

350


26

Discussion 351
























27





28























processes 399


29

























30



as well 425












437










31


447











458

BiFC Assay 459











32









YFP signals 477

478

Split-Luc Assay 479














33







498















point 513


34

514








522









531







35

537













times 550

551










36







566

Acknowledgement 567










577

Figure legends 578




37
















of GAL4 596

597






blotting 603


38





608


background 610








618










39




2pad4-5 630



633




22degC 637



control gene 640


22degC 642









40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

15





16









3F) 246

247















17





18



background 266

267





















19





20




and ACA8 293

294









of the protein 303

304










21





22





23








326

















24





25




signatures 348

349

350


26

Discussion 351
























27





28























processes 399


29

























30



as well 425












437










31


447











458

BiFC Assay 459











32









YFP signals 477

478

Split-Luc Assay 479














33







498















point 513


34

514








522









531







35

537













times 550

551










36







566

Acknowledgement 567










577

Figure legends 578




37
















of GAL4 596

597






blotting 603


38





608


background 610








618










39




2pad4-5 630



633




22degC 637



control gene 640


22degC 642









40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

16









3F) 246

247















17





18



background 266

267





















19





20




and ACA8 293

294









of the protein 303

304










21





22





23








326

















24





25




signatures 348

349

350


26

Discussion 351
























27





28























processes 399


29

























30



as well 425












437










31


447











458

BiFC Assay 459











32









YFP signals 477

478

Split-Luc Assay 479














33







498















point 513


34

514








522









531







35

537













times 550

551










36







566

Acknowledgement 567










577

Figure legends 578




37
















of GAL4 596

597






blotting 603


38





608


background 610








618










39




2pad4-5 630



633




22degC 637



control gene 640


22degC 642









40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

17





18



background 266

267





















19





20




and ACA8 293

294









of the protein 303

304










21





22





23








326

















24





25




signatures 348

349

350


26

Discussion 351
























27





28























processes 399


29

























30



as well 425












437










31


447











458

BiFC Assay 459











32









YFP signals 477

478

Split-Luc Assay 479














33







498















point 513


34

514








522









531







35

537













times 550

551










36







566

Acknowledgement 567










577

Figure legends 578




37
















of GAL4 596

597






blotting 603


38





608


background 610








618










39




2pad4-5 630



633




22degC 637



control gene 640


22degC 642









40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

18



background 266

267





















19





20




and ACA8 293

294









of the protein 303

304










21





22





23








326

















24





25




signatures 348

349

350


26

Discussion 351
























27





28























processes 399


29

























30



as well 425












437










31


447











458

BiFC Assay 459











32









YFP signals 477

478

Split-Luc Assay 479














33







498















point 513


34

514








522









531







35

537













times 550

551










36







566

Acknowledgement 567










577

Figure legends 578




37
















of GAL4 596

597






blotting 603


38





608


background 610








618










39




2pad4-5 630



633




22degC 637



control gene 640


22degC 642









40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

19





20




and ACA8 293

294









of the protein 303

304










21





22





23








326

















24





25




signatures 348

349

350


26

Discussion 351
























27





28























processes 399


29

























30



as well 425












437










31


447











458

BiFC Assay 459











32









YFP signals 477

478

Split-Luc Assay 479














33







498















point 513


34

514








522









531







35

537













times 550

551










36







566

Acknowledgement 567










577

Figure legends 578




37
















of GAL4 596

597






blotting 603


38





608


background 610








618










39




2pad4-5 630



633




22degC 637



control gene 640


22degC 642









40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

20




and ACA8 293

294









of the protein 303

304










21





22





23








326

















24





25




signatures 348

349

350


26

Discussion 351
























27





28























processes 399


29

























30



as well 425












437










31


447











458

BiFC Assay 459











32









YFP signals 477

478

Split-Luc Assay 479














33







498















point 513


34

514








522









531







35

537













times 550

551










36







566

Acknowledgement 567










577

Figure legends 578




37
















of GAL4 596

597






blotting 603


38





608


background 610








618










39




2pad4-5 630



633




22degC 637



control gene 640


22degC 642









40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

21





22





23








326

















24





25




signatures 348

349

350


26

Discussion 351
























27





28























processes 399


29

























30



as well 425












437










31


447











458

BiFC Assay 459











32









YFP signals 477

478

Split-Luc Assay 479














33







498















point 513


34

514








522









531







35

537













times 550

551










36







566

Acknowledgement 567










577

Figure legends 578




37
















of GAL4 596

597






blotting 603


38





608


background 610








618










39




2pad4-5 630



633




22degC 637



control gene 640


22degC 642









40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

22





23








326

















24





25




signatures 348

349

350


26

Discussion 351
























27





28























processes 399


29

























30



as well 425












437










31


447











458

BiFC Assay 459











32









YFP signals 477

478

Split-Luc Assay 479














33







498















point 513


34

514








522









531







35

537













times 550

551










36







566

Acknowledgement 567










577

Figure legends 578




37
















of GAL4 596

597






blotting 603


38





608


background 610








618










39




2pad4-5 630



633




22degC 637



control gene 640


22degC 642









40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

23








326

















24





25




signatures 348

349

350


26

Discussion 351
























27





28























processes 399


29

























30



as well 425












437










31


447











458

BiFC Assay 459











32









YFP signals 477

478

Split-Luc Assay 479














33







498















point 513


34

514








522









531







35

537













times 550

551










36







566

Acknowledgement 567










577

Figure legends 578




37
















of GAL4 596

597






blotting 603


38





608


background 610








618










39




2pad4-5 630



633




22degC 637



control gene 640


22degC 642









40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

24





25




signatures 348

349

350


26

Discussion 351
























27





28























processes 399


29

























30



as well 425












437










31


447











458

BiFC Assay 459











32









YFP signals 477

478

Split-Luc Assay 479














33







498















point 513


34

514








522









531







35

537













times 550

551










36







566

Acknowledgement 567










577

Figure legends 578




37
















of GAL4 596

597






blotting 603


38





608


background 610








618










39




2pad4-5 630



633




22degC 637



control gene 640


22degC 642









40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

25




signatures 348

349

350


26

Discussion 351
























27





28























processes 399


29

























30



as well 425












437










31


447











458

BiFC Assay 459











32









YFP signals 477

478

Split-Luc Assay 479














33







498















point 513


34

514








522









531







35

537













times 550

551










36







566

Acknowledgement 567










577

Figure legends 578




37
















of GAL4 596

597






blotting 603


38





608


background 610








618










39




2pad4-5 630



633




22degC 637



control gene 640


22degC 642









40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

26

Discussion 351
























27





28























processes 399


29

























30



as well 425












437










31


447











458

BiFC Assay 459











32









YFP signals 477

478

Split-Luc Assay 479














33







498















point 513


34

514








522









531







35

537













times 550

551










36







566

Acknowledgement 567










577

Figure legends 578




37
















of GAL4 596

597






blotting 603


38





608


background 610








618










39




2pad4-5 630



633




22degC 637



control gene 640


22degC 642









40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

27





28























processes 399


29

























30



as well 425












437










31


447











458

BiFC Assay 459











32









YFP signals 477

478

Split-Luc Assay 479














33







498















point 513


34

514








522









531







35

537













times 550

551










36







566

Acknowledgement 567










577

Figure legends 578




37
















of GAL4 596

597






blotting 603


38





608


background 610








618










39




2pad4-5 630



633




22degC 637



control gene 640


22degC 642









40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

28























processes 399


29

























30



as well 425












437










31


447











458

BiFC Assay 459











32









YFP signals 477

478

Split-Luc Assay 479














33







498















point 513


34

514








522









531







35

537













times 550

551










36







566

Acknowledgement 567










577

Figure legends 578




37
















of GAL4 596

597






blotting 603


38





608


background 610








618










39




2pad4-5 630



633




22degC 637



control gene 640


22degC 642









40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

29

























30



as well 425












437










31


447











458

BiFC Assay 459











32









YFP signals 477

478

Split-Luc Assay 479














33







498















point 513


34

514








522









531







35

537













times 550

551










36







566

Acknowledgement 567










577

Figure legends 578




37
















of GAL4 596

597






blotting 603


38





608


background 610








618










39




2pad4-5 630



633




22degC 637



control gene 640


22degC 642









40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

30



as well 425












437










31


447











458

BiFC Assay 459











32









YFP signals 477

478

Split-Luc Assay 479














33







498















point 513


34

514








522









531







35

537













times 550

551










36







566

Acknowledgement 567










577

Figure legends 578




37
















of GAL4 596

597






blotting 603


38





608


background 610








618










39




2pad4-5 630



633




22degC 637



control gene 640


22degC 642









40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

31


447











458

BiFC Assay 459











32









YFP signals 477

478

Split-Luc Assay 479














33







498















point 513


34

514








522









531







35

537













times 550

551










36







566

Acknowledgement 567










577

Figure legends 578




37
















of GAL4 596

597






blotting 603


38





608


background 610








618










39




2pad4-5 630



633




22degC 637



control gene 640


22degC 642









40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

32









YFP signals 477

478

Split-Luc Assay 479














33







498















point 513


34

514








522









531







35

537













times 550

551










36







566

Acknowledgement 567










577

Figure legends 578




37
















of GAL4 596

597






blotting 603


38





608


background 610








618










39




2pad4-5 630



633




22degC 637



control gene 640


22degC 642









40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

33







498















point 513


34

514








522









531







35

537













times 550

551










36







566

Acknowledgement 567










577

Figure legends 578




37
















of GAL4 596

597






blotting 603


38





608


background 610








618










39




2pad4-5 630



633




22degC 637



control gene 640


22degC 642









40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

34

514








522









531







35

537













times 550

551










36







566

Acknowledgement 567










577

Figure legends 578




37
















of GAL4 596

597






blotting 603


38





608


background 610








618










39




2pad4-5 630



633




22degC 637



control gene 640


22degC 642









40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

35

537













times 550

551










36







566

Acknowledgement 567










577

Figure legends 578




37
















of GAL4 596

597






blotting 603


38





608


background 610








618










39




2pad4-5 630



633




22degC 637



control gene 640


22degC 642









40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

36







566

Acknowledgement 567










577

Figure legends 578




37
















of GAL4 596

597






blotting 603


38





608


background 610








618










39




2pad4-5 630



633




22degC 637



control gene 640


22degC 642









40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

37
















of GAL4 596

597






blotting 603


38





608


background 610








618










39




2pad4-5 630



633




22degC 637



control gene 640


22degC 642









40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

38





608


background 610








618










39




2pad4-5 630



633




22degC 637



control gene 640


22degC 642









40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

39




2pad4-5 630



633




22degC 637



control gene 640


22degC 642









40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

40

650













663


mutants 665









41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

41




676

677










687


responses 689








42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

42




699

700



molecules 703


705





EDS1 and PAD4 710


712

713

714

715

716


43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations

43

717

718

719

720








































































































































Parsed Citations

Reviewer PDF

Parsed Citations







































































































































Parsed Citations

Reviewer PDF

Parsed Citations

calcium pumps and interacting bon1 protein modulate ...€¦ · 12/07/2017 · the bon1 protein...

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