Multiple ER-to-nucleus stress signaling pathways become active … · The IRE1/bZIP60 pathway of unfolded protein response (UPR) is activated by potyviruses and potexviruses, limiting

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Multiple ER-to-nucleus stress signaling pathways become active during Plantago asiatica 1

mosaic virus and Turnip mosaic virus infection in Arabidopsis thaliana. 2

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Mathieu Gayrala, Omar Arias Gaguancelaa, Evelyn Vasquezb, Venura Heratha, Mingxiong Panga, 4

Francisco Javier Florezb,c, Martin B Dickmand, Jeanmarie Verchota,d 5

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a Texas A&M Agrilife Research and Extension Center in Dallas. 17360 Coit Rd, Dallas TX 75252, 7

USA 8

b Departamento de Ciencias de la Vida y la Agricultura, Universidad de las Fuerzas 9

Armadas-ESPE, Sangolquí, Pichincha, Ecuador 10

c Centro de Investigación de Alimentos, CIAL, Facultad de Ciencias de la Ingeniería e Industrias, 11

Universidad Tecnológica Equinoccial-UTE, Quito, Pichincha, Ecuador 12

dDepartment of Plant Pathology and Microbiology, Institute for Plant Genomics and 13

Biotechnology, Texas A&M University, College Station TX, USA 14

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Corresponding author: Jeanmarie Verchot. [email protected] 972-952-9224 17

not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted September 30, 2019. . https://doi.org/10.1101/786137doi: bioRxiv preprint

https://doi.org/10.1101/786137

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Summary 18

Endoplasmic reticulum (ER) stress due to biotic or abiotic stress activates the unfolded protein 19

response (UPR) to restore ER homeostasis. The UPR relies on multiple ER-to-nucleus signaling 20

factors which mainly induce the expression of cytoprotective ER-chaperones. The inositol 21

requiring enzyme (IRE1) along with its splicing target, bZIP60, restrict potyvirus, and potexvirus 22

accumulation. Until now, the involvement of the alternative UPR pathways and the role of UPR to 23

limit virus accumulation have remained elusive. Here, we used the Plantago asiatica mosaic 24

virus (PlAMV) and the Turnip mosaic virus (TuMV) to demonstrate that the potexvirus triple gene 25

block 3 (TGB3) protein and the potyvirus 6K2 protein activate the bZIP17, bZIP28, bZIP60, 26

BAG7, NAC089 and NAC103 signaling in Arabidopsis thaliana. Using the corresponding knock- 27

out mutant lines, we demonstrated that these factors differentially restrict local and systemic 28

virus accumulation. We show that bZIP17, bZIP60, BAG7, and NAC089 are factors in PlAMV 29

infection, whereas bZIP28 and bZIP60 are factors in TuMV infection. TGB3 and 6K2 transient 30

expression in leave reveal that these alternative pathways induce BiPs expression. Finally, using 31

dithiothreitol (DTT) and tauroursodeoxycholic acid (TUDCA) treatment, we demonstrated that the 32

protein folding capacity significantly influences PlAMV accumulation. Together, these results 33

indicate that multiple ER-to-nucleus signaling pathways are activated during virus infection and 34

restrict virus accumulation through increasing protein folding capacity. 35

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Keywords: ER-to-nucleus signaling, Unfolded Protein Response, Plantago asiatica mosaic 37

virus, Turnip mosaic virus, Protein folding capacity. 38

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Significance statement 40

The IRE1/bZIP60 pathway of unfolded protein response (UPR) is activated by potyviruses and 41

potexviruses, limiting their infection, but the role of alternative UPR pathways is unknown. This 42

study reveals the activation of multiple ER-to-nucleus signaling pathways by the Plantago 43

asiatica mosaic virus and the Turnip mosaic virus. We identify additional signaling pathways 44

serve to restrict virus accumulation through increased protein folding capacity. 45


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Introduction 46

The endoplasmic reticulum (ER) and Golgi network house the cellular machinery for 47

protein synthesis, maturation, and delivery to the intended subcellular compartments. 48

Environmental challenges such as heat, drought, and pathogen attack can drastically alter the 49

protein maturation process and cause a bottleneck of malformed proteins to accumulate in the 50

ER. The unfolded protein response (UPR) describes alternative ER-to-nucleus signaling 51

pathways leading to increased nuclear gene expression (Bao et al., 2018, Nawkar et al., 2018). 52

Transmembrane sensors embedded in the ER respond to the accumulation of aberrant proteins 53

to increase the synthesis of chaperones and the endoplasmic-reticulum-associated protein 54

degradation machinery to refold or decay proteins to alleviate any bottleneck of protein 55

maturation in the ER and recover ER homeostasis (Angelos et al., 2017). When ER stress is 56

chronic, and cells cannot return to homeostasis, there is heightened autophagy and cell death 57

(Srivastava et al., 2018). 58

One pathway in plants involves the inositol-requiring enzyme (IRE1) and the basic 59

leucine zipper 60 (bZIP60) transcription factor. Arabidopsis thaliana has two copies of IRE1 60

known as IRE1a and IRE1b that catalyze the splicing of bZIP60 mRNAs (Deng et al., 2013). In 61

the nucleus, the truncated bZIP60 transcription factor upregulates the expression of several 62

ER-resident chaperones including protein disulfide isomerase (PDI), calnexin (CNX), calreticulin 63

(CRT) and the HSP70-like binding immunoglobulin protein (BiP) (Iwata et al., 2008). A separate 64

branch of the IRE1-led pathway targets mRNAs of secreted proteins in a process called 65

Regulated IRE1-Dependent Decay of mRNAs (RIDD). The RIDD activity of IRE1b is linked to the 66

activation of autophagy in response to ER stress (Liu et al., 2012, Bao et al., 2018). 67

The second major pathway is mediated by bZIP17 and bZIP28. Under normal conditions, 68

bZIP17 and bZIP28 reside in the ER membrane (Angelos et al., 2017, Kim et al., 2018). The 69

Bcl-2–associated athanogene 7 (BAG7) is an ER-localized co-chaperone that under regular 70

conditions associates with bZIP28 and BiP in the ER (Williams et al., 2010, Li et al., 2017) 71

(Srivastava et al., 2013). The BAG7 and bZIP28 can be activated in cells following heat stress or 72

chemical stress, while bZIP17 is reported to be activated by salt stress. Upon activation, the 73

BAG7/bZIP28/BiP complex dissociates, and the bZIP28 and BAG7 are available to move 74

between the ER and the nucleus(Williams et al., 2010, Li et al., 2017). The SITE-1 Protease 75

(S1P) and S2P remove the transmembrane domains releasing the bZIP17 and bZIP28 76

transcription factors to enter the nucleus (Liu et al., 2007b, Liu et al., 2007a, Iwata et al., 2017) 77

and activate expression of BiPs and other chaperones, similar to bZIP60 (Nawkar et al., 2018). 78


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The transmembrane domain of BAG7 is also proteolytically removed, and the truncated BAG7 79

translocates to the nucleus where it principally interacts with WRKY28, independent of bZIP28. 80

The BAG7/WRKY28 induce transcription of stress-responsive chaperones, including BAG7 81

itself, which protects the cell and ensures its survival (Li et al., 2017). 82

Generally, bZIP17, bZIP28, and bZIP60 can form homo- and heterodimers (Deppmann et 83

al., 2004) that further increase the possible regulatory combinations. The bZIP factors recognize 84

G-box core promoter sequences (CACGTG). The flanking sequences near each G-box 85

determines recognition specificity by each factor (Ezer et al., 2017). We know very little about 86

which stretches of sequences containing G-boxes are differential targets for homo-and 87

heterodimers of these bZIP factors and cannot yet precisely predict the transcription pattern of 88

genes by examining promoter sequences (Ezer et al., 2017). Nevertheless, experiments have 89

demonstrated that the bZIP60 and bZIP28 recognize the promoters of at least two NAC 90

(NAM/ATAF/CUC) transcription factors (Nawkar et al., 2018). NAC103 is activated by bZIP60 91

through the UPRE-III cis-elements and upregulates the expression of genes that are 92

categorically described as “pro-survival” factors (Sun et al., 2013). NAC089 is localized in the ER 93

membrane and is regulated by bZIP28 and bZIP60 heterodimers through the UPRE and ERSE-I 94

cis-elements (Yang et al., 2014). The silencing of NbbZIP28 or NbNAC089 in Nicotiana 95

benthamiana increases plant susceptibility to the Tobacco mosaic virus (TMV) and Cucumber 96

mosaic virus (CMV) (Shen et al., 2017, Li et al., 2018). During ER stress, NAC089 promotes 97

transcriptional activation of BAG6 (Yang et al., 2014). BAG6 is a co-chaperone with a 98

calmodulin-binding domain and can activate autophagy required for basal immunity against the 99

necrotrophic fungus Botrytis cinerea (Li et al., 2016). 100

Upon entry into the host cells, Plantago asiatica mosaic virus (PlAMV) (genus: 101

Potexvirus) and Turnip mosaic virus (TuMV) (genus: Potyvirus), induce rearrangements of 102

organellar membranes, including the ER (Verchot, 2016). Viruses principally use the ER as a 103

replication site and guide for cell-to-cell movement, forming vesicles or spherules surrounding 104

viral replication complexes (Tilsner et al., 2013, Cabanillas et al., 2018). These 105

vesicles/spherules concentrate viral proteins necessary for replication and protection against 106

cellular defenses. The ER is essential for potyviruses and potexviruses as a guide for cell-to-cell 107

movement through plasmodesmata (Verchot-Lubicz et al., 2010, Grangeon et al., 2013). The 108

movement from cell-to-cell permits the virus to spread inside the leaves, called local infection. 109

Then virus loads into sieve elements and spreads throughout the plant via the phloem, called 110

systemic infection. The potyvirus membrane-binding protein 6K2 and potexvirus triple gene block 111

3 (TGB3) are essential in these mechanisms. Several studies reported that potexviruses and 112


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potyvirus infection induce ER stress and the related UPR signaling (Ye et al., 2011, Ye et al., 113

2013, Verchot, 2014, Zhang et al., 2015, Gaguancela et al., 2016, Verchot, 2016). Each viral 114

protein of TuMV and Potato virus Y (PVY)(genus: Potyvirus) as well as PlAMV and Potato virus 115

X (PVX) (genus: Potexvirus) protein, was transiently expressed in N. benthamiana or 116

Arabidopsis leaves using agro-delivery of binary constructs and it was demonstrated that the 117

potyvirus 6K2 and potexvirus TGB3 proteins function as viral inducer of UPR via the 118

IRE1/bZIP60 pathway (Zhang et al., 2015, Gaguancela et al., 2016). We also used infectious 119

clones of PlAMV and TuMV which have GFP introduced into the viral genomes to inoculate 120

plants and investigate how these viruses interact with the UPR machinery (Ye et al., 2011, Ye et 121

al., 2013, Gaguancela et al., 2016). GFP expression from the viral genome can be used to 122

visualize and quantify virus accumulation in knockout (KO) mutant plants that have defects in the 123

UPR machinery and wild-type Col-0 plants. This powerful technology has enabled us to 124

demonstrate that PlAMV-GFP and TuMV-GFP accumulate to higher levels in local and systemic 125

leaves in ire1a/ire1b and bzip60 mutants than wild-type Col-0 plants (Zhang et al., 2015, 126

Gaguancela et al., 2016). Moreover, we reported that TGB3 preferentially engages IRE1a, 127

whereas 6K2 preferentially engages IRE1b in Arabidopsis plants. In this study, we investigate 128

whether bZIP17 and bZIP28 are engaged in UPR responses to infection by PlAMV-GFP and 129

TuMV-GFP in Arabidopsis plants. Our results reveal that multiple ER-to-nucleus stress signaling 130

pathways respond to TGB3 and 6K2 proteins. These alternative UPR pathways converge to 131

mainly restrict PlAMV-GFP and TuMV-GFP accumulation in plants through enhancing the 132

protein folding capacity of the ER. 133

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Results 135

Potexvirus TGB3 and potyvirus 6K2 induce expression of bZIP60, bZIP28, and bZIP17. 136

Increased accumulation of bZIP60, bZIP28, or bZIP17 transcripts is typically reported as 137

evidence of UPR activation (Iwata and Koizumi, 2012, Fanata et al., 2013). Since research has 138

well established that the potexvirus TGB3 and potyvirus 6K2 proteins activate bZIP60-led 139

upregulation of UPR related genes, we investigated whether bZIP28 and bZIP17 led branch 140

pathways respond to delivery of these viral proteins. We established that quantitative RT-PCR 141

(qRT-PCR) could be used to detect changes in transcript levels of bZIP60 and other UPR related 142

chaperones at 2- and 5- days post infiltration (dpi) following agro-delivery of binary vectors 143

expressing the PlAMV TGB3, PVX TGB3, PVY 6K2, or TuMV 6K2 genes (Ye et al., 2011, Ye et 144

al., 2013, Zhang et al., 2015). In this study, binary constructs expressing PlAMV TGB3, PVX 145


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TGB3, PVY 6K2, and TuMV 6K2 were delivered by agro-infiltration to individual Col-0 leaves 146

(n=3 plants). The treatment of mock controls was with A. tumefaciens cultures that did not 147

contain the constructs expressing the viral genes. Multiple infiltrated leaves were harvested from 148

each plant and pooled for RNA extraction at 2 and 5 dpi. The qRT-PCRs were performed with 149

the three experimental replicates to obtain the average level of RNA accumulation relative to the 150

mock-treated control within each experiment. Experiments were repeated multiple times to 151

confirm the reproducibility of individual experiments. At two dpi, bZIP17, bZIP28, and bZIP60 152

expression levels were between 2- and 11-fold higher than the control, following expression of 153

each viral TGB3 or 6K2 gene (Figure 1a; P<0.05). At 5 dpi, bZIP60 expression levels remained 154

elevated in all samples (P<0.05) while bZIP17 transcripts declined to control levels for most 155

treatments except in PVX TGB3 treated leaves (Figure 1b; P<0.05). The level of bZIP28 156

transcripts also dropped but remained above the control levels in PlAMV TGB3, and PVY 6K2 157

treated leaves (Figure 1b; P<0.05). 158

159

bZIP60 and bZIP17 significantly reduce PlAMV-GFP accumulation. 160

We conducted a series of experiments to learn whether bZIP28 and bZIP17 play a role in 161

local infection by promoting or restricting virus accumulation in the inoculated leaves. 162

PlAMV-GFP infectious clones were used to inoculate bzip60, bzip17, bzip28, bzip60/bzip17 and 163

bzip60/bzip28 KO lines, as well as wild-type Col-0 plants. Immunoblot detection of the viral coat 164

protein (CP), as well as GFP fluorescence were used to detect virus infection in the inoculated 165

leaves. We examined leaves every day and determined that GFP fluorescence appears at 4 dpi. 166

We identified 5 dpi as the optimum time to isolate leaves for analysis (Figure S1). Immunoblots 167

revealed higher levels of CP in bzip60, bzip17, bzip60/bzip17, and bzip60/bzip28 leaves than in 168

bzip28 or Col-0 leaves (Figure 2a). Image J was used to quantify GFP fluorescence value (FV) in 169

individual leaves (n=6). Based on the average FVs, PlAMV-GFP accumulation was significantly 170

higher in the bzip60, bzip17, bzip60/17 and bzip28/bzip60 leaves (between 3.1- and 9.4-fold; 171

P<0.05) than in the Col-0 leaves (Figure 2b). The FV on bzip28 leaves were not different from 172

Col-0 (Figure 2b). These data suggest that bZIP60 and bZIP17 significantly contribute to 173

restricting PlAMV-GFP local infection. 174

To learn if these bZIP factors play a role in promoting or restricting systemic infection, we 175

first conducted a time course to study GFP systemic accumulation until 19 days. GFP 176

fluorescence in Col-0 plants unloaded from the midrib and primary branching veins into the first 177

upper leaves at 12 dpi, and the fluorescence spread throughout several upper leaves 19 days 178

(Figure S1). While there was a continued spread of GFP beyond 19 dpi, the time between 10 and 179


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19 dpi provided a linear range for GFP FVs that allowed comparisons between wild-type and KO 180

lines. By including 10 dpi as a time point, we were able to determine if the systemic infection was 181

earlier or delayed in the KO lines compared to the Col-0 plants. In these and subsequent 182

experiments, Image J was used to quantify systemic GFP fluorescence images taken at 10, 12, 183

17, and 19 dpi (n=6). The FVs were plotted and statistically analyzed (Figure 2c; P<0.05). The 184

systemic FVs due to PlAMV-GFP increased at a high rate in bzip60, bzip17, and bzip60/bzip17 185

plants and were statistically different from the average FVs calculated in Col-0 plants (Figure 2c; 186

P<0.05). It is interesting to note that PlAMV-GFP accumulation is significantly higher in 187

bzip60/bzip17 than in bzip17 plants. However, there was no significant difference between 188

systemic FVs in bzip60 and bzip60/bzip17 plants, suggesting that the bZIP60 and bZIP17 act 189

independently. The average FVs in Col-0, bzip28, and bipz60/bzip28 plants were not significantly 190

different from each other (Figure 2c; P<0.05). We first observed GFP at 10 dpi in the upper 191

leaves of bzip60, bzip17, and bzip60/zip17 plants but at 12 dpi in Col-0, bzip28, and bzip60/28 192

plants (Figure 2d). These combined results indicate that bZIP60 and bZIP17 contribute to 193

restricting the local and systemic infection of PlAMV-GFP, whereas bZIP28 does not limit 194

PlAMV-GFP infection. 195

196

bZIP60 and bZIP28 significantly reduce TuMV-GFP accumulation. 197

We conducted similar experiments to learn whether bZIP28 and bZIP17, alongside 198

bZIP60, promote or restrict TuMV-GFP local and systemic infection. We examined GFP 199

fluorescence each day to identify the optimum duration to measure GFP and coat protein levels 200

that is nearest to the time of gene induction, as direct indicators of virus accumulation in wild type 201

and mutant plants. The onset of TuMV-GFP infection in the inoculated leaves is slower than 202

PlAMV-GFP. Although we first observed GFP at 6 dpi, we identified 8 dpi was preferable for 203

immunoblot analysis of CP levels and fluorescence quantification (Figure S1). Virus CP 204

accumulation was higher in bzip60, bzip28, bzip60/bzip17, and bzip60/bzip28 infected leaves 205

than in Col-0 infected leaves (Figure 2e). The average FVs in the TuMV-GFP inoculated leaves 206

of bzip60, and bzip28 plants were significantly higher than in Col-0 plants (Figure 2f; P<0.05, 207

n=6), whereas the average FVs in bzip17 leaves were not different from the average FVs in 208

Col-0 leaves. These data suggest that bZIP60 and bZIP28, but not bZIP17, contribute to 209

establishing local infection. 210

To learn if these bZIP factors play a role in promoting or restricting TuMV-GFP systemic 211

infection, we conducted time-course experiments to study GFP systemic accumulation over 19 212

days. GFP fluorescence in Col-0 plants appeared to be unloading into the first upper leaves at 12 213


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dpi, and the fluorescence continued to spread through 19 dpi, similar to PlAMV-GFP (Figure S1). 214

We measure GFP fluorescence at 10, 12, 17, and 19 dpi using the Image J software, and the 215

average FVs were statistically compared (n=6). The average GFP FVs were significantly higher 216

in bzip60, bzip28, bzip60/bzip28, and bzip60/bzip17 plants than in bzip17 or Col-0 plants (Figure 217

2g; P<0.05). It is interesting to note that bzip60, bzip28, bzip60/bzip28 are not significantly 218

different, suggesting that effects of the mutations in bZIP60 and bZIP28 are not additive. At 10 219

dpi, GFP appeared systemically in bzip60, bzip28, bzip60/bzip28 and bzip60/bzip17 plants, but 220

appeared at 12 dpi in the upper leaves of bzip17 and Col-0 plants (Figure 2h). Finally, these 221

results reveal that bZIP60 and bZIP28 restricts TuMV-GFP local and systemic infection. 222

223

BAG7 reduces the local accumulation of PlAMV-GFP but not TuMV-GFP. 224

BAG7 is a recently discovered hallmark of UPR during heat stress and is involved in two 225

separate activities. First, BAG7 is engaged in the retention of bZIP28 in the ER. Second, BAG7 226

translocates to the nucleus where it participates with WRKY29 to upregulate genes involved in 227

heat stress resistance (Li et al., 2017). We hypothesized that if bZIP28 228

plays a role in restricting TuMV-GFP but not PlAMV-GFP, then BAG7 may also be engaged in 229

regulating virus accumulation. First, Col-0 and bag7 plants were inoculated with PlAMV-GFP and 230

at 5 dpi, the average FV was 6.9-fold higher in the bag7 than in the Col-0 inoculated leaves 231

(Figure 3a; P<0.05; n=6). Immunoblots also showed that the CP levels were relatively higher in 232

bag7 compared to Col-0 leaves (Figure 3a). These data indicate that BAG7 is engaged in 233

restricting PlAMV-GFP local infection. As in previous experiments, we recorded and statistically 234

analyzed FVs in systemic leaves between 10 and 19 dpi (Figure 3b; n=12). In these experiments, 235

there was no difference in FVs in the upper leaves of Col-0 and bag7 plants (Figure 3b and c; 236

P<0.05). These results demonstrated that BAG7 is essential for restricting PlAMV-GFP local 237

infection in the inoculated leaves but does not limit systemic infection. 238

In TuMV-GFP inoculated and systemic leaves, the FVs and CP levels were similar in 239

bag7 and Col-0 plants (Figure 3d, e and f; P<0.05) demonstrating that BAG7 does not restrict 240

local or systemic TuMV-GFP infection. 241

242

TGB3 and 6K2 induce NAC089 and NAC103 expression. 243

The NAC089 and NAC103 encode transcription factors and are among the canonical 244

UPR genes that relay ER stress signals from bZIP60 and bZIP28. NAC089 regulates 245

downstream genes involved in programmed cell death and proteasome degradation of proteins, 246

such as BAG6, while NAC103 activates downstream UPR genes including CRT1 and CNX1 and 247


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PDI5 (Sun et al., 2013, Yang et al., 2014). To gain evidence that the downstream 248

UPR-responsive genes NAC089 and NAC103 are sensitive to the potexvirus TGB3 or potyvirus 249

6K2 proteins, binary plasmids containing these PlAMV TGB3, PVX TGB3, TuMV 6K2 and PVY 250

6K2 genes were agro-infiltrated to Col-0 as well as bzip60, bzip28 and bzip17 plants. Then we 251

performed qRT-PCR to examine changes in transcript accumulation at 2 dpi. Elevated levels of 252

NAC089 ranged between 8- and 37-fold in Col-0 plants treated with each viral elicitor (Figure 4a; 253

P<0.05). However, in the bzip60, bzip28, and bzip17 plants, the NAC089 transcripts were 254

reduced (Figure 4a; P<0.05). In bzip60/bzip17 and bzip60/bzip28 plants, there was no induction 255

following treatment with each of these viral elicitors, suggesting an additive effect of the 256

combined genes. 257

In Col-0 plants, NAC103 was induced by approximately 2.5-fold following expression of 258

the TGB3 or 6K2 proteins (Figure 4b; P<0.05). In bzip17 and bzip28 plants, NAC103 expression 259

ranged from 3- to 8-fold above the control leaves, except in the case of PVY 6K2 which did not 260

show a significant change in bzip28 plants. Gene induction was not observed in bzip60, 261

bzip60/bzip17 and bzip60/bzip28 plants (Figure 4b; P<0.05). These combined data suggest that 262

TGB3 and 6K2 induced NAC089 and NAC103 expression. 263

264

NAC089, but not BAG6, reduces the systemic accumulation of PlAMV-GFP. 265

NAC089 relays UPR gene activation from bZIP60, bZIP28, and bZIP17, to upregulate ER 266

stress-responsive genes, including BAG6 (Yang et al., 2014). BAG6 is a co-chaperone that is 267

conserved across eukaryotes involved in protein quality control and proteasome elimination of 268

abnormal proteins (Yamamoto et al., 2017). The human BAG6 aids the folding of 269

aggregation-prone proteins, polyubiquitinated proteins, and transmembrane proteins (Kawahara 270

et al., 2013). The Arabidopsis BAG6 contributes to autophagy that is associated with disease 271

resistance to Botrytis cinerea (Li and Dickman, 2016, Li et al., 2016). We observed the 272

accumulation of PlAMV-GFP and TuMV-GFP in the systemic leaves of nac089 and bag6 plants 273

and quantified FVs (Figure 5; n=6). We did not conduct experiments to test the role of NAC103 in 274

virus infection because there are no available homozygous mutant lines (https://abrc.osu.edu) 275

with altered NAC103 expression. 276

As in previous experiments, GFP was a reporter of PlAMV-GFP and TuMV-GFP systemic 277

infection in nac089 and bag6 plants. Among PlAMV-GFP inoculated plants, the FVs were 278

significantly higher in nac089 than in Col-0 plants (Figure 5b; P<0.05). However, the average 279

FVs in Col-0 and bag6 plants were not significantly different (Figure 5b; P>0.05). These results 280

suggest that NAC089, like bZIP60 and bZIP17, although to a lesser extent, reduce PlAMV-GFP 281


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systemic accumulation, whereas BAG6 is not a factor of PlAMV-GFP infection. Regarding 282

TuMV-GFP infection, the FVs in systemic leaves were unaffected in nac089, bag6 compared to 283

Col-0 plants (Figure 5d; P>0.05). Finally, these results suggest that NAC089 has a role in 284

restricting PlAMV-GFP systemic accumulation and that BAG6 is not involved in the systemic 285

accumulation of either virus. 286

287

Increased protein folding capacity reduces PlAMV-GFP accumulation. 288

Cells gain tolerance to ER stress by increasing the protein folding capacity of the ER 289

through enhanced expression of chaperones. BiPs are among the primary targets of UPR 290

signaling pathways converging on their activation in response to ER stress. Because Arabidopsis 291

BiP1 and BiP2 share 97% nucleotide and 99% amino acid sequence identities, we could not 292

sperately quantify these transcript levels using qRT-PCR. BiP3 is 80% identical to BiP1/2, and 293

PCR primers can differentially detect these transcripts (Noh et al., 2003, Srivastava et al., 2013). 294

At 5 dpi, the BiP1/2 transcripts were higher following agro-delivery of TuMV or PVY 6K2 (Figure 295

6a; P<0.05) but not following expression of the PlAMV or PVX TGB3 genes. However, BiP3 296

transcripts were elevated 3- to 4-fold above the control in response to expression of PlAMV 297

TGB3, PVX TGB3, PVY 6K2, and TuMV 6K2 (Figure 6a; P<0.05). 298

To examine the importance of the protein folding capacity in virus accumulation, we 299

quantified virus-GFP fluorescence in infected plantlets grown on MS medium with added DTT 300

and TUDCA. DTT causes significant ER stress in plant cells by reducing protein disulfide bond 301

formation and reducing the protein folding capacity. TUDCA is a chemical chaperone that 302

alleviates ER stress when applied to plants because it mitigates protein aggregation and 303

stabilizes protein conformation (Zhang et al., 2015, Fernández‐Bautista et al., 2017, Uppala et 304

al., 2017). We were unable to infect young plantlets with TuMV-GFP and conducted these 305

experiments only with PlAMV-GFP. We inoculated 10-day-old Col-0 seedlings with PlAMV-GFP 306

and transferred them to MS medium alone, with added 0.1 mM DTT, or with added 0.5 mM 307

TUDCA (Figure 6b and c). At 15 dpi, we measured the fluorescence in crude extracts and 308

calculated the average FV relative to the average sample fresh weight for each treatment (Figure 309

6c). The FV of PlAMV-GFP infected plantlets grown on DTT containing medium were higher than 310

FV of infected plantlets grown on MS medium alone (Figure 6c; P<0.05). These data suggest 311

that compromising the protein folding capacity of the cell favors virus infection. On the other 312

hand, PlAMV infected plantlets grown on the TUDCA containing medium showed lower FV than 313


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the controls (Figure 6c; P<0.05) suggesting that increasing the protein folding capacity leads to a 314

decrease in virus accumulation. 315

We also grew bzip17, bzip28 and bzip60 infected plantlets with PlAMV-GFP on MS 316

media alone, with 0.1 mM DTT or with 0.5 mM TUDCA. These infected plantlets grew slowly on 317

DTT containing medium and we could not generate an adequate fluorescence dataset. Thus, we 318

only analyzed infected seedlings grown on MS and TUDCA containing medium. PlAMV-GFP 319

fluorescence was lower in bzip17, bzip28, and bzip60 plants grown on TUDCA medium 320

compared to the fluorescence obtained from plants grown in regular MS media (Figure 6d; 321

P<0.05). These combined data demonstrate that TUDCA can mitigate mutations affecting the 322

UPR machinery during PlAMV-GFP infection. Finally, this result shows that expanding the 323

protein folding capacity leads to decreased PlAMV-GFP accumulation and suggests that the 324

maintenance of the functional protein folding machinery is vital for plants to restrict virus 325

infection. 326

327

Discussion 328

This study demonstrates that the bZIP17/bZIP28 pathway, alongside the IRE1/bZIP60 329

pathways, restrict PlAMV and TuMV infection in Arabidopsis plants. The PlAMV TGB3 and TuMV 330

6K2 proteins trigger the bZIP28/bZIP17 pathway and the IRE1/bZIP60 pathway of the UPR, 331

which increases the expression of NAC089 transcription factors. These alternative pathways 332

converge to activate expression of chaperones like BiPs, that are essential for the proper 333

function of the cell, but also restrict virus infection. 334

Data presented here demonstrates bZIP60, bZIP17, and BAG7 serve to limit early events 335

in PlAMV-GFP infection. The higher accumulation of PlAMV-GFP in bzip17 and bzip60 than in 336

Col-0 plants, clearly demonstrated that both bZIP17 and bZIP60 have a role in restricting local 337

and systemic infection. In Figure 2 PlAMV-GFP accumulation in the inoculated and systemic 338

leaves in the bzip17 and bzip60 plants were not significantly different, indicating that both factors 339

are essential for restrict PlAMV accumulation. Moreover, systemic infection was not statistically 340

different in the bzip60/bzip17 relative to the bzip60 plants indicating that these factors do not act 341

additively. Since bZIP60 and bZIP17 form heterodimers for inducing target genes (Deppmann et 342

al., 2004), these two factors may act synergistically to induce specific genes that restrict 343

PlAMV-GFP accumulation. We were surprised by the results indicating that PlAMV-GFP 344

accumulation is unaltered mainly in bzip28 and bzip28/bzip60 compared to Col-0 plants. Since 345

PlAMV-GFP CP and FVs are typically higher in bzip60 plants, we expected to observe higher 346

PlAMV-GFP accumulation in both bzip60/bzip28 and bzip60 plants. It is possible that bZIP28 347


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and bZIP60 redundantly upregulate unknown factor(s) providing positive support for PlAMV 348

infection and that defects in both genes overcome the defect in bzip60 plants. Such factors 349

would likely not be downstream of NAC089 (which also serves to limit PlAMV infection) but 350

would likely involve another downstream signal. 351

Given that it is well established that the bZIP17/bZIP28 and IRE1/bZIP60 pathways relay 352

information for nuclear gene expression that activate pro-survival and pro-death pathways in 353

plants, it is reasonable to consider that these ER stress sensors might also initiate signaling 354

cascades that can activate cellular responses that may limit or promote virus infection. In this 355

model, bZIP60 and bZIP17 synergistically induce genes restricting PlAMV infection, whereas 356

bZIP60 and bZIP28 independently induce genes supporting PlAMV infection (Figure S2). In 357

bzip60 and bzip17 plants, bZIP17 or bZIP60 alone do not induce genes restricting virus infection, 358

whereas bZIP28 induces genes that promote higher PlAMV accumulation. In Col-0 and bzip28 359

plants, bZIP60 and bZIP17 may together induce genes restricting virus accumulation, while 360

bZIP60 may act alone to induce genes supporting PlAMV infection. Finally, in bzip60/bzip28 361

plants, the phenotype observed in bzip60 plants is alleviated by the absence of supporting genes 362

induction by bZIP28 (Figure S2). 363

This study also showed that bZIP60 and bZIP28 are principally important for limiting 364

TuMV-GFP accumulation in the inoculated and systemic leaves (Figure S3). The results in 365

Figure 2 indicate that bZIP28 and bZIP60 are essential, although not additive, to limit TuMV-GFP 366

accumulation. The GFP FVs measured in systemic leaves were comparable between bzip60, 367

bzip28, and bzip60/bzip28 plants, suggesting that these factors may synergistically activate 368

genes that limit infection. The bZIP17 and NAC089 likely do not contribute to the restriction of 369

TuMV-GFP accumulation since GFP FVs were unaltered in the bzip17 or nac089 plants 370

compared to Col-0 plants. Based on these results we propose a model in which bZIP60 and 371

bZIP28, but not bZIP17 are virus-limiting factors, that may have overlapping target genes (Figure 372

S3). 373

The bZIP28 resides in a bZIP28/BAG7/BiPs complex in the ER under normal conditions, 374

and this complex dissociates during ER stress, sending bZIP28 and BAG7 into the nucleus 375

where they separately regulate gene expression (Li et al., 2017). Surprisingly loss of BAG7 376

seems to hamper PlAMV-GFP infection in the inoculated leaves whereas bZIP28 is not a factor 377

during PlAMV infection. Further experiments are needed to test whether stress tolerance 378

associated with BAG7 is the result of BAG7 chaperone functions in the ER as well as 379

BAG7-WRKY29 interactions in the nucleus. The hypothesis that BAG7-WRKY29 gene regulation 380

is a factor in virus infection is particularly intriguing because WRKY29 is engaged in 381


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pattern-triggered immunity (Asai et al., 2002). Overexpressing WRKY29 may enhance disease 382

resistance to Fusarium graminearum infection and other pathogens (Sarowar et al., 2019). It is 383

reasonable to consider that the PlAMV TGB3 protein might activate UPR in a manner that 384

produces cytoprotective chaperones while also managing anti-viral immunity. We also postulated 385

that ER-to-nucleus signaling via the bZIP28 branch of the UPR would be less restricted in bag7 386

plants (Williams et al., 2010). Notably, loss of BAG7 expression did not alter the levels of 387

TuMV-GFP in systemic leaves suggesting the difference between bZIP17 and bZIP28 in 388

potyvirus and potexvirus restriction is linked to their own activation. Finally, for both PlAMV-GFP 389

and TuMV-GFP BAG7 seems to act independent of bZIP28 in regulating virus infection. 390

Other research shows that bZIP transcription factors form heterodimers to expand the 391

diversity of ER stress-induced genes that they can activate. Many reports have shown that 392

bZIP17, bZIP28, and bZIP60 co-regulate specific genes in a manner that can expand the pattern 393

of tissue expression, the timing of gene induction, or the magnitude of the response (Liu and 394

Howell, 2010, Henriquez-Valencia et al., 2015, Angelos et al., 2017, Kim et al., 2018, Ruberti et 395

al., 2018). These bZIP transcription factors can also act alone, as homodimers, to activate 396

specific downstream genes. For example, bZIP60 directly binds to the cis-element pUPRE-III 397

(Sun et al., 2013), whereas only bZIP17 can activate some specific downstream transcription 398

responses in response to salt stress (Liu et al., 2007b). Our results support a model in which 399

bZIP60 act independent of bZIP28 for support PlAMV infection or, can act synergistically with 400

bZIP17 or bZIP28 to limit PlAMV and TuMV infection, respectively (Figure 7). Consequently, the 401

different affinities of bZIP17, bZIP28 and bZIP60 to cis-regulatory elements of UPR genes could 402

explain the differences observed between PlAMV and TuMV infection in the KO mutant plants 403

and reveals a fine-tuning of UPR in plants following virus infection. 404

Most reported studies of UPR signaling in Arabidopsis have typically used ER 405

stress-inducing agents such as heat, tunicamycin, or DTT. Investigations to uncover the 406

molecular basis of adaptive UPR typically examine root and shoot growth of seedlings following 407

short term or chronic treatments with these ER stress-inducing agents. The results of such 408

studies indicate that bZIP28 and bZIP60 act in parallel to modulate common sets of ER 409

stress-responsive genes engaged in adaptive UPR and ER stress recovery as well as chronic 410

ER stress (Angelos et al., 2017, Ruberti et al., 2018). Prior studies characterized bZIP28 and 411

bZIP60 as having partially redundant roles in UPR and stress recovery in roots, but function in 412

parallel in shoot tissues (Ruberti et al., 2018). By using identified viral elicitors of UPR and by 413

infecting Arabidopsis plants with PlAMV-GFP and TuMV-GFP, we were able to contrast the 414

transcriptional responses to ER stress and the requirements for bZIP factors to limit virus 415


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14

accumulation. Until now, bZIP17 was only known for its ability to engage in salt stress responses 416

and vegetative growth. This study reports the involvement of bZIP17 in virus-induced ER stress 417

and suggests that bZIP17 is also involved in maintaining ER homeostasis under chronic 418

virus-induced ER stress (Figure 7). 419

This study also examined the roles of two NAC factors that are known to relay information 420

from the bZIP factors for UPR gene activation (Nawkar et al., 2018). The results indicate that 421

NAC089 and NAC103 respond to TGB3 and 6K2 transient expression. PlAMV-GFP, but not 422

TuMV-GFP accumulation was elevated in nac089 than in Col-0 plants, suggesting that NAC089 423

restricts PlAMV infection. NAC089 modulates BAG6 gene expression, and our data shows that 424

BAG6 is not a factor in regulating PlAMV-GFP and TuMV-GFP accumulation. BAG6 was 425

particularly interesting because of its function as a co-chaperone engaged in protein turnover in 426

mammals and its role in plants in regulating autophagy and programmed cell death in response 427

to fungal pathogens (Yang et al., 2014, Li and Dickman, 2016, Li et al., 2016). We carried out a 428

brief analysis of ATG8 lipidation in ire1a/ire1b, bzip60, bzip28, bzip17, nac089 and bag6 plants 429

which did not reveal differences in autophagy processes following PlAMV-GFP and TuMV-GFP 430

infection suggesting that autophagy is not linked to UPR during these infection (Figure. S4). 431

Overall, the results in this study suggest that the role of NAC089 during PlAMV infection is 432

probably linked to regulating ER stress and cellular homeostasis rather than autophagy and cell 433

death. 434

The UPR is mainly responsible for increasing the protein folding capacity of the ER 435

through enhanced expression of the chaperones such as BiP, CRT, CNX, and PDI. We 436

previously demonstrated that TGB3 activates expression of CRT2 and PDI in Arabidopsis and N. 437

benthamiana (Ye et al., 2011) and this study showed that TGB3 and 6K2 induce expression of 438

BiPs. Overexpression of N. tabacum BiP-like protein 4 protects against viral-induced necrosis 439

(Leborgne-Castel et al., 1999, Ye and Verchot, 2011, Ye et al., 2013). We show that decreasing 440

or increasing the folding capacity of Arabidopsis plants through DTT and TUDCA application 441

leads to a significant increase or decrease of PlAMV-GFP accumulation, respectively. Using 442

TUDCA to mitigate defects in UPR signaling during PlAMV infection provided further evidence 443

that maintaining healthy protein folding activities is essential for limiting virus infection. 444

Altogether, these results suggest that during virus infection, UPR works to maintain a functional 445

protein folding machinery for repress virus accumulation (Figure 7). The role of the protein 446

folding machinery in virus inhibition is not clear, but one can imagine that the virus actively 447

highjacks the protein folding machinery from its normal focus on cellular proteins to fold viral 448

proteins. Under these conditions, increasing the protein folding capacity would allow the cell to 449


https://doi.org/10.1101/786137

15

protect homeostatic protein production and ensure proper production of defense proteins. 450

However, regarding the synergic action of bZIP factors involve in UPR associated virus inhibition 451

and differences observed between PlAMV and TuMV, the role of UPR is probably not limited to 452

protein folding capacity and further experiments are needed to delineate the role of UPR in 453

repressing virus accumulation. 454

In conclusion, this paper brings new knowledge on UPR and details the function of the 455

Arabidopsis UPR signaling during potyvirus and potexvirus infection that could be useful for 456

increase plant virus resistance. We provide evidence of the role of bZIP17 under chronic ER 457

stress led by virus infection. We reveal that the two UPR arms seem to be mainly associated with 458

virus inhibition, at the exception of downstream factors of bZIP28 during PlAMV infection and 459

probably repress virus accumulation by increasing protein folding capacity. 460

461

Experimental procedures 462

Plant materials. 463

We obtained from the ABRC (The Ohio State University, Columbus, OH, USA) 464

Arabidopsis thaliana ecotype Columbia-0 (Col-0) and knockout independent homozygous 465

transfer DNA (T-DNA) insertion lines; bzip17 (SALK_104326), bzip28-2 (SALK_132285), 466

bzip60-2 (SAIL_283_B03),nac089 (SALK_201394), bag6-1 (SALK_047959), bag7 467

(Salk_065883), bzip60-2/28-2, bzip60-2/17, and ire1a-2/1b-4. We verified all lines by PCR and 468

maintained them in the laboratory (Williams et al., 2010, Gaguancela et al., 2016, Li et al., 2016). 469

Arabidopsis plants were grown in a long day (16h) or short day (12h) photoperiod at 23°C. 470

471

Agrobacterium delivery of virus genes and infectious clones. 472

We delivered Agrobacterium (GV3101) cultures harboring the pMDC32 and pGWB505 473

binary vectors containing PVY 6K2, TuMV 6K2, PVX TGB3 or PlAMV TGB3 genes by infiltration 474

to the underside of Arabidopsis leaves as previously reported (Gaguancela et al., 2016) 475

(ThermoFisher, Richardson, TX, USA). The pGWB505 constructs have GFP fused to the 3' 476

end of each viral gene. Control Agrobacterium (referred to as mock) harboring the empty 477

pMDC32 or the GFP expressing pXF7FNF2.0 were infiltrated to Arabidopsis leaves. The 478

GFP-tagged infectious clones of TuMV and PlAMV were reported previously (Gaguancela et al., 479

2016). We verified each viral cDNA insertion in each plasmid by sequencing. Culture 480

suspensions harboring the relevant expression constructs were prepared using a solution of 10 481

mM MES-KOH (pH 5.6), 10 mM MgCl2, and 200 μM acetosyringone and adjusted to OD600 = 482


https://doi.org/10.1101/786137

16

0.7 to 1.0. A 1-ml needle-free syringe was used to infiltrate the Agrobacterium suspensions into 483

leaves of 3-week-old Arabidopsis seedlings grown under short-day conditions. Fig. S1 shows 484

representative timelines for PlAMV-GFP and TuMV-GFP infection in Arabidopsis plants. 485

486

RNA extraction and real-time quantitative RT-PCR. 487

We plunged leaf punches into liquid nitrogen and then extracted the RNA using the 488

Maxwell LEV simplyRNA purification kit (Promega Corp., Madison WI, USA). The cDNA 489

synthesis was carried out using the High-capacity cDNA reverse transcription kit (ThermoFisher, 490

Richardson, TX, USA) and random primers. For the qPCR assays, we verified the efficiencies of 491

all primers by endpoint PCR and gel electrophoresis. The transcript abundance was quantified 492

using the Power SYBR Green II PCR master mix (ThermoFisher) in the ABI 7500 PCR and 493

Step-One Plus or the QuantSudio 3 machines (Applied Biosystems, Foster City, CA, USA). The 494

relative increase of cellular transcripts was calculated using the comparative cycle threshold (CT) 495

method, which employs the equation 2-ddCT (Livak and Schmittgen, 2001). The endogenous 496

control was ubiquitin-10 or 18S. Statistical analysis was carried out using the Student t-test to 497

validate the results. 498

499

Immunoblot analysis. 500

For detecting viral proteins, immunoblot analysis was carried out using virus-infected 501

inoculated leaves, harvested at 5 or 8 dpi for PlAMV-GFP or TuMV-GFP, respectively. 502

PlAMV-GFP fluorescence appeared earlier than TuMV-GFP fluorescence in inoculated leaves 503

(Fig. S1). Total protein was extracted using standard methods (Ye et al., 2011) and quantified 504

using the Pierce™ Coomassie Plus assay (ThermoFisher, Rockford, IL, USA). Then nine µg of 505

protein per sample were loaded onto 15% SDS-PAGE gels. For ATG8 detection, we harvested 506

systemically infected leaves at 25 dpi, and total protein was extracted using a buffer comprised 507

of 50 mM NaH2PO4 (pH 7.0), 200 mM NaCl, 10 mM MgCl2, 10% glycerol, 0.2% 508

β-mercaptoethanol and protease inhibitor cocktail (Sigma-Aldrich Co., St. Louis, MO, USA). 509

Then for each sample, 15 µg of protein were loaded onto 15% SDS-PAGE gels containing 6M 510

urea. In both experiments, the Trans-Blot® Turbo™ Transfer System (Bio-Rad Laboratories, 511

Hercules, CA, USA) was used for electroblot transfer of proteins to PVDF membranes where 512

were later probed with antisera detecting the viral CPs (Agdia, Elckhardt, IN, USA) or Atg8 513

(Abcam, Cambridge, UK). 514

515

Analysis of local and systemic infection. 516


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17

Analysis of local and systemic infection was optimized and reported (Gaguancela et al., 517

2016). To analyze PlAMV-GFP or TuMV-GFP in the inoculated leaves, we acquired fluorescent 518

images at 5 dpi and 8 dpi, respectively. To analyze systemic PlAMV-GFP or TuMV-GFPinfection, 519

we acquired fluorescent images at intervals between 10 and 19 dpi. Image J software quantified 520

the fluorescent intensity. The results represent the average fluorescence value (FV) for at least 6 521

plants. For comparisons of inoculated leaves, reported the average FV for each treatment 522

relative to the control. We plotted the FV values representing systemic infection and used 523

ANOVA (P<0.05) to validate the results. 524

525

Chaperone protection assays. 526

Arabidopsis seeds were sterilized with 50% bleach and 1% Triton X-100 for 10 min and 527

then washed five times with sterile water. We stratified seeds for three days at 4°C, and then 528

germinated on solid (0.8% agar) ½- strength Murashige and Skoog medium including 2% 529

glucose (½-MS) in a growth chamber for 10 days under long-day conditions. Seedlings were 530

then vacuum infiltrated with the infectious clones of PlAMV-GFP (OD600=0.5 to 0.7) and 531

transferred to liquid ½-MS medium for 24h. Seedlings were washed with ½-MS medium 532

containing 100 µg/mL Timentin and then cultivated on solid ½-MS medium containing 100 µg/mL 533

timentin plus 01.mM DTT or 0.5mM TUDCA. Plantlets were harvested at 15 dpi and ground in 534

liquid nitrogen before GFP extraction in PBS buffer. We quantified GFP fluorescence using a 535

Fluoroskan FL microplate fluorometer (Ex/Em=485 nm/538 nm), and the results were expressed 536

in arbitrary fluorescence units per fresh weight (FLU/mg) and were averaged for three to four 537

plants. Statistical analysis was conducted using the Student t-test (P<0.05). 538

539

Acknowledgments 540

We thank Steven Howell for providing the homozygous mutant lines used in this study. This 541

research was funded by NSF/USDA-AFRI joint award number 1759034. 542

543

Conflict of interest 544

The authors declare no conflicts of interest. 545

546

Supporting information 547

Figure S1. Representative images are capturing the timeline progression of PlAMV-GFP and 548

TuMV-GFP infection in Col-0 plants. 549


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18

Figure S2. Proposed model for PlAMV infection UPR in mutant plants. 550

Figure S3. Proposed model for TuMV infection UPR in mutant plants. 551

Figure S4. ATG8 lipidation following PlAMV-GFP and TuMV-GFP infection. 552

553

Table S1. Primers used in this study. 554

555

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Uppala, J.K., Gani, A.R. and Ramaiah, K.V. (2017) Chemical chaperone, TUDCA unlike PBA, 671

mitigates protein aggregation efficiently and resists ER and non-ER stress induced 672

HepG2 cell death. Scientific reports, 7, 3831. 673

Verchot-Lubicz, J., Torrance, L., Solovyev, A.G., Morozov, S.Y., Jackson, A.O. and Gilmer, 674

D. (2010) Varied movement strategies employed by triple gene block-encoding viruses. 675

Molecular plant-microbe interactions, 23, 1231-1247. 676

Verchot, J. (2014) The ER quality control and ER associated degradation machineries are vital 677

for viral pathogenesis. Frontiers in plant science, 5, 66. 678

Verchot, J. (2016) How does the stressed out ER find relief during virus infection? Current 679

opinion in virology, 17, 74-79. 680

Williams, B., Kabbage, M., Britt, R. and Dickman, M.B. (2010) AtBAG7, an Arabidopsis 681

Bcl-2-associated athanogene, resides in the endoplasmic reticulum and is involved in the 682

unfolded protein response. Proceedings of the National Academy of Sciences of the 683

United States of America, 107, 6088-6093. 684

Yang, Z.T., Wang, M.J., Sun, L., Lu, S.J., Bi, D.L., Sun, L., Song, Z.T., Zhang, S.S., Zhou, 685

S.F. and Liu, J.X. (2014) The membrane-associated transcription factor NAC089 686

controls ER-stress-induced programmed cell death in plants. PLoS genetics, 10, 687

e1004243. 688

Ye, C., Dickman, M.B., Whitham, S.A., Payton, M. and Verchot, J. (2011) The unfolded 689

protein response is triggered by a plant viral movement protein. Plant physiology, 156, 690

741-755. 691


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Ye, C. and Verchot, J. (2011) Role of unfolded protein response in plant virus infection. Plant 692

signaling & behavior, 6, 1212-1215. 693

Ye, C.M., Chen, S., Payton, M., Dickman, M.B. and Verchot, J. (2013) TGBp3 triggers the 694

unfolded protein response and SKP1‐dependent programmed cell death. Molecular plant 695

pathology, 14, 241-255. 696

Zhang, L., Chen, H., Brandizzi, F., Verchot, J. and Wang, A. (2015) The UPR Branch 697

IRE1-bZIP60 in plants plays an essential role in viral infection and is complementary to 698

the only UPR pathway in yeast. PLoS Genetics, 11, e1005164. 699

700

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Figure Legend 702

Figure 1. TGB3 and 6K2 induces transcription of bZIP17 bZIP28 and bZIP60 genes in 703

Arabidopsis Col-0 leaves. 704

qRT-PCR results are providing relative RNA levels of bZIP17, bZIP28, and bZIP60 following 705

treatments with each viral factor at 2 (a) and 5 dpi (b). Error bars represent the standard 706

deviation (SD); asterisks indicate significant differences to the mock control: Student’s t-test; 707

P<0.05; n=3. 708

709

Figure 2. Enhanced local and systemic infection of PlAMV-GFP and TuMV-GFP in bzip60, 710

bzip17, bzip28, and double mutant plants compared to Col-0 plants. 711

Immunoblots are detecting PlAMV (a) and TuMV (e) CPs in the inoculated leaves at 5 dpi. 712

Ponceau S-stained membranes below the blots show equal protein loading. The labels identify 713

each bzip mutant host below each lane. M= molecular weight marker; “0” is mock. 714

Representative images of PlAMV-GFP (b) and TuMV-GFP (f) inoculated leaves at 5 and 8 dpi 715

respectively. The average fluorescence value (FV) and SD is reported below each image, and 716

the asterisks indicate statistical differences: Student’s t-test; P<0.05; n=6. Scatter plots show the 717

average FVs, SDs, and trend lines for mutant and Col-0 plants inoculated with PlAMV-GFP (c) 718

and TuMV-GFP (g). Letters on the right of each line represents the statistical relatedness of the 719

FVs for each treatment and at each time point as determined by ANOVA; P<0.05; n=6. 720

Representative images of plants infected with PlAMV-GFP (d) and TuMV-GFP (h) at 10, 12, 17, 721

and 19 dpi. Red boxes surround the plants at 10 or 12 dpi showing the first signs of systemic 722

fluorescence, which in some cases was a small area of a single leaf and in other cases was a 723

broad area. 724

725

Figure 3. Local and systemic infection of PlAMV-GFP and TuMV-GFP in bag7 and Col-0 plants. 726

Representative images of PlAMV-GFP (a) and TuMV-GFP (d) inoculated leaves at 5 and 8 dpi, 727

respectively. The average FVs, the SDs, and statistical relatedness are reported at the bottom of 728

each panel. Asterisks indicate statistical differences: Student’s t.test; P<0.05; n=6. Immunoblot 729

analysis detecting PlAMV (a) and TuMV (d) CPs in the inoculated leaves, respectively. Ponceau 730

S-stained membrane below the immunoblot shows total equal protein loading. Representative 731

images of plants infected with PlAMV-GFP (b) and TuMV-GFP (e) at 12, 17and 19 dpi. Scatter 732

plot values and trend lines represent the average FV and SDs for each mutant and Col-0 line 733

inoculated with PlAMV-GFP (c) and TuMV-GFP (f). Letters next to each trend line represent the 734

statistical relatedness of the FVs for each treatment: Student’s paired t.test; P<0.05; n=12. 735


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736

Figure 4. Overexpression of NAC089 and NAC103 in wild-type and bZIP mutants Arabidopsis 737

leaves following agro-delivery of TGB3 and 6K2 genes. 738

Bar graphs depict the average relative NAC089 (a) and NAC103 (b) transcript levels in samples 739

harvested at 2 dpi. Agro-delivery of each viral factor is identified in the chart legend in panel (a). 740

Error bars represent SD and asterisks indicate significant differences between treatments with 741

viral factors and the mock: Student’s t.test; P<0.05; n=3. 742

743

Figure 5. Local and systemic infection of PlAMV-GFP and TuMV-GFP in nac089 and bag6, and 744

Col-0 plants. 745

Representative images of Col-0, nac089, and bag6 plants infected with PlAMV-GFP (a) or 746

TuMV-GFP (d). Scatter plot values and trend lines represent the average FVs and SDs for each 747

mutant line and Col-0 inoculated with PlAMV-GFP (b) and TuMV-GFP (e). Letters next to each 748

trend line represent the statistical relatedness of the FVs for each treatment: Student’s paired 749

t.test; P<0.05; n=6. Percentage of plants that are infected by PlAMV-GFP (c) or TuMV-GFP (f) at 750

10 to 19 dpi. 751

752

Figure 6. TUDCA represses PlAMV-GFP accumulation. 753

qRT-PCR results are providing relative RNA levels of BiP1/2 (a), BiP3 (b) following treatments 754

with each viral factor identified by color in the chart legend. Error bars represent SD and 755

asterisks indicate significant differences to the mock control: Student’s t-test; P<0.05; (n=3). (b) 756

Bright-field and fluorescent images of Col-0 plantlets grown on MS medium, 0.1mM of DTT or 757

0.5mM of TUDCA containing medium and infected with PlAMV-GFP at 15 dpi. (c) Fluorescence 758

from PlAMV-GFP infected Col-0 plantlets grown on MS medium, 0.1mM of DTT or 0.5mM of 759

TUDCA containing medium at 15 dpi. The results are expressed in arbitrary fluorescence units 760

per fresh weight (FLU/mg). The error bars represent SD and asterisks indicate significant 761

difference from plantlet on MS medium: Student’s t.test; P<0.05; n=3-4. (e) Fluorescence from 762

PlAMV-GFP infected bzip17, bzip28 and bzip60 mutant plantlets grown on MS medium or 763

0.5mM of TUDCA containing medium at 15 dpi (FLU/mg). The error bars represent SD and 764

asterisks indicate a significant difference between treatment and non-treated samples: Student’s 765

t-test; P<0.05; n=3-4. 766

767

Figure 7. Proposed model for the function of UPR during virus infection. 768


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During poty- and potexvirus infection, the 6K2 and TGB3 viral proteins, respectively, induce the 769

two arms of UPR. The IRE1 splices the bZIP60 mRNA producing a truncated bZIP60 770

transcription factor. bZIP17 and bZIP28 move to the Golgi apparatus, S1P and S2P release the 771

cytosolic part which goes to the nucleus. Once in the nucleus, bZIP17, bZIP28 and bZIP60 772

induce transcription of chaperone proteins that increase the ER folding capacity leading to 773

repression of virus accumulation. 774


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Multiple ER-to-nucleus stress signaling pathways become active … · The IRE1/bZIP60 pathway of unfolded protein response (UPR) is activated by potyviruses and potexviruses, limiting