1

RESEARCH ARTICLE 1 2

Synergy Among Exocyst and SNARE Interactions Identifies a Functional 3

Hierarchy in Secretion during Vegetative Growth 4

5

6 7

Emily R Larson1,4,5

, Jitka Ortmannová2,4,6

, Naomi A Donald1, Jonas Alvim

1, Michael R Blatt

1§8 and Viktor Žárský

2,3 9

10 1 Laboratory of Plant Physiology and Biophysics, Bower Building, University of Glasgow, Glasgow 11 G12 8QQ, United Kingdom 12 2 Institute of Experimental Botany, Academy of Sciences of the Czech Republic, 165 02 Prague 6, 13 Czech Republic 14 3 Department of Experimental Plant Biology, Faculty of Science, Charles University, 128 44 Prague 2, 15 Czech Republic 16 4 These authors made equal contributions to the project 17 5 Current address: Root Development Research Group, School of Biological Sciences, University of 18 Bristol, Bristol Life Sciences Building, 24 Tyndall Avenue, Bristol, BS8 1TQ, United Kingdom 19 6 Current address: Uppsala Biocenter, SLU, Department of Plant Biology, PO Box 7080, SE-19 750 20 07 Uppsala, Sweden 21 §Author for correspondence: michael.blatt@glasgow.ac.uk 22

23 Short title: Plant exocyst–SNARE interactions 24

25 One-sentence summary: Direct interactions between exocyst and SNARE subunits hierarchically 26 coordinate secretion at the plasma membrane for plant growth. 27

28 §The author responsible for distribution of materials presented in this article in accordance with the 29 policy described in the Instructions for Authors (www.plantcell.org) is Prof. M.R. Blatt 30 (michael.blatt@glasgow.ac.uk) 31

32 33

ABSTRACT 34

Vesicle exocytosis underpins signaling and development in plants and is vital for cell 35

expansion. Vesicle tethering and fusion are thought to occur sequentially, with tethering mediated by 36

the exocyst and fusion driven by assembly of SNARE (Soluble NSF Attachment Protein Receptor) 37

proteins from the vesicle membrane (R-SNAREs or Vesicle Associated Membrane Proteins 38

[VAMPs]) and the target membrane (Q-SNAREs). Interactions between exocyst and SNARE protein 39

complexes are known, but their functional consequences remain largely unexplored. We now identify 40

a hierarchy of interactions leading to secretion in Arabidopsis thaliana. Mating-based split-ubiquitin 41

screens and in vivo Förster Resonance Energy Transfer (FRET) analyses showed that exocyst EXO70 42

subunits bind preferentially to cognate plasma membrane SNAREs, notably SYP121 and VAMP721. 43

The exo70A1 mutant affected SNARE distribution and suppressed vesicle traffic similarly to the 44

dominant-negative truncated protein SYP121C, which blocks secretion at the plasma membrane. 45

These phenotypes are consistent with the epistasis of exo70A1 in the exo70A1 syp121 double mutant, 46

Plant Cell Advance Publication. Published on July 22, 2020, doi:10.1105/tpc.20.00280

©2020 The author(s).

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which shows decreased growth similar to exo70A1 single mutants. However, the exo70A1 47

vamp721 mutant showed a strong, synergy, suppressing growth and cell expansion beyond the 48

phenotypic sum of the two single mutants. These data are best explained by a hierarchy of SNARE 49

recruitment to the exocyst at the plasma membrane, dominated by the R-SNARE and plausibly with 50

the VAMP721 longin domain as a nexus for binding. 51

INTRODUCTION 52

Exocytosis depends on the spatiotemporal formation, delivery and fusion of membrane vesicles that 53

distribute their cargos to the plasma membrane and apoplast. Proteins that regulate exocytotic 54

mechanics include the exocyst complex, which tethers and docks vesicles to target membranes 55

(Terbush et al., 1996; Zarsky et al., 2009; Vukasinovic and Zarsky, 2016), and SNARE (Soluble NSF 56

Attachment Protein Receptor) protein complexes that drive the final steps of vesicle fusion (Jahn and 57

Scheller, 2006; Bassham and Blatt, 2008; Sudhof and Rothman, 2009). Structural protein models 58

suggest the exocyst assembles as a set of helical bundles (Picco et al., 2017; Mei et al., 2018) that are 59

evolutionary conserved and comprise eight subunits (Terbush et al., 1996; Zarsky et al., 2013), SEC3, 60

SEC5, SEC6, SEC8, SEC10, SEC15, EXO70 and EXO84. The EXO70 family of subunits in land 61

plants shows substantial gene expansion, with 23 paralogues in Arabidopsis thaliana that may reflect 62

specialization in the maintenance of different tethering domains (Synek et al., 2006; Cvrckova et al., 63

2012). 64

SNARE proteins are classified by the Q or R amino acid residue found at the core of the 65

interacting assembly and localize to vesicle (R-SNAREs or Vesicle Associated Membrane Proteins, 66

VAMPs) and target (Qa-, Qb-, and Qc-SNAREs) membranes (Fasshauer et al., 1998; Blatt and Thiel, 67

2003; Jahn and Scheller, 2006). SNARE function is conserved within eukaryotes, but these gene 68

families are similarly expanded in land plants consistent with evolutionary specialization (Sanderfoot 69

et al., 2000; Blatt and Thiel, 2003; Pratelli et al., 2004). The Qa-SNAREs, SYP121 and SYP122 have 70

overlapping functions at the plasma membrane and are associated with accelerated traffic during 71

growth as well as specialized functions in abiotic and biotic stress resistance (Grefen et al., 2015; 72

Karnik et al., 2017; Waghmare et al., 2018). SYP121 and SYP122 assemble with cognate R-SNAREs 73

VAMP721 and VAMP722 as functional complexes that fuse vesicles with the plasma membrane 74

(Kwon et al., 2008; Karnik et al., 2013; Karnik et al., 2015). 75

Current models differentiate temporally and mechanistically between vesicle tethering 76

mediated by the exocyst and fusion mediated by the SNAREs to highlight the sequential functions of 77

these protein complexes (Cai et al., 2007; Mei and Guo, 2019). Nonetheless, with the spatial and 78

functional proximities of the two complexes, increasing evidence for interactions between subunits of 79

the separate complexes has surfaced. Several exocyst subunits in yeast interact with SNAREs in vitro 80

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or affect their localization within the cell (Sivaram et al., 2005; Morgera et al., 2012; Shen et al., 81

2013; Yue et al., 2017), leading to the idea of a coordination between exocyst and SNARE complexes 82

at the plasma membrane (Picco et al., 2017; Mei and Guo, 2019). In Arabidopsis, the exocyst 83

EXO70B1 and EXO70B2 subunits interact in vitro with the SNAP33 subunit of the plasma membrane 84

SNARE complex (Pecenkova et al., 2011), although the significance of these interactions in vivo is 85

unknown. 86

One difficulty is the paucity of in vivo data that might speak to the physiological 87

consequences of exocyst and SNARE subunit interactions, especially in plants. Inhibiting SNARE 88

function has been shown to disrupt exocytosis in Arabidopsis and other plant models (Geelen et al., 89

2002; Tyrrell et al., 2007; Karnik et al., 2013; Grefen et al., 2015), and impairing exocyst function 90

affects growth and, in some instances, SNARE distributions within cells (Fendrych et al., 2013; Synek 91

et al., 2017). These observations are consistent with the concept of serial processing from tethering 92

through to vesicle fusion. What has been missing is evidence arising from an interaction between one 93

or more exocyst and SNARE subunits that surpasses any functional consequences of the subunits in 94

isolation. 95

We report here the interactions of members of the EXO70 exocyst subfamily with SNARE 96

proteins that drive vesicle fusion at the plasma membrane and the consequences for secretory vesicle 97

traffic. We show that the omnipresent EXO70A1 exocyst subunit requires the so-called longin 98

regulatory domain of the R-SNARE VAMP721 for interaction. Most important, we report that the 99

loss of a subset of interactors among exocyst and SNARE proteins affecting vesicle traffic leads to 100

synergistic impacts on cell expansion and vegetative growth that are unique to EXO70 interactions 101

with the R-SNARE. The findings demonstrate a functional hierarchy in interactions between 102

EXO70A1 with VAMP721 that suggest a handover of vesicles between exocyst and SNARE 103

complexes that leads to vesicle fusion and establishes a landmark for further exploration of secretory 104

mechanics in plants. 105

106

RESULTS 107

The exocyst and SNARE proteins physically interact 108

EXO70A1 associates dynamically with the plasma membrane, where it has been suggested to 109

provide a landmark for vesicle docking. Indeed, VAMP721 localization is altered in the exo70A1 110

mutant (Fendrych et al., 2013), consistent with this idea. To assess whether direct interactions 111

between the exocyst and one or more SNAREs may underpin these processes, we used a yeast 112

mating-based split-ubiquitin system (mbSUS) (Grefen et al., 2015; Zhang et al., 2018). The mbSUS 113

approach offers a number of advantages over yeast two-hybrid methods in allowing for analysis of 114

interactions between full-length proteins, especially with integral membrane and membrane-115

associated proteins, and in screening weighted against false-positive interactions that facilitates 116

4

comparisons of relative binding specificities (Zhang et al., 2015; Xing et al., 2016; Zhang et al., 117

2018). We tested for interactions with four EXO70 subunits, including EXO70A1 that is widely 118

expressed in the vegetative plant (Synek et al., 2006), EXO70H1 and two EXO70B subunits that have 119

been associated with pathogen defense and autophagy (Pecenkova et al., 2011; Kulich et al., 2013; 120

Wang et al., 2020), challenging these with several plasma membrane-associated SNAREs (Figure 1 121

and Supplemental Figure 1). Strong yeast growth was recovered, notably with EXO70A1 when paired 122

with the plasma membrane Qa-SNAREs SYP121 and SYP122, the plasma membrane R-SNAREs 123

VAMP721, VAMP722, VAMP724 and VAMP727, and to a lesser extent with the cognate Qbc-124

SNARE SNAP33. 125

Increasing methionine concentrations in the medium, to suppress bait expression and test the 126

strength of interactions, showed that EXO70A1 has a modest preference for SYP121 over SYP122, 127

and for VAMP721 over VAMP722. It also showed no appreciable interaction with the endomembrane 128

R-SNARE VAMP723, even though the R-SNARE was strongly expressed in each of the assays129

(Supplemental Figure 2), consistent with a specificity for components assembling in plasma 130

membrane SNARE core complexes. Like EXO70A1, the cognate SNARES SYP121 and VAMP721 131

are widely expressed throughout the plant (Bassham and Blatt, 2008; Karnik et al., 2017; Waghmare 132

et al., 2018). As these two SNAREs generally interacted most strongly with the EXO70 subunits, 133

especially with the widely expressed EXO70A1 subunit of the exocyst, we focused on these 134

interactors. 135

To assess the characteristics of exocyst binding with the R-SNARE, we compared interactions 136

of EXO70A1 with VAMP721 and VAMP723. These R-SNAREs are 89% sequence-identical and 137

exhibit over 94% similarity at the amino-acid level [for R-SNARE protein alignment see 138

Supplemental Figure S1 of Zhang et al. (2015)]. Previous studies showed that domain exchanges 139

between the longin domains of these R-SNAREs is sufficient to alter their binding with the plasma 140

membrane K+ channels KAT1 and KC1 and affect channel gating, effects that were traced to a single 141

amino acid residue that differs between the two R-SNAREs (Zhang et al., 2015; Zhang et al., 2017). 142

Furthermore, a functional mapping of the putative binding surface exposed a secondary binding site 143

for the K+ channels that is common to both R-SNAREs (Zhang et al., 2017), thus arguing against 144

substantial conformational differences between the two R-SNAREs and their mutations. 145

We used the same constructs of VAMP721 and VAMP723 incorporating the respective 146

domain exchanges (Zhang et al., 2015; Zhang et al., 2017) to explore the R-SNARE domains required 147

for interaction with the EXO70A1 subunit bait. We observed yeast growth in mbSUS assays with 148

VAMP723 only when the VAMP721 longin domain was substituted for that of VAMP723; 149

conversely, yeast growth was lost when the longin domain of VAMP723 was substituted for the 150

longin domain in VAMP721. A similar loss of interaction was also observed with the other EXO70 151

subunits, indicating that the VAMP721 longin domain incorporates an essential structural component 152

5

determining EXO70 binding (Figure 1B and Supplemental Figures 3 and 4) much as it does for 153

VAMP721 binding with the K+ channels. The finding is of interest, because both R-SNARE partners 154

have been proposed as plasma membrane landmarks affecting the final stages of vesicle fusion 155

(Grefen et al., 2015; Karnik et al., 2017; Zhang et al., 2017). 156

To validate the EXO70 interactions in vivo, we used Förster Resonance Energy Transfer 157

(FRET) after transiently transforming Nicotiana benthamiana leaves with mCherry-EXO70A1 and 158

the several GFP-VAMP protein fusions under control of the 35S promoter. The constructs were 159

incorporated within the pFRETgc-2in1 multi-cistronic vector to ensure equal genetic loads as well as 160

providing markers for expression on a cell-by-cell basis (Hecker et al, 2015). These experiments were 161

combined with mCherry-SYP121 and GFP-VAMP721 and with mCherry-EXO70A1 with free 2xGFP 162

as positive and negative controls, respectively. To accommodate differences in fluorescence 163

intensities of the protein fusions, we used the PixFRET method (Feige et al., 2005) that corrects for 164

spectral bleed-through relative to fluorophore intensity and for bias arising from differences in 165

fluorophore emission that would otherwise affect comparisons of FRET across interacting partner 166

sets. Analysis of the resulting FRET images showed a strong signal with the SYP121-VAMP721 167

positive control and a lower, but highly significant signal when the R-SNARE was paired with 168

EXO70A1, but no appreciable signal between EXO70A1 and VAMP723 or the negative free GFP 169

control, despite the comparable levels of donor and acceptor fluorescence (Figure 2). These data 170

support the capacity for EXO70A1 and VAMP721 to interact in planta and are consistent with the 171

mbSUS findings on heterologous expression in yeast. 172

173

Impaired exocyst function inhibits secretion at the plasma membrane 174

To explore the contributions of EXO70A1 subunit to secretory vesicle traffic, we used the 175

tetracistronic expression vector pTecG–2in1–CC (Karnik et al., 2013; Grefen et al., 2015; Zhang et 176

al., 2015). The pTecG–2in1–CC vector incorporates, within a single backbone, expression cassettes 177

for secreted YFP (secYFP) to monitor traffic, GFP-HDEL as a marker for transformation and for 178

ratiometric quantification of secretion, and two additional cassettes for expressing proteins of interest. 179

Using this vector ensured equal genetic loads for the encoded proteins, enabling the essential controls 180

for quantification of their actions on a cell-by-cell basis, which would not be possible by other means 181

(Grefen and Blatt, 2012). Previous studies showed that blocking secretory traffic yielded a substantial 182

increase in the cellular YFP/GFP fluorescence ratio recorded by confocal fluorescence imaging 183

(Karnik et al., 2013) and concomitant and quantitative loss in secYFP protein collected from the 184

apoplast (Grefen et al., 2015). 185

We transiently expressed the pTecG–2in1–CC vector with secYFP and GFP-HDEL alone in 186

wild-type and exo70A1 Arabidopsis seedling roots to visualize and quantify secretion at the plasma 187

membrane. As a positive control for secretory block, seedlings were transiently transformed with the 188

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same vector carrying the cytoplasmic domain of SYP121 (SYP121∆C) that functions as a dominant 189

negative fragment and suppresses secretion at the plasma membrane (Tyrrell et al., 2007; Grefen et 190

al., 2015). Expressing SYP121∆C in wild-type and exo70A1 mutant seedlings provided a basis for 191

comparing the impact on secretion in each genetic background. 192

We quantified a secretory block as the ratio of YFP to GFP fluorescence in live imaging of 193

the transiently-transformed seedlings by confocal microscopy (Figure 3). In the absence of SYP121∆C, 194

we observed a significant block of secretion in the exo70A1 mutant compared to the wild-type 195

seedlings, evidenced by the elevated mean in the YFP/GFP ratio. Seedlings transformed to express the 196

SYP121∆C fragment in addition to the fluorescent proteins yielded a similar mean YFP/GFP ratio in 197

the exo70A1 mutant. By contrast, the mean YFP/GFP ratio was statistically different and much higher 198

on expressing the SYP121∆C fragment in wild-type than in its absence. These results showed that the 199

impaired exocyst function in the exo70A1 mutant inhibits secretion to the plasma membrane with an 200

efficacy comparable to that of the SYP121∆C fragment. Furthermore, given the absence of further 201

secretory suppression in the presence of the SYP121∆C fragment, the results suggest that this 202

inhibition functionally overlaps with that introduced by the Qa-SNARE fragment. 203

204

SNARE protein localization is altered in the exo70A1 mutant 205

Both the exocyst and a subset of SNARE proteins associate dynamically with the plasma 206

membrane. The exocyst forms a peripheral membrane complex (Fendrych et al., 2013; Synek et al., 207

2014; Kulich et al., 2018). The Qa- and R-SNAREs are integral membrane proteins; whereas SYP121 208

localizes to the plasma membrane (Lipka et al., 2007; Honsbein et al., 2009; Grefen et al., 2015; 209

Waghmare et al., 2019), the R-SNAREs, including VAMP721, localize to the vesicle membrane and 210

associate transiently with the plasma membrane (Uemura et al., 2005; Zhang et al., 2011; Zhang et al., 211

2015). We therefore asked if the loss of one interacting partner could affect the localization of the 212

other as a possible mechanism to explain secretory block. 213

For this purpose, SNARE and exocyst GFP protein fusion constructs were stably transformed 214

into the reciprocal exocyst and SNARE mutant backgrounds. We quantified their distributions as 215

ratios between the fluorescence signal present at the cell periphery, delineated as the first 2 m from 216

the cell surface, and in the interior of cells within the hypocotyl and roots of transgenic plants, 217

normalizing the fluorescence distribution to the total fluorescence signal in each case. In the exo70A1 218

mutant, we observed a pronounced shift in VAMP721-GFP away from the cell periphery (Figure 219

4A,C), much as has been reported also for the sec84b exocyst mutant (Fendrych et al., 2013). 220

Similarly, VAMP722-GFP and SYP121-GFP distributions were reduced at the cell periphery in 221

exo70A1 mutants compared to the wild type (Figure 4A,B). However, the GFP-EXO70A1 signal, 222

which localized primarily to the cell periphery, was not significantly affected in each of the SNARE 223

mutant backgrounds (Figure 4C,D). These results suggest that EXO70A1 is important either for 224

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localization of the SNAREs or their maintenance at the cell periphery, especially of the R-SNAREs. 225

This interpretation is consistent with the idea that EXO70A1 is important for exocyst targeting 226

(Fendrych et al., 2013; Kalmbach et al., 2017) and its loss delocalizes and destabilizes the exocyst 227

complex, thereby affecting SNARE complex assembly, SNARE protein delivery or recycling. The 228

interpretation is also supported by evidence from yeast that suggests exocyst subunits can bind both 229

vesicle- and plasma membrane-associated proteins (Shen et al., 2013; Yue et al., 2017; Mei and Guo, 230

2019; Rossi et al., 2020). 231

232

Differential effects of exocyst-SNARE interactions on plant growth 233

Single mutants of several plasma membrane SNAREs normally show subtle defects in 234

Arabidopsis; these weak phenotypes are generally thought to reflect functional redundancies. For 235

example, the single R-SNARE mutants vamp721 and vamp722 are largely indistinguishable from the 236

wild type and only the double vamp721vamp722 mutant is lethal (Zhang et al., 2011). Similarly the 237

single Qa-SNARE mutants syp121 and syp122 exhibit strong phenotypes that surface only when 238

challenged with abiotic and biotic stress (Zhang et al., 2008; Eisenach et al., 2012; Waghmare et al., 239

2018), again reflecting functional overlaps as well as potential specificities among non-canonical 240

protein partners. The single mutant exo70A1 also exhibits a range of phenotypes, including reduced 241

vegetative growth and cell-specific patterning (Synek et al., 2006; Fendrych et al., 2010; Kalmbach et 242

al., 2017; Vukasinovic et al., 2017). These characteristics are consistent with the effects of the 243

exo70A1 mutant in secretory traffic and SNARE localization, despite the apparent lack of any additive 244

effect over that of the SYP121∆C dominant negative fragment (Figure 3 and 4). 245

Vesicle tethering by the exocyst is thought to occur before the SNAREs assemble to drive 246

vesicle fusion, but both are necessary for cargo secretion (Bassham and Blatt, 2008; Ravikumar et al., 247

2017). We therefore hypothesized that loss of both EXO70A1 and single SNARE gene expression 248

should exhibit defects synergistic to those of the single mutant parents if the functions of the gene 249

products are closely connected. The exo70A1 null mutant is male lethal and is impaired in stigma 250

receptivity (Synek et al., 2006). Therefore we crossed exo70A1+/- heterozygote with the homozygotic 251

mutants vamp721-/- and syp121-/- and examined the segregation of the double mutant in the F2 252

generations. Growth of the syp121 mutant is normally only marginally retarded compared to the wild 253

type (Bassham and Blatt, 2008; Eisenach et al., 2012). Like the exo70A1 single mutant, we found that 254

growth of the double exo70A1 syp121 mutant showed a significant but modest reduction in vegetative 255

growth in the F2 generation, suggesting that the exo70A1 mutation is epistatic to that of syp121. 256

By contrast, the F2 population of the exo70A1+/- and vamp721-/- cross yielded a segregating 257

population of extremely dwarfed plants, each of which was identified as a exo70A1 vamp721 double 258

mutant (Figure 5A,B). The exo70A1 vamp721 double mutant plants could be maintained for up to 10 259

weeks when grown under a short-day light cycle, but the plants remained severely dwarfed, showed 260

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late lethality, and failed to flower. We therefore maintained the double mutant as a exo70A1+/-261

vamp721-/- segregating population. 262

To examine the mutant lines in greater detail, we grew seedlings on agar plates for 10 d and 263

examined the characteristics of early development following germination. The exo70A1 vamp721 264

double mutants showed severely stunted growth in the root and cotyledon expansion was much 265

reduced compared to the wild-type seedlings and the single mutant parents (Figure 5C). These 266

phenotypes were evident 7-10 d after germination. Transient transformation by co-cultivation must be 267

started with younger seedlings, precluding direct assays of traffic using the pTecG–2in1–CC vector 268

with secYFP and GFP-HDEL (see Figure 3). Nonetheless, we noted that the exo70A1 vamp721 269

double mutants produced small, delicate leaves with morphologically similar structure. Quantitative 270

analysis of the epidermal cells, which are important drivers in leaf expansion (Walter et al., 2009), 271

showed that in the exo70A1 vamp721 double mutants these cells were significantly smaller than those 272

from leaves of the wild-type and single mutant plants (Figure 5D,E). These observations are 273

consistent with suppressed cell expansion arising from the impaired traffic of the exo70A1 vamp721 274

double mutant, and they implicate specific overlaps in the progression of exocyst-SNARE interactions 275

that occur in the events leading up to secretion. 276

277

DISCUSSION 278

Models of exocytosis have generally divided the process into three stages, vesicle tethering, 279

docking, and fusion, each thought to occur as part of a serial progression with distinct sets of proteins 280

that regulate each step (Cai et al., 2007; Bassham and Blatt, 2008; Zarsky et al., 2009). Major protein 281

assemblies contributing to this progression include exocyst and SNARE complexes, each of which 282

relies on interactions between a discrete set of protein subunits and on their correct cellular location 283

for function. Even so, there is growing evidence that these assemblies are not isolated interaction 284

networks (Shen et al., 2013; Mei and Guo, 2019; Rossi et al., 2020) nor, as we now show, are their 285

functional impacts. Here, we present evidence in vitro and in vivo that point to direct interactions in 286

Arabidopsis between members of the exocyst complex EXO70 subunit family that are important for 287

vesicle tethering and recruitment to the plasma membrane, and the SNAREs that drive the final stages 288

of membrane fusion. Furthermore, the functional bias evident among these interactions leads to a 289

clear hierarchy between exocyst-SNARE binding partners with differential consequences for SNARE 290

protein localization, secretion, cellular expansion and vegetative growth. Thus, our findings point both 291

to an overlap between the exocyst and SNAREs, and to a temporal 'handshake' between subunits of 292

these protein complexes that is necessary for vesicles to progress through the final stages of tethering 293

and fusion. 294

295

Exocyst - R-SNARE interactions engender a functional synergy in membrane traffic 296

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A singular feature of the EXO70-SNARE interactions we uncovered through yeast mating-297

based split-ubiquitin screening is its R-SNARE bias. Each of the major, plasma membrane-associated 298

SNAREs – the Qa-SNARE SYP121, the R-SNAREs VAMP721, VAMP722, VAMP724 and 299

VAMP727 and to lesser extents the Qa-SNARE SYP122 and Qbc-SNARE SNAP33 – bound 300

differentially with several EXO70 subunits, but not with the endomembrane R-SNARE VAMP723 301

(Figure 1, Supplemental Figure 1). These data also highlighted an overall pattern among the EXO70 302

subunits that favored the plasma membrane R-SNAREs, as demonstrated by VAMP721. In secretory 303

traffic assays, we found no evidence for an additional impact of the cognate Qa-SNARE SYP121 in 304

secretory trafficking assays (Figure 3) when combined with the exo70a1 mutant. The block of traffic 305

in the the exo70a1 mutant was comparable to that of the dominant-negative SYP121C fragment that 306

competes with the full-length Qa-SNAREs SYP121 and SYP122 to suppress secretion (Tyrrell et al., 307

2007; Grefen et al., 2015), and their combination yielded no evidence of additive interaction. 308

Similarly, the strongest effect in complementary protein localization assays was of the exo70a1 309

mutant on VAMP721 (Figure 4). 310

Most telling, however, we uncovered a functional synergy of EXO70A1 with the R-SNARE. 311

Crossings to yield the exo70a1 syp121 double mutant resulted in mutant plants with characteristics 312

similar to the effects of the exo70a1 mutant; by contrast, the exo70a1 vamp721 double mutant was 313

strongly impaired in cell expansion, exhibited severe dwarfing in vegetative growth, and failed to 314

transit to flowering, even though the vamp721 mutant showed no impact on cell expansion or 315

vegetative growth (Figure 5) (Zhang et al., 2011; Zhang et al., 2015; Zhang et al., 2017). The effects 316

of EXO70A1 with the Qa-SNARE can be understood as the result of the sequential actions of the 317

exocyst and Qa-SNARE, so that the exocyst subunit mutation dominates over events engaging the Qa-318

SNARE. Such is not true for the effects of the EXO70 subunit mutation with that of the R-SNARE. In 319

this case, the strong synergy evident in vivo is best understood in context of a mutual interdependence 320

in the roles of the two proteins. Most important, this synergy implies a clear hierarchy among 321

interactions between the Qa-SNARE SYP121 and the R-SNARE VAMP721 with EXO70A1. 322

How is the spatio-temporal overlap of EXO70 and R-SNARE binding enfolded within the 323

respective roles of these subunits within the functions of their respective complexes? In vivo and 324

cryo-electron microscopy analyses of the exocyst complex indicate a topologically optimal space 325

between the exocyst complex, plasma membrane, and tethered vesicle membrane in yeast that could 326

accommodate exocyst and SNARE proteins in association (Picco et al., 2017; Mei et al., 2018). This 327

proximity of exocyst and SNARE complexes fits well with the evidence of direct physical interaction, 328

the requirement for EXO70A1 in SNARE distributions (Figures 2 and 4), and with previous evidence 329

for a close complementarity in protein localizations in yeast and plants (Fendrych et al., 2013; Shen et 330

al., 2013). The exocyst topology suggests that the EXO70 subunit is exposed to the cytosol when the 331

exocyst complex is anchored to the plasma membrane. Such exposure would certainly offer a surface 332

10

for interaction with the R-SNARE and its cytosolic longin domain as the vesicle membrane 333

approaches its target. Indeed, recent findings implicate vesicle tethering in yeast that depends on R-334

SNARE binding with Sec6 associated in complex with EXO70: Rossi, et al (2020) developed an in 335

vitro assay using vesicle clustering to monitor tethering, incorporating components of the exocyst, to 336

show that clustering is enhanced by the vesicle-localized R-SNARE Snc2 and strongly promoted with 337

EXO70 mutants that bypass Rho GTPase activation. These results, and reconstructions from single-338

particle electron microscopy, indicate a conformational change of the holocomplex on activation by 339

the Rho GTPase that exposes a binding site within the Sec6-EXO70 complex for the R-SNARE. 340

Although interactions in yeast between the R-SNARE and exocyst appear dominated by binding to 341

the exocyst Sec6 subunit, in vitro assays have indicated a direct interaction between the EXO70 342

subunit and Snc2 (Shen et al., 2013). Assuming similar topological relationships for Arabidopsis, the 343

functional synergy uncovered for EXO70A1 and VAMP721 leads us to posit that these binding events 344

precede SYP121 introduction and the assembly of VAMP721 with the Qa-SNARE leading to vesicle 345

fusion. 346

347

EXO70 and SNARE redundancies 348

It is perhaps surprising that, unlike the parental exo70A1 and vamp721 mutants, the exo70A1 349

vamp721 double mutant exhibited so profound a set of phenotypes allied to secretory traffic. The 350

EXO70 subfamily in Arabidopsis incorporates 23 members and, although the majority of these differ 351

in developmental and tissue-specific expression, there are nonetheless overlaps (Cvrckova et al., 352

2012). Similarly, VAMP721 and VAMP722 are near sequence-identical and, with the exception of 353

the associated interactions with the Sec1/Munc18 protein SEC11 that distinguish their binding 354

between the plasma membrane Qa-SNAREs SYP121 and SYP122 (Karnik et al., 2015), the two R-355

SNAREs appear functionally redundant. Nonetheless, the several plasma membrane Qa- and R-356

SNAREs, although cross-interactive and seemingly redundant in function, show very different 357

phenotypes. Consider SYP121 and SYP122 that both bind the R-SNAREs VAMP721 and VAMP722. 358

The single syp121 and syp122 mutant plants appear similar to the wild type when grown under 359

permissive conditions (Zhang et al., 2007), and the dominant-negative SYP121C and SYP122C 360

fragments are equally effective in secretory traffic block in vivo (Tyrrell et al., 2007; Grefen et al., 361

2015). Yet the two Qa-SNARE null mutants show very different phenotypes in pathogen defence and 362

abiotic stress resistance (Collins et al., 2003; Zhang et al., 2007; Eisenach et al., 2012) and the Qa-363

SNAREs mediate the traffic of different subsets of secretory cargo (Rehman et al., 2008; Waghmare 364

et al., 2018). These, and associated studies (Reichardt et al., 2011; Hashimoto-Sugimoto et al., 2013; 365

Xia et al., 2019; Zhang et al., 2019) implicate a functional network of trafficking and related 366

regulatory protein partners that is substantially more dynamic than may be deduced from an analysis 367

of gene families. 368

11

Our present data do not speak directly to the functional specificities among the several plasma 369

membrane SNAREs with EXO70A1. Even so, when compared with VAMP722, we note that the 370

peripheral localization of VAMP721 was most strongly affected in the exo70a1 mutant (Figure 4), 371

which may indicate more subtle functional differences between the two R-SNAREs that have yet to 372

be resolved. Furthermore, the strong phenotypic synergy in the exo70A1 vamp721 double mutant that 373

we identified indicates that the presence of VAMP722 is not sufficient to compensate for the loss of 374

VAMP721. One simple explanation is that EXO70A1 and VAMP721 may be specialized as a 375

functional unit, analogous to that implicated in yeast (Shen et al., 2013; Rossi et al., 2020), for the 376

delivery of a unique set of cargos critical to the growth of the plant, much as is now evident from 377

analysis of the distribution of cargos between the Qa-SNAREs SYP121 and SYP122 (Waghmare et 378

al., 2018). 379

Finally, our data focus attention to the longin domain of the R-SNARE. The longin domain of 380

VAMP721 was previously shown to be a key regulator in SNARE protein interactions (Uemura et al., 381

2005). It is essential for selective binding with non-canonical K+ channel partners that have been 382

suggested to coordinate secretion with K+ uptake (Zhang et al., 2015) and, as we demonstrate, it is 383

important for the interactions with several EXO70 subunits (Figure 1, Supplemental Figures 1 and 3). 384

Thus, the VAMP721 longin domain may function as the nexus in a progression of hand-offs between 385

multiple binding partners. We speculate that the exocyst complex could participate in targeting 386

VAMP721-loaded vesicles to the plasma membrane through direct interactions between SNAREs and 387

the exocyst during vesicle tethering and/or recruitment to the plasma membrane (Figure 6). Important 388

questions now will be to identify the domains within the plant EXO70 and SNARE proteins that are 389

critical for their interactions and to determine their dynamics in context of vesicle delivery to the 390

plasma membrane. 391

392

METHODS 393

Plant growth conditions, crosses and genotyping 394

Seeds from wild-type (Col-0) and mutant Arabidopsis thaliana were stratified in the dark at 395

4C and then germinated in long-day conditions in growth chambers (60% relative humidity, 16 h:8 h 396

(light:dark), 22°C) on half-strength Murashige and Skoog (MS) medium solidified with 1% agar. 397

Sealed plates were placed in long-day light conditions (150 mol m-2s-1 PAR) before seedlings were 398

evaluated for mutant phenotypes and collected for genotyping or were transplanted to single pots or 399

15-holder trays for further growth and observation. 400

To generate the exo70A1 vamp721 double mutant, the heterozygous exo70A1+/- 401

(SALK_135462) mutant was used as a maternal parent in a cross with the homozygous vamp721 402

mutant (SALK_037273). The F1 generation was allowed to self-fertilize and the F2 seed was collected 403

for further analysis. Tissue was harvested from individual F1 and F2 plants of the ex70A1+/- x vamp721 404

12

cross for PCR analysis using gene-specific and T-DNA primers (Supplemental Table 1). Double 405

heterozygous F1 mutants were identified and allowed to set seed. Since the exo70A1 single mutant is 406

infertile, a segregating population of exo70A1+/-vamp721-/- was maintained for isolating double 407

mutants. For the exo70A1 syp121 double mutant, the exo70A1+/- (SALK_135462) line was crossed 408

with the syp121 null mutant carrying a point mutation that encodes a premature stop codon and does 409

not produce a transcript but introduces a MluI restriction site that is identifiable as a band shift on 410

digestion (Pajonk et al., 2008).The double mutant was propagated as an exo70A1+/-syp121-/- line. 411

412

Yeast and plant vector construction 413

All plasmids were constructed using the Gateway cloning system (Thermo Fisher Scientific, 414

UK). Details for all yeast and plant expression vectors are included in publications as cited in the text 415

and will be found at www.psrg.org.uk. 416

417

Mating-based split-ubiquitin system (mbSUS) 418

Haploid yeast strains for bait and prey, THY.AP4 and THY.AP5 respectively, were used to 419

express pMetYOst-Dest or pNX35-Dest constructs of exocyst subunits or SNARE proteins and 420

analyse protein-protein interactions as described previously (Zhang et al., 2017). Ten to 15 yeast 421

colonies were selected and inoculated into selective media (CSM-LM for THY.AP4 and CSM-MTU for 422

THY.AP5) for overnight growth at 180 rpm and 28°C. Liquid cultures were harvested and 423

resuspended in YPD medium. Yeast mating was performed in sterile PCR tubes by mixing equal 424

aliquots of yeast containing bait and prey construct. Aliquots of 5 µl were dropped on YPD plates and 425

incubated at 28°C overnight. Colonies were transferred from YPD onto CSM-LTUM plates and 426

incubated at 28°C for 2-3 days. Diploid colonies were selected and inoculated in liquid CSM-LTUM 427

media and grown at 180 rpm 28°C overnight before harvesting by centrifugation and resuspension in 428

sterile water. Serial dilutions at OD600 1.0 and 0.1 in water were dropped, 5 µL per spot, on CSM-429

LTUMAH plates with added methionine. Plates were incubated at 28°C and images were taken after three 430

days. Yeast were also dropped on CSM-LTUM control plates to confirm mating efficiency and cell 431

density, and growth was imaged after 24 h at 28°C. To confirm construct expression, mated yeast was 432

grown overnight in 5 ml selective medium and harvested by centrifugation at 5,000xg. Pelleted yeast 433

was resuspended in diluted lysis buffer (1/1 v/v, 10% SDS, 4 mM EDTA, 0.2% Triton-X 100, 0.01% 434

bromophenol blue, 20 mM DTT, 20% Glycerol and 100 mM Tris, pH 6.8.), sonicated 2x for 30 s and 435

boiled at 100°C for 5 min. Prepared protein samples were loaded on 10% polyacrylamide gels and 436

blotted onto a nitrocellulose membrane. The membrane was blocked overnight (1x PBS, 0.25% 437

Tween, 5% low fat milk) and then incubated with primary α-HA or α-VP16 antibodies at 1:2000 438

dilution. Proteins were detected using the secondary α-mouse or α-rabbit antibodies (Promega, 439

Madison WI, USA). 440

13

441

Transformations for FRET and secretion analysis 442

Nicotiana benthamiana plants with fully expanded true leaves were used for transient 443

transformation with Agrobacterium tumefaciens strain GV3101 carrying the multicistronic vector 444

pFRETgc-2in1-NN that enabled simultaneous expression of GFP and mCherry protein fusions 445

(Hecker et al, 2015). Overnight cultures of GV3101 expressing these constructs were diluted in 446

infiltration buffer (10 mM MgCl2, 100 μM acetosyringone) to OD600 0.07 and infiltrated with a 447

syringe into the abaxial surface of N. benthamiana leaves. A Zeiss LSM 880 confocal microscope 448

with 40x water immersion objective (Zeiss, Jena) was used for image acquisition of transformed N. 449

benthamiana leaves. For FRET, the GFP constructs were excited at 488 nm and detected at 505–530 450

nm. mCherry constructs were detected at 600–620 nm after excitation at 552 nm to assess fluorophore 451

expression and at 488 nm to collect the FRET signal. FRET image analysis was performed using the 452

PixFRET tool (Feige et al., 2005) in ImageJ (Schindelin et al., 2012) applied to image stacks across 453

the entire fields of view. PixFRET corrects for spectral bleedthrough and for variations in donor and 454

acceptor signals using the product of their fluorescence signals on a pixel-by-pixel basis. FRET 455

analysis of each experiment was typically applied to 3-5 randomly selected images for each construct 456

pair. 457

Secretion assays used the tetracistronic vector pTecG-2in1-CC (Karnik et al., 2013) that 458

incorporates four expression cassettes, each driven by a 35S promoter, two carrying the coding 459

sequences for secreted YFP (secYFP) and GFP-HDEL, and the remaining two cassettes providing 460

Gateway recombination sites for proteins of interest. Assays were carried out without and with 461

SYP121ΔC incorporated in one of the latter cassettes as described previously (Karnik et al., 2013). 462

Arabidopsis seed was sterilized and grown in 0.05 MS liquid medium for 2-3 d until root and 463

cotyledons emerged. Seedlings were then cocultivated with Agrobacterium carrying the multicistronic 464

constructs for an additional 3-4 d before imaging. Live root epidermal tissue was imaged under a 465

Leica SP8-SMD confocal microscope with a 20x/0.85NA objective (Leica Microsystems, Milton 466

Keynes, UK) using the 470-nm and 514-nm laser lines for excitation and fluorescence was collected 467

over the bandwidths 490-530 and 520-565 nm for the GFP and YFP protein fusions, respectively. 468

Roots and roothairs were routinely assayed near the root base to ensure imaging of live cells and cell 469

viability was checked by monitoring of cytoplasmic streaming in root hairs. GFP and YFP signals 470

were corrected for background post-collection using the mean signals from untransformed seedlings 471

before calculating YFP/GFP fluorescence ratios. 472

473

Protein localization 474

The dynamic study of the lateral membranes of root epidermal cells of the elongation zone 475

was performed on a Nikon TE200e with a Yokogawa Andor spinning disc unit with 40x and 63x oil 476

14

immersion objectives (Nikon, Tokyo). The images were processed by Fiji/ImageJ software and the 477

periphery/internal ratio was calculated as previously described after demarking a 2-m wide perimeter 478

at the cell surface corresponding to the peripheral zone (Fendrych et al., 2010). 479

480

Statistics 481

Statistical analyses were conducted with the SigmaPlot (Systat Software, San Jose, USA) 482

utility for analysis of variance (ANOVA) with post-hoc Holm-Sidak analysis. 483

484

Accession numbers 485

Genes used in this study can be found in TAIR (www.arabidopsis.org) under the following 486

identifiers: SYP121, At3g11820; VAMP721, At1g04750; VAMP723, At2g33110; EXO70A1, 487

At5g03540. 488

489

490

Acknowledgements 491

We thank Jana Šťovíčková from the Laboratory of Cell Biology for technical help with plant 492

harvesting, and Martin Potocký and Tamara Pečenková for the critical reading. This work was 493

supported by the Imaging Facility of the Institute of Experimental Botany, CAS, and MEYS CR 494

(grants LM2018129 Czech-BioImaging, OPVVV455 CZ.02.1.01/0.0/0.0/16_013/0001775 and OPPK 495

CZ.2.16/3.1.00/21519), by grants BB/L019025/1, BB/N006909/1, BB/P011586/1 and BB/N01832X/1 496

from the BBSRC and grant IE150953 from the Royal Society to MRB. 497

498

Author Contributions 499

MRB, VŽ, ERL, and JO developed the project; ERL and JO designed the experiments; ERL, 500

JO, and NAD performed the experiments; ERL, JO, JA and MRB analyzed and interpreted the data; 501

ERL and JO wrote the manuscript with MRB and VŽ. 502

503

504

Supplemental Data 505

Supplemental Figure 1. EXO70 homologs interact differentially with several SNAREs. 506

Supplemental Figure 2. Immunoblot analysis for EXO70 interaction with several SNAREs. 507

Supplemental Figure 3. The longin domain of VAMP721 is essential for EXO70 interaction. 508

Supplemental Figure 4. Immunoblot analysis for EXO70 interaction with VAMP721, VAMP723 and 509

their chimeras. 510

Supplemental Table 1. Gene-specific primers for PCR analysis of parental lines and crosses. 511

512

15

513

Figure 1. Exocyst EXO70 subunits interact differentially with SNAREs associated with 514

secretion at the plasma membrane 515

(A) Yeast mating-based Split-Ubiquitin Screen (mbSUS) assay for interaction of the OST4-anchored516

EXO70A1-Cub bait and Nub prey fusions with the SNARE proteins as indicated. Yeast dropped as 517

1.0x and 0.1x dilutions of 0.6 OD600 yeast suspensions and grown on selective synthetic medium 518

lacking Leu, Trp, Ura, Met (CSM-LTUM) as a control for mating, and on the same medium additionally 519

lacking Ala and His (CSM-LTUMAH) as a test for bait and prey interaction. NubG and NubI were 520

included as negative and positive prey controls, respectively. Growth on 5 and 50 M Met to suppress 521

bait expression tests for interaction specificity. Immunoblots confirming bait and prey expression are 522

included in Supplemental Figure 3. 523

(B) mbSUS assay for EXO70A1 interactions as in (A) but with VAMP721/VAMP723 chimeras as524

indicated (left). Comparison of reciprocal domain exchanges indicate that the VAMP721 longin 525

domain is required for interaction. Mated yeast dropped as 1.0x and 0.1x dilutions of 0.6 OD600 yeast 526

suspensions. Immunoblots confirming bait and prey expression are included in Supplemental Figure 527

4. Both (A) and (B) are one of three independent experiments, all yielding similar results.528

529

Figure 2. VAMP721 and EXO70A1 interact in vivo 530 (A) Förster Resonance Energy Transfer (FRET) analysis on co-expressing mCherry-EXO70A1 and531

GFP-VAMP721, GFP-VAMP723 and free GFP (2xGFP) in transiently transformed N. benthamiana 532

leaf epidermis. Co-expression used the pFRETgc-2in1-NN multicistronic vector and quantification 533

was with the PixFRET tool (Feige et al., 2005; Schindelin et al., 2012). The combinations of 534

mCherry-SYP121 with GFP-VAMP721 and mCherry-SYP121 with 2xGFP were included as positive 535

and negative controls, respectively. Images in the panel rows (top to bottom) are projections of image 536

stacks analysed for FRET, the GFP (donor) and mCherry (acceptor) fluorescence signals. The FRET 537

analysis is pseudo-color-coded to the scale shown (right). A FRET signal was evident between 538

EXO70A1 and VAMP721 but not VAMP723 or 2xGFP. Scale bar, 50 μm. 539

(B) Quantification of FRET efficiency (above) calculated from PixFRET greyscale outputs with540

correction for the relative intensities of GFP (donor) and mCherry (acceptor) fluorescence signals. 541

Data are means ±SE of n 8 independent experiments, with measurements in each experiment taken 542

from the entire image planes of 3-5 randomly selected image sets. Quantification of GFP (donor) and 543

mCherry (acceptor) fluorescence signals (below) are for the same data sets and show no significant 544

difference in donor fluorescence. No significant differences between mCherry-EXO70A1 545

fluorescence signals were observed, and only the mCherry-SYP121 fluorescence was stronger. Note 546

that PixFRET (Feige et al., 2005; Schindelin et al., 2012) corrects for variation in donor and acceptor 547

16

between data sets as the product of their fluorescence signals. Lower case letters indicate significance 548

at P<0.01; upper case letters indicate significance at P<0.05. 549

550

Figure 3. The exo70A1 mutation impairs secretion 551 Transient expression of secretory YFP (secYFP) in the hypocotyl and root of 5-d old wild-type (WT) 552

and exo70A1 Arabidopsis seedlings. To ensure equal genetic loads, seedlings were transformed with 553

the pTecG-2in1-CC multicistronic vector (Karnik et al., 2013) carrying cassettes to express secYFP, 554

GFP-HDEL as an expression and ratiometric marker. The lower set of images show the results of 555

including the dominant-negative SYP121C fragment as a test for secretory block. When expressed 556

with GFP-HDEL alone, secYFP yielded a modest signal relative, consistent with its loss to the 557

medium on secretion (Grefen et al., 2015). When expressed additionally with SYP121C an increase 558

in secYFP signal relative to GFP-HDEL was evident, indicating secretory block and secYFP retention 559

in the tissue (Tyrrell et al., 2007; Grefen et al., 2015). Note the retention of secYFP in the exo70A1 560

mutant, even in the absence of SYP121C expression. Scale bar, 50 µm. YFP to GFP fluorescence 561

ratios calculated after subtracting the autofluorescence signals from untransformed seedlings. Data are 562

means ±SE, each of n18 independent transformants, with letters indicating statistical significance on 563

post-hoc Holm-Sidak analysis (P<0.001). 564

565 Figure 4. SNARE localization is affected in the exo70A1 mutant 566 (A) Representative images of VAMP721, VAMP722, and SYP121 localized as GFP fusions in wild-567

type and exo70A1 mutant Arabidopsis, showing a redistribution of the SNAREs in the exocyst 568

mutant. (B) SYP121, VAMP721 and VAMP722 localization in wild-type (black) and exo70A1 mutant 569

(grey) Arabidopsis, including data in (A), as the ratio of fluorescence between the periphery and 570

cytosol indicating a shift of SNARE distribution to the cytosol in the absence of EXO70A1. Data are 571

means ±SE, each of n36 randomly selected images from n6 independent experiments, with letters 572

indicating pairwise statistical significance from post-hoc Holm-Sidak analysis (P<0.001). 573

(C) Representative images of GFP-tagged EXO70A1 in wild-type and syp121, vamp721, and 574

vamp722 mutant Arabidopsis, showing no appreciable change in its peripheral localisation. 575

(D) EXO70A1 localization in wild-type and syp121, vamp721, and vamp722 mutant Arabidopsis as 576

the ratio of fluorescence between the periphery and cytosol. Data are means ±SE, each of n36 577

randomly selected images from n6 independent experiments. No statistically significant differences 578

from wild type were evident. Scale bars in (A) and (C), 10 μm. 579

580 Figure 5. The exo70A1 vamp721 double mutant synergistically suppresses growth 581 (A) Representative Arabidopsis plants grown ove 4 weeks under 8:16 L:D from F2 seed germinated 582

on soil to isolate the double mutants from crosses of the SNARE mutants with exo70A1. Scale, 2 cm. 583

syp121 and vamp721 single mutants were visually indistinguishable from the wild type while the 584

17

exo70A1 mutant was 40-50% smaller than wild type. exo70A1 syp121 double mutants were visually 585

indistinguishable from the exo70A1 single mutant, while the exo70A1 vamp721 double mutant 586

showed a severe suppression of growth unique from the wild type and single mutant parents. Plants in 587

every case were genotyped as in (C). 588

(B) Rosette diameter of Arabidopsis grown over 4 (light grey bars) and 8 weeks (dark grey bars) as in589

(A). Data are means ±SE of n10 plants for each line. Letters indicate significant differences with 590

post-hoc Holm-Sidak analysis (P<0.01). 591

(C) Seedling and root development of wild type, exo70a1 and vamp721 single mutants, and exo70a1592

vamp721 double mutants grown on 0.5 MS and 1% agar medium for 10 d. Note the modest reduction 593

in growth and smaller epinastic cotyledons of the exo70A1 mutant and much stronger growth 594

suppression of the exo70A1 vamp721 double mutant. Right: Seedling genotypes confirmed by PCR 595

using gene-specific primers (Supplemental Table 1) and a T-DNA left boarder primer to amplify 596

wild-type and mutant allele products. V1 and A1 indicate the wild type alleles of VAMP721 and 597

EXO70A1, respectively; v1 and a1 indicate the T-DNA mutant alleles of vamp721 and exo70A1, 598

respectively. Scale bar, 1 cm. 599

(D,E) Epidermal cell size analysis of mature leaves of 8 week-old plants of single- and double-mutant 600

Arabidopsis. Leaf epidermal peels (D) were analyzed for epidermal cell surface area (green; scale bar, 601

50 μm) and data assembled (E) in a box and whisker plot. Cell area of exo70a1 vamp721 plants were 602

significantly smaller than those of the wild-type and parental lines. Data are means ±SE of n150 603

cells chosen at random from n8 plants for each line. Note the logarithmic y-axis scale. Letters 604

indicate significant differences with post-hoc Holm-Sidak analysis (P<0.001). 605

606 Figure 6. Model of exocyst and R-SNARE binding in sequential recruitment and SNARE 607 complex assembly for secretion 608 Cartoon of EXO70A1 and VAMP721 synergy in vesicle fusion. Peripherally localized EXO70A1 is 609

indicated by the exposed (orange) face of an oval exocyst complex (purple). SYP121 and SYP122 are 610

shown in green and blue, respectively, with membrane anchor, longer H3 (SNARE) and shorter Habc 611

domains. VAMP721 and VAMP722 are shown in red and yellow, respectively, with only a membrane 612

anchor and SNARE domain. Other SNARE and associated proteins are omitted for clarity. 613

(A) Normal (wild-type) secretion engages EXO70A1 in sequentially recruiting VAMP721 (a) and614

SYP121 from Qa-SNARE 'icebergs' (b) leading to vesicle fusion (c). 615

(B) The exo70a1 mutant impairs secretion by slowing trans SNARE assembly (d).616

(C) The exo70a1 syp121 double mutant similarly slows secretion with the remaining excess of Qa-617

SNARE SYP122 available for trans SNARE assembly (e). 618

(D) The exo70a1 vamp721 double mutant strongly suppresses secretion by reducing R-SNARE619

availability as well as eliminating exocyst recruitment for trans SNARE assembly. 620

18

621

622

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Figure 1. Exocyst EXO70 subunits interact differentially with SNAREs associated with secretion at the plasma membrane (A) Yeast mating-based Split-Ubiquitin Screen (mbSUS) assay for interaction of the OST4-anchored EXO70A1-Cub bait and Nub prey fusions with the SNARE proteins as indicated. Yeastdropped as 1.0x and 0.1x dilutions of 0.6 OD600 yeast suspensions and grown on selectivesynthetic medium lacking Leu, Trp, Ura, Met (CSM-LTUM) as a control for mating, and on the same

medium additionally lacking Ala and His (CSM-LTUMAH) as a test for bait and prey interaction. NubG and NubI were included as negative and positive prey controls, respectively. Growth on 5 and 50 µM Met to suppress bait expression tests for interaction specificity. Immunoblots confirming bait and prey expression are included in Supplemental Figure 3. (B) mbSUS assay for EXO70A1 interactions as in (A) but with VAMP721/VAMP723 chimeras asindicated (left). Comparison of reciprocal domain exchanges indicate that the VAMP721 longindomain is required for interaction. Mated yeast dropped as 1.0x and 0.1x dilutions of 0.6 OD600yeast suspensions. Immunoblots confirming bait and prey expression are included inSupplemental Figure 4. Both (A) and (B) are one of three independent experiments, all yieldingsimilar results.

Figure 2. VAMP721 and EXO70A1 interact in vivo (A) Förster Resonance Energy Transfer (FRET) analysis on co-expressing mCherry-EXO70A1and GFP-VAMP721, GFP-VAMP723 and free GFP (2xGFP) in transiently transformed N.benthamiana leaf epidermis. Co-expression used the pFRETgc-2in1-NN multicistronic vector andquantification was with the PixFRET tool (Feige et al., 2005; Schindelin et al., 2012). Thecombinations of mCherry-SYP121 with GFP-VAMP721 and mCherry-SYP121 with 2xGFP wereincluded as positive and negative controls, respectively. Images in the panel rows (top to bottom)are projections of image stacks analysed for FRET, the GFP (donor) and mCherry (acceptor)fluorescence signals. The FRET analysis is pseudo-color-coded to the scale shown (right). AFRET signal was evident between EXO70A1 and VAMP721 but not VAMP723 or 2xGFP. Scalebar, 50 μm.(B) Quantification of FRET efficiency (above) calculated from PixFRET greyscale outputs withcorrection for the relative intensities of GFP (donor) and mCherry (acceptor) fluorescence signals.Data are means ±SE of n ³8 independent experiments, with measurements in each experimenttaken from the entire image planes of 3-5 randomly selected image sets. Quantification of GFP(donor) and mCherry (acceptor) fluorescence signals (below) are for the same data sets and showno significant difference in donor fluorescence. No significant differences between mCherry-EXO70A1 fluorescence signals were observed, and only the mCherry-SYP121 fluorescence wasstronger. Note that PixFRET (Feige et al., 2005; Schindelin et al., 2012) corrects for variation indonor and acceptor between data sets as the product of their fluorescence signals. Lower caseletters indicate significance at P<0.01; upper case letters indicate significance at P<0.05.

Figure 3. The exo70A1 mutation impairs secretion Transient expression of secretory YFP (secYFP) in the hypocotyl and root of 5-d old wild-type (WT) and exo70A1 Arabidopsis seedlings. To ensure equal genetic loads, seedlings were transformed with the pTecG-2in1-CC multicistronic vector (Karnik et al., 2013) carrying cassettes to express secYFP, GFP-HDEL as an expression and ratiometric marker. The lower set of images show the results of including the dominant-negative SYP121DC fragment as a test for secretory block. When expressed with GFP-HDEL alone, secYFP yielded a modest signal relative, consistent with its loss to the medium on secretion (Grefen et al., 2015). When expressed additionally with SYP121DC an increase in secYFP signal relative to GFP-HDEL was evident, indicating secretory block and secYFP retention in the tissue (Tyrrell et al., 2007; Grefen et al., 2015). Note the retention of secYFP in the exo70A1 mutant, even in the absence of SYP121DC expression. Scale bar, 50 µm. YFP to GFP fluorescence ratios calculated after subtracting the autofluorescence signals from untransformed seedlings. Data are means ±SE, each of n³18 independent transformants, with letters indicating statistical significance on post-hoc Holm-Sidak analysis (P<0.001).

Figure 4. SNARE localization is affected in the exo70A1 mutant (A) Representative images of VAMP721, VAMP722, and SYP121 localized as GFP fusions inwild-type and exo70A1 mutant Arabidopsis, showing a redistribution of the SNAREs in the exocystmutant. (B) SYP121, VAMP721 and VAMP722 localization in wild-type (black) and exo70A1mutant (grey) Arabidopsis, including data in (A), as the ratio of fluorescence between theperiphery and cytosol indicating a shift of SNARE distribution to the cytosol in the absence ofEXO70A1. Data are means ±SE, each of n³36 randomly selected images from n³6 independentexperiments, with letters indicating pairwise statistical significance from post-hoc Holm-Sidakanalysis (P<0.001).(C) Representative images of GFP-tagged EXO70A1 in wild-type and syp121, vamp721, andvamp722 mutant Arabidopsis, showing no appreciable change in its peripheral localisation.(D) EXO70A1 localization in wild-type and syp121, vamp721, and vamp722 mutant Arabidopsisas the ratio of fluorescence between the periphery and cytosol. Data are means ±SE, each ofn³36 randomly selected images from n³6 independent experiments. No statistically significantdifferences from wild type were evident. Scale bars in (A) and (C), 10 μm.

Figure 5. The exo70A1 vamp721 double mutant synergistically suppresses growth (A) Representative Arabidopsis plants grown ove 4 weeks under 8:16 L:D from F2 seed germinated on soil to isolate the double mutants from crosses of the SNARE mutants with

exo70A1. Scale, 2 cm. syp121 and vamp721 single mutants were visually indistinguishable from the wild type while the exo70A1 mutant was 40-50% smaller than wild type. exo70A1 syp121 double mutants were visually indistinguishable from the exo70A1 single mutant, while the exo70A1 vamp721 double mutant showed a severe suppression of growth unique from the wild type and single mutant parents. Plants in every case were genotyped as in (C). (B) Rosette diameter of Arabidopsis grown over 4 (light grey bars) and 8 weeks (dark grey bars)as in (A). Data are means ±SE of n³10 plants for each line. Letters indicate significant differenceswith post-hoc Holm-Sidak analysis (P<0.01).(C) Seedling and root development of wild type, exo70a1 and vamp721 single mutants, andexo70a1 vamp721 double mutants grown on 0.5 MS and 1% agar medium for 10 d. Note themodest reduction in growth and smaller epinastic cotyledons of the exo70A1 mutant and muchstronger growth suppression of the exo70A1 vamp721 double mutant. Right: Seedling genotypesconfirmed by PCR using gene-specific primers (Supplemental Table 1) and a T-DNA left boarderprimer to amplify wild-type and mutant allele products. V1 and A1 indicate the wild type alleles ofVAMP721 and EXO70A1, respectively; v1 and a1 indicate the T-DNA mutant alleles of vamp721and exo70A1, respectively. Scale bar, 1 cm.(D,E) Epidermal cell size analysis of mature leaves of 8 week-old plants of single- and double-mutant Arabidopsis. Leaf epidermal peels (D) were analyzed for epidermal cell surface area(green; scale bar, 50 μm) and data assembled (E) in a box and whisker plot. Cell area of exo70a1vamp721 plants were significantly smaller than those of the wild-type and parental lines. Data aremeans ±SE of n³150 cells chosen at random from n³8 plants for each line. Note the logarithmicy-axis scale. Letters indicate significant differences with post-hoc Holm-Sidak analysis (P<0.001).

Figure 6. Model of exocyst and R-SNARE binding in sequential recruitment and SNARE complex assembly for secretion Cartoon of EXO70A1 and VAMP721 synergy in vesicle fusion. Peripherally localized EXO70A1 is indicated by the exposed (orange) face of an oval exocyst complex (purple). SYP121 and SYP122 are shown in green and blue, respectively, with membrane anchor, longer H3 (SNARE) and shorter Habc domains. VAMP721 and VAMP722 are shown in red and yellow, respectively, with only a membrane anchor and SNARE domain. Other SNARE and associated proteins are omitted for clarity. (A) Normal (wild-type) secretion engages EXO70A1 in sequentially recruiting VAMP721 (a) andSYP121 from Qa-SNARE 'icebergs' (b) leading to vesicle fusion (c).(B) The exo70a1 mutant impairs secretion by slowing trans SNARE assembly (d).(C) The exo70a1 syp121 double mutant similarly slows secretion with the remaining excess ofQa-SNARE SYP122 available for trans SNARE assembly (e).(D) The exo70a1 vamp721 double mutant strongly suppresses secretion by reducing R-SNAREavailability as well as eliminating exocyst recruitment for trans SNARE assembly.

IN A NUTSHELL Background: Cells use vesicles to transport cargo throughout the cell and to the plasma membrane, which is critical for plant cell growth and function. Several different proteins help control and regulate vesicle traffic to deliver cellular cargo to its functional locations. During secretion, SNARE proteins located on the vesicle and plasma membrane interact to fuse the two membranes together and deposit the protein cargo. While SNAREs are required for membrane fusion, they rely on other mechanisms to get the vesicles in proximity to their target membranes. The exocyst is a well-studied tethering complex that helps deliver vesicles to the plasma membrane. Although it has been hypothesized that SNARE and exocyst protein complexes work in the same secretory pathways, there is a lack of in vivo evidence of interactions between either protein subunits or complexes in plants. Question: We hypothesized that there are direct interactions between SNARE and exocyst proteins that support cargo delivery at the plasma membrane, and that these interactions could happen in a sequential manner to regulate vesicle delivery and fusion. Findings: Using split-ubiquitin yeast mating and FRET analyses, we identified direct interactions between the EXO70A1 exocyst subunit and SNAREs on both the vesicle (VAMP721) and plasma membrane (SYP121). We found that the exo70a1 mutant has reduced secretion, consistent with the redistribution of the vesicle SNARE VAMP721 away from the plasma membrane in the exo70a1 mutant background. These results suggest that the exocyst helps deliver or recruit vesicles to the plasma membrane through its interactions with SNARE proteins. In the syp121 exo70a1 double mutant, syp121 was epistatic to exo70a1 but surprisingly, the vamp721 exo70a1 double mutant had synergistic phenotypes, including severe growth suppression. We believe that these results indicate a functional hierarchy between vesicle and plasma membrane SNAREs and highlight the consequences of these interactions on plant growth.

Next steps: We know the protein domain of VAMP721 that is important for its interactions with EXO70A1, we would now like to know what part of EXO70A1 regulates its interactions with SNARE proteins and how they support the dynamics of vesicle delivery at the plasma membrane.

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DOI 10.1105/tpc.20.00280; originally published online July 22, 2020;Plant Cell

Viktor ZarskyEmily R Larson, Jitka Ortmannová, Naomi Alice Donald, Jonas Chaves Alvim, Mike R. Blatt and

during Vegetative GrowthSynergy Among Exocyst and SNARE Interactions Identifies a Functional Hierarchy in Secretion

 This information is current as of December 20, 2020

 

Supplemental Data /content/suppl/2020/11/04/tpc.20.00280.DC2.html /content/suppl/2020/07/23/tpc.20.00280.DC1.html

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