2 and contributes to innate immunity · 2 and contributes to innate immunity 3 an quoc phama,...

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Arabidopsis Lectin Receptor Kinase P2K2 is a second plant receptor for extracellular ATP 1

and contributes to innate immunity 2

An Quoc Phama, Sung-Hwan Choa, Cuong The Nguyena,b, and Gary Staceya,1 3

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Affiliations: 5

a Divisions of Plant Science and Biochemistry, C.S. Bond Life Science Center, University of 6

Missouri, Columbia, MO 65211, USA 7

b Cuu Long Delta Rice Research Institute, Cantho, Vietnam 8

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1 Correspondence: [email protected] 10

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Keywords: Extracellular ATP (eATP), P2K1, DORN1, P2K2, LecRK, Pseudomonas syringae, 12

receptors 13

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Running title: P2K2 is an eATP receptor. 15

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One-sentence summary: A receptor kinase that can bind to ATP with high affinity plays a 17

partially redundant role during plant immunity. 18

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Author Contributions: 20

A.Q.P. and G.S. designed the experiments. A.Q.P, S.-H. C. and C. T. N. performed the 21

experiments. A.Q.P, S.-H. C. and G.S wrote the paper. 22

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Plant Physiology Preview. Published on April 28, 2020, as DOI:10.1104/pp.19.01265

Copyright 2020 by the American Society of Plant Biologists

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Abstract 32

In animals, extracellular adenosine 5’-triphosphate (eATP) is a well-studied signaling molecule 33

that is recognized by plasma membrane-localized P2-type purinergic receptors. However, in 34

contrast, much less is known about purinergic signaling in plants. P2 receptors play critical roles 35

in a variety of animal biological processes including immune system regulation. The first plant 36

purinergic receptor, Arabidopsis P2K1 (L-type lectin receptor kinase-I.9), was shown to 37

contribute to plant defense against bacterial, oomycete and fungal pathogens. Here, we 38

demonstrate the isolation of a second purinergic receptor, P2K2, by complementation of an 39

Arabidopsis p2k1 mutant. P2K2 (LecRK-I.5) has 74% amino acid similarity to P2K1. The P2K2 40

extracellular lectin domain binds to ATP with higher affinity than P2K1 (Kd = 44.47 15.73 41

nM). Interestingly, p2k2 and p2k1 p2k2 mutant plants showed increased susceptibility to the 42

pathogen Pseudomonas syringae, with the double mutant showing a stronger phenotype. In vitro 43

and in planta studies demonstrate that P2K2 and P2K1 interact and cross-phosphorylate upon 44

eATP treatment. Thus, similar to animals, plants possess multiple purinergic receptors. 45

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3

Introduction 50

Adenosine 5'-triphosphate (ATP) is well-known as the source of cellular energy in all organisms. 51

Under normal conditions, cells maintain mM levels of intracellular ATP but also nM levels of 52

extracellular ATP (eATP). In response to the appropriate stimulus, ATP is released from the 53

cytosol to the extracellular matrix and becomes an essential signaling molecule for growth, 54

development, and stress responses (Khakh and Burnstock, 2009; Tanaka et al., 2010; Cho et al., 55

2017). In mammals, P2X (ion channels) and P2Y (G-protein coupled) receptors are plasma 56

membrane purinoreceptors, which can bind eATP and trigger various downstream signaling 57

cascades. Humans possess 7 P2X receptors and 8 P2Y receptors (Burnstock, 2018). The action 58

of these multiple P2X and P2Y receptors explains the critical roles of eATP in various biological 59

processes in animals such as inflammation, neurotransmission, immune response, cell 60

proliferation, cell differentiation and cell death (Burnstock and Verkhratsky, 2010; Cekic and 61

Linden, 2016; Diezmos et al., 2016; Ferrari et al., 2016). 62

Plants lack canonical P2X and P2Y receptors but still respond to eATP by, for example, 63

triggering an increase of cytosolic calcium (Ca2+[cyt]), as well as nitric oxide (NO) and reactive 64

oxygen species (ROS) (Reichler et al., 2009; Tanaka et al., 2010). Indeed, eATP signaling has 65

been implicated in a variety of plant processes, including root growth (Tang et al., 2003; 66

Weerasinghe et al., 2009; Clark et al., 2010), stress responses (Thomas et al., 2000; Song et al., 67

2006) and pollen germination (Reichler et al., 2009; Rieder and Neuhaus, 2011). 68

The question of how plants recognize eATP was answered by the cloning of the first plant eATP 69

receptor, DORN1 (DOes not Response to Nucleotides 1), defining a new kinase class of 70

purinoreceptor (Choi, Tanaka et al. 2014). In keeping with the animal P2 receptor nomenclature, 71

we now prefer the designation of P2K1 for this first plant purinergic receptor. The P2K1 protein 72

was previously referred to as L-type lectin receptor kinase I.9 (LecRK-I.9) based on sequence 73

comparisons of various Arabidopsis thaliana LecRK proteins. Subsequent studies from our lab 74

showed that P2K1, upon eATP addition, regulates stomatal aperture by direct phosphorylation of 75

NADPH oxidase (RBOHD) (Chen et al., 2017). P2K1 also phosphorylates protein 76

acyltransferases (i.e., PAT5 and 9) to regulate their activity (Chen et al., 2019). 77

The LecRK protein family members contain a lectin-like ectodomain, a single transmembrane 78

domain and an intracellular serine/threonine kinase domain. In Arabidopsis, the LecRK family 79

contains 45 members. Thirty-eight are divided into nine subclades, while seven singleton 80



4

members do not belong to any clade (Bouwmeester and Govers, 2009). The diversity of the 81

LecRK family in plants was proposed to be the result of tandem- and whole-genome duplication 82

(Hofberger et al., 2015). As a result of duplication events, proteins can diverge to have diverse 83

functions or may share similar functions (Moore and Purugganan, 2003). For example, in 84

AtLecRK clade I, P2K1 (LecRK-I.9) is an ATP receptor, while LecRK-I.8 was shown to be a 85

nicotinamide adenine dinucleotide (NAD+) sensor (Choi et al., 2014; Wang et al., 2017). In clade 86

IX, LecRK-IX.1 and LecRK-IX.2 share analogous function, regulating Phytophthora resistance 87

and plant cell death (Wang et al., 2015). 88

Under normal growth conditions, only P2K1 (LecRK-I.9), LecRK-IV.1, and LecRK-VIII.1 are 89

expressed at a high level in most plant tissues, whereas the other LecRK genes show lower 90

expression and only in specific tissues. However, the expression of individual LecRK genes was 91

found to be responsive to specific hormone treatments, abiotic stresses, elicitor treatments, or 92

pathogen infection, suggesting a functional role for these receptors under these specific 93

conditions (Bouwmeester and Govers, 2009). While the specific functions as well as ligands of 94

most LecRK family members remain unknown, there appears to be a general association of these 95

receptors with the plant response to both abiotic and biotic stress. LecRK-I.3 was reported to be 96

an active kinase that is regulated by ethylene in response to salt stress (He et al., 2004). LecRK-97

IV.3, which is strongly induced by abscisic acid, methyl jasmonate, salicylic acid or stress 98

treatments, plays critical roles in both abiotic and biotic stresses responses (Huang et al., 2013). 99

Besides P2K1 (LecRK-I.9), LecRK-V.2 and LecRK-VII.1 were also reported to play essential 100

roles in regulating stomatal closure (Yekondi et al., 2018). Recently, it was reported that P2K1 101

is involved in modulation of jasmonic acid (JA)-mediated gene expression for plant defense 102

responses (Tripathi et al., 2018). 103

Animals possess multiple P2X and P2Y receptors, consistent with the wide and varying 104

functions that purinergic signaling plays in physiology (Puchałowicz et al., 2014). Given that 105

eATP was shown to mediate a variety of plant processes (Tanaka et al., 2010), it seemed 106

reasonable to suggest that plants also encode multiple purinergic receptors. In this study, we 107

identify P2K2 (LecRK-I.5) as a second Arabidopsis extracellular ATP receptor by screening 108

various AtLecRK clade I RLKs for their ability to complement an Arabidopsis p2k1 mutant. The 109

P2K2 lectin ectodomain binds ATP with higher affinity than P2K1 and is an active kinase with 110

the ability to auto-phosphorylate and trans-phosphorylate other proteins. A variety of assays 111

showed that P2K1 and P2K2 can interact. Similar to p2k1 mutant plants, plants defective in 112



5

P2K1 or P2K1/P2K2 function are significantly more susceptible to pathogen infection. 113

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Results 115

Identification of the second extracellular ATP receptor, P2K2, by screening all members of 116

the LecRK clade I 117

Among 9 subclades of the LecRK family of Arabidopsis, the first plant eATP receptor P2K1 118

(LecRK-I.9) belongs to clade I, which has 11 members (Bouwmeester and Govers, 2009) 119

(Supplemental Fig. S1). Compared to P2K1, all of the other LecRK clade I members are 120

expressed at relatively low levels in Arabidopsis under normal conditions but can be induced by 121

various stimuli (Bouwmeester and Govers, 2009). Among clade I members, LecRK-I.5 122

(At3g45430) and LecRK-I.10 (At5g60310) are the most closely related to P2K1, based on 123

sequence comparisons. 124

In order to test whether other LecRK clade I members, in addition to P2K1, could function in 125

eATP perception, we ectopically expressed each of the clade I LecRKs genes in the Arabidopsis 126

p2k1-3 mutant, which expresses the calcium reporter protein aequorin, and then monitored the 127

cytosolic calcium (Ca2+[cyt]) response upon eATP addition. Among the LecRKs tested, ectopic 128

expression of LecRK I.8 showed a lethal phenotype while LecRK I.10 showed no expression 129

(Supplemental Fig. S2). Only LecRK-I.5 (P2K2) could partially restore the Ca2+[cyt] response of 130

the p2k1-3 mutant plants (Fig. 1). These results led us to test the Ca2+[cyt] response of p2k2 (lecrk-131

I.5) mutant plants to eATP (Supplemental Fig. S3). The results indicated that the loss of P2K2 132

function reduced the ability of the plants to elevate Ca2+[cyt] levels in response to ATP, although 133

this defect was not as severe as found with p2k1 mutant plants. The calcium response of the 134

p35S:P2K2 over-expression plants was higher than the wild-type (Fig. 1 and Supplemental Fig. 135

S3). Given these results, we renamed LecRK-I.5 as P2K2, reflecting a functional annotation for 136

this protein beyond the strictly sequence based assignment of LecRK-I.5. 137

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ATP binds to the P2K2 extracellular domain 139

If P2K2 is a bona fide purinergic receptor, then it should bind ATP with a physiologically 140

relevant affinity. In order to test this experimentally, we purified the P2K2 ectodomain 141

(Supplemental Fig. S4) and conducted in vitro binding studies using radiolabeled γ32P-ATP. The 142



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P2K2 protein showed a typical saturation curve for ATP binding with high affinity (Kd = 44.47 ± 143

15.73 nM and Bmax = 625.8 ± 86.48 pmol/mg) (Fig. 2). 144

To determine whether P2K2 could bind other nucleotides or ligands, in vitro competitive binding 145

assays were performed. We tested the ability of unlabeled ligands to compete against the binding 146

of radiolabeled γ32P-ATP. Unlabeled ATP and ADP were found to be strong competitors (ATP 147

Kj = 31.7 nM; ADP Kj = 97.3) (Fig. 2). Unlabeled GTP was a weak competitor, while the other 148

ligands, AMP, adenosine, adenine ITP, CTP, TTP, UTP, showed no competition (Fig. 2). These 149

results differ from our previous studies with P2K1 and suggest that P2K2 may have higher 150

specificity for ATP and ADP than shown for P2K1 (Choi et al., 2014). 151

152

The P2K2 intracellular domain has strong kinase activity, which is essential for 153

downstream signaling. 154

The P2K2 intracellular domain is predicted to have kinase activity. In order to confirm this 155

prediction, we cloned the P2K2 intracellular domain (Supplemental Fig. S4), including the 156

putative kinase domain (P2K2-KD). Subsequent purification of the P2K2-KD demonstrated 157

strong autophosphorylation activity in vitro, as well as the ability to trans-phosphorylate myelin 158

basic protein (MBP) (Fig. 3). Comparison of the sequence of P2K1-KD and P2K2-KD allowed 159

us to predict critical amino acid residues for kinase activity. Specific mutations were generated 160

within the P2K2-KD sequence using site direct mutagenesis, specifically p2k2D467N (kinase 161

activation motif mutation) and p2k2D525N [similar to the p2k1-1 mutant version (Choi et al., 162

2014)]. Subsequent in vitro assays demonstrated a lack of kinase activity for the p2k2D467N 163

protein and very low kinase activity for the p2k2D525N protein (Fig. 3). Electrophoretic separation 164

of the wild-type P2K2 and kinase domain mutant P2K2 proteins demonstrated differences in 165

their mobility, which we attributed to their relative ability to auto-phosphorylate (Fig. 3). 166

Consistent with this notion, treatment of the wild-type and kinase mutant forms of P2K2 with 167

lambda protein phosphatase (PPase), to release the phosphate group, resulted in all three proteins 168

showing similar electrophoretic mobility (Supplemental Fig. S5). 169

In order to ascertain whether P2K2 kinase activity was critical for function, we expressed the 170

wild-type P2K2 protein (as control) and the full-length p2k2D467N and p2k2D525N mutant versions 171

in the p2k1-3 mutant background and assayed the ability of the resulting transgenic plants to 172

elevate Ca2+[cyt] upon ATP addition. Ectopic expression of the full-length p2k2D467N or p2k2D525N 173



7

mutant proteins in p2k1-3 mutant plants failed to complement the p2k1 phenotype (Fig. 3). We, 174

therefore, conclude that P2K2 kinase function, similar to the results obtained with P2K1 (Choi et 175

al., 2014), is required for P2K2 receptor function. 176

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P2K2 self-associates and also interacts with P2K1 on the plasma membrane 178

Like P2K1, P2K2 has a putative transmembrane domain (Supplemental Fig. S4). To determine 179

whether P2K2 localizes to the plasma membrane, we fused full-length P2K2 with YFP (yellow 180

fluorescence protein) and expressed P2K2-YFP or free YFP in Arabidopsis protoplasts. We used 181

FM4-64 for plasma membrane staining. The resulting data indicate, again like P2K1, that P2K2-182

YFP localizes to the plasma membrane (Fig. 4 and Supplemental Fig. S6). 183

Plasma membrane receptors often form homodimer or heterodimer complexes. For example, 184

P2K1 was previously shown to self-associate on the plasma membrane (Chen et al., 2017). 185

Therefore, we performed bimolecular fluorescence complementation (BiFC) assays in 186

Arabidopsis protoplasts expressing P2K2-YFPn and P2K2-YFPc, as well as P2K2-YFPn and 187

P2K1-YFPc. The data indicate that P2K2 co-localized with the FM4-64 plasma membrane 188

marker (Fig. 4). Full-length P2K2 and P2K1 were cloned into pCambia-Nluc or pCambia-Cluc 189

and the resulting luciferase fusion constructs were co-expressed with P2K2-Cluc in Nicotiana 190

benthamiana leaves. Consistent with the BiFC results, P2K2-Nluc and P2K1-Nluc showed a 191

strong interaction signal in the split-luciferase assay when it was co-expressed with P2K2-Cluc, 192

but not with empty vector controls (Fig. 4). Furthermore, the strength of the P2K2-P2K2 and 193

P2K2-P2K1 interactions were increased by the exogenous addition of ATP (Fig. 4). Our in vitro 194

pull-down experiments also showed that P2K2-KD-His directly interacted with GST-P2K2-KD 195

and GST-P2K1-KD but not with GST-LYK5-KD, which served as our negative control (Fig. 4). 196

Coupled with our previous results regarding P2K1, the data indicate that both P2K1 and P2K2 197

can self-associate but also have the ability to form heterocomplexes within the plasma 198

membrane. In our experiments, these associations were enhanced upon the addition of ATP (Fig. 199

4). 200

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P2K1 can phosphorylate P2K2, but not vice versa 202

The data above indicate that both P2K1 and P2K2 can auto-phosphorylate, as well as trans-203

phosphorylate other proteins. Hence, there is the possibility that these two proteins could interact 204



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and trans-phosphorylate each other. In order to test this, we incubated kinase-dead versions of 205

GST-P2K1D525N, D572N-KD and GST-P2K2D467N-KD proteins with kinase-active versions of GST-206

P2K1-KD and GST-P2K2-KD and assayed for phosphorylation using radiolabeled 32P-ATP. 207

GST-P2K1-KD and GST-P2K2-KD phosphorylated GST-p2k2D467N-KD in vitro (Fig. 5). 208

However, P2K1 and P2K2 failed to trans-phosphorylate the two kinase-dead versions of P2K1, 209

GST-p2k1D525N-KD and GST-p2k2D572N-KD (Fig 5). The data indicate that P2K1 is incapable of 210

transphosphorylation of itself but also cannot be phosphorylated by P2K2. The data suggest that, 211

if P2K1 and P2K2 form an active heterocomplex, then activation of P2K1 is likely the initial step 212

leading to transphosphorylation of P2K2, as well as other downstream proteins. 213

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P2K2 plays a partially redundant role with P2K1 during plant immunity 215

Previous reports indicated that P2K1 positively regulates plant defense against Pseudomonas 216

syringae (Balague et al., 2017; Chen et al., 2017), Phytophthora infestans, Phytophthora 217

brassicae (Gouget et al., 2006; Bouwmeester et al., 2011; Bouwmeester et al., 2014) and Botrytis 218

cinerea (Tripathi et al., 2018). Given that P2K1 and P2K2 interact and that P2K2 can partially 219

complement the P2K1 phenotype, it is likely that these two proteins may function redundantly in 220

ATP signaling, either in separate complexes or in association with one another. Therefore, we 221

hypothesized that P2K2 might also be involved in pathogen resistance. To test the role of P2K2 222

directly, we examined plant susceptibility to P. syringae upon flood inoculation. The level of 223

bacterial infection was monitored by bio-luminescence and confirmed by direct counting to 224

quantify the level of pathogen colonization (Fig. 6). The p2k1-3, p2k2, and p2k1p2k2 double 225

mutant plants showed significantly greater colonization than wild-type plants (Fig. 6). Consistent 226

with the visual assays, direct bacterial counts showed that p2k1-3, p2k2, and p2k1p2k2 double 227

mutant plants were significantly more susceptible to bacterial infection compared to the wild 228

type, whereas the P2K2 complemented line showed no significant difference to the wild type 229

(Fig. 6). Interestingly, those plants ectopically expressing P2K2 showed elevated resistance to 230

bacterial infection relative to the wild type (Fig. 6). These results are consistent with P2K2 231

playing an important role in plant innate immunity. The fact that the p2k1 p2k2 double mutant 232

showed the highest level of susceptibility suggests that, at least for this specific phenotype, the 233

two receptors show some level of functional redundancy. 234



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To further understand how P2K2 contributes to bacterial pathogen resistance, we measured the 235

activity or expression of a few genes known to respond to bacterial infection. We used wild-type 236

(Col-0 background) plants as the positive control and the p2k1-3 mutant line as a negative 237

control while testing the pathogen response of p2k2 single mutant and p2k1p2k2 double mutant 238

plants. A key aspect of the pathogen response pathway is the activation of MAPK signaling (Bi 239

and Zhou, 2017). Therefore, after ATP treatment, we measured activation of MPK3 and MPK6 240

in p2k2 and p2k1 p2k2 mutant plants, relative to controls. As shown in Fig. 7, wild-type plants 241

showed strong phosphorylation of MPK3 and MPK6 upon ATP treatment, whereas the p2k1-3, 242

p2k2 or p2k1 p2k2 mutant plants exhibited lower phosphorylation. Interestingly, the phenotype 243

of the p2k1 p2k2 double mutant was stronger than each of the single mutants, suggesting that the 244

two receptors are at least partially redundant in function. In addition, we measured the expression 245

of MYC2 and ZAT10, previously reported to be regulated by P2K1 and known to respond to 246

pathogen infection (Balague et al., 2017; Tripathi et al., 2018; Jewell et al., 2019). The 247

expression of both genes was significantly reduced in the p2k2 and p2k1 p2k2 mutant plants after 248

ATP treatment (Fig. 7). Taken together, the results suggest that P2K2, in addition to P2K1, is a 249

critical component of ATP signaling through the pathogen response pathway. 250

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Discussion 252

The first evidence that eATP plays a signaling role in plants was found in 1973 when exogenous 253

addition of ATP stimulated faster closure of the specialized leaves of the Venus fly trap 254

(Dionaea muscipula) (Jaffe, 1973). About four decades after this report, the first plant 255

extracellular ATP receptor was identified, P2K1 (LecRK-I.9) (Choi et al., 2014). The p2k1 256

mutant plants are unable to recognize exogenous ATP and activate various downstream 257

responses. In both animals and plants, eATP appears to play a variety of important roles. Hence, 258

it seemed reasonable to consider the possibility that both animals and plants possess multiple 259

eATP receptors. The identification of P2K2 (LecRK-I.5) confirms this possibility. As expected, 260

P2K2 can bind ATP (Fig. 2) with high affinity, roughly equivalent to that measured previously 261

for P2K1 (Choi et al., 2014) and consistent with ATP levels required to induced measurable 262

physiological responses (Tanaka et al., 2010). Interestingly, in vitro competitive binding data 263

indicate that P2K2 has a higher specificity for ATP and ADP than demonstrated for P2K1 (Fig. 264

2) (Choi et al., 2014). The physiological relevance of this is unknown but could be important, 265

especially when P2K1 and P2K2 may function in planta as a heteromeric complex (Fig. 4). 266



10

P2K1 and P2K2 are likely the result of a tandem genome duplication. Therefore, P2K1 and 267

P2K2 were suggested to be ohnologous genes (Hofberger et al., 2015) (paralogous genes that 268

diverged at the same time through whole-genome duplication). Ohnologous genes can share 269

similar functions, but, more often, diverge and develop different functions (Moreira and López-270

García, 2011; Hofberger et al., 2015). Our current data argue that both P2K1 and P2K2 have a 271

similar function in that they bind and respond to eATP and can, at least partially complement one 272

another (Fig. 1, Fig. 2 and Supplemental Fig. S7) (Choi et al., 2014). 273

Our data suggest the possibility that P2K1 and P2K2 may function in planta as part of a 274

heteromeric complex. However, it should be noted that, under normal growth conditions, P2K1 275

is strongly expressed in most tissues, which is not the case for P2K2 (Bouwmeester and Govers, 276

2009). Our initial mutant screen for plants unable to increase intracellular calcium levels upon 277

ATP addition yielded largely mutants in P2K1 (Choi et al., 2014). Therefore, P2K1 appears to be 278

the primary eATP receptor in Arabidopsis. The expression of P2K2, although quite low under 279

normal growth conditions, is induced under stress conditions, including upon the addition of 280

ATP or pathogen treatment (Supplemental Fig. S8) (Bouwmeester and Govers, 2009; Balague et 281

al., 2017). Therefore, the possibility remains that a heteromeric complex of P2K1 and P2K2 282

could function during such stress conditions, perhaps as a means to increase the specificity 283

and/or intensity of the response. Such an occurrence would be similar to the situation in animals, 284

in which heteromeric purinergic receptor complexes are well documented (Nicke et al., 1998; 285

Virginio et al., 1998; Kawate et al., 2009). Given that P2K1 can trans-phosphorylate P2K2 but 286

not vice versa, if such a heteromeric complex is formed, it seems likely that activation of P2K1 is 287

the primary event, leading to trans-phosphorylation of P2K2 and other downstream signaling 288

components. 289

Plants recognize conserved molecular patterns derived from pathogens [i.e., pathogen-associated 290

molecular patterns (PAMPs)] resulting in the activation of an innate immune response [called 291

PAMP-triggered immunity (PTI)] (Jones and Dangl, 2006). These PAMPs are recognized by 292

specific pattern recognition receptors (PRRs). In a similar manner, specific PRRs can also 293

recognize molecules released from plants due to cellular damage (termed damage-associated 294

molecular patterns, DAMPs) (Gust et al., 2017). The responses of plants to DAMPs are similar 295

to PTI responses (Yamaguchi and Huffaker, 2011). Consequently, plants trigger a variety of 296

secondary, intracellular events, such as cytosolic calcium increase, ROS generation, and protein 297

phosphorylation (such as MAPKs and CDPKs). As a result, various transcription factors (such as 298



11

MYBs, MYCs, and ZATs) are activated that contribute to pathogen resistance (Bigeard et al., 299

2015). In both animals and plants, eATP is defined as a DAMP (Choi et al., 2014; Tanaka et al., 300

2014; Tripathi and Tanaka, 2018). Recognition of eATP by its receptors triggers very similar 301

responses as seen in the plant response to well-characterized PAMPs (Choi et al., 2014; Chen et 302

al., 2017). Indeed, given that PAMPs appear to induce the release of ATP, we previously 303

proposed that at least a portion of the PTI response is actually due to purinergic signaling (Choi 304

et al., 2014; Chen et al., 2017). It is now clear that both P2K1 and P2K2 contribute to the ability 305

of ATP to induce plant immune responses. An important role of P2K2 in innate immunity is 306

consistent with an increase in P2K2 expression upon either ATP or pathogen treatment 307

(Supplemental Fig. S8). The data are consistent with a model in which P2K2, normally 308

expressed at a low basal level, is induced upon pathogen challenge (perhaps specifically due to 309

eATP), resulting in an elevation of purinergic signaling and the formation of a stress-specific, 310

heteromeric P2K1-P2K2 complex. 311

Humans possess 7 P2X receptors and 8 P2Y receptors and, therefore, is it possible for plants to 312

function with only two purinergic receptors, P2K1 and P2K2? We are cognizant of the fact that 313

the identification of these two plant receptors relied on the ability to detect an increase in 314

intracellular calcium upon ATP addition. Hence, if other receptors exist that are not coupled to 315

this calcium response, our assays would not have detected them. The fact that P2K2 is not 316

expressed except under stress conditions likely explains why this particular receptor was not 317

identified in our initial mutant screens, suggesting that other receptors may only be detectable 318

under very specific growth conditions. There are 45 LecRK proteins encoded within the 319

Arabidopsis genome, 60 L-type LecRKs in soybean (Glycine max) (Liu et al., 2018), 50 L-type 320

LecRKs in Populus (Yang et al., 2016), 84 L-type LecRKs in wheat (Triticum aestivum) 321

(Shumayla et al., 2016), 72 L-type LecRKs in rice (Oryza sativa) (Vaid et al., 2012), and 53 L-322

type LecRKs in foxtail millet (Setaria italica) (Zhao et al., 2016). If only a fraction of these 323

LecRK proteins function in purinergic signaling, then the field is indeed very rich in future 324

discoveries as the community continues to explore all of the various functions that eATP plays in 325

plant growth and development. 326

327

Materials and methods 328

Phylogenetic tree analysis 329



12

11 LecRK clade I protein sequences were collected from the Arabidopsis Information Resource 330

website (TAIR https://www.arabidopsis.org/). The AGI numbers are: LecRK-I.1 331

(AT3G45330.1); LecRK-I.10 (AT5G60310.1); LecRK-I.11 (AT5G60320.1); LecRK-I.2 332



(AT5G60280.1); LecRK-I.9 (AT5G60300.3). The sequences alignment was performed using 335

Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/). The phylogenetic tree was generated 336

in MEGA7.0 by using the Maximum Likelihood method based on the JTT matrix-based model. 337

338

Plant materials 339

All Arabidopsis thaliana plants used in this study are aequorin-expressing lines in the Col-0 340

background. Wild-type and p2k1-3 mutants (SalK_042209) with Aequorin (AEQ) stable 341

transgenic lines were described in our previous studies (Choi et al., 2014; Chen et al., 2017). The 342

lecrk-i.5 T-DNA mutant, p2k2 (GK-777H06), was obtained from the Arabidopsis Biological 343

Resource Center (ABRC, Ohio State University, Columbus, OH) and crossed with wild-344

type/AEQ and p2k1-3/AEQ to generate p2k2/AEQ and p2k1p2k2/AEQ. Homozygosity for the T-345

DNA insertion and the aequorin transgene in F2 progeny was confirmed by PCR-based 346

genotyping and RT-qPCR using the specific primers listed in Supplemental Table S1. 347

Arabidopsis seeds were sterilized and submerged in autoclaved water and incubated at 4oC for 4-348

7 days before sowing onto half-strength Murashige and Skoog medium plates (1% (w/v) sucrose, 349

0.4% (w/v) phytagel, 2.56 mM MES (pH 5.7). The seedlings were grown under long-day 350

conditions in a growth chamber with the setting of 16 h light, 8 h dark, 22oC, 70% humidity, and 351

100 µE cm-2sec-1 light intensity. 352

353

Molecular complementation and ectopic expression 354

Full-length genomic DNA of clade I LecRK members were amplified using specific primers 355

(Supplemental Table S1) from chromosomal DNA of wild-type (Col-0). The PCR products were 356

cloned into the pDONR-Zeo vector using Gateway BP Clonase II enzyme mix (Invitrogen). 357

Target sequences were then cloned into pGWB14 destination vectors using Gateway LR Clonase 358

II Enzyme Mix (Invitrogen). These constructs were electroporated into Agrobacterium 359

tumefaciens GV3101 for transformation into p2k1-3/AEQ using the floral dipping method (Davis 360


https://www.ebi.ac.uk/Tools/msa/clustalo/


13

et al., 2009). The stable transgenic lines were selected on medium that contained hygromycin B 361

at a concentration of 25 µg/ml. Target gene insertion events were confirmed using specific 362

forward primers of target genes and HA reverse primer (Supplemental Table S1). Full-length 363

genomic DNA of P2K2 (At3g45430) with and without the ~1.5 kb 5'-flanking region were 364

amplified using specific primers (Supplemental Table S1) from wild-type genomic DNA. The 365

products were cloned into the pDONR-Zeo vector. Target sequences were then cloned into 366

pGWB13 or pGWB14 destination vectors using LR cloning. These constructs were 367

electroporated into A. tumefaciens GV3101 for transformation into wild-type/AEQ, p2k2/AEQ, 368

or p2k1/AEQ using the floral dipping method (Davis et al., 2009). The stable transgenic lines 369

were confirmed by hygromycin resistance and qPCR using the appropriate specific primer set 370

(Supplemental Table S1). The T2 transgenic lines were used for calcium assay while T3 371

transgenic lines were used for other experiments. 372

373

Cytoplasmic calcium assay 374

Five-day-old Arabidopsis seedlings were individually transferred into separated wells of white 375

96-well plates. Each of the wells contained 50 µl of CTZ buffer, including 10 µM coelenterazine 376

(Nanolight technology, Pinetop, AZ), 2 mM MES, 10 mM CaCl2, pH 5.7. The plates were 377

incubated at room temperature overnight in the dark, followed by the addition of 50 µl of 2x 378

treatment solution directly applied into the wells using a multi-channel pipette. This process 379

takes approximately 10-20 seconds before measurements can be initiated. The production of 380

luminescence was immediately measured using an image-intensifying CCD camera (Photek 216; 381

Photek, Ltd.). After these measurements, the remaining unchelated aequorin was estimated by 382

applying discharging buffer, including 2 M CaCl2 and 20% (v/v) ethanol, and luminescence was 383

measured by the CCD camera. The photon counting data were normalized and converted into 384

calcium concentration using the procedures of (Mithofer and Mazars, 2002). 385

386

Plasmid construction and protein purification 387

The full-length genomic DNA of P2K2 in the pDONR-Zeo vector was used as the template for 388

further cloning. The extracellular domain and kinase domain of P2K2 were amplified using 389

gene-specific primers (Supplemental Table S1) by PCR. The PCR products were digested by 390

restriction enzymes BamHI and XhoI for extracellular domain cloning. After gel extraction and 391



14

purification, the DNA products were cloned into the pGEX-2T vector (in the case of 392

extracellular domain cloning) and pET-41a or pET-28a vectors (in the case of kinase domain 393

cloning) using T4 DNA ligase enzyme. To generate mutant versions, the wild-type P2K2 394

extracellular domain in pGEX-5X-1 of the kinase domain in pET-41a was used as a template. 395

The constructs used for protein purification were transformed into Escherichia coli BL21-AI 396

(Invitrogen). The bacteria were cultured in LB medium to reach OD600 = 0.6-0.9 at 37oC. The 397

bacteria were shaken at 25oC for 30 minutes before the target proteins were induced by addition 398

of 0.1 mM IPTG at 25oC, for 6 hours. The culture was then centrifuged at 4500 rpm for 10 399

minutes and 4oC. After decanting the LB medium, the bacterial pellet for GST-fused protein 400

purification was resuspended in TBS buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 401

0.1% (v/v) Triton X-100, 1X protease inhibitor (Pierce), and 2 mM PMSF; while cells for HIS-402

fused protein purification were resuspended in HIS-lysis buffer containing 50 mM sodium 403

phosphate pH 7.5, 300 mM NaCl, 8 mM Imidazole, 0.05% (v/v) NP-40, 1X protease inhibitor 404

(Pierce), and 2 mM PMSF. The cells were broken by sonication followed by centrifugation at 405

12,000 rpm at 4oC for 10 minutes. The supernatant was then applied to glutathione sepharose 4B 406

R10 resin (GE healthcare) for GST-fused proteins, while TALON Metal Affinity Resin 407

(Clontech) was used for HIS-fused proteins. The supernatant was incubated with gentle rotation 408

with the resin for 2 hours at 4oC; the resin was collected by centrifugation at 5000 rpm for 5 409

minutes at 4oC. The resin was washed three times with 1X TBS buffer (for GST-fused proteins) 410

or HIS washing buffer containing 50 mM sodium phosphate pH 7.5, 300 mM NaCl, and 8 mM 411

Imidazole. After removing the washing buffer, elution buffer containing 50 mM Tris-HCl pH 412

8.0, 150 mM NaCl, 25 mM reduced glutathione (for GST-fused proteins) or 150 mM Imidazole 413

(for His-fused proteins), and 10% (v/v) glycerol was added. Purified proteins were stored at -414

80oC until use. 415

416

In vitro ATP binding assay 417

The purified P2K2 extracellular domain protein was mixed in a 50 µl reaction including 10 mM 418

HEPES (pH 7.5), 5 mM MgCl2, in the presence or absence of 100-fold unlabeled ATP (for the 419

specific binding assay) or unlabeled other nucleotides (for the competitive binding assay), and 420

γ32P-ATP (PerkinElmer, 800 Ci mmol-1). The reactions were incubated at 4oC for 30 minutes. 421

After that, the reactions were loaded onto Sephadex G-25 gel filtration columns (GE Healthcare). 422



15

The free nucleotides were trapped in the column while the bound radioligand went through the 423

column and was collected by scintillation vials. After mixing with scintillation cocktail (MP 424

Biomedicals), the signal of bound radioligand was measured using liquid scintillation counting 425

(Tri-Carb 2810TR, PerkinElmer). The data were analyzed using GraphPad Prism 7. 426

427

In-vitro kinase assay 428

The kinase assays were performed with minor modifications as described by (Choi et al., 2014). 429

For the kinase assay, 5 µg of GST or GST-fusion proteins were mixed with or without 2 µg 430

myelin basic protein in kinase assay buffer (50 mM Tris-HCl pH 7.5, 50 mM KCl,10 mM 431

MgCl2, 4 µM ATP, 0.2 ul γ32P-ATP (PerkinElmer; specific activity 6000 Ci mmol-1)) and 432

incubated for 30 mins at 30oC. After 10% SDS-PAGE, the gels were exposed for 3 h for 433

autoradiography. After the kinase assay, samples were treated with 0.5 µl of Lambda protein 434

phosphatase (NEB). Results were detected by 10% SDS-PAGE and Coomassie brilliant blue 435

staining. 436

437

Bimolecular fluorescence complementation (BiFC) 438

Full-length genomic DNA of P2K2 from the pDONR-Zeo, described above, was cloned into 439

pAM-PAT-35s:YFP:GW (for identification of P2K2 sub-cellular localization) and pAM-PAT-440

35s:YFPn (for BiFC assay) destination vectors. For the free YFP control, a 66 bp fragment of 441

YFP was sub-cloned into the pDONR-Zeo vector and then cloned into pAM-PAT-35s:YFP:GW, 442

pAM-PAT-35s:YFPn:GW, or pAM-PAT-35s:YFPc:GW. The full-length cDNA of P2K1 in 443

pAM-PAT-35s:YFPc:GW was described from our previous study (Chen et al., 2017). These 444

constructs were transformed or co-transformed into Arabidopsis protoplasts. The protoplast 445

isolation and transformation were performed as previously described (Cao et al., 2016). The YFP 446

fluorescence was observed under a Leica DM 5500B compound microscope using a Leica 447

DFC290 color digital camera. FM4-64 dye (Invitrogen, T3166) was directly added into the W5 448

solution containing protoplasts for plasma membrane staining before visualization under the 449

fluorescence microscope. 450

451

Split-luciferase complementation assay 452



16

The vector pCambia-NLuc and pCambia-CLuc were kindly provided by Dr. Jian-Min Zhou 453

(Chen et al., 2008). EFR, P2K1 and P2K2 in pDONR-Zeo constructs were used for LR reactions 454

to generate EFR, P2K1 and P2K2 in pCambia-NLuc and P2K2 in pCambia-CLuc. These 455

constructs were electroporated into the A. tumefaciens GV3101. These constructs were co-456

infiltrated into 3-week-old N. benthamiana leaves following a protocol of (Li, 2011). After three 457

days, the infiltrated leaves were treated with 200 µM ATP or 2 mM MES (pH 5.7) or non-458

treated. After 30 minutes of treatment, the leaves were sprayed with 1 mM luciferin (Goldbio) 459

and kept in the dark for 6 minutes before being observed under a CCD imaging apparatus 460

(Photek 216; Photek, Ltd.). 461

462

GST-HIS pull-down assay 463

The LYK5 in pGEX-5X-1 was kindly provided by Dr. Dongqin Chen (Chen et al., 2017). The 464

p2k1-1 kinase-dead and wild-type P2K1 kinase domain in pGEX-5X-1 was kindly provided by 465

Dr. Jeongmin Choi (Choi et al., 2014). The in vitro pull-down assay was performed as described 466

by Chen et al. 2018 with minor modifications. In brief, 2 µg of HIS-fused and GST-fused 467

proteins were mixed in 1 ml pull-down buffer containing 50 mM Tris-HCl pH 7.5, 100 mM 468

NaCl, and 0.5% (v/v) Triton X-100, followed by incubation for 1 hour at 4oC. After that, 25 469

µl glutathione resin was added and incubated for one hour at 4oC. The resin was washed at 470

least ten times with pull-down buffer. The proteins were eluted using GST elution buffer 471

containing 50 mM Tris-HCl pH 8.0, 150 mM NaCl, and 25 mM reduced glutathione. The results 472

were detected by SDS-PAGE, followed by immunoblotting using anti-HIS (SAB1305538, 473

Sigma) and anti-GST (A01380-40, GeneScript) antibody. 474

475

Bacterial inoculation assay 476

The assay was modified from the Arabidopsis seedling flood inoculation assay (Ishiga et al., 477

2011). In detail, 3-week-old Arabidopsis seedlings grown in square Petri dishes were used for 478

this assay. Fourty ml of P. syringae pv. tomato DC3000 Lux (Fan et al., 2008) (OD600 = 0.002) 479

bacterial suspension in sterile water with 0.025% (v/v) Silwet L-77 was dispensed into the dishes 480

for 2-3 mins. After removing the bacterial suspension, the plants were incubated in a growth 481

chamber. After one day post-inoculation, the seedlings, without roots, were collected and the 482

weight was determined followed by washing with sterile water for 5 minutes. Bacterial growth 483



17

was visualized and analyzed under a CCD camera (Photek 216; Photek, Ltd.). The seedling 484

tissue was ground in 10 mM MgCl2, diluted serially, and dropped onto King B agar plates 485

containing rifampicin and kanamycin. The number of colonies (CFU) was counted and analyzed 486

after incubation at room temperature for two days. 487

488

MAPK assay 489

Ten-day-old Arabidopsis seedlings were incubated in sterile water at room temperature 490

overnight. After treatment with 200 µM ATP, total protein was extracted from whole seedlings 491

by homogenization in RIFA buffer containing 10 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM 492

EDTA, 0.1% (v/v) NP-40, 1 mM DTT, 0.5% (w/v) sodium deoxycholate, 2 mM PMSF, and 1X 493

protein inhibitor (Pierce). The clear lysate was mix with 5X Laemmli loading buffer, containing 494

10% (w/v) SDS, 50% (v/v) glycerol, 0.01% (w/v) bromophenol blue, 10% (v/v) beta-495

mercaptoethanol, 0.3 M Tris-HCl pH 6.8, and heated in boiling water 10 minutes. The total 496

extracted proteins were separated by 10% SDS-PAGE and transferred to polyvinylidene 497

difluoride (PVDF) membranes. The transblots were blocked by 5% (w/v) skim milk in TBST 498

buffer and then incubated with rabbit anti-phospho-p44/p42 MAPK antibody (Cell signaling 499

technology). After washing three times with TBST, the immunoblots were incubated with 500

horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (Jackson ImmunoResearch 501

Laboratories). After washing three times with TBST, the final signal on immunoblots was 502

visualized using a Fuji LAS3000 luminescence imaging system (Fujifilm). 503

504

RT-qPCR assay 505

Five-day-old Arabidopsis seedlings were incubated in sterile water at room temperature 506

overnight. After treatment with 200 µM ATP, samples were collected for total RNA extraction 507

using Trizol reagent (Invitrogen). One microgram of total RNA was treated with Turbo DNA-508

free DNase (Ambion). The RNA was then used for cDNA synthesis using the M-MLV kit 509

(Promega). SYBR Green and the 7500 Realtime PCR system (Applied Biosystems) was used 510

to perform the quantitative PCR with specific primer sets defined in Supplemental Table S1. 511

RNA levels were normalized against the expression of the reference gen, SAND (At2g28390) 512

(Choi et al., 2014). 513

514



18

Statistical Analyses 515

The average and standard error of all results were calculated and one-way anova and student’s 516

t-tests were performed using IBM SPSS statistics version 25. The The inhibition constant (Ki), 517

dissociation constant (Kd), maximum binding capacity (Bmax) and model goodness of fit (R2) 518

values in in vitro ATP binding assay were calculated using a one site – fit Ki non-linear 519

regression model, GraphPad Prism 6. 520

Accession Numbers 521

Sequence data from this article can be found in Tair (https://www.arabidopsis.org/) using 522

accession number: LecRK-I.1, At3g45330.1; LecRK-I.10, At5g60310.1; LecRK-I.11, 523

At5g60320.1; LecRK-I.2, At3g45390.1; LecRK-I.3, At3g45410.1; LecRK-I.4, 524

At3g45420.1; LecRK-I.5, At3g45430.1; LecRK-I.6, At3g45440.1; LecRK-I.7, At5g60270.1; 525

LecRK-I.8, At5g60280.1; LecRK-I.9, At5g60300.3; EFR, At5g20480.1; LYK5, At2g33580.1; 526

SAND, At2g28390.1. 527

528

Acknowledgements: Research was supported by the National Institute of General Medical 529

Sciences of the National Institutes of Health (grant no. R01GM121445), the Next-Generation 530

BioGreen 21 Program Systems and Synthetic Agrobiotech Center, Rural Development 531

Administration, Republic of Korea (grant no. PJ01116604) and through the 3rd call of the ERA-532

NET for Coordinating Action in Plant Sciences, with funding from the US National Science 533

Foundation (grant 1826803). 534

535

Supplemental Data 536

Supplemental Figure S1: Molecular phylogenetic analysis of AtLecRK clade I members. 537

Supplemental Figure S2: Ectopic expression of other clade-I lecRKs in p2k1-3 mutant plants 538

cannot restore the ability to response to eATP. 539

Supplemental Figure S3: P2K2 could play a partially redundant role with P2K1 during ATP-540

induced responses. 541

Supplemental Figure S4: Domain structure of P2K2. 542

Supplemental Figure S5: P2K2 kinase domains, wild-type, and mutant versions were treated 543

with Lambda protein phosphatase (PPase). 544


https://www.arabidopsis.org/


19

Supplemental Figure S6: P2K2 localized to the plasma membrane in the root. 545

Supplemental Figure S7: Expression of P2K1 and P2K2 in mutant lines. 546

Supplemental Figure S8: ATP treatment triggered elevated expression of P2K2. 547

Supplemental Table S1: List of primers. 548

549

Figure legends 550

Figure 1. P2K2 (LecRK-I.5) plays a critical role in the eATP-triggered cytosolic calcium 551

response. (A) Ectopic expression of P2K2 (p35S:LecRK-I.5) in p2k1-3 mutant plants confers 552

partial complementation of the eATP-triggered cytosolic calcium response phenotype. 1 to 5 553

represent independent transgenic plants (B) eATP-triggered calcium response of the p2k2 T-554

DNA mutant and two independent P2K2 overexpression lines. The bar graphs show total 555

cytosolic [Ca2+] after 200 µM ATP treatment over 400 seconds. The wild-type (Col-0) and p2k1-556

3 mutant were used as controls. Data represent means ± SEs, n = 8; The panels with different 557

letters were considered statistically significant (P<0.05, ANOVA). Experiments were repeated at 558

least three times with similar results. 559

560

Figure 2. P2K2 binds ATP. (A) In vitro binding of 32P-labeled ATP to the ectodomain of P2K2 561

wild-type version. After incubating purified ectodomain of P2K2 with indicated concentrations 562

of 32P-labeled ATP, bound ATP and free ATP were separated by gel filtration chromatography. 563

Data were calculated as a mean of specific binding with SE of three replications. The 564

dissociation constant (Kd), maximum binding capacity (Bmax) and model goodness of fit (R2) 565

were calculated by a non-linear regression model analysis using GraphPad Prism 7. (B) 566

Competitive binding assay for P2K2. 25 nM 32P-labeled ATP and 10 nM to 10 mM of unlabeled 567

nucleotides were used for the assay. After gel filtration chromatography, the results were 568

obtained by measuring specific binding of 32P-labeled ATP. The inhibition constant (Ki) values 569

were calculated using a one site – fit Ki non-linear regression model, GraphPad Prism 7. Data are 570

represented as mean of two replicates. 571

572

Figure 3. P2K2 has strong kinase activity which plays a critical role in the eATP-triggered 573

calcium response. (A) P2K2 phosphorylates MBP (myelin basic protein) in vitro. Purified GST-574

P2K2-KD (kinase domain) recombinant protein was incubated with MBP. GST (glutathione S-575



20

transferase) and P2K1-KD were used as controls. Autophosphorylation and trans-576

phosphorylation were measured by incorporation of 32P-labeled ATP. (B) Kinase activity of the 577

P2K2-KD and its mutant versions (p2k2D467N and p2k2D525N). Autoradiographs (top panels) show 578

the kinase activity of indicated proteins. Coomassie blue panels (bottom panels) show the 579

loading control. P2K2-KD with MBP was used as a control. Experiments were repeated at least 580

three times with similar results. (C) Ectopic expression of the kinase-dead mutant P2K2 in the 581

p2k1-3 mutant line failed to complement the eATP-triggered cytosolic calcium response 582

phenotype. The wild-type (Col-0), p2k1-3 mutant and ectopic expression of P2K2 in the p2k1-3 583

mutant were used as controls. 1 and 2 represent independent transgenic lines. Data represent 584

means ± SEs, n = 8; The panels with different letters were considered statistically significant 585

(P<0.05, ANOVA). Experiments were repeated at least three times with similar results. 586

587

Figure 4. P2K2 is localized to the plasma membrane, self-associates and interacts with 588

P2K1. (A) Fluorescence microscope images of Arabidopsis protoplasts transiently expressing 589

the indicated constructs. Bright field shows non-fluorescence protoplasts. The protoplast plasma 590

membrane was labeled with the FM4-64 dye. Chlorophyll was detected by auto-fluorescence. 591

Free YFP was used as a control for P2K2 localization. EFR-YFPn was used as control for BiFC 592

assay. Bar = 20 µm. (B) Split-luciferase assay image of N. benthamiana leaves co-infiltrated 593

with the agrobacterial strains containing P2K2-NLuc/CLuc, P2K1-NLuc and EFR-NLuc. Circles 594

indicate leaf panels that were infiltrated with Agrobacterium containing each construct. ATP: 595

leaves infiltrated with 200 µM ATP; Mock: leaves infiltrated with 2 mM MES (pH 5.7). 596

Asterisks denote values significantly different from P2K2/EFR (top, n = 4) or mock treatment 597

(bottom, n = 7) (*P<0.05, Student’s t-test). (C) P2K2 directly interacts with P2K1 and itself in 598

vitro. Purified GST-P2K1-KD, GST-P2K2-KD and GST-LYK5-KD (negative control) 599

recombinant proteins were incubated with or without His-P2K2-KD for one hour. The results of 600

GST-bead pull-down were detected by anti-His and anti-GST immunoblot (two top panels). The 601

His-P2K2-KD loading control was detected by anti-His immunoblot (bottom panels). All 602

experiments were repeated with similar results. 603

604

Figure 5. P2K1 can phosphorylate P2K2. (A) P2K1 and P2K2 can phosphorylate a kinase 605

dead version of P2K2. Purified GST-p2k2D467N-KD (kinase dead) or GST-LYK5-KD (negative 606

control) was incubated with GST-P2K1-KD, GST-P2K2-KD or GST (negative control) in an in 607



21

vitro kinase assay. (B) P2K1 and P2K2 cannot phosphorylate kinase dead versions of P2K1. 608

Purified GST-p2k1D525N-KD, GST-p2k1D572N-KD or GST-p2k2D467N-KD (positive control) was 609

incubated with GST-P2K1-KD, GST-P2K2-KD or GST in an in vitro kinase assay. 610

Autophosphorylation and trans-phosphorylation were detected by incorporation of 32P labeled-611

ATP (top panels). The protein loading was measured by Coomassie brilliant blue (CBB) staining 612

(bottom panels). LD: Ladder. These experiments were repeated three times with similar results. 613

614

Figure 6. P2K2 plays a critical role in plant resistance to P. syringae DC3000. Wild-type 615

(Col-0) and the p2k3-1 mutant were used as controls to compare to p2k2, p2k1 p2k2, 616

pP2K2:P2K2 (the p2k2 complemented line using the P2K2 native promoter) and p35S:P2K2 (the 617

P2K2 overexpression line) plants. Fourteen-day-old seedlings were flood inoculated with a P. 618

syringae DC3000 lux suspension (OD600 = 0.002) containing 0.025% (v/v) Silwet L-77. At one 619

day after inoculation, (A) bright field photographs were taken by a normal camera while bacteria 620

invasion was detected by a CCD camera (the bio-luminescence panels); (B) bacterial 621

colonization was determined by plate counting. Values represent the mean ± SEs, n = 6 622

(biological replicates). ANOVA with multiple comparisons analysis was calculated by GraphPad 623

Prism 7. Means with different letters are significantly different (P< 0.05). The experiment was 624

repeated with similar results. 625

626

Figure 7. The p2k2 and p2k1 p2k2 mutants are defective in ATP-induced downstream 627

responses. (A) The p2k1-3 (a negative control), p2k2 and p2k1 p2k2 mutant plants showed weak 628

MPK3 and MPK6 phosphorylation in response to 200 µM ATP, compared with wild-type (Col-629

0) plants (a positive control). The phosphorylation of MPK3 and MPK6 was detected by using 630

antibody against phospho-p44/p42 mitogen-activated protein kinase (top panel). Coomassie 631

brilliant blue (CBB) staining was used as the loading control (bottom panel). (B) and (C) Wild-632

type (Col-0) and p2k1-3 mutant plants were used as controls to compare to p2k2 and p2k1 p2k2 633

mutant plants. Ten-day-old seedlings were treated with 200 µM ATP for 0, 30 or 120 minutes 634

and RNA was collected for RT-qPCR analysis. Expression of (B) MYC2 and (C) ZAT10 were 635

normalized against the SAND reference gene. The results are relative to expression levels of 636

mock-treated plants (set as 1). The bar graphs are means of three biological replicates; the error 637

bars are SEs. Asterisks show the significant difference to wild-type at the same time points 638

(*P<0.05, Student’s t-test). 639



22

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Figure 1. P2K2 (LecRK-I.5) plays a critical role in the eATP-triggered cytosolic calcium

response. (A) Ectopic expression of P2K2 (p35S:LecRK-I.5) in p2k1-3 mutant plants confers

partial complementation of the eATP-triggered cytosolic calcium response phenotype. 1 to 5

represent independent transgenic plants (B) eATP-triggered calcium response of p2k2 T-DNA

mutant and two independent P2K2 overexpression lines. The bar graphs show total cytosolic

[Ca2+] after 200 µM ATP treatment over 400 seconds. The wild-type (Col-0) and p2k1-3 mutant

were used as controls. Data represent means ± SEs, n = 8; The panels with different letters were

considered statistically significant (P<0.05, Anova). Experiments were repeated at least three

times with similar results.



Figure 2. P2K2 binds ATP. (A) In vitro binding of 32P-labeled ATP to ectodomain of P2K2

wild-type version. After incubating purified ectodomain of P2K2 with indicated concentrations

of 32P-labeled ATP, bound ATP and free ATP were separated by gel filtration chromatography.

Data were calculated as a mean of specific binding with SE of three replications. The

dissociation constant (Kd), maximum binding capacity (Bmax) and model goodness of fit (R2)

were calculated by a non-linear regression model analysis using GraphPad Prism 7. (B)

Competitive binding assay for P2K2. 25 nM 32P-labeled ATP and 10 nM to 10 mM of unlabeled

nucleotides were used for the assay. After gel filtration chromatography, the results were

obtained by measuring specific binding of 32P-labeled ATP. The inhibition constant (Ki) values

were calculated using a one site – fit Ki non-linear regression model, GraphPad Prism 7. Data

represented as mean of two replicates.



Figure 3. P2K2 has strong kinase activity which plays a critical role in the eATP-triggered

calcium response. (A) P2K2 phosphorylates MBP (myelin basic protein) in vitro. Purified GST-

P2K2-KD (kinase domain) recombinant protein was incubated with MBP. GST (glutathione S-

transferase) and P2K1-KD were used as controls. Autophosphorylation and trans-

phosphorylation were measured by incorporation of 32P-labeled ATP. (B) Kinase activity of the

P2K2-KD and its mutant versions (p2k2D467N and p2k2D525N). Autoradiographs (top panels) show

the kinase activity of indicated proteins. Coomassie blue panels (bottom panels) show the

loading control. P2K2-KD with MBP was used as a control. Experiments were repeated at least

three times with similar results. (C) Ectopic expression of the kinase-dead mutant P2K2 in the

p2k1-3 mutant line failed to complement the eATP-triggered cytosolic calcium response



phenotype. The wild-type (Col-0), p2k1-3 mutant and ectopic expression of P2K2 in the p2k1-3

mutant were used as controls. 1 and 2 represent independent transgenic lines. Data represent

means ± SEs, n = 8; The panels with different letters were considered statistically significant

(P<0.05, ANOVA). Experiments were repeated at least three times with similar results.



Figure 4. P2K2 is localized to the plasma membrane, self-associates and interacts with

P2K1. (A) Fluorescence microscope images of Arabidopsis protoplasts transiently expressing

the indicated constructs. Bright field shows non-fluorescence protoplasts. The protoplast plasma

membrane was labeled with the FM4-64 dye. Chlorophyll was detected by auto-fluorescence.

Free YFP was used as a control for P2K2 localization. EFR-YFPn was used as control for BiFC

assay. Bar = 20 µm. (B) Split-luciferase assay image of N. benthamiana leaves co-infiltrated

with the agrobacterial strains containing P2K2-NLuc/CLuc, P2K1-NLuc and EFR-NLuc. Circles

indicate leaf panels that were infiltrated with Agrobacterium containing each construct. ATP:

leaves infiltrated with 200 µM ATP; Mock: leaves infiltrated with 2 mM MES (pH 5.7).

Asterisks denote values significantly different from P2K2/EFR (top, n = 4) or mock treatment

(bottom, n = 7) (*P<0.05, Student’s t-test). (C) P2K2 directly interacts with P2K1 and itself in



vitro. Purified GST-P2K1-KD, GST-P2K2-KD and GST-LYK5-KD (negative control)

recombinant proteins were incubated with or without His-P2K2-KD for one hour. The results of

GST-bead pull-down were detected by anti-His and anti-GST immunoblot (two top panels). The

His-P2K2-KD loading control was detected by anti-His immunoblot (bottom panels). All

experiments were repeated with similar results.



Figure 5. P2K1 can phosphorylate P2K2. (A) P2K1 and P2K2 can phosphorylate a kinase

dead version of P2K2. Purified GST-p2k2D467N-KD (kinase dead) or GST-LYK5-KD (negative

control) was incubated with GST-P2K1-KD, GST-P2K2-KD or GST (negative control) in an in

vitro kinase assay. (B) P2K1 and P2K2 cannot phosphorylate kinase dead versions of P2K1.

Purified GST-p2k1D525N-KD, GST-p2k1D572N-KD or GST-p2k2D467N-KD (positive control) was

incubated with GST-P2K1-KD, GST-P2K2-KD or GST in an in vitro kinase assay.

Autophosphorylation and trans-phosphorylation were detected by incorporation of 32P labeled-

ATP (top panels). The protein loading was measured by Coomassie brilliant blue (CBB) staining

(bottom panels). LD: Ladder. These experiments were repeated three times with similar results.



Figure 6. P2K2 plays a critical role in plant resistance to P. syringae DC3000. Wild-type

(Col-0) and p2k3-1 were used as controls to compare to p2k2, p2k1p2k2, pP2K2:P2K2 (the p2k2

complemented line using P2K2 native promoter) and p35S:P2K2 (the P2K2 overexpression

line). Fourteen-day-old seedlings were flood inoculated with a P. syringae DC3000 lux

suspension (OD600 = 0.002) containing 0.025% (v/v) Silwet L-77. At one day after inoculation,



(A) Bright field photographs were taken by normal camera while bacteria invasion was detected

by a CCD camera (the bio-luminescence panels); (B) bacterial colonization was determined by

plate counting. Values represent the mean ± SEs, n = 6 (biological replicates). ANOVA with

multiple comparisons analysis was calculated by GraphPad Prism 7. Means with different letters

are significantly different (P< 0.05, Anova). Experiment was repeated with similar results.



Figure 7. The p2k2 and p2k1p2k2 mutants are defective in ATP-induced downstream

responses. (A) The p2k1-3 (a negative control), p2k2 and p2k1p2k2 mutant plants showed weak

MPK3 and MPK6 phosphorylation in response to 200 µM ATP, compared with wild-type (Col-

0) plants (a positive control). The phosphorylation of MPK3 and MPK6 was detected by using

antibody against phospho-p44/p42 mitogen-activated protein kinase (top panel). Coomassie

brilliant blue (CBB) staining was used as loading control (bottom panel). (B) and (C) Wild-type

(Col-0) and p2k1-3 mutant plants were used as controls to compare to p2k2 and p2k1p2k2 mutant

plants. 10-day-old seedling plants were treated with 200 µM ATP for 0, 30 or 120 minutes and

RNA collected for qRT-PCR analysis. Expression of (B) MYC2 and (C) ZAT10 were normalized

against the SAND reference gene. The results are relative to expression levels of mock treated

plants (set as 1). The bar graphs are means of three biological replicates; the error bars are SEs.

Asterisks show the significant difference to wild-type at the same time points (*P<0.05,

Student’s t-test).



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https://www.ncbi.nlm.nih.gov/pubmed?term=Tripathi D%2C Zhang T%2C Koo AJ%2C Stacey G%2C Tanaka K %282018%29 Extracellular ATP Acts on Jasmonate Signaling to Reinforce Plant Defense%2E Plant physiology&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Vaid N%2C Pandey PK%2C Tuteja N %282012%29 Genome%2Dwide analysis of lectin receptor%2Dlike kinase family from Arabidopsis and rice%2E Plant Mol Biol&dopt=abstract

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https://scholar.google.com/scholar?as_q=A lectin receptor kinase as a potential sensor for extracellular nicotinamide adenine dinucleotide in Arabidopsis thaliana.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Wang&as_ylo=2017&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Wang Y%2C Cordewener JH%2C America AH%2C Shan W%2C Bouwmeester K%2C Govers F %282015%29 Arabidopsis Lectin Receptor Kinases LecRK%2DIX%2E1 and LecRK%2DIX%2E2 Are Functional Analogs in Regulating Phytophthora Resistance and Plant Cell Death%2E Molecular plant%2Dmicrobe interactions %3A MPMI&dopt=abstract

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