Short title: MEKK2 and CRCK3 in plant cell death regulation · 1 1 Short title: MEKK2 and CRCK3 in plant cell death regulation 2 3 *Corresponding authors: 4 Ping He 5 Tel: 979-458-1368

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Short title: MEKK2 and CRCK3 in plant cell death regulation 1

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*Corresponding authors: 3

Ping He 4 Tel: 979-458-1368 5 E-mail: [email protected] 6

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Xiaofeng Wang 8 Tel: 086-18092867224 9 E-mail: [email protected] 10

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RNAi-based screen reveals concerted functions of MEKK2 and CRCK3 in plant cell 13

death regulation 14

15 Yong Yang1,2, Jun Liu3, Chuanchun Yin2, Luciano de Souza Vespoli2, Dongdong Ge2, 16 Yanyan Huang3, Baomin Feng3, Guangyuan Xu3, Ana Marcia E. de A. Manhães2, Shijuan 17 Dou3,4, Cameron Criswell3, Libo Shan2, Xiaofeng Wang1#, and Ping He3# 18

19 1State Key Laboratory of Crop Stress Biology in Arid Areas, College of Horticulture, 20 Northwest A&F University, No.3, Taicheng Road, Yangling, Shaanxi 712100, China 21 22 2Department of Plant Pathology & Microbiology, Institute for Plant Genomics & 23 Biotechnology, Texas A&M University, College Station, TX 77843, USA 24 25 3Department of Biochemistry & Biophysics, Institute for Plant Genomics & Biotechnology, 26 Texas A&M University, College Station, TX 77843, USA 27 28 4 College of Life Sciences, Hebei Agricultural University, No.2596, Lekainan Street, Baoding, 29 Hebei 071001, China 30

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One-sentence summary: A transient RNAi-based genetic screen helps elucidate the 32 mechanisms of plant cell death control. 33 34 Author contributions: 35

Y.Y., J.L., L.S., X.W., and P.H. designed the research; Y.Y., J.L., C.Y., L.S.V., D.G., Y.H., B.F., 36

G.X., A.A.M., S.D., and C.C. performed the research; Y.Y., J.L., L.S., X.W., and P.H. 37

analyzed the data; Y.Y., L.S., and P.H. wrote the paper with the inputs from all authors. 38

Plant Physiology Preview. Published on March 12, 2020, as DOI:10.1104/pp.19.01555

Copyright 2020 by the American Society of Plant Biologists

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39 Funding information: 40

This work was supported by National Science Foundation (NSF) (MCB-1906060) and 41

National Institutes of Health (NIH) (R01GM092893) to P.H.; NIH (1R01GM097247) and the 42

Robert A. Welch foundation (A-1795) to L.S.; the National Natural Science Foundation of 43

China (31672142) to X.W.; Y.Y., C.Y., Y.H., and D.G. are partially supported by China 44

Scholarship Council (CSC). 45 46



3

Abstract 47

A wide variety of intrinsic and extrinsic cues lead to cell death with unclear mechanisms. The 48

infertility of some death mutants often hurdles the classical suppressor screens for death 49

regulators. We have developed a transient RNAi-based screen using a virus-induced gene 50

silencing (VIGS) approach to understand diverse cell death pathways in Arabidopsis 51

(Arabidopsis thaliana). One death pathway is due to the depletion of a MAP kinase (MAPK) 52

cascade, consisting of MEKK1, MKK1/2, and MPK4, which depends on a 53

nucleotide-binding site leucine-rich repeat (NLR) protein SUMM2. Silencing of MEKK1 by 54

VIGS resembles the mekk1 mutant with autoimmunity and defense activation. The 55

RNAi-based screen towards Arabidopsis T-DNA insertion lines identified SUMM2, MAPK 56

kinase kinase 2 (MEKK2), and Calmodulin-binding receptor-like cytoplasmic kinase 3 57

(CRCK3) to be vital regulators of RNAi MEKK1-induced cell death, consistent with the 58

reports of their requirement in the mekk1-mkk1/2-mpk4 death pathway. Similar with MEKK2, 59

overexpression of CRCK3 caused dosage- and SUMM2-dependent cell death, and the 60

transcripts of CRCK3 were upregulated in mekk1, mkk1/2, and mpk4. MEKK2-induced cell 61

death depends on CRCK3. Interestingly, CRCK3-induced cell death also depends on MEKK2, 62

consistent with the biochemical data that MEKK2 complexes with CRCK3. Furthermore, the 63

kinase activity of CRCK3 is essential, whereas the kinase activity of MEKK2 is dispensable, 64

for triggering cell death. Our studies suggest that MEKK2 and CRCK3 exert concerted 65

functions in the control of NLR SUMM2 activation and MEKK2 may play a structural role, 66

rather than function as a kinase, in regulating CRCK3 protein stability. 67

68

69



4

INTRODUCTION 70

71

Plants and microbes are engaged in a continuous battle for survival. Plants have evolved 72

sophisticated defense mechanisms to detect the telltale molecules produced by pathogens and 73

to initiate defense responses to ward off potential infections (Peng et al., 2018). In the first 74

layer, plasma membrane-localized pattern recognition receptors (PRRs) detect conserved 75

pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs), and induce 76

pattern-triggered immunity (PTI) (Yu et al., 2017; Saijo et al., 2018). Adapted pathogens 77

deliver an arsenal of effectors into host cells to damp host immune responses and promote 78

parasitism (Dou and Zhou, 2012; Macho and Zipfel, 2015). Facing these challenges, plants 79

have evolved a second layer of defenses whereby a plethora of intracellular 80

nucleotide-binding site leucine-rich repeat (NBS-LRR) receptors (NLRs) detect those 81

effectors directly or indirectly and initiate effector-triggered immunity (ETI) (Cui et al., 2015), 82

which sometimes results in localized programmed cell death (PCD) called hypersensitive 83

response (Coll et al., 2011). 84

Activation of mitogen-activated protein kinase (MAPK) cascades is one of the earliest 85

responses in plant immune signaling (Meng and Zhang, 2013). A conventional MAPK 86

cascade is assembled by three interlinked kinases: MAPKs (MPKs) are activated by MAPK 87

kinases (MAPKKs, or MKKs), which are further phosphorylated and activated by MAPK 88

kinase kinases (MAPKKKs, or MEKKs) (Zhang et al., 2018). Generally, activated MPKs 89

phosphorylate downstream targets, such as transcriptional factors and enzymes, to regulate 90

gene expression and other cellular activities. In Arabidopsis (Arabidopsis thaliana), one 91

MAPK cascade consisting of MAPKKK3/5, MKK4/5, and MPK3/6 plays diverse roles in 92

plant immune responses, such as ethylene and camalexin synthesis, and stomatal immunity 93

(Tena et al., 2011; Devendrakumar et al., 2018). MPK3/6 were reported to regulate ethylene 94

production at both transcriptional and post-transcriptional levels. Activated MPK6 95

phosphorylates and stabilizes 1-AMINOCYCLOPROPANE-1-AARBOXYLIC ACID (ACC) 96

SYNTHASE 2 (ACS2) and ACS6, two rate-limiting enzymes in ethylene biosynthesis, 97

leading to enhanced ACS proteins and increased ethylene production (Liu and Zhang, 2004). 98

Besides, MPK3/6 also phosphorylate WRKY33, a transcription factor that binds to the 99



5

promoters of ACS2 and ACS6, to regulate the expression of ACS2 and ACS6 (Li et al., 2012). 100

Another MAPK cascade comprising MEKK1, MKK1/2, and MPK4 is also activated during 101

plant defense responses (Gao et al., 2008; Qiu et al., 2008). This signaling pathway was 102

considered to negatively regulate plant immunity. Plants carrying the active form of MPK4 103

showed compromised disease resistance to bacterial pathogens and MPK4 activity negatively 104

regulates certain responses of plant PTI and ETI (Berriri et al., 2012). In addition, MPK4 105

phosphorylates a trihelix transcription factor ARABIDOPSIS SH4-RELATED3 (ASR3) to 106

suppress the expression of a subset of flg22-induced genes (Li et al., 2015). 107

The Arabidopsis mekk1, mkk1/2, and mpk4 mutants exhibit autoimmune phenotypes, 108

described as dwarfism, spontaneous cell death, constitutively activated defense responses, 109

and accumulation of reactive oxygen species (ROS) (Petersen et al., 2000; Ichimura et al., 110

2006; Nakagami et al., 2006; Suarez-Rodriguez et al., 2007; Gao et al., 2008). The cell death 111

of mekk1, mkk1/2, and mpk4 mutants could be suppressed by loss-of-function mutations in 112

coiled-coil (CC)-type NLR SUPPRESSOR OF MKK1 MKK2 2 (SUMM2) (Zhang et al., 113

2012). Recently, it was reported that the mekk1 cell death could be conditionally suppressed 114

by a mutation in the Toll/Interleukin-receptor (TIR)-type NLR RPS6 (Takagi et al., 2019), 115

suggesting that the MEKK1-MKK1/2-MPK4 pathway regulates the activation of both 116

CC-NLR (CNL) and TIR-NLR (TNL). MEKK1 belongs to a tandemly duplicated gene 117

family with MEKK2 and MEKK3, and the mutations in MEKK2, also called SUMM1, 118

suppressed the autoimmunity and defense responses of mekk1, mkk1/2, and mpk4 (Kong et al., 119

2012; Su et al., 2013). MEKK2 is also a substrate of MPK4 (Kong et al., 2012). Furthermore, 120

the abundance of MEKK2 transcripts is tightly associated with the autoimmunity observed in 121

the mekk1, mkk1/2, and mpk4 mutants (Su et al., 2013). MPK4 also associates and 122

phosphorylates CALMODULIN-BINDING RECEPTOR-LIKE CYTOPLASMIC KINASE 3 123

(CRCK3), which is required for the autoimmune phenotype of mekk1, mkk1/2, and mpk4 124

(Zhang et al., 2017). Both MEKK2 and CRCK3 function upstream of SUMM2, and CRCK3 125

is proposed to be guarded by SUMM2 for defense activation (Kong et al., 2012; Zhang et al., 126

2017). 127

We previously reported a RNAi-based genetic screen by virus-induced gene silencing 128

(VIGS) to identify suppressors of cell death mediated by two closely related receptor-like 129



6

kinases (RLKs): BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED RECEPTOR 130

KINASE 1 (BAK1), also called SOMATIC EMBRYOGENESIS RECEPTOR KINASE 3 131

(SERK3) and SERK4 (de Oliveira et al., 2016; Yu et al., 2019). In this study, we explored the 132

possibility to apply this screen to understand the mechanism in mekk1 cell death. We report 133

here that silencing of MEKK1 by VIGS resulted in autoimmune phenotypes resembling the 134

mekk1 mutant. With this RNAi-based genetic screen of Arabidopsis T-DNA insertion lines, 135

we identified several mutants as suppressors of MEKK1-mediated cell death and the 136

corresponding genes for these mutants were named LETHALITY SUPPRESSORS OF 137

MEKK1 (LETUM, or LET). Genetic analysis revealed that LET4 is MEKK2, LET5 is SUMM2, 138

and LET6 encodes CRCK3. Biochemical and genetic analysis indicates that the kinase 139

activity of MEKK2 is dispensable but the kinase activity of CRCK3 is important to regulate 140

MEKK1-mediated cell death. Similar with MEKK2, CRCK3 transcription is upregulated in 141

the mekk1, mkk1/2, and mpk4 mutants, and overexpression of CRCK3 triggered 142

dosage-dependent cell death. Furthermore, MEKK2 associates with CRCK3 and positively 143

regulates the protein homeostasis of CRCK3, which is essential to elicit NLR 144

SUMM2-dependent cell death. In addition, neither MEKK2, CRCK3, nor SUMM2 is 145

required for RNAi BAK1/SERK4-or BAK1-INTERACTING RECEPTOR-LIKE KINASE 1 146

(BIR1)-mediated cell death, suggesting diverse signaling pathways involved in cell death 147

regulation. Thus, our RNAi-based genetic screen is a useful tool to understand plant cell 148

death regulation, and our studies reveal the concerted action of MEKK2 and CRCK3 in the 149

control of NLR SUMM2 activation. 150

151

152



7

RESULTS 153

154

Silencing of MEKK1 activates spontaneous cell death and defense responses 155

156

MEKK1 was previously reported to play an important role in plant defense responses (Asai et 157

al., 2002). The mekk1 mutant exhibited a seedling-lethal phenotype, accompanied with 158

constitutive defense responses, such as elevated accumulation of hydrogen peroxide (H2O2) 159

and defense-related genes (Ichimura et al., 2006; Nakagami et al., 2006; Suarez-Rodriguez et 160

al., 2007; Gao et al., 2008). We tested whether silencing of MEKK1 by 161

Agrobacterium-mediated virus-induced gene silencing (VIGS) could phenocopy the mekk1 162

mutants. As shown in Fig. 1A and B, at two weeks after inoculation, Arabidopsis Col-0 163

wild-type (WT) plants inoculated with Agrobacteria carrying a tobacco rattle virus 164

(TRV)-based VIGS vector targeting MEKK1 exhibited a severe cell death phenotype with a 165

reduced plant stature, and curly and collapsed leaves, similar to the mekk1 mutant (Fig. 1, A 166

and B). The plants inoculated with control (Ctrl) vector carrying GFP behaved similarly with 167

non-inoculated plants (Fig. 1A). VIGS of a chloroplast development gene, CLOROPLASTOS 168

ALTERADOS 1 (CLA1) (Gao et al., 2011), induced the leaf albino phenotype, which was 169

included as a visible marker for silencing efficiency (Fig. 1B). We further carried out the 170

trypan blue staining to examine the cell death and 3,3’-diaminobenzidine (DAB) staining to 171

determine the accumulation of H2O2 in MEKK1-silenced and Ctrl plants. Compared with the 172

leaves from Ctrl plants, the leaves from MEKK1-silenced plants displayed extensive cell 173

death and accumulation of H2O2 (Fig. 1C). Defense marker genes, including 174

pathogenesis-related 1 (PR1) and PR2 were constitutively expressed in the mekk1 mutant 175

(Ichimura et al., 2006). Analysis of the expression of PR1 and PR2 by reverse transcription 176

quantitative PCR (RT-qPCR) revealed that their expression was drastically increased in the 177

MEKK1-silenced plants (Fig. 1D). Taken together, these data indicate that silencing of 178

MEKK1 via VIGS resulted in constitutively activated cell death and spontaneous defense 179

responses, similar to the mekk1 mutants. 180

181

The mekk2 and summ2 mutants suppress the cell death triggered by silencing of MEKK1, 182



8 but not BAK1/SERK4 or BIR1 183



9

184

The availability of the sequence-indexed T-DNA insertion library in Arabidopsis has greatly 185

advanced plant biology research (Alonso et al., 2003). The fast and efficient VIGS to 186

transiently silence endogenous genes enabled us to carry out multiple suppressor screens of 187

Arabidopsis T-DNA insertion lines, and to compare the divergence and convergence of cell 188

death signaling pathways mediated by key immune regulators, such as BAK1/SERK4 and 189

MEKK1. Multiple candidates that showed suppression of the cell death caused by 190

MEKK1-silencing were isolated from screening of ~10,000 Arabidopsis T-DNA insertion 191

lines. The corresponding genes for these mutants were named as LETHALITY 192

SUPPRESSORS OF MEKK1 (LETUM, or LET for short). Of the eight mutants we identified, 193

the let4 mutant (SALK_150039C) potently suppressed the dwarfism and lethality induced by 194

silencing of MEKK1 (Fig. 1B). The cell death suppression was not due to the impaired RNA 195

silencing machinery in the let4 mutant because silencing of CLA1 induced leaf albino 196

phenotype in both WT and let4 (Fig. 1B). The let4 mutant, which was renamed as mekk2, 197

harbors a T-DNA fragment inserted at 385 base pair (bp) upstream of the start codon of 198

At4g08480, encoding MEKK2. Several ethyl methanesulfonate (EMS) mutagenized mutant 199

alleles of MEKK2 have been identified as suppressors of mkk1/2 cell death, thus MEKK2 was 200

also named as SUMM1 (Kong et al., 2012). The mekk2 T-DNA insertion mutant 201

(SALK_150039C) was also able to suppress mpk4-mediated cell death (Su et al., 2013). 202

Because MEKK1 and MEKK2 are closely linked (~12 kb apart), it is not feasible or very 203

tedious to test whether mekk2 could suppress mekk1 cell death by generating the mekk1mekk2 204

double mutant with genetic crosses (Kong et al., 2012). Our VIGS approach could silence 205

genes independent of their genetic distance, and we convincingly show here that the mutation 206

in mekk2 suppressed mekk1 cell death. 207

Staining results indicated that over-accumulation of H2O2 and dead cells induced by 208

silencing of MEKK1 were abolished in the mekk2 mutant compared to WT plants (Fig. 1C). 209

Additionally, constitutive expression of PR1 and PR2 was also markedly suppressed in the 210

mekk2 mutant when MEKK1 was silenced (Fig. 1D). Consistent with the previous reports 211

(Kong et al., 2012; Su et al., 2013), the cell death and defense activation induced by silencing 212

of MEKK1 also depends on MEKK2, which further supports the feasibility to use VIGS 213



10

approach to understand mekk1-mediated cell death. 214

The let5 mutant identified from this screen is SALK_062374, which suppressed the 215

growth defect and cell death caused by silencing of MEKK1 (Fig. 1E). The let5 mutant 216

(SALK_062374) was originally annotated to bear a T-DNA insertion in At3G15720, which 217

encodes a pectin lyase-like superfamily protein. However, recent TDNA-Seq with next 218

generation sequencing identified additional T-DNA insertions in the SALK_062374 genome 219

(https://www.arabidopsis.org/servlets/TairObject?id=4664962&type=germplasm). One 220

T-DNA insertion is located in the CC region of SUMM2 (At1G12280) (Supplemental Fig. S1, 221

A and B). To confirm whether there is a T-DNA insertion in the SUMM2 gene in let5, we 222

PCR amplified SUMM2 full length genomic DNA in the WT and let5 mutant. The SUMM2 223

gene could be amplified from WT, but not let5 (Fig. 1F), suggesting that let5 carries a T-DNA 224

insertion in SUMM2. We thus named let5 as summ2-9 (Supplemental Fig. S1A). 225

We have shown before that components required for BAK1/SERK4 cell death may not 226

be involved in MEKK1 or BIR1 cell death (de Oliveira et al., 2016). We tested here whether 227

MEKK2 and SUMM2 are involved in BAK1/SERK4- or BIR1-mediated cell death by VIGS 228

assays. Although the mekk2 or summ2 mutants suppressed RNAi MEKK1 cell death, they did 229

not affect the cell death caused by RNAi BAK1/SERK4 or BIR1 (Fig. 1G). The data 230

strengthen the notion that diverse mechanisms underlie cell death regulation mediated by 231

MEKK1, BAK1/SERK4, and BIR1. 232

233

The MEKK2 kinase mutant is able to activate cell death responses 234

235

MEKK2 shows 64% identity with MEKK1 at the amino acid level (Supplemental Fig. S2). 236

The conservation of the kinase domain of MEKK1 and MEKK2 is particularly high 237

(Supplemental Fig. S2). When expressed in Arabidopsis protoplasts, MEKK1 strongly 238

activated MAPKs as shown by α-pERK antibody that detects phosphorylated MAPKs 239

(Supplemental Fig. S3). However, we did not observe the activation of MAPKs by MEKK2, 240

although MEKK2 proteins were expressed well and even stronger than MEKK1 241

(Supplemental Fig. S3). Those observations prompted us to test whether the kinase activity of 242

MEKK2 is required for its function in cell death control. Overexpression of MEKK2 led to 243



11 constitutive cell death and defense responses (Kong et al., 2012; Su et al., 2013). We 244



12

generated a binary vector harboring the full-length coding sequence of MEKK2 under the 245

control of a double cauliflower mosaic virus 35S promoter with a double HA epitope at its 246

C-terminus (2x35S::MEKK2-HA). We further generated a MEKK2 kinase mutant (KM) 247

variant with the conserved lysine (K) residue in the kinase ATP-binding loop mutated to 248

methionine (K529M) (2x35S::MEKK2KM-HA). The 2x35S::MEKK2-HA and 249

2x35S::MEKK2KM-HA constructs were introduced into the WT Col-0 background. As 250

previously reported (Kong et al., 2012; Su et al., 2013), we observed that overexpression of 251

MEKK2 transgenic plants were small and dwarfed with reduced plant architecture and cell 252

death apparent in leaves (Fig. 2A). Interestingly, MEKK2KM transgenic plants were also small 253

and dwarfed, morphologically indistinguishable from MEKK2 transgenic plants (Fig. 2B). 254

The severity of the dwarfism and cell death was positively associated with an increasing level 255

of MEKK2 or MEKK2KM protein expression (Fig. 2, A and B). As the phenotypes varied 256

among the individual transgenic plants, we screened and carefully characterized a large 257

number of transgenic plants. We obtained 42 transgenic plants carrying 2x35S::MEKK2-HA 258

which showed positive signals by α-HA immunoblots. We further classified them into three 259

categories according to the phenotypic severity: 21.4% (9 out of 42) plants exhibited severe 260

dwarf and cell death phenotypes, 57.1% (24 out of 42) showed moderate dwarf phenotypes, 261

and 21.4% (9 out of 42) exhibited weak dwarfism (Fig. 2A). Similarly, for 262

2x35S::MEKK2KM-HA transgenic plants, among 56 transgenic plants with positive signals by 263

α-HA immunoblots, 16.0% (9 out of 56) were severe dwarf with cell death, 55.4% (31 out of 264

56) were moderate dwarf, and 28.6% (16 out of 56) were weak dwarf (Fig. 2B). Similarly, 265

overexpression of MEKK2KM under the control of a double cauliflower mosaic virus 35S 266

promoter with a double FLAG epitope at its C-terminus (2x35S::MEKK2KM-FLAG) also 267

induced plant growth defects and cell death (Fig. 2C). Among 57 transgenic plants with 268

positive signals by α-FLAG immunoblots, 17.5% (10 out of 57) were severe dwarf with cell 269

death, 52.6% (30 out of 57) were moderate dwarf, and 29.8% (17 out of 57) were weak dwarf 270

(Fig. 2C). These results indicate that both WT MEKK2 and the kinase mutant MEKK2KM 271

could trigger cell death when overexpressed in plants. It is likely that the kinase activity of 272

MEKK2 is not required for its function in regulating plant cell death. 273

274



13

The genomic DNA of CRCK3 complements let6 in regulating MEKK1-mediated cell 275

death 276

277

The let6 mutant (SALK_039370) isolated during this VIGS-based suppressor screen also 278

suppressed the cell death phenotypes induced by silencing of MEKK1 (Fig. 3, A and B). The 279

T-DNA fragment is located in the third intron of At2G11520 (Supplemental Fig. S4A), which 280

was recently identified as SUMM3 (CRCK3), and let6 was named as summ3-17 (Zhang et al., 281

2017). Similar with mekk2 and summ2, let6/summ3-17 did not affect the cell death induced 282

by RNAi BAK1/SERK4 or BIR1 (Fig. 1G). To confirm that CRCK3 is LET6, we transformed 283

the full-length cDNA of CRCK3 (referred as cCRCK3) under the control of 35S promoter 284

tagged with a double FLAG epitope at its C-terminus (p35S::cCRCK3-FLAG) into the 285

let6/summ3-17 mutant. More than 70 transformants were obtained from the hygromycin 286

resistant screens. The presence of cCRCK3 construct was confirmed by PCR analysis in the 287

transgenic lines (Supplemental Fig. S4B). To our surprise, cCRCK3-FLAG proteins were 288

undetectable by immunoblots in all the transgenic lines. In addition, none of transgenic lines 289

could complement the cell death phenotype induced by RNAi MEKK1 in summ3-17 290

(Supplemental Fig. S4C). 291

It is known that introns can affect gene expression in various ways (Le Hir et al., 2003; 292

Rose, 2008). There are six introns in the gDNA region of CRCK3. We generated a construct 293

harboring the full-length genomic DNA of CRCK3 containing all six introns with a 294

C-terminal GFP tag under the control of 35S promoter, and transformed 35S::CRCK3-GFP 295

into summ3-17 plants. The CRCK3-GFP proteins could be detected by immunoblots in these 296

transgenic lines, and the cell death phenotype induced by MEKK1 silencing was restored to a 297

level similar to or even more severe than that of WT Col-0 (Fig. 3, C and D), suggesting that 298

the introns of CRCK3 might be important for its expression in planta. 299

300

The kinase activity of CRCK3 is important for its function in regulating cell death 301

302

To determine whether the kinase activity of CRCK3 is necessary for its function in the 303

MEKK1-mediated cell death pathway, we mutated a conserved lysine residue in the 304



14 ATP-binding loop of CRCK3 to glutamic acid (K253E, CRCK3 kinase mutant CRCK3KM) 305



15

and generated transgenic lines expressing CRCK3KM in the summ3-17 mutant background. 306

Two representative lines with CRCK3KM-GFP protein expression level similar to 307

CRCK3-GFP were used for the subsequent complementation analysis (Fig. 3D). Three weeks 308

after silencing of MEKK1, the CRCK3KM-GFP transgenic lines only showed a weak cell 309

death phenotype, whereas CRCK3-GFP transgenic plants showed a much more severe cell 310

death phenotype (Fig. 3C), suggesting that unlike the WT CRCK3, CRCK3KM was not able to 311

complement the summ3-17 mutant for RNAi MEKK1-triggered cell death. We examined 312

whether CRCK3KM bears reduced kinase activity in plants. We first expressed CRCK3-FLAG 313

and CRCK3KM-FLAG in protoplasts, and immunoprecipitated for an in vitro kinase assay. As 314

shown in Fig. 3E, CRCK3KM-FLAG exhibited a substantially reduced phosphorylation level 315

compared to CRCK3-FLAG (Fig. 3E). Taken together, the data indicate that CRCK3KM could 316

not fully complement the phenotype of summ3-17, and the kinase activity of CRCK3 is 317

required for its full function in the MEKK1-mediated cell death pathway. 318

319

Overexpression of CRCK3 induces a kinase activity-dependent cell death in a protein 320

dosage-dependent manner 321

322

When we introduced the 35S::CRCK3-GFP construct into the WT Col-0 background, we 323

observed that transgenic plants with elevated CRCK3-GFP protein expressions showed 324

dwarfism and cell death. Of the 73 transgenic lines obtained in the T1 generation, seven 325

displayed phenotypes that resembled WT (like L15 and L5 in Fig. 4A), 40 exhibited stunted 326

growth and twisted leaves (like L11 in Fig. 4A), while 26 plants were small (Like L3 and L12 327

in Fig. 4A). Trypan blue and DAB staining indicated the cell death and H2O2 accumulation in 328

the leaves of plants exhibiting dwarf phenotypes (Fig. 5, A and B). Immunoblot analysis 329

indicated that the level of plant dwarfism and cell death was positively associated with the 330

protein level of CRCK3-GFP in transgenic plants: lines expressing low or moderate level of 331

CRCK3 proteins resembled WT phenotype and those with high level of CRCK3 proteins 332

exhibited severe dwarf phenotype (Fig. 4B). These results indicate that overexpression of 333

CRCK3 leads to constitutively activated cell death in a protein dosage-dependent manner. 334

The kinase activity of CRCK3 is important for its function in regulating 335



16 MEKK1-mediated cell death (Fig. 3). To investigate whether the kinase activity of CRCK3 is 336



17 also important in CRCK3 overexpression-induced cell death, we introduced 337



18

35S::CRCK3KM-GFP into the WT background and found that none of the transgenic lines 338

displayed abnormal growth phenotypes (Fig. 4A). Interestingly, immunoblot analysis 339

indicated that the proteins of CRCK3KM-GFP accumulated to a relatively lower level than 340

those of WT CRCK3-GFP (Fig. 4B). We could not identify 35S::CRCK3KM-GFP plants with 341

protein levels similar to 35S::CRCK3-GFP plants with strong cell death. The data suggest 342

that the kinase activity of CRCK3 is required for its function in the activation of cell death. 343

To further determine whether overexpression of CRCK3 could promote cell death, we 344

silenced MEKK1 by VIGS in 35S::CRCK3-GFP/WT transgenic plants L15 and L5, which 345

phenotypically resembled WT Col-0. Substantially, L15 and L5 transgenic plants displayed 346

more severe cell death than WT plants when MEKK1 was silenced (Fig. 4C), further 347

supporting the important role of CRCK3 level in mekk1 cell death. 348

349

Both SUMM2 and MEKK2 are required for cell death caused by overexpression of 350

CRCK3 351

352

SUMM2 is a NLR functioning genetically downstream of MEKK2 and CRCK3 (Zhang et al., 353

2012; Zhang et al., 2017). We next investigated whether cell death caused by overexpression 354

of CRCK3 requires SUMM2 by transforming 35S::CRCK3-GFP into the summ2-8 mutant, 355

which is morphologically similar to WT plants. Unlike the transgenic lines in the WT 356

background, the 35S::CRCK3-GFP transgenic lines in the summ2-8 mutant background 357

exhibited normal plant growth phenotypes similar to WT or summ2-8 plants (Fig. 5A). In 358

addition, the extensive cell death and over-accumulation of H2O2 were not observed in the 359

35S::CRCK3-GFP transgenic lines in the summ2-8 background (Fig. 5B). The protein levels 360

of CRCK3-GFP in the summ2-8 mutant were comparable with those in WT plants (Fig. 5C). 361

These data indicate that CRCK3 overexpression-induced cell death depends on SUMM2. 362

It has been reported that CRCK3 is required for the autoimmune phenotypes induced by 363

overexpression of MEKK2 (Zhang et al., 2017), suggesting that CRCK3 functions genetically 364

downstream of MEKK2 in the mekk1 cell death pathway. Thus, it is plausible to speculate 365

that the autoimmune phenotypes triggered by overexpression of CRCK3 are independent of 366

MEKK2. Surprisingly, when we transformed 35S::CRCK3-GFP into mekk2 plants, all of 367



19

35S::CRCK3-GFP/mekk2 transgenic plants (more than sixty independent transgenic lines 368

were isolated and characterized) showed similar growth phenotypes as WT plants (Fig. 5A), 369

and no cell death or over-accumulation of H2O2 was detected in the transgenic lines (Fig. 5B). 370

The data indicate that CRCK3-induced autoimmune phenotypes also genetically depend on 371

MEKK2. Together, CRCK3 and MEKK2 are intricately linked with each other for the cell 372

death inducibility, pinpointing the possibility of a protein complex containing both CRCK3 373

and MEKK2. In addition, the protein levels of CRCK3-GFP in the mekk2 mutant were 374

slightly lower than those in the WT or the summ2-8 mutant (Fig. 5C). 375

376

CRCK3 transcription is upregulated in the mekk1, mkk1/2, and mpk4 mutants 377

378

Apparently, similar with MEKK2, the expression level of CRCK3 is critical for cell death 379

inducibility. It has been shown that the cell death and autoimmune phenotypes in the mekk1, 380

mkk1/2, and mpk4 mutants are associated with the upregulation of MEKK2 transcripts (Su et 381

al., 2013). A modest increase in MEKK2 transcription could induce defense responses (Su et 382

al., 2013). We therefore investigated whether CRCK3 transcripts are also mis-regulated in the 383

mekk1, mkk1/2, and mpk4 mutants (Fig. 6A). Consistent with the previous report (Su et al., 384

2013), the transcripts of MEKK2 were upregulated about two-fold higher in the mekk1, 385

mkk1/2, and mpk4 mutants compared to WT plants (Fig. 6B). Similarly, the transcripts of 386

CRCK3 were also about two-fold higher in the mekk1, mkk1/2, and mpk4 mutants compared 387

to those in WT plants (Fig. 6C). The mekk1/2/3 mutant, which bears a deletion of MEKK1, 388

MEKK2, and MEKK3, and suppresses the mekk1 cell death phenotype (Su et al., 2013), 389

showed a similar expression level of CRCK3 as WT plants (Fig. 6C). Thus, the increased 390

CRCK3 transcripts in the mekk1 mutant are also likely suppressed by the mekk2 mutation. 391

The data suggested that the transcript levels of both CRCK3 and MEKK2 are associated with 392

mekk1, mkk1/2, and mpk4 cell death, and the expression of CRCK3 and MEKK2 might be 393

co-regulated. Indeed, the expression pattern of CRCK3 and MEKK2 is very similar as 394

observed in the Arabidopsis eFP Brower (Supplemental Fig. S5) 395

396

MEKK2 associates with and stabilizes CRCK3 397



20 398



21

The genetic interaction between CRCK3 and MEKK2 led us to hypothesize that MEKK2 399

forms a complex with CRCK3. To test whether MEKK2 associates with CRCK3, a 400

co-immunoprecipitation (Co-IP) assay was performed. MEKK2-FLAG and CRCK3-HA were 401

transiently expressed in Arabidopsis protoplasts, and the proteins were immunoprecipitated 402

with α-FLAG affinity beads. We then examined whether CRCK3 was in the precipitates by 403

immunoblotting with an α-HA antibody. As shown in Fig. 7A, the CRCK3-HA proteins were 404

detected only in the sample containing MEKK2-FLAG, not in the negative control. 405

Furthermore, an in vitro pull-down assay was performed with MEKK2-FLAG transiently 406

expressed in Arabidopsis protoplasts as the bait against the cytoplasmic domain of CRCK3 407

fused with GST at its N-terminus (GST-CRCK3CD). As shown in Fig. 7B, GST-CRCK3CD, 408

but not GST protein itself, was able to pull down MEKK2-FLAG. Taken together, the data 409

suggest that CRCK3 and MEKK2 form a complex. 410

The observation that the protein level of CRCK3-GFP in mekk2 was lower than that in 411

WT plants (Fig. 5C) prompted us to test whether MEKK2 may positively regulate the protein 412

accumulation of CRCK3. When we co-expressed CRCK3-HA together with MEKK2-FLAG 413

in protoplasts for the Co-IP assay, we observed an increased CRCK3-HA protein level in the 414

presence of MEKK2-FLAG compared to the vector control (Fig. 7A, 3rd panel). We further 415

transiently co-expressed CRCK3-FLAG with MEKK2-HA or a vector control in Nicotiana 416

benthamiana, and the increased CRCK3-FLAG protein level was detected in the presence of 417

MEKK2-HA compared with the control (Fig. 7C), suggesting that MEKK2 regulates CRCK3 418

protein accumulation. We then examined whether CRCK3 is degraded in 419

proteasome-dependent manner. The seedlings of the 35S::CRCK3-GFP/WT transgenic plants 420

were treated with MG132, a 26S proteasome inhibitor, for different times, and subjected to 421

immunoblot analyses. As shown in Figure 7D, MG132 treatments stabilized the accumulation 422

of CRCK3 proteins, suggesting that CRCK3 stability is regulated by 26S 423

proteasome-mediated degradation. 424

We further tested the importance of CRCK3 protein level in MEKK1-mediated cell 425

death. We generated transgenic plants carrying CRCK3-GFP under its native promoter in the 426

summ3-17 mutant (pCRCK3::CRCK3-GFP/summ3), and silenced MEKK1 by VIGS (Fig. 7E). 427

Transformation of pCRCK3::CRCK3-GFP into the summ3 mutant restored the RNAi 428



22

MEKK1-induced cell death as the WT plants (Fig. 7E). Importantly, the protein level of 429

CRCK3-GFP was substantially increased (~4-6 fold) upon silencing of MEKK1 in all 430

transgenic lines examined (Fig. 7F). Notably, there was only about a two-fold increase of 431

CRCK3 transcripts in the mekk1 mutant compared to WT plants (Fig. 6C). Apparently, 432

CRCK3 proteins are stabilized or translationally increased in the MEKK1-silenced plants. 433

Thus, CRCK3 protein homeostasis plays a crucial role in the MEKK1 cell death pathway. 434

435

436



23

Discussion 437

438

Modifier and suppressor screens are powerful forward genetic approaches to understand 439

signaling pathways and uncover genetic interactions in a particular biological process. 440

However, the beauty of this approach can be diminished when it is used to understand the 441

functions of some essential genes due to the lethality of the null mutants. In plants, VIGS is a 442

widely used RNAi approach for functional analysis of individual genes by knocking down 443

the expression of endogenous genes, avoiding lethality caused by complete loss-of-functions 444

(Burch-Smith et al., 2004; Senthil-Kumar and Mysore, 2011). In addition, this approach 445

could silence multiple members of a gene family spontaneously to eliminate functional 446

redundancy. We have used VIGS to silence two functionally redundant receptor-like kinases 447

BAK1 and SERK4, which resembled the bak1/serk4 mutant plants (de Oliveira et al., 2016). 448

VIGS could also be readily deployed in different genetic backgrounds, which could 449

substantially expedite the tedious and time-consuming process of generating higher order 450

mutants. For examples, we have silenced two closely related cysteine-rich protein kinases 451

CRK22 and CRK28 in the crk29 mutant to reveal their redundant functions in plant immunity 452

(Yadeta et al., 2017). By making a random cDNA library of certain plant species, VIGS could 453

also be used as a high throughput and fast forward genetic screen for identifying components 454

in a biological process (Lu et al., 2003; Li et al., 2015). In Arabidopsis, the availability of 455

sequence index T-DNA insertion library makes it possible to identify the mutants that 456

suppress or enhance the phenotype caused by VIGS of gene(s). The causal mutations could 457

be easily identified based on the information of T-DNA insertions. With this approach, we 458

have shown that protein glycosylation is important in bak1/serk4 cell death (de Oliveira et al., 459

2016). Thus, this unbiased and highly efficient genetic screen combines the features of both 460

forward and reverse genetics, and provides an alternative to uncover the pathways and 461

mechanisms regulating plant cell death, and embryonic or postembryonic seedling lethality 462

caused by one or multiple redundant genes. 463

Understanding the mekk1-mkk1/2-mpk4 cell death pathway was mainly achieved through 464

the suppressor screen of the mkk1/2 mutant, which is able to produce seeds when grown at 28℃ 465

(Gao et al., 2009; Kong et al., 2012; Zhang et al., 2017). Interestingly, certain mutants could 466



24

suppress mkk1/2 cell death, but did not affect mekk1 or mpk4 cell death (Lian et al., 2018), 467

suggesting the independent functions of individual components in the 468

MEKK1-MKK1/2-MPK4 cascade. Unlike mkk1/2, the genetic mutants of mekk1 are unable 469

to produce enough seeds for a suppressor screen. We show here that silencing of MEKK1 by 470

VIGS in WT plants resembles the mekk1 mutant with autoimmune phenotypes. We further 471

used this approach to screen the Arabidopsis T-DNA insertion library and identified MEKK2, 472

SUMM2, and CRCK3 as specific regulators of RNAi MEKK1, but not BAK1/SERK4 nor 473

BIR1-induced cell death. Previous identification of these components in the 474

mekk1-mkk1/2-mpk4 cell death pathway supports the feasibility of our approach to 475

understand the conserved and specific functions of MEKK1 in regulating cell death and 476

others. 477

MEKK2 resides in a tandem repeat region with MEKK1 and shares 64% amino acid 478

identity with MEKK1 (Supplemental Fig. S2). MEKK1 is indispensable for MPK4 activation, 479

consistent with the fact that MEKK1 functions as a MAPKKK for the activation of MPK4 480

(Ichimura et al., 2006; Nakagami et al., 2006; Suarez-Rodriguez et al., 2007). Consistently, 481

MEKK1 has strong kinase activity (Asai et al., 2002) (Supplemental Fig. S3). Surprisingly, 482

the kinase activity of MEKK1 might not be required for the activation of MPK4 since the 483

kinase-impaired mutant of MEKK1K361M could complement the mekk1 defects in terms of 484

MPK4 activation (Suarez-Rodriguez et al., 2007). In addition, MEKK1K361M could 485

complement the lethality of the mekk1 mutant, suggesting that the kinase activity of MEKK1 486

might not be essential for its function in cell death control (Suarez-Rodriguez et al., 2007). 487

We show here that MEKK2 has little kinase activity in our assay conditions and the 488

kinase-impaired mutant MEKK2K529M did not affect its cell death inducibility when 489

overexpressed in Arabidopsis WT plants, suggesting that kinase activity of MEKK2 may not 490

be required for its function in cell death control (Fig. 2). Thus, both MEKK1 and MEKK2 491

may confer roles as structural or scaffold proteins, rather than functional kinases, in the 492

MEKK1-MKK1/2-MPK4 cascade. Apparently, MPK4 kinase activation is associated with its 493

function in plant immunity and cell death control (Berriri et al., 2012; Su et al., 2013). Thus, 494

it is tantalizing to hypothesize that another MAPKKK, which might be scaffolded by 495



25

MEKK1 or MEKK2, may form a conventional MAPK cascade together with MKK1/2 and 496

MPK4 in activating the downstream signaling. 497

CRCKs are calcium-dependent CaM-binding receptor-like cytoplasmic kinases (RLCKs) 498

(Yang et al., 2004). RLCKs play important roles in plant immunity and development (Lin et 499

al., 2013; Liang and Zhou, 2018). The Arabidopsis genome encodes three CRCKs (Yang et al., 500

2004). However, their biological functions and connections with Ca2+/CaM still remain 501

elusive. It has been shown that Ca2+/CaM can stimulate CRCK1 kinase activity (Yang et al., 502

2004). Ca2+ signaling is essential in the activation of plant NLRs (Gao et al., 2013). It will be 503

interesting to determine in the future whether Ca2+ or CaM is involved in CRCK3-mediated 504

NLR SUMM2 activation. Several RLCKs, such as AVRPPHB SUSCEPTIBLE 1 (PBS1) and 505

PBS1-LIKE 2 (PBL2), have been proposed to act as guardees or decoys for the activation of 506

NLR immune receptors (Shao et al., 2003; Wang et al., 2015). CRCK3 was hypothesized as a 507

guardee of SUMM2 and the phosphorylation level of CRCK3 by MPK4 is sensed by 508

SUMM2 for activation (Zhang et al., 2017). We show here that overexpression of CRCK3 led 509

to SUMM2-dependent cell death. Thus, in addition to phosphorylation, the homeostasis of 510

CRCK3 is important for NLR SUMM2 activation. 511

Transcriptional reprogramming is one of the most important responses in plant defense 512

(Li et al., 2016). The transcripts of both MEKK2 and CRCK3 are upregulated in the mekk1, 513

mkk1/2, and mpk4 mutants, and the abundance of MEKK2 and CRCK3 is tightly associated 514

with autoimmune phenotypes (Su et al., 2013) (Fig. 6, B and C). However, it is still unclear 515

whether the upregulation of MEKK2 and CRCK3 is responsible for or the outcome of the 516

autoimmune phenotypes observed in the mekk1, mkk1/2, and mpk4 mutants. The 517

transcriptional regulation of MEKK2 and CRCK3 appears to be complicated. Although 518

MEKK2 is a substrate of MPK4 (Kong et al., 2012), the MEKK2 transcript level is regulated 519

by MPK4 activity since overexpression of constitutively active MPK4 rescued the mekk1 cell 520

death phenotype and restored the elevated MEKK2 expression of the mekk1 mutant to the WT 521

level (Su et al., 2013). Similarly, the transcript level of CRCK3 might be regulated by 522

MEKK2 since the increased CRCK3 expression in the mekk1 mutant was restored in the 523

mekk1/2/3 mutant (Fig. 6C). Thus, multiple positive feedback regulations may exist in the 524

mekk1-mkk1/2-mpk4 cell death pathway. It is also possible that the expression of MEKK2 and 525



26

CRCK3 might be regulated at the post-transcriptional or translational levels. Some RNA 526

processing-associated proteins, such as MODIFIER OF snc1 (MOS), are involved in the 527

regulation of NLR protein SNC1-mediated autoimmune phenotypes (Palma et al., 2007). 528

Identification and functional studies of other LETs have great potential to uncover the 529

detailed molecular mechanisms underlying NLR SUMM2 activation and regulation at 530

transcriptional, posttranscriptional, and posttranslational levels. 531

Co-IP and pull-down assays revealed that CRCK3 associates with MEKK2 in vivo and in 532

vitro (Fig. 7, A and B), indicating that they exist in a protein complex in plants, and may 533

require each other for functionality. Consistently, we observed that CRCK3 cell death 534

inducibility depends on MEKK2 (Fig. 5). MEKK2 cell death inducibility also depends on 535

CRCK3 (Zhang et al., 2017). Notably, MEKK2 positively regulates the protein stability of 536

CRCK3 (Fig. 7C). Considering the dispensable role of kinase activity in MEKK2 cell death 537

inducibility, MEKK2 is likely a nonfunctional kinase, and it may function as a scaffold to 538

stabilize CRCK3. It is possible that CRCK3 undergoes constant turnover in WT plants, 539

whereas, in the mekk1 mutant, CRCK3 transcripts are increased and proteins are stabilized 540

(Fig. 6C and 7F), partly due to the increased expression of MEKK2 scaffolding or stabilizing 541

the complex. Notably, the pseudokinase RKS1 is required for RLCK PBL2 and NLR 542

ZAR1-mediated immunity and to form the PBL2-RKS1-ZAR1 resistosome (Wang et al., 543

2019; Wang et al., 2019). The activated NLR ZAR1 may form pores in the plasma membrane, 544

leading to cell death and oxidative stress. It is tempting to hypothesize that 545

CRCK3-MEKK2-SUMM2 also assemble into a similar resistosome that is activated by the 546

depletion of MEKK1-MKK1/2-MPK4 cascade, ultimately triggering cell death. 547

548

MATERIALS AND METHODS 549

550

Plant Materials and Growth Conditions 551

The Arabidopsis (Arabidopsis thaliana) mutant lines used here were described previously: 552

mekk2 (SALK_150039C), summ2-8 (SAIL_1152_A06), summ2-9 (SALK_062374), 553

summ3-17 (SALK_039370), mekk1 (SALK_052557), mpk4-2 (SALK_056245), mkk1/2, 554

mekk1/2/3 in the Col-0 background (Zhang et al., 2012; Su et al., 2013; Zhang et al., 2017). 555



27

Arabidopsis T-DNA insertion lines and library were obtained from Arabidopsis Biological 556

Resource Center (ABRC). Arabidopsis and Nicotiana benthamiana plants were grown on soil 557

(LP5 RSi, Sun Gro Horticulture) in a growth room at 23°C, 50% relative humidity and 75 μE 558

m-2s-1 light with a 12-hr light/12-hr dark photoperiod. 559

560

Plasmid Construction and Generation of Transgenic Plants 561

The VIGS constructs for MEKK1, BAK1/SERK4, BIR1, and CLA1 were reported previously 562

(de Oliveira et al., 2016). To generate pMDC32-2x35S::cCRCK3-FLAG and 563

pMDC32-2x35S::MEKK2-HA, the coding sequences of CRCK3 and MEKK2 were amplified 564

by PCR from Col-0 cDNA and introduced into a plant gene expression vector pHBT 565

containing an HA tag at the C-terminus by Xba I/Sma I or BamH I/Stu I restriction enzyme 566

digestions. The fragments were released by Bgl II/Sma I or BamH I/Stu I enzyme digestions 567

and subcloned into modified pMDC32-FLAG or pMDC32-HA vectors. To make 568

pCB302-35S::CRCK3-GFP and pCB302-35S::CRCK3-FLAG, the genomic fragment of 569

CRCK3 was amplified from Col-0 genomic DNA and cloned into pHBT by Bgl II/Sma I 570

enzyme digestions. The CRCK3 fragments were subcloned into modified pCB302-GFP or 571

pCB302-FLAG vectors by Nhe I/Sma I digestions. The point mutations of MEKK2KM and 572

CRCK3KM were generated using site-directed mutagenesis. To generate 573

pCB302-pCRCK3::CRCK3-GFP, the promoter of CRCK3 was amplified from Col-0 574

genomic DNA and cloned into pCB302 by Sac I/BamH I enzyme digestions, then the 575

CRCK3-GFP fragments from pHBT-p35S::CRCK3-GFP was released by Bgl II/EcoR I 576

digestion and subcloned into this modified pCB302 vector. The E. coli expression vector 577

GST-CRCK3CD was generated by PCR amplifying the CRCK3 cytosolic domain (146-510 578

amino acids) from pHBT-35S::cCRCK3-HA, and subcloning it into a modified pGST vector 579

using a one-step cloning kit (Vazyme Biotech) by BamH I and Stu I digestion. All DNA 580

fragments cloned into vectors were confirmed via Sanger sequencing. The primers for 581

cloning and point mutations are listed in Supplemental Table S1. 582

Transgenic plants were generated via the Agrobacterium-mediated floral-dip method. 583

The transformants were screened with the herbicide BASTA (Bayer; resistance conferred by 584

the pCB302 vector) or the antibiotic hygromycin for the pMDC32 vector. 585



28

586

Agrobacterium-mediated virus-induced gene silencing assay 587

The VIGS assay was performed as described previously (de Oliveira et al., 2016). In brief, 588

the Agrobacterium strain GV3101 containing pYL156-RNA1, pYL156-MEKK1, 589

pYL156-BAK1/SERK4, pYL156-BIR1, pYL156-CLA1, or pYL156-GFP (the vector control) 590

was grown overnight in LB medium (50 μg/ml kanamycin, 50 μg/ml gentamycin, 10 mM 591

MES, and 20 μM acetosyringone). The bacteria were harvested by centrifugation at room 592

temperature and resuspended in infiltration buffer (10 mM MES, 10 mM MgCl2, and 200 μM 593

acetosyringone). Bacterial cultures containing pYL156-MEKK1, pYL156-BAK1/SERK4, 594

pYL156-BIR1, pYL156-CLA1, or pYL156-GFP were mixed with pYL156-RNA1 cultures at 595

the 1:1 ratio, individually. The first pair of true leaves of 10-day-old plants were 596

hand-infiltrated using a needleless syringe with the mixed bacterial cultures. The albino 597

phenotype (CLA1-silencing) or cell death phenotypes showed up two weeks post infiltration. 598

599

Trypan blue and DAB staining 600

Staining was performed as described previously, with minor modifications (Zhou et al., 2019). 601

For trypan blue staining, the leaves from five-week-old plants were immersed in boiled 602

latophenol (lactic acid/glycerol/liquid phenol/distilled water, 1:1:1:1) with 0.25 mg/ml trypan 603

blue for 30 s. For 3,3’-diaminobenzidine (DAB) staining, the leaves from five-week-old 604

plants were immersed in 1 mg/ml DAB solution under vacuum pressure for 2 hr, followed by 605

an overnight incubation at room temperature in the dark. The trypan blue or DAB stained 606

leaves were destained with destain buffer (ethanol/lactic acid/liquid phenol, 2:1:1) at 65°C for 607

1 hr, and washed three times with 75% (v/v) ethanol. The destained leaves were 608

photographed under the microscope. 609

610

Co-IP and transient expression assays 611

Arabidopsis protoplasts were transfected with different plasmids and incubated overnight. 612

Samples were lysed in Co-IP buffer (100 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl, pH 7.5, 613

2 mM NaF, 2 mM Na3VO4, 1 mM DTT, 0.5% v/v Triton X-100, 10% v/v glycerol, and 1x 614

protease inhibitor). The supernatant was collected after centrifugation at 13,000 rpm at 4°C 615



29

for 15 min and incubated with α-FLAG affinity beads (Sigma) at 4°C for 2 hr with gentle 616

shaking. The beads were collected and washed three times with Co-IP washing buffer (20 617

mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 1% v/v Triton X-100). The 618

immunoprecipitated and input proteins were analyzed by immunoblot with the indicated 619

antibodies. 620

The transient expression assays in Nicotiana benthamiana were carried out as described 621

previously (Feng et al., 2016). Briefly, Agrobacterium strain GV3101 containing binary 622

vectors was cultured overnight at 28°C. Bacteria were harvested and resuspended with 623

infiltration buffer (10 mM MES, 10 mM MgCl2, and 200 μM acetosyringone) for inoculation. 624

The leaf samples were harvested at 30 hr post-infiltration (hpi). 625

626

RNA isolation and reverse transcription quantitative PCR (RT-qPCR) analysis 627

Total RNAs were prepared using the TRIzol reagent (Invitrogen). RNase-free DNase I (New 628

England Biolabs) was used to remove contaminating genomic DNA. Complementary DNAs 629

(cDNAs) were synthesized with M-MuLV Reverse Transcriptase (New England Biolabs) and 630

oligo(dT) primers. RT-qPCR analysis was performed using iTaq Universal SYBR green 631

Supermix (Bio-Rad) with an 7900HT Fast Real-Time PCR system (Applied Biosystems). The 632

expression of each gene was normalized to the expression of UBQ10. 633

634

Protein isolation and in vitro pull-down assay 635

GST and GST-CRCK3CD were purified from E. coli with a standard glutathione agarose 636

beads (Thermo Scientific). MEKK2-FLAG was transiently expressed in protoplasts overnight, 637

and lysed with 250 μl of extraction buffer (10 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM 638

EDTA, 10% v/v glycerol, 0.5% v/v Triton X-100, and 1:200 complete protease inhibitor 639

cocktail from Sigma). For the pull down assay, about 10 μg GST or GST-CRCK3CD proteins 640

were mixed with the MEKK2-FLAG cell lysate at 4°C for 1 hr with gentle shaking, 641

subsequently incubated with 20 μl of glutathione agarose beads at 4°C for another 2 hr with 642

gentle shaking. The beads were harvested by centrifugation and washed five times with the 643

washing buffer (10 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM EDTA, 10% v/v glycerol, 0.5% 644

v/v Triton X-100). The bound proteins were released from beads by boiling in 50 μl of 2x 645



30

SDS protein loading buffer for 10 min and detected by an immunoblot with an α-FLAG 646

antibody. 647

648

In vitro kinase assay 649

GFP-FLAG, CRCK3-FLAG, and CRCK3KM-FLAG were transiently expressed in protoplasts 650

for overnight, and purified by α-FLAG agarose. The proteins were incubated with 20 μl of 651

kinase assay buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 5 mM EGTA, 100 mM NaCl, 1 652

mM DTT and 1 μl [γ-32P]ATP) at room temperature for 3 hr with gentle shaking. The 653

reactions were stopped by adding 4× SDS protein loading buffer. The phosphorylation of 654

proteins was analyzed by autoradiography after separation with 10% SDS-PAGE. 655

656

Accession numbers 657

Sequence data from this article can be found in The Arabidopsis Information Resource (TAIR) 658

or GenBank/EMBL databases under the following accession numbers: CLA1(AT4G15560), 659

CRCK3 (AT2G11520), MEKK1 (AT4G08500), MEKK2(AT4G08480), SUMM2 660

(AT1G12280), MKK1(AT4G26070), MKK2(AT4G29810), MPK4(AT4G01370), BIR1 661

(AT5G48380), BAK1 (AT4G33430), SERK4 (AT2G13790), PR1 (AT2G14610), PR2 662

(AT3G57260), ACTIN2 (AT3G18780), and UBQ10 (AT4G05320). 663

664

SUPPLEMENTAL DATA 665

666

Supplemental Figure S1. SALK_062374 is summ2-9. 667

Supplemental Figure S2. Sequence alignment of MEKK1 and MEKK2. 668

Supplemental Figure S3. MEKK2 does not have detectable kinase activity. 669

Supplemental Figure S4. Overexpression of CRCK3 derived from cDNA cannot 670

complement summ3-17 phenotype. 671

Supplemental Figure S5. The expression pattern of CRCK3 and MEKK2. 672

Supplemental Table S1. Primers for gene cloning, point mutation, genotyping, and 673

RT-qPCR. 674

675



31

676

ACKNOWLEDGEMENTS: 677

We thank Arabidopsis Biological Resource Center (ABRC), Drs. Patrick Krysan (University 678

of Wisconsin, USA) and Yuelin Zhang (University of British Columbia, Canada) for 679

Arabidopsis seeds, and members in He and Shan laboratories for comments. 680

681

Figure Legends: 682

Figure 1. The mekk2 and summ2 mutants suppress the cell death mediated by silencing 683

of MEKK1. 684

(A) Silencing of MEKK1 by virus-induced gene silencing (VIGS) triggers cell death 685

phenotype resembling the mekk1 mutant. Col-0 WT and mekk1 were grown on soil for 3 686

weeks (left two panels). Plant phenotypes of Col-0 WT plants are shown two weeks after 687

VIGS of a vector control or MEKK1. Bar = 1 cm. 688

(B) The let4/mekk2 mutant suppresses growth defects triggered by RNAi-MEKK1. Plant 689

phenotypes of WT and mekk2 after VIGS of MEKK1. CLA1 is a visible marker for VIGS 690

efficiency. Plant pictures were digitally extracted and placed on a black background in A, B. 691

Bar = 1 cm. 692

(C) The mekk2 mutant suppresses cell death and H2O2 accumulation triggered by 693

RNAi-MEKK1. Plant true leaves after VIGS of MEKK1 were stained with trypan blue for cell 694

death (left) and 3,3’-diaminobenzidine (DAB) for H2O2 accumulation (right). Bar = 0.5 cm. 695

(D) The mekk2 mutant suppresses PR1 and PR2 expression triggered by RNAi-MEKK1. The 696

expression of PR1 and PR2 was normalized to the expression of UBQ10. The data are shown 697

as mean ± SE from three independent repeats. The different letters denote statistically 698

significant difference according to one-way ANOVA followed by Tukey test (p<0.05). 699

(E) The let5/summ2-9 mutant suppresses growth defects triggered by RNAi-MEKK1. Plant 700

phenotypes of WT and let5/summ2-9 after VIGS of MEKK1 or CLA1. Bar = 1 cm. 701

(F) PCR analysis of SUMM2 in let5.The top panel shows that SUMM2 was not amplified in 702

the let5 mutant with primers amplifying full length genomic DNA. The bottom panel shows 703

ACTIN2 control. 704



32

(G) The mekk2, summ2, and crck3 mutants specifically suppressed RNAi MEKK1, but not 705

BAK1/SERK4 or BIR1 cell death. Plant phenotypes of Col-0 plants and mutants are shown 706

two weeks after VIGS of a vector control, MEKK1, BAK1/SERK4, BIR1, or CLA1. Bar = 1 707

cm. 708

709

Figure 2. Overexpression of both WT and kinase mutant of MEKK2 induces cell death. 710

(A) Overexpression of MEKK2 induces dwarf phenotype. Four-week-old soil-grown plants 711

were photographed. Transgenic plants of 35S::MEKK2-HA in WT show severe dwarfism, 712

and were grouped into three categories. Each category was calculated as a percentage of the 713

total dwarf plants. Bottom shows the protein expression of MEKK2-HA from the above 714

plants detected by immunoblot (IB) with an α-HA antibody. Coomassie Brilliant Blue (CBB) 715

staining was used as the loading control. Bar = 1 cm. 716

(B) Overexpression of MEKK2KM, a kinase-dead mutant (Lys529 to Met), triggers plant 717

dwarfism. Five-week-old soil-grown plants were photographed. Transgenic plants 718

of 35S::MEKK2KM-HA in WT show severe dwarfism, and were grouped into three categories. 719

Each category was calculated as a percentage of the total dwarf plants. Immunoblots with an 720

α-HA antibody show MEKK2KM-HA protein expression and CBB staining was used as the 721

loading control (bottom). Bar = 1 cm. 722

(C) Overexpression of MEKK2KM-FLAG triggers plant dwarfism. Five-week-old soil-grown 723

plants were photographed. Transgenic plants of 35S::MEKK2KM-FLAG in WT show severe 724

dwarfism, and were grouped into three categories. Each category was calculated as a 725

percentage of the total dwarf plants. Immunoblots with an α-FLAG antibody show 726

MEKK2KM-FLAG protein expression and CBB staining was used as the loading control 727

(right). Plant pictures were digitally extracted and placed on a black background in A, B, C. 728

Bar = 1 cm. 729

730

Figure 3. Kinase activity of CRCK3 is important for its function in MEKK1-mediated 731

cell death. 732

(A) The let6/summ3-17 mutant suppresses MEKK1-silencing induced growth defect. Bar = 1 733

cm. 734



33

(B) The summ3-17 mutant suppresses cell death (left) and H2O2 accumulation (right) 735

triggered by RNAi-MEKK1. Plant true leaves after VIGS of MEKK1 were stained with trypan 736

blue for cell death and DAB for H2O2 accumulation. Bar = 0.5 cm. 737

(C) Phenotype of five-week-old Arabidopsis WT, summ3-17, and indicated transgenic lines 738

after silencing of MEKK1. L15 and L8 are lines expressing GFP-tagged WT CRCK3 genomic 739

DNA in the summ3-17 mutant. L5 and L6 are lines expressing the kinase dead CRCK3KM-GFP 740

(Lys253 to Glu) in the summ3-17 mutant. Plant pictures were digitally extracted and placed on a 741

black background in A, C. Bar = 1 cm. 742

(D) Immunoblot analysis of the protein levels of CRCK3-GFP and CRCK3KM-GFP in the 743

transgenic lines in (C). Ponceau staining of RubisCo (RBC) was used as the loading control. 744

(E) CRCK3 has kinase activity. CRCK3-FLAG or CRCK3KM-FLAG was expressed in 745

Arabidopsis protoplasts, and immunoprecipitated with α-FLAG agarose beads for an in vitro 746

kinase assay. The autoradiograph (top) shows kinase activity, and the immunoblot (bottom) 747

shows protein expression. GFP-FLAG was included as a control. 748

749

Figure 4. Kinase activity of CRCK3 is required for its cell death inducibility. 750

(A) Phenotypes of four-week-old WT, CRCK3-GFP, and CRCK3KM-GFP transgenic lines in 751

WT background. Bar = 1cm. 752

(B) Immunoblot analysis of CRCK3-GFP and CRCK3KM-GFP proteins in the indicated 753

transgenic lines in (A) with an α-GFP antibody. Ponceau staining of RBC was used as the 754

protein loading control. 755

(C) Silencing of MEKK1 causes more severe cell death in CRCK3-overexpressing plants. 756

Phenotypes of Col-0 WT, and CRCK3-GFP/WT L15 and L5 (two independent lines) 757

transgenic plants are shown two weeks after VIGS of a vector control or MEKK1. Plant 758

pictures were digitally extracted and placed on a black background in A, C. Bar = 1 cm. 759

760

Figure 5. Overexpression of CRCK3 induces a SUMM2 and MEKK2-dependent cell 761

death. 762



34

(A) Phenotypes of representative transgenic lines with overexpression of CRCK3-GFP in WT, 763

summ2-8, and mekk2 backgrounds. Pictures were taken from four-week-old soil-grown plants. 764

Plant pictures were digitally extracted and placed on a black background. Bar = 1 cm. 765

(B) Cell death (upper panel) and H2O2 production (lower panel) of leaves from transgenic lines 766

in (A). Leaves from five-week-old plants were used for trypan blue staining for cell death and 767

DAB staining for H2O2 production. Bar = 0.5 cm. 768

(C) Immunoblot analysis of the expression of CRCK3-GFP proteins in the representative 769

transgenic lines in (A). CRCK3 protein levels were examined via an α-GFP antibody. Ponceau 770

staining of RBC serves as the protein loading control. 771

772

Figure 6. The transcript levels of CRCK3 are up-regulated in the mekk1, mkk1/2, and 773

mpk4 mutants. 774

(A) Phenotypes of 16-day-old WT, mekk1, mkk1/2, mpk4, and mekk1/2/3. Bar = 1 cm. 775

(B) and (C) Expression levels of MEKK2 and CRCK3 in the indicated seedlings as determined 776

by RT-qPCR. Values were normalized to the expression levels of UBQ10. The data are shown 777

as mean ± SE from three independent repeats. 778

779

Figure 7. MEKK2 associates with and stabilizes CRCK3. 780

(A) MEKK2 associates with CRCK3 in the co-immunoprecipitation (Co-IP) assay. 781

MEKK2-FLAG and CRCK3-HA were transiently expressed in Arabidopsis protoplasts, 782

immunoprecipitated with α-FLAG affinity beads (IP: α-FLAG), and immunoblotted with an 783

α-HA (IB: α-HA) or α-FLAG (IP: α-FLAG) antibody (top two panels). The protein inputs 784

were detected before immunoprecipitation by an α-HA or α-FLAG immunoblot, respectively 785

(bottom two panels). 786

(B) MEKK2 interacts with CRCK3 in a pull-down assay. Arabidopsis protoplasts transiently 787

expressing MEKK2-FLAG were incubated with GST or GST-CRCK3CD. The interaction 788

between MEKK2 and CRCK3 was detected by α-FLAG immunoblot after 789

immunoprecipitation with glutathione-agarose beads (top). The protein levels of MEKK2 and 790

CRCK3CD were detected with an α-FLAG immunoblot or CBB staining, respectively. 791

(C) Immunoblot analysis of CRCK3-FLAG protein levels in N. benthamiana. CRCK3-FLAG 792



35

was transiently co-expressed in N. benthamiana with either a control vector or MEKK2-HA. 793

Total protein extract was subjected to immunoblot analysis using an α-FLAG or α-HA 794

antibody. The protein loading is shown by Ponceau staining of RBC. 795

(D) MG132 stabilizes CRCK3. Ten-day-old 35S::CRCK3-GFP/WT seedlings were treated 796

with 2 μM MG132 for 0, 1, 2, or 3 hr. Total protein extracts were analyzed by 797

immunoblotting using an α-GFP antibody. CBB staining was used as a loading control. 798

(E) Phenotype of five-week-old Arabidopsis WT, summ3-17, and 799

pCRCK3::CRCK3-GFP/summ3-17 complementation lines after silencing of MEKK1. L16, 800

L17, L31, and L32 are lines expressing GFP-tagged CRCK3 genomic DNA driven by its 801

native promoter in the summ3-17 mutant. Plant pictures were digitally extracted and placed 802

on a black background. Bar = 1 cm. 803

(F) Silencing of MEKK1 by VIGS increased CRCK3 protein accumulation. The CRCK3 804

protein levels in the representative transgenic lines in (D) were examined via an α-GFP 805

immunoblot. The band intensities were quantified using ImageJ software and labeled 806

underneath the gel. The protein loading is shown by CBB staining. 807

The above experiments were repeated three times with similar results. 808

809

810



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https://scholar.google.com/scholar?as_q=Proteolytic Processing of SERK3/BAK1 Regulates Plant Immunity, Development, and Cell Death&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhou&as_ylo=2019&as_allsubj=all&hl=en&c2coff=1


Documents

Short title: MEKK2 and CRCK3 in plant cell death regulation · 1 1 Short title: MEKK2 and CRCK3 in plant cell death regulation 2 3 *Corresponding authors: 4 Ping He 5 Tel: 979-458-1368