Cell Host & Microbe
Article
The Calcium-Dependent ProteinKinase CPK28 Buffers Plant Immunityand Regulates BIK1 TurnoverJacqueline Monaghan,1 Susanne Matschi,2 Oluwaseyi Shorinola,1,3 Hanna Rovenich,1,4 Alexandra Matei,1,5
Cecile Segonzac,1,6 Frederikke Gro Malinovsky,1,7 John P. Rathjen,1,8 Dan MacLean,1 Tina Romeis,2 and Cyril Zipfel1,*1The Sainsbury Laboratory, Norwich Research Park, NR4 7UH Norwich, UK2Department of Plant Biochemistry, Dahlem Centre of Plant Sciences, Freie Universitat Berlin, 14195 Berlin, Germany3Present address: Department of Crop Genetics, John Innes Centre, Norwich Research Park, NR4 7UH Norwich, UK4Present address: Laboratory of Phytopathology, Wageningen University, 6708 PB Wageningen, The Netherlands5Present address: Max Planck Institute for Terrestrial Microbiology, 35043 Marburg, Germany6Present address: Institute of Agriculture and Environment, Massey University, Palmerston North 4410, New Zealand7Present address: DNRF Center DynaMo, Copenhagen Plant Science Center, Department of Plant and Environmental Sciences,
Faculty of Science, University of Copenhagen, 1871 Frb. C, Denmark8Present address: Research School of Biology, Australian National University, Acton 0200, Australia
*Correspondence: [email protected]://dx.doi.org/10.1016/j.chom.2014.10.007
SUMMARY
Plant perception of pathogen-associated molecularpatterns (PAMPs) triggers a phosphorylation relayleading to PAMP-triggered immunity (PTI). Despiteincreasing knowledge of PTI signaling, how immunehomeostasis is maintained remains largely un-known. Here we describe a forward-genetic screento identify loci involved in PTI and characterizethe Arabidopsis calcium-dependent protein kinaseCPK28 as a negative regulator of immune signaling.Genetic analyses demonstrate that CPK28 at-tenuates PAMP-triggered immune responses andantibacterial immunity. CPK28 interacts with andphosphorylates the plasma-membrane-associatedcytoplasmic kinase BIK1, an important convergentsubstrate of multiple pattern recognition receptor(PRR) complexes. We find that BIK1 is ratelimiting in PTI signaling and that it is continuouslyturned over to maintain cellular homeostasis.We further show that CPK28 contributes to BIK1turnover. Our results suggest a negative regula-tory mechanism that continually buffers immunesignaling by controlling the turnover of this keysignaling kinase.
INTRODUCTION
Innate immunity is characterized by the ability of cells to sense
invading pathogens and initiate robust defense responses. In
plants, this is achieved through a multilayered surveillance sys-
tem involving both surface-localized and cytosolic immune re-
ceptors (Dodds and Rathjen, 2010). The first layer is mediated
by plasma-membrane-localized pattern recognition receptors
(PRRs) that bind pathogen-associated molecular patterns
Cell Host &
(PAMPs) leading to PAMP-triggered immunity (PTI) (Zipfel,
2014). One of the earliest physiological changes following PRR
activation is a rapid burst of reactive oxygen species (ROS),
mediated in Arabidopsis by the NADPH oxidase RBOHD (Nuhse
et al., 2007; Torres et al., 2002; Zhang et al., 2007). PRR activa-
tion additionally triggers activation of mitogen-activated protein
kinases (MAPKs) and calcium (Ca2+)-dependent protein kinases
(CDPKs), resulting in the expression of defense genes (Tena
et al., 2011). These and other responses result in broad-spec-
trum basal disease resistance (Dangl et al., 2013; Dodds and
Rathjen, 2010).
All known plant PRRs are receptor kinases (RKs) or receptor-
like proteins (RLPs) that form larger complexes with additional
proteins at the plasma membrane (Bohm et al., 2014; Macho
and Zipfel, 2014). RKs contain a cytosolic kinase domain and
a variable ectodomain featuring leucine-rich repeats (LRRs),
LysM motifs, or other ligand-binding domains. Classical PRRs
include the Arabidopsis LRR-RKs FLS2 and EFR that bind bac-
terial flagellin (or the minimal epitope flg22) and elongation factor
Tu (EF-Tu; or the minimal epitope elf18), respectively (Gomez-
Gomez and Boller, 2000; Zipfel et al., 2006). In Arabidopsis,
fungal chitin is perceived by the LysM-RK CERK1 (Liu et al.,
2012; Miya et al., 2007; Wan et al., 2008). Additional
PRRs perceive endogenous damage-associated molecular pat-
terns (DAMPs) released under stress conditions (Zipfel, 2014).
For example, the small endogenous peptide AtPep1 binds
Arabidopsis LRR-RKs PEPR1 and PEPR2, triggering immune re-
sponses (Krol et al., 2010; Yamaguchi et al., 2010; Yamaguchi
et al., 2006).
Immediately following ligand binding, FLS2, EFR, and PEPR1
associate with the LRR-RK BAK1/SERK3 and related SERK pro-
teins (Chinchilla et al., 2007; Heese et al., 2007; Liebrand et al.,
2014; Postel et al., 2010; Ranf et al., 2011; Roux et al., 2011;
Sun et al., 2013). Complex formation leads to PRR and BAK1
phosphorylation, which initiates immune signaling (Schulze
et al., 2010; Schwessinger et al., 2011; Sun et al., 2013). Impor-
tantly, CERK1 homodimerizes upon chitin binding and does not
require BAK1 or SERK proteins for signaling (Gimenez-Ibanez
Microbe 16, 605–615, November 12, 2014 ª2014 Elsevier Inc. 605
Figure 1. Restoration of PAMP Responses
in bak1-5 mob1
(A) ROS assay after treatment with 100 nM flg22,
100 nM elf18, or 1 mM AtPep1 expressed as rela-
tive light units (RLU) over time. Values are means ±
SDs (n = 8).
(B) Seedling growth in media containing 1 mM
flg22, 1 mM elf18, or 1 mM AtPep1 normalized
against growth without peptides. Relative values
are means + SD (n = 10). Significantly different
groups (p < 0.0001) are indicated with lower-
case letters based on ANOVA analysis. Experi-
ments were repeated at least three times with
similar results. Refer to Figure S1 for additional
information.
Cell Host & Microbe
CPK28 Buffers BIK1-Mediated Plant Immunity
et al., 2009; Liu et al., 2012; Schulze et al., 2010; Shan et al.,
2008).
Plant receptor-like cytoplasmic kinases (RLCKs) have
emerged as key immune regulators acting immediately down-
stream of PRR complexes (Lin et al., 2013; Wu and Zhou,
2013). Multiple RKs in Arabidopsis, including FLS2, EFR,
PEPR1, CERK1, and BAK1, associate with the plasma-mem-
brane-associated RLCK BIK1 and related PBL proteins (Lin
et al., 2014; Liu et al., 2013; Lu et al., 2010; Zhang et al., 2010).
BIK1 is directly phosphorylated in vitro by FLS2, PEPR1, and
BAK1 and reciprocally phosphorylates BAK1 and FLS2 (Lin
et al., 2014; Liu et al., 2013; Lu et al., 2010; Xu et al., 2013; Zhang
et al., 2010). PAMP perception triggers hyperphosphorylation of
BIK1 (Benschop et al., 2007; Liu et al., 2013; Lu et al., 2010;
Zhang et al., 2010), which most likely activates the kinase to
phosphorylate downstream substrates. Recent work demon-
strated that BIK1 interacts with and phosphorylates RBOHD to
enable PAMP-triggered ROS production and stomatal immunity
(Kadota et al., 2014; Li et al., 2014). In addition to BIK1,
Arabidopsis PBL27 regulates chitin-induced MAPK activation
and callose deposition downstream of CERK1 (Shinya et al.,
2014). Furthermore, the RLCK BSK1 also associates with FLS2
and bsk1 mutant plants are impaired in flg22 signaling (Shi
et al., 2013).
Despite these recent advances, our knowledge of the
molecular events occurring downstream of PRR activation is
limited. In an effort to identify regulators of PTI, we designed
a sensitized forward-genetic screen in the immune-deficient
bak1-5 mutant background. The unique dominant-negative
bak1-5 mutant is characterized by a single amino acid substi-
tution in the kinase domain that renders BAK1 specifically
defective in immune signaling but does not affect its function
in brassinosteroid signaling or cell death control (Schwes-
singer et al., 2011). Using bak1-5 facilitated the recovery of
mutants with an enhanced PAMP-triggered ROS burst. Here,
606 Cell Host & Microbe 16, 605–615, November 12, 2014 ª2014 Elsevier Inc.
we describe two allelic mutants isolated
from our modifier of bak1-5 (mob)
screen caused by mutations in the
gene encoding the Ca2+-dependent
protein kinase CPK28. We show that
CPK28 facilitates BIK1 turnover and
negatively regulates BIK1-mediated im-
mune responses triggered by several
PAMPs. Our work reveals a regulatory mechanism that consti-
tutively buffers BIK1 turnover to ensure optimal immune
outputs.
RESULTS
mob Mutants Restore PAMP Responsiveness in bak1-5
To identify loci involved in PTI, we mutagenized bak1-5 with
ethyl-methyl sulfonate and screened the M2 for mob mutants
that regained responsiveness to flg22 in a ROS burst assay
adapted for agar plates (Figure S1A available online). Here
we focus on bak1-5 mob1, which partially restored flg22-,
elf18-, and AtPep1-triggered ROS compared to bak1-5 (Fig-
ure 1A). Prolonged exposure to these peptides is linked
to growth inhibition in wild-type seedlings (Gomez-Gomez
et al., 1999; Krol et al., 2010; Zipfel et al., 2006); however,
this effect is strongly impaired in bak1-5 (Roux et al., 2011;
Schwessinger et al., 2011). Although bak1-5 mob1 remained
insensitive to flg22 in seedling growth inhibition assays, sensi-
tivity to elf18 and AtPep1 was regained (Figure 1B). Thus,
mob1 restores signaling triggered by multiple immunogenic
peptides in bak1-5.
Genetic analysis of F2 progeny from bak1-5 mob1 back-
crossed to bak1-5 indicated that mob1 segregates as a single,
recessive locus (51/222 plants had regained elf18-triggered
ROS; x2 = 0.486; p = 0.4855). To establish complementation
groups, bak1-5 mob1 was crossed with other recessive
mob mutants, and F1 individuals were analyzed for seedling
sensitivity to AtPep1. One mutant, bak1-5 mob2, which also
restored PAMP-triggered ROS (Figure S1B) and seedling
growth inhibition (Figure S1C), was not able to complement
bak1-5 mob1 (Figure S1D). As segregation of AtPep1 sensi-
tivity was not observed in the F2 progeny of an independent
cross (80 individuals tested), we conclude that mob1 and
mob2 are allelic.
A
B
C
D
E
F
G
Figure 2. MOB1/MOB2 Encodes the Ca2+-
Dependent Protein Kinase CPK28
(A) Map position of mob1 on the bottom arm of
Chromosome 5 based on linkage analysis using
segregating F2 progeny from bak1-5 mob1
crossed to Ler-0.
(B) Illumina sequencing of bulked F2 segregants
from bak1-5 mob1 backcrossed to bak1-5 called
SNPs in three genes in the mapped region.
(C) Exact positions of SNPs in bak1-5 mob1.
Position: genomic position (in bp); Ref: base
sequence in bak1-5; Seq: base sequence in bak1-
5 mob1; AGI: gene number; Gene: gene name;
Change: amino acid residue substitution.
(D and E) Positions of CPK28 mutations in mob1
and mob2 indicating both genomic and peptide
transitions.
(F and G) Seedling growth assay on MS media
containing 100 nM elf18 (F) or 1 mM AtPep1 (G)
normalized against growth in control media.
Relative values are means + SD (n = 10). Signifi-
cantly different groups (p < 0.0001) are indicated
with lower-case letters based on one-way ANOVA
analysis.
Cell Host & Microbe
CPK28 Buffers BIK1-Mediated Plant Immunity
MOB1/MOB2 Encodes the Ca2+-Dependent ProteinKinase CPK28To identify the causative mutations in mob1 and mob2, we
combined classical map-based cloning with whole-genome
sequencing. We mapped mob1 to the bottom arm of Chro-
mosome 5 between markers MPA24 (26.3 Mbp) and K9I9
(26.9 Mbp) using linkage analysis of F2 segregants from bak1-5
mob1 (in Col-0) crossed to Ler-0 (Figure 2A). In parallel, we
bulked F2 segregants from bak1-5 mob1 backcrossed to bak1-
5 and sequenced the mutant and parental genomes using
Illumina High-Seq. Four nonsynonymous C-T transitions were
uniquely identified in three genes located within the mapped re-
gion of bak1-5 mob1 (Figure 2B); two were in CPK28/At5g66210
(Figures 2C and 2D) and the others were in AtC3H68/At5g66270
and MAN7/At5g66460 (Figure 2C). Sanger sequencing of
genomicCPK28, AtC3H68, andMAN7 in bak1-5 mob2 identified
a homozygous nonsynonymous G-A transition in CPK28 (Fig-
ure 2E) but no SNPs in AtC3H68 or MAN7. The mutations in
bak1-5 mob1 resulted in a CPK28 protein with amino acid sub-
stitutions S245L and A295V (Figure 2D), while the mutation in
bak1-5 mob2 resulted in an early stop codon predicted to result
in a truncated protein (Figure 2E).
To confirm that we had isolated mutant alleles of CPK28, we
stably transformed bak1-5 mob1 plants with genomic CPK28
and found that two independent bak1-5 mob1/pCPK28:CPK28
transgenic lines complemented seedling insensitivity to elf18
and AtPep1 (Figures 2F and 2G). We also obtained cpk28-1
and cpk28-3 insertion alleles (Figure S2A) and crossed
them to bak1-5. Similar to bak1-5 mob1, bak1-5 cpk28-1 and
bak1-5 cpk28-3 seedlings regained sensitivity to AtPep1 (Fig-
ure S2B). Thus, the phenotypes observed in the allelic mutants
mob1 and mob2 are caused by mutations in CPK28. We
renamed these alleles cpk28-4 and cpk28-5, respectively
(Figure S2A).
Cell Host &
CPK28 was previously shown to regulate vegetative stage
transition (Matschi et al., 2013). We obtained cpk28-4 and
cpk28-5 single mutants by crossing to Col-0 and observed
rosette development over a 47 day timeframe. Like cpk28-1
and cpk28-3, mature cpk28-4 and cpk28-5 mutants displayed
a stage transition defect but were phenotypically indistinguish-
able from Col-0 in the juvenile phase (Figure S2C).
Loss of CPK28 Results in Enhanced PAMP-TriggeredSignaling and Antibacterial ImmunityTo assess the role of CPK28 in PTI, we characterized PAMP-
induced responses in cpk28-1 and cpk28-3. Both alleles
produced significantly more ROS compared to Col-0 after treat-
ment with flg22, elf18, AtPep1, or chitin (Figure 3A), indicating
enhanced responsiveness to a broad range of PAMPs. This was
an induced response, as untreated cpk28 plants did not produce
apoplastic ROS (Figure S2D). The cpk28 mutants additionally
displayed enhanced sensitivity to elf18 and AtPep1 in seedling
growth inhibition assays, while intriguingly, sensitivity to flg22
wasnotaffected (Figure3B). Thismay reflect thresholddifferences
in seedling growth inhibition and ROS assays mediated by
different PRRs. In parallel to the ROS burst, PRR activation also
triggers MAPK cascades (Segonzac et al., 2011; Xu et al., 2014;
Zhang et al., 2007). We observed slightly enhanced PAMP-trig-
gered MPK4/11 activation in the cpk28 mutants compared to
Col-0 (Figure S2E), while MPK3/6 activation was unaffected.
Importantly, ligand-dependent association between FLS2
and BAK1 was maintained in the cpk28 mutants (Figure S2F).
Furthermore, accumulation of BAK1 and FLS2 was similar in all
genotypes (Figure S2F), as was basal expression of FLS2,
EFR, CERK1, BAK1, BIK1, and RBOHD (Figure S2G).
As PTI signaling results in resistance against pathogens,
we surface-inoculated plants with the virulent bacterium
Pseudomonas syringae pv. tomato (Pto) DC3000 and monitored
Microbe 16, 605–615, November 12, 2014 ª2014 Elsevier Inc. 607
Figure 3. Loss of CPK28 Results in Enhanced PTI Signaling and Antibacterial Immunity
(A) ROS assay following treatment with 100 nM flg22, 100 nM elf18, 1 mM AtPep1, or 100 mg/ml chitin. Values are means ± SD (n = 8).
(B) Seedling inhibition in media containing 100 nM flg22, 100 nM elf18, or 1 mM AtPep1. Relative values are means + SD (n = 10).
(C) Growth of the virulent bacterium Pto DC3000 3 days postinfection (dpi) with a bacterial suspension of 108 cfu/ml. Values are means + SD (n = 4). All
experiments were repeated at least three times with similar results. Significant differences are designated by asterisks (***p < 0.0001; **p < 0.005; n.s. is not
significant) based on unpaired Student’s t tests comparing the means to Col-0. Refer to Figures S2 and S3 for additional information.
Cell Host & Microbe
CPK28 Buffers BIK1-Mediated Plant Immunity
bacterial titers after 3 days. We observed significantly restricted
bacterial growth incpk28mutants compared toCol-0 (Figure 3C),
indicating that enhanced PTI signaling in cpk28 mutants results
in increased immunity.
CPK28 belongs to Arabidopsis CDPK subgroup IV and is
closely related to CPK16 and CPK18 (Boudsocq and Sheen,
2013; Hamel et al., 2014) (Figure S3A). Publically available micro-
array data (Schmid et al., 2005; Winter et al., 2007) indicates that
whileCPK28 is expressed in many tissue types,CPK16 is almost
exclusively expressed in pollen, and CPK18 does not appear to
be expressed in any tissues (Figure S3B). Consistently, the elf18-
and AtPep1-triggered ROS burst was not affected in cpk16 and
cpk18 insertion alleles compared to Col-0 (Figures S3C and
S3D). We therefore do not expect biological redundancy among
these CDPKs.
CPK28 Kinase Activity Is Required to Attenuate PTISignalingThe cpk28-4 mutations S245L and A295V are located within the
kinase domain of CPK28 (Figure 4A). To test if these residues
are important for kinase activity,wepurifiedwild-type andmutant
CPK28 variants N-terminally tagged with maltose-binding
protein (MBP) fromEscherichia coli andperformed kinase assays
in vitro. Wild-type MBP-CPK28 readily incorporated radioactive
phosphate, while the catalytically dead variantMBP-CPK28D188A
(Matschi et al., 2013) did not (Figure 4B), indicating that CPK28
autophosphorylates in vitro. The cpk28-4 mutant variant MBP-
CPK28S245L/A295V was unable to incorporate radioactive phos-
608 Cell Host & Microbe 16, 605–615, November 12, 2014 ª2014 Els
phate, suggesting compromised kinase function (Figure 4B). To
determine the contribution of S245 and A295 to CPK28 kinase
activity, we individuallymutated both residues and could demon-
strate that MBP-CPK28A295V was catalytically inactive, while
MBP-CPK28S245L retained enzymatic activity (Figure 4B). We
conclude that residueA295 is important forCPK28 kinase activity
in vitro.
To determine if kinase activity is necessary for CPK28 function
in PTI signaling, we stably transformed cpk28-1 plants with wild-
type or mutant CPK28 variants driven by the 35S promoter
and C-terminally tagged with yellow fluroescent protein (YFP)
and tested two independent lines for complementation.
Importantly, all transgenic lines expressed the recombinant pro-
teins to comparable levels (Figure S4A). Both the wild-type
35S:CPK28-YFP and mutant variant 35S:CPK28S245L-YFP com-
plemented enhanced elf18- and AtPep1-triggered ROS in
cpk28-1, while the kinase-dead variants 35S:CPK28D188A-YFP,
35S:CPK28S245L/A295V-YFP, and 35S:CPK28A295V-YFP did not
(Figures 4C and S4B). Therefore, kinase activity is necessary
for CPK28 function in PTI, and the A295V substitution is causa-
tive of the cpk28-4 phenotype.
CDPKs are modular proteins, containing a highly conserved
kinase domain, an autoinhibitory pseudosubstrate domain,
multiple Ca2+-binding EF-hand motifs, and a variable N-terminal
domain (Liese and Romeis, 2013). To determine if CPK28 kinase
activity is sufficient for its function in PTI, we tested if a constitu-
tively active variant of CPK28, containing only the variable
N-terminal domain and the kinase domain (thus referred to as
evier Inc.
A B
C
Figure 4. CPK28 Kinase Activity Is Required to Attenuate PTI Signaling
(A) CPK28 protein organization illustrating the kinase domain, autoinhibitory domain (AID), and Ca2+-binding domain. A multiple sequence alignment generated
using ClustalW and Boxshade II of the region containing the cpk28-4 (red) and cpk28-5 (blue) residue transitions across Arabidopsis CDPKs. Divergent (white),
similar (gray), and conserved (black) residues are indicated.
(B) Autoradiograph showing incorporation of radioactive phosphate in recombinant MBP-CPK28 compared to mutant variants in vitro. Membranes were stained
with Coomassie brilliant blue (CBB) as a loading control.
(C) ROS assay following treatment with 100 nM elf18 in Col-0 and cpk28-1 compared to transgenic lines generated in cpk28-1. All constructs are driven by 35S
and C-terminally tagged with YFP (‘‘35S:CPK28-YFP’’) with the indicated mutations. Values represent means of total photon counts over 60 min + SD (n = 8).
Significantly different groups (p < 0.05) are indicated with lower-case letters based on one-way ANOVA analysis. These experiments were repeated at least three
times with similar results. Refer to Figure S4 for additional information.
Cell Host & Microbe
CPK28 Buffers BIK1-Mediated Plant Immunity
‘‘VK’’), could complement cpk28-1. Indeed, 35S:CPK28VK-YFP
(Matschi et al., 2013) complemented enhanced elf18- and
AtPep1-triggered seedling inhibition (Figure S4C) and ROS burst
(Figure S4D) in cpk28-1. Together, these results indicate that
CPK28 kinase function is necessary and sufficient for its function
in PTI signaling.
Overexpression of CPK28 Inhibits PTI Signaling andImmunityWe next tested the physiological effects of ectopically overex-
pressingCPK28. We generated CPK28-OE lines by transforming
Col-0 with 35S:CPK28-YFP and selected two independent lines
with strongly increased CPK28 expression for analysis (Fig-
ure S5A). Both CPK28-OE1 and CPK28-OE4 were morphologi-
cally similar to Col-0, although CPK28-OE1 was slightly smaller
(Figure S4B). Remarkably, both CPK28-OE lines exhibited a
severely reduced ROS burst after treatment with flg22, elf18,
AtPep1, or chitin (Figure 5A). Both CPK28-OE lines were signifi-
cantly less sensitive to both elf18 and AtPep1 in seedling
growth assays but remained as sensitive as Col-0 to flg22 (Fig-
ure 5B). In addition, PAMP-induced activation of MPK4/11 was
reduced in the CPK28-OE lines compared to Col-0 (Figure S5C),
as was elf18-triggered expression of the marker genes FRK1,
At1g51890, and NHL10 (Figure S5D).
Cell Host &
Importantly, FLS2-BAK1 complex formation was comparable
between Col-0 and the CPK28-OE lines (Figure S5E), and the
overexpression of CPK28 did not result in impaired steady-state
expression of several relevant defense genes (Figure S5F).
The CPK28-OE lines additionally supported significantly
higher bacterial growth compared to Col-0 after spray-infection
with Pto DC3000 (Figure 5C). Accordingly, the CPK28-OE lines
were impaired in flg22-induced stomatal closure (Figure S5G).
Together, these results indicate that overexpression of CPK28
impairs PTI signaling and immunity.
CPK28 Associates to the Plasma Membrane viaMyristoylationAnalysis of cpk28-1/35S:CPK28-YFP tissue using confocal mi-
croscopy revealed that CPK28-YFP localizes to the plasma
membrane (Figure S6A). Similar to many CDPKs, CPK28 con-
tains a predicted N-terminal myristoylation motif at position G2
(Boudsocq and Sheen, 2013). To determine if CPK28 localization
is the consequence of myristoylation, we created a G2A variant
and transiently expressed 35S:CPK28G2A-YFP in Nicotiana
benthamiana alongside 35S:CPK28-YFP. The mutant variant
clearly lost membrane localization (Figure S6B), indicating that
CPK28 is targeted to the plasma membrane via myristoylation
on residue G2.
Microbe 16, 605–615, November 12, 2014 ª2014 Elsevier Inc. 609
Figure 5. Overexpression of CPK28 Inhibits PTI Signaling and Antibacterial Immunity
(A) ROS assay following treatment with 100 nM flg22, 100 nM elf18, 1 mM AtPep1, or 100 mg/ml chitin. Values are means ± SD (n = 8).
(B) Seedling inhibition assay in media containing either 100 nM flg22, 100 nM elf18, or 100 nM AtPep1. Relative values are means ± SD (n = 10).
(C) Growth of the virulent bacterium Pto DC3000 3 dpi with a bacterial suspension of 108 cfu/ml. Values are means ± SD (n = 4). Significant differences are
designated by asterisks (***p < 0.0001; **p < 0.005; *p < 0.01; n.s. is not significant) based on unpaired Student’s t tests comparing the means to Col-0. All
experiments were repeated at least three times with similar results. Refer to Figure S5 for additional information.
Cell Host & Microbe
CPK28 Buffers BIK1-Mediated Plant Immunity
CPK28 Associates with and Phosphorylates BIK1Our genetic analyses indicated a broad effect on BAK1-depen-
dent (flg22, elf18, and AtPep1) and BAK1-independent (chitin)
signaling cascades, suggesting that CPK28 may target a down-
stream regulator of several PRR complexes.
The plasma-membrane-associated kinase BIK1 is a key regu-
lator required for signaling through several PRRs.We thus tested
whether CPK28 and BIK1 could associate by coexpressing
35S:CPK28-YFP and 35S:BIK1-HA in N. benthamiana and
conducting coimmunoprecipitation (coIP) assays. A signal for
BIK1-HA was clearly observed in the CPK28-YFP immunopre-
cipitate (Figure S6C), indicating that CPK28 and BIK1 can asso-
ciate. We confirmed this association in Arabidopsis transgenic
plants expressing pBIK1:BIK1-HA and 35S:CPK28-YFP gener-
ated through crossing (Figure 6A).
As BIK1 associates with the FLS2-BAK1 complex (Lu et al.,
2010; Zhang et al., 2010) and the NADPH oxidase RBOHD
(Kadota et al., 2014; Li et al., 2014), we were interested to
know if CPK28 also associates with these proteins. We found
that FLS2 and BAK1 did not associate with CPK28-YFP in coIP
assays using cpk28-1/35S:CPK28-YFP tissue treated with or
without flg22 (Figure S6D). However, association was observed
between FLAG-RBOHD and CPK28-YFP when coexpressed in
N. benthamiana (Figure S6E). Notably, CPK28 associates with
RBOHD and BIK1 before and after treatment with flg22 (Figures
6A, S6C, and S6E), suggesting that CPK28 constitutively associ-
ates with the BIK1/RBOHD complex but not with FLS2 or BAK1.
610 Cell Host & Microbe 16, 605–615, November 12, 2014 ª2014 Els
Because BIK1 acts upstream of RBOHD and is required for
its function (Kadota et al., 2014; Li et al., 2014), we focused
on characterizing the interaction between CPK28 and BIK1.
To test if CPK28 can phosphorylate BIK1, we performed
in vitro kinase assays. MBP-CPK28 trans-phosphorylated both
the wild-type MBP-BIK1 protein and the catalytically inactive
variant MBP-BIK1K105A/K106A (Lu et al., 2010) (Figure 6B). Impor-
tantly, MBP-BIK1K105A/K106A did not incorporate radioactive
phosphate on its own or when incubated with the kinase-dead
variant MBP-CPK28D188A, indicating that trans-phosphorylation
was the consequence of incubation with CPK28. These results
suggest that CPK28 associates with and phosphorylates BIK1.
BIK1 Accumulation Is Regulated by CPK28 and the 26SProteasomeWe noted that overexpression of BIK1 in Col-0/pBIK1:BIK1-HA
plants (Kadota et al., 2014; Zhang et al., 2010) (Figure S7A)
resulted in a strongly enhanced elf18- and AtPep1-triggered
ROS burst (Figures 7A, 7D, S7B, and S7D), agreeing with its
role as a key positive regulator of RBOHD (Kadota et al., 2014;
Li et al., 2014). We generated CPK28-OE1/pBIK1:BIK1-HA lines
though crossing and found that the increased burst after elf18 or
Pep1 treatment was almost completely suppressed (Figures 7A
and S7B), supporting a role for CPK28 in negatively regulating
BIK1-mediated signaling. Interestingly, we found that while
PAMP-triggered BIK1-HA hyperphosphorylation was main-
tained, BIK1-HA protein accumulation was strongly reduced in
evier Inc.
Figure 6. CPK28 Associates with and Phosphorylates BIK1
(A) CoIP assay before and after treatment with 200 nM flg22 for 10 min. WB
analysis using a-GFP and a-HA on total protein extracts (‘‘input’’) and on eluted
proteins after IP with a-GFP magnetic beads.
(B) Autoradiograph showing incorporation of radioactive phosphate in the
indicated recombinant proteins following kinase assays in vitro. Both experi-
ments were repeated three times with similar results. CBB stains are included
as controls. Refer to Figure S6 for additional information.
Cell Host & Microbe
CPK28 Buffers BIK1-Mediated Plant Immunity
CPK28-OE1/pBIK1:BIK1-HA compared to control plants (Fig-
ure 7B). BIK1 transcript levels were comparable between
CPK28-OE1/pBIK1:BIK1-HA and Col-0/pBIK1:BIK1-HA (Fig-
ure S7A), indicating that reduced BIK1-HA accumulation is
caused either by reduced protein synthesis or increased protein
turnover. BAK1 accumulation was not affected (Figure 7B), sug-
gesting that the overall translational machinery is functional.
Thus, reduced BIK1-HA accumulation in CPK28-OE1/pBIK1:
BIK1-HA is probably due to increased protein degradation.
A common protein degradation pathway is mediated by the
ubiquitin/proteasome system (Nelson et al., 2014). Chemical
inhibition of the proteasome using MG-132 (Rock et al., 1994)
resulted in strongly enhanced accumulation of BIK1 in both
Col-0/pBIK1:BIK1-HA and CPK28-OE1/pBIK1:BIK1-HA, while
BAK1 accumulation was unaffected (Figure 7C). Moreover, we
observed laddering of BIK1-HA in MG-132-treated samples
(Figure S7C), indicative of polyubiquitination.
We next generated cpk28-1/pBIK1:BIK1-HA lines through
crossing. These plants were hyperresponsive to elf18 and
AtPep1 in ROS burst assays, indicating additive genetic effects
of cpk28-1 andBIK1 overexpression (Figures 7D andS7D).While
BIK1 expression was similar between Col-0/pBIK1:BIK1-HA and
cpk28-1/pBIK1:BIK1-HA (Figure S7E), higher BIK1 accumulation
was detected in cpk28-1/pBIK1:BIK1-HA (Figure 7E). Impor-
tantly, BAK1 levels were unaffected in these lines (Figure 7E).
Our results suggest that CPK28 constitutively regulates BIK1
accumulation, thereby buffering the amplitude of PTI signaling.
DISCUSSION
The temporal and physical dynamics of receptor complexes,
together with phosphorylation initiated by kinases within these
complexes, underpin ligand-triggered signaling events that
must be tightly controlled (Lemmon and Schlessinger, 2010;
Cell Host &
Scott and Pawson, 2009). This is particularly true for immune re-
sponses whose suboptimal or supraoptimal induction can have
detrimental effects on cellular homeostasis and survival (Kondo
et al., 2012; Sasai and Yamamoto, 2013). In this study, we iden-
tified CPK28 as a negative regulator of plant immunity. We show
that CPK28 interacts with and phosphorylates BIK1, a key regu-
latory kinase associated with PRR complexes and involved in
PAMP-triggered signal transduction. We find that BIK1 is contin-
uously degraded by the 26S proteasome and that CPK28 con-
tributes to BIK1 turnover. Our results reveal a regulatory mecha-
nism that ensures an optimal amplitude of immune responses.
CDPKs contain a Ca2+-binding domain and represent a unique
subclass of kinases found in plants, green algae, and some
protists (Hamel et al., 2014; Hrabak et al., 2003). Available
biochemical and structural data suggest that Ca2+ binding to
CDPKs results in a conformational change that releases autoin-
hibition and exposes the active site (Harper et al., 2004; Liese
and Romeis, 2013). PAMP perception results in a Ca2+ influx
that is thought to contribute to immune signaling through
activation of Ca2+-binding proteins such as CDPKs (Romeis
and Herde, 2014). Several CDPKs have been implicated in
response to biotic and abiotic stresses, mostly as positive regu-
lators (Boudsocq and Sheen, 2013; Romeis and Herde, 2014).
In-gel kinase assays demonstrated that PAMP perception
results in phosphorylation of multiple CDPKs (Boudsocq et al.,
2010). In particular, CPK5 and related CDPKs CPK4, CPK6,
and CPK11 participate in immunity and phosphorylate the
NADPH oxidase RBOHD (Boudsocq et al., 2010; Dubiella
et al., 2013; Gao et al., 2013; Kadota et al., 2014). In contrast,
we find that CPK28 is a negative regulator of PTI signaling.
All of our experiments were performed using juvenile cpk28
plants, which are indistinguishable from wild-type plants up to
5 weeks postgermination (Figure S2C). Previous work showed
that mature cpk28 plants accumulate high levels of anthocyanin
and exhibit severely reduced stem elongation linked to impaired
gibberellic acid biosynthesis (Matschi et al., 2013). A similar
observation was noted in N. attenuata plants silenced for
NaCDPK4 and NaCDPK5, two CDPKs orthologous to CPK28
(Heinrich et al., 2013; Yang et al., 2012). Juvenile NaCDPK4/
NaCDPK5-silenced plants overaccumulate defense-related
metabolites, including jasmonic acid, when treated with oral se-
cretions from the insect herbivore Manduca sexta and exhibit
enhanced resistance against insect feeding (Yang et al., 2012).
Inactivation of the Medicago truncatula CPK28 ortholog
MtCDPK1 has also been linked to enhanced defense responses,
causing reduced root colonization by both the mycorrhizal fun-
gusGlomus versiforme and the rhizobial symbiontSinorhizobium
meliloti (Ivashuta et al., 2005). Together with our characterization
of cpk28 mutants, which exhibit enhanced PAMP-triggered
responses, it is tempting to speculate that CPK28 orthologs
broadly function as negative regulators of immunity across plant
species, potentially by regulating RLCKs.
Negative regulation of PRR complexes attenuates PTI
signaling. In the absence of PAMPs, complex formation between
FLS2 and BAK1 is inhibited by the BAK1-associated LRR-RK
BIR2 (Halter et al., 2014). BAK1 is additionally negatively
regulated by a specific PP2A isoform whose activity is inhibited
upon PAMP perception (Segonzac et al., 2014). Following PAMP
perception, ligand-bound FLS2 is endocytosed (Robatzek et al.,
Microbe 16, 605–615, November 12, 2014 ª2014 Elsevier Inc. 611
Figure 7. BIK1 Accumulation Is Regulated
by CPK28 and the 26S Proteasome
(A) ROS assay following treatment with 100 nM
elf18. Values are means ± SD (n = 8).
(B) WB analysis of BIK1-HA before and after
treatment with 100 nM elf18 for 20 min.
(C) WB analysis of BIK1-HA after treatment for 8 hr
with either 1% DMSO (�) or 100 mM MG-132 (+).
(D) ROS assay following treatment with 100 nM
elf18. Values are means ± SD (n = 8).
(E) WB analysis of BIK1-HA before and after
treatment with 100 nM elf18 for 20 min. All mem-
branes were probed with a-BAK1 and stained with
CBB as loading controls. Densiometry values
(representing means ± SE from three independent
experiments) are plotted beneath the blots; a-HA
band intensities were normalized against a-BAK1
band intensities from the same samples. Asterisks
indicate statistical significance (*p < 0.05; **p <
0.005; ***p < 0.0001) based on an unpaired
Student’s t test comparing the means to the con-
trol. All experiments were repeated at least three
times with similar results. Refer to Figure S7 for
additional information.
Cell Host & Microbe
CPK28 Buffers BIK1-Mediated Plant Immunity
2006) and degraded by the E3 ubiquitin ligases PUB12 and
PUB13 (Lu et al., 2011). Our results suggest that CPK28-medi-
ated phosphorylation of BIK1 facilitates its turnover, possibly
by influencing its ubiquitination and consequent degradation
via the 26S proteasome (Figure S7F). CPK28-mediated phos-
phorylation may result in the recruitment of a currently unknown
E3 ligase leading to BIK1 degradation. Potential candidates
include PUB22, PUB23, and PUB24, which have been shown
to negatively control PAMP-induced ROS and PTI marker gene
expression in Arabidopsis (Trujillo et al., 2008). Future research
will indicate if this working model is correct.
BIK1 has emerged over the last few years as a key convergent
signaling component downstream of multiple PRRs. Consis-
tently, BIK1 and related proteins are required for signaling medi-
ated by multiple PAMPs and immunity against bacterial and
fungal pathogens (Laluk et al., 2011; Liu et al., 2013; Lu et al.,
2010; Veronese et al., 2006; Zhang et al., 2010). In addition,
BIK1 and related proteins are direct substrates of two unrelated
bacterial effector proteins, the P. syringae cysteine protease
AvrPphB (Zhang et al., 2010) and the Xanthomonas campestris
uridine 50-monophosphate transferase AvrAC (Feng et al.,
2012), illustrating the key role played by BIK1 in immune
signaling. We found that increasing BIK1 levels (either in Col-0/
pBIK1:BIK1-HA or cpk28-1 plants) results in enhanced PTI
signaling, suggesting that BIK1 is rate limiting. Since BIK1
directly activates RBOHD (Kadota et al., 2014; Li et al., 2014),
612 Cell Host & Microbe 16, 605–615, November 12, 2014 ª2014 Elsevier Inc.
reduced BIK1 levels (as observed upon
CPK28 overexpression) would result in
a reduced pool of activated RBOHD
and an impaired ROS burst (Figure 5A).
We therefore propose that by regulating
BIK1 levels, CPK28 buffers immune
signaling to tailor the appropriate ampli-
tude of immune outputs. Future work
will aim to uncover the details surround-
ing the interplay between phosphorylation and ubiquitination in
BIK1 turnover and how this regulation is controlled upon PAMP
perception. Of particular interest will be the identification of
BIK1 residues that are specifically phosphorylated by CPK28
to affect its turnover, in contrast to residues phosphorylated by
the PRR complex that lead to BIK1 activation.
EXPERIMENTAL PROCEDURES
Growth Conditions and Plant Materials
Arabidopsis thaliana plants were grown on soil as one plant per pot in
controlled rooms maintained at 20�C–22�C with a 10 hr photoperiod or as
seedlings on sterile Murashige and Skoog (MS) media supplemented with vi-
tamins and 1% sucrose (Duchefa) in similar rooms with a 16 hr photoperiod.
Assays using soil-grown plants were performed 3 to 4 weeks postgermination
(wpg) prior to reproductive transition, while assays using plate-grown seed-
lingswere performed 2wpg. T-DNA insertionmutants cpk28-1 (GABI_523B08)
and cpk28-3 (WiscDsLox_264D03) were obtained from the European Arabi-
dopsis Stock Centre in Nottingham (NASC) and genotyped to homozygosity
using allele-specific primers. The bak1-5 mutant was previously described
(Schwessinger et al., 2011), as were Col-0/pBIK1:BIK1-HA (Kadota
et al., 2014; Zhang et al., 2010), cpk28-1/35S:CPK28-YFP, and cpk28-1/
35S:CPK28D188A-YFP (Matschi et al., 2013). We generated plants containing
multiple homozygous transgenes through crosses and allele-specific genotyp-
ing in subsequent generations. Agrobacterium tumefaciens strains GV3101-
pMP90 or GV3101-pMP90RK were used for stable and transient plant trans-
formation. Stable lines with single inserts, as indicated by 3:1 segregation on
selection plates in the T2, were carried forward to homozygosity in the T3and used for further analysis. Please refer to the Supplemental Information
Cell Host & Microbe
CPK28 Buffers BIK1-Mediated Plant Immunity
for details about additional plant lines, genotyping primers, molecular cloning
methods, and transient transformation of N. benthamiana.
Identification of mob Mutants
Bak1-5 seeds were mutagenized with ethyl-methyl sulfonate (Sigma Aldrich).
Roughly 40,000 M2 seeds were surface sterilized and sown on 1% MS agar
plates alongside Col-0 controls (Figure S1A). After stratification for 3 days at
4�C, the plates were transferred to light for 9 days. Seedlings were then
submerged in elicitor solution containing 100 nM flg22, 1 mM of the luminol
derivative L-012 (Wako Chemicals), and 200 mg/ml horseradish peroxidase
(Sigma Aldrich). The ROS burst was qualitatively scored over 45 min using
a charge-coupled device camera fitted to a computer monitor (Photek Ltd.,
East Sussex). Positive seedlings were rinsed with water, transferred to soil,
retested as adult plants, and confirmed in subsequent generations.
Map-Based Cloning and Whole-Genome Sequencing
The bak1-5 mob1 mutant (in Col-0) was crossed to Ler-0. Approximately
1,000 F2 segregants were genotyped for bak1-5 using the Col-0/Ler-0 indel
markers F17M5 and T16L1, which flank the BAK1 locus. Homozygous
bak1-5 segregants were phenotyped for mob1, and linkage analysis was
performed using an array of genome-wide markers designed in-house or by
the Arabidopsis Mapping Platform (Hou et al., 2010). For whole-genome
sequencing, F2 plants from bak1-5 mob1 crossed to bak1-5 were scored
for elf18-induced ROS and seedling inhibition, and positive segregants
were bulked prior to isolation of genomic DNA. The Beijing Genomics Institute
(Hong Kong) prepared Illumina-adapted libraries and sequenced bak1-5
mob1 as well as bak1-5 as a reference using the High-Seq 2000 platform.
We identified unique SNPs in the bak1-5 mob1 genome through comparison
with the reference sequence. Further details are available in the Supplemental
Information.
Bioassays
Peptide sequences for flg22, elf18, and AtPep1 have been previously
described (Felix et al., 1999; Huffaker et al., 2006; Kunze et al., 2004) and
were synthesized by EZBiolab (Indiana). Chitin was purchased from Sigma
Aldrich. PAMP-triggered ROS burst, seedling growth inhibition, MAPK activa-
tion, and pathogen assays were performed as previously described (Schwes-
singer et al., 2011). Minor modifications are outlined in the Supplemental
Information.
Immunoprecipitation and Western Blots
Proteins were extracted from frozen plant tissue in buffer containing 50 mM
Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 5 mM dithiothreitol, 1% pro-
tease inhibitor cocktail (Sigma Aldrich), 2 mM Na2MoO4, 2.5 mM NaF,
1.5 mM activated Na3VO4, 1 mM phenylmethanesulfonyl fluoride and 1%
Igepal, and normalized using Bradford Reagent (Biorad). The following
beads were used for coIPs as indicated: a-rabbit Trueblot agarose beads
(eBioscience), a-GFP mMACs magnetic beads (Miltenyi Biotec), and GFP-
Trap agarose beads (ChromoTek). After the IP, beads were washed at least
three times in buffer containing 1x TBS, 1% protease inhibitor cocktail
(Sigma Aldrich), and 0.5% Igepal. When indicated, 1% DMSO or 100 mM
MG-132 (Calbiochem) was vacuum infiltrated into leaves 7 hr prior to harvest-
ing. Samples were boiled for 5 min in Laemmli sample buffer (LSB) and sub-
ject to SDS-PAGE and western blot (WB) analysis. WB quantification was
performed using ImageJ software (http://imagej.net/). Please refer to the
Supplemental Information for details about all antibodies used in WBs.
In Vitro Kinase Assays
MBP-BIK1 and MBP-CPK28 variants were expressed and purified from
Escherichia coli strain BL21 using constructs outlined in the Supplemental In-
formation. A total of 2 mg of kinase protein and 2 mg of substrate protein (or sim-
ply 2 mg of kinase protein for autophosphorylation assays) were incubated
together for 30 min with gentle shaking at 30�C in buffer containing 50 mM
Tris-HCl (pH 7.5), 25 mM MnCl2, 5 mM dithiothreitol, 5 mM cold ATP, and
183 KBq radioactive [32P]-g-ATP. Samples were denatured in LSB at 70�Cfor 10 min. Following SDS-PAGE, the proteins were transferred to polyvinyli-
dene difluoride membranes, and incorporation of radioactive ATP was
analyzed using a phosphoimager (Fuji Film FLA-5000).
Cell Host &
Statistical Analyses
Statistically significant groups were determined by Students’ t tests or
one-way ANOVAs, followed by Tukey’s multiple comparison post hoc test
using GraphPad Prism 5.1 (http://www.graphpad.com/scientific-software/
prism/).
SUPPLEMENTAL INFORMATION
Supplemental Information includes seven figures and Supplemental Experi-
mental Procedures and can be found with this article online at http://dx.doi.
org/10.1016/j.chom.2014.10.007.
ACKNOWLEDGMENTS
We thank Vardis Ntoukakis and Yasuhiro Kadota for critically reading the
manuscript and all members of the Zipfel laboratory for helpful discussions,
invaluable advice, and technical knowledge. We thank Matthew Smoker and
Jodie Pike for creating transgenic lines and the JIC horticultural staff for
growing plants and maintaining growth facilities. This research was funded
by grants from The Gatsby Foundation and The European Research Council
to C.Z., TheGermanResearch Foundation to T.R., and TheGatsby Foundation
and the UK Biotechnology and Biological Sciences Research Council (grant
BB/E017134/1) to J.P.R. and C.Z. J.M. was the recipient of a Long-Term
Fellowship from the European Molecular Biology Organization, O.S. is part
of the JIC/TSL Rotation PhD Programme, H.R. was funded by an Erasmus
Mundus Scholarship, and A.M. was funded by an Erasmus-SMP grant from
the German Academic Exchange Service.
Received: May 17, 2014
Revised: August 11, 2014
Accepted: September 11, 2014
Published: November 12, 2014
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Supplemental Information
The Calcium-Dependent Protein
Kinase CPK28 Buffers Plant Immunity
and Regulates BIK1 Turnover
Jacqueline Monaghan, Susanne Matschi, Oluwaseyi Shorinola, Hanna Rovenich, Alexandra Matei, Cécile Segonzac, Frederikke Gro Malinovsky, John P. Rathjen, Dan MacLean, Tina Romeis, and Cyril Zipfel
B
A C
~40,000 M2 seeds sown on agar plates
M1 generation bulked in small pools
EMS-mutagenesis of bak1-5
9-day-old seedlings submerged in
flg22 elicitor solution and ROS
measured with a CCD camera
900+ mutants with regained ROS
transferred to soil and grown for 3 weeks
flg22- and elf18-induced ROS measured
in 2 leaf discs from each mutant
M3 progeny of 100+ mutants harvested
1º Screen
flg22-, elf18- and AtPep1-triggered ROS
measured in M3 generation
2º Screen
3º Screen
10 mob mutants taken for further study
a
b
a
a
a a
b b
b
flg22
elf18
AtPep1
D
flg22
elf18
AtPep1
Figure S1 (Related to Figure 1): The modifier of bak1-5 screen and characterization of bak1-5
mob2.
(A) Outline of the mob screen. (B) ROS assay following treatment with 100 nM flg22, 100 nM elf18, or
1 μM AtPep1. Values are means ± standard deviation (n=8). (C) Seedling inhibition assay after growth
in media containing 1 μM flg22, 1 μM elf18 , or 1 μM AtPep1 normalized against growth in control
media. Relative values are means + standard deviation (n=6). Significantly different groups (p <
0.0001) are indicated with lower-case letters based on one-way ANOVA analysis and Tukey’s multiple
comparison post-test. (D) Seedling growth in control media or media containing 1 μM AtPep1. The
bak1-5 mob1 x bak1-5 mob2 cross was repeated once and the F2 was similarly tested. All other
experiments were repeated at least three times with similar results.
water
A
B
D
AtPep1
b
a a a a
C
E
G
F
Figure S2 (Related to Figure 3): Loss of CPK28 results in enhanced PTI signaling.
(A) Gene structure of CPK28/At5g66210 showing the positions of exons (boxes), introns (lines), T-
DNA insertion alleles, and mob mutations. (B) Seedling inhibition in media containing 1 μM AtPep1.
Relative values are means + standard deviation (n=10). Significantly different groups (p < 0.0001) are
indicated with lower-case letters based on one-way ANOVA analysis and Tukey’s multiple
comparison post-test. (C) Rosette morphology and shoot elongation in the indicated genotypes at 22,
35, and 47 days after germination under long-day (LD) conditions (16 h light/ 8 h dark), as well as 40
days under short-day (SD) conditions (10 h light/14 h dark). (D) Basal ROS assay using water. Values
are means ± standard deviation (n=8). (E) Western blot (WB) using α-p42/p44-erk against
phosphorylated MPK6, MPK3, and MPK4/11 over a time-course following treatment with 100 nM
flg22, 100 nM elf18, 1 μM AtPep1 or 100 μg/mL chitin. Membranes were stained with CBB as a
loading control. (F) Co-immunoprecipitation (IP) assay between FLS2 and BAK1 before and after
treatment with 200 nM flg22 for 10 minutes. WB analysis using the native antibodies α-FLS2 and α-
BAK1 on total protein extracts (‘input’) and on eluted proteins after IP with α-BAK1/Trueblot. (G)
Quantitative real-time RT-PCR analysis of FLS2, EFR, CERK1, BAK1, BIK1, and RBOHD expression
in the indicated genotypes. Values are means + standard deviation (n=3), relative to ACTIN2.
CPK16 (At2g17890)
CPK18 (At4g36070)
CPK28 (At5g66210)
CPK subgroup IVA
B
C
D elf18 AtPep1
b b
a a a ac a
a a a ac a
Figure S3 (Related to Figure 3): Analysis of Arabidopsis CDPK sub-group IV.
(A) CPK28 is closely related to CPK16 and CPK18. The tree is based on ones generated in
(Boudsocq and Sheen, 2012; Hamel et al., 2014) and shown here for illustrative purposes only. (B)
Expression values of CPK16, CPK18, and CPK28 in different Arabidopsis tissue types based on
publically available microarray data, accessed through eFP browser (Winter et al., 2007). (C) Gene
structure of CPK16/At2g17890 and CPK18/At4g36070 showing the positions of exons (boxes),
introns (lines) and T-DNA insertion alleles (triangles). (D) ROS assay following treatment with 100 nM
elf18 or 1 μM AtPep1. Values represent means of total photon counts over 60 minutes + standard
deviation (n=8). Significantly different groups (p < 0.05) are indicated with lower-case letters based on
one-way ANOVA analysis and Tukey’s multiple comparison post-test. This assay was repeated twice
with similar results.
A
B
a
b
a
c b
b
a a
c bc
AtPep1
elf18
AtPep1
C D
elf18
AtPep1
b
b
b
b
a a
a a
a
a a
c
Figure S4 (Related to Figure 4): CPK28 kinase activity is necessary and sufficient for its
function in PAMP signaling.
(A) WB analysis using α-GFP on total protein extracts from the transgenic lines analysed in Fig 4C
and Fig S4B indicate similar accumulation of wild-type and mutant variants. All constructs are driven
by 35S and C-terminally tagged with YFP (‘35S-CPK28-YFP’) with the indicated mutations.
Membranes were stained with CBB as a loading control. (B) ROS assay following treatment with 1 μM
AtPep1 in Col-0 and cpk28-1 compared to transgenic lines generated in cpk28-1. All constructs are
driven by 35S and C-terminally tagged with YFP (‘35S-CPK28-YFP’) with the indicated mutations.
Values represent means of total photon counts over 60 minutes + standard deviation (n=8). (C)
Seedling inhibition in media containing 100 nM elf18 or 1 μM AtPep1 in Col-0 and cpk28-1 compared
to a homozygous cpk28-1/35S:CPK28VK-YFP transgenic line. Relative values are means + standard
deviation (n=8). (D) ROS assay following treatment with 100 nM elf18 or 1 μM AtPep1. Values
represent means of total photon counts over 60 minutes + standard deviation (n=8). Significantly
different groups (p < 0.05) are indicated with lower-case letters based on one-way ANOVA analysis
and Tukey’s multiple comparison post-test. These assays were repeated three times with similar
results.
A
B
D
*** ***
C
FRK1 At1g51890 NHL10
E
F
G
*** n.s. *
Figure S5 (Related to Figure 5): Characterization of CPK28-OE lines.
(A) CPK28 expression is strongly enhanced in both CPK28-OE1 and CPK28-OE4, as indicated by
quantitative real-time PCR. Values are means + standard deviation (n=3), relative to ACTIN2.
Asterisks indicate statistical significance (*** p < 0.0001) determined by Student’s t-test compared to
Col-0. (B) Rosette morphology and shoot elongation in the indicated genotypes at 22, 35, and 47 days
after germination under long-day (LD) conditions (16 h light/ 8 h dark). (C) Western blot (WB) using α-
p42/p44-erk against phosphorylated MPK6, MPK3, and MPK4/11 before and after 10 minutes
treatment with 100 nM flg22, 100 nM elf18, 1 μM AtPep1 or 100 μg/mL chitin. Membranes were
stained with CBB as a loading control. (D) Quantitative real-time RT-PCR of FRK1, At1g51890 and
NHL10 after treatment with 100 nM elf18 in the indicated genotypes. Values are means + standard
deviation (n=3), relative to U-BOX. (E) Co-immunoprecipitation (IP) assay of FLS2-BAK1 complex
formation after treatment with 200 nM flg22 for 10 minutes. WB analysis using the native antibodies α-
FLS2 and α-BAK1 on total protein extracts (‘input’) and on eluted proteins after IP with α-
BAK1/Trueblot agarose beads. (F) Quantitative real-time RT-PCR of FLS2, EFR, CERK1, BAK1,
BIK1, and RBOHD expression in the indicated genotypes. Values are means + standard deviation
(n=3), relative to ACTIN2. (G) Stomatal aperture measurements in the indicated genotypes after
treatment with 5 μM flg22 for 1 hr. Values represent means + standard deviation (n=15-30 stomata).
Asterisks indicate statistical significance (*** p < 0.0001, * p < 0.01, n.s. is not significant) determined
by Student’s t-test comparing the mock and flg22 treatments. All experiments were repeated at least
three times with similar results.
A
C
B
D E
Figure S6 (Related to Figure 6): CPK28 localizes to the plasma membrane and associates with
BIK1 and RBOHD.
(A) Confocal microscopy of cells from cpk28-1/35S:CPK28-YFP plants in isotonic (water) and
hypertonic (2.5% NaCl) conditions. Arrows indicate CPK28-YFP signal at the detaching membrane
due to plasmolysis. (B) Confocal microscopy of N. benthamiana leaves co-expressing free mCherry
with 35S:CPK28-YFP or 35S:CPK28G2A-YFP. Arrows indicate cytoplasmic streaming. (C) Co-
immunoprecipiation (co-IP) assay showing that CPK28-YFP and BIK1-HA associate in Nicotiana
benthamiana, irrespective of treatment with 200 nM flg22 for 10 minutes. Western blot (WB) analysis
using α-GFP and α-HA on total protein extracts (‘input’) and on eluted proteins after IP with α-GFP
magnetic beads. (D) WB analysis using α-FLS2, α-BAK1 and α-GFP on total protein extracts (‘input’)
and on eluted proteins after IP with either GFP-Trap beads or α-BAK1/Trueblot beads from cpk28-
1/35S:CPK28-YFP transgenic plants. (E) Co-IP assay showing that CPK28-YFP and FLAG-RBOHD
associate in N. benthamiana, irrespective of treatment with 200 nM flg22 for 10 minutes. WB analysis
using α-GFP and α-FLAG on total protein extracts (‘input’) and on eluted proteins after IP with α-GFP
magnetic beads. All experiments were repeated three times with similar results.
a a
b
b
A B
a
a
BIK1
BIK1
AtPep1
AtPep1
C D E
F
Col-0
CPK28-OE1
Col-0/pBIK1:BIK1-HA
CPK28-OE1/pBIK1:BIK1-HA
Col-0
cpk28-1
Col-0/pBIK1:BIK1-HA
cpk28-1/pBIK1:BIK1-HA
Figure S7 (Related to Figure 7): BIK1 accumulation is regulated by CPK28 and the 26S
proteasome.
(A) BIK1 expression as demonstrated by quantitative real-time RT-PCR. Values are means +
standard deviation (n=3), relative to U-BOX. (B) ROS assay following treatment with 1 μM AtPep1.
Values are means ± standard deviation (n=8). (C) Western blot (WB) of BIK1-HA after 8 hours
treatment with 1% DMSO (-) or 100 µM MG-132 (+). Black arrows indicate typical BIK1-HA migration
and grey arrows mark laddering indicative of poly-ubiquitination. The lower blot is a short exposure
similar to what is shown in Figure 7C and the upper blot is a long exposure from the same membrane.
CBB staining is included as a loading control. (D) ROS assay following treatment with 1 μM AtPep1.
Values are means ± standard deviation (n=8). (E) BIK1 expression as demonstrated by quantitative
real-time RT-PCR. Values are means + standard deviation (n=3), relative to U-BOX. Significantly
different groups (p < 0.0001) are indicated with lower-case letters based on one-way ANOVA analysis
and Tukey’s multiple comparison post-test. All experiments were repeated three times with similar
results. (F) Conceptual model illustrating that CPK28 negatively regulates PAMP signaling and
contributes to BIK1 turnover. Our data suggest that a currently unknown E3 ligase mediates BIK1
ubiquitination and continual degradation to ensure optimal immune outputs. We propose that CPK28
may have additional, currently unknown, targets.
SUPPLEMENTAL EXPERIMENTAL PROCEDURES
Genetic analysis
To determine heritance, bak1-5 mob1 and bak1-5 mob2 were back-crossed to bak1-
5. Segregating F2 plants were scored based on elf18-induced ROS or seedling
inhibition. Both mutations were found to be recessive, as 23% and 17% of the F2
populations, respectively, contained plants with the mob phenotype. All assays in
Figures 1, 2 and S1 were conducted using F3 generation backcrossed bak1-5 mob1
or bak1-5 mob2 plants. Allelism was determined by crossing bak1-5 mob1 with bak1-
5 mob2 and analysing the F1 for non-complementation, and further confirmed
through an independent cross.
Whole-genome sequencing
222 F2 plants from the cross bak1-5 mob1 with bak1-5 were scored for elf18-induced
ROS. 51 plants with increased ROS were identified and confirmed in the F3
generation to restore elf18-ROS and elf18-seedling inhibition. 5 seedlings from each
of the positive F3 parents were bulked and ground to a fine powder in liquid nitrogen.
Tissues were equilibrated in buffer containing 50 mM Tris-HCl (pH 8.0), 200 mM
NaCl, 2 mM EDTA for 30 min at 37°C with occasional mixing, and for a further 20 min
at 37°C with 0.2 mg/mL RNAse. Roughly 10 ng of genomic DNA was then extracted
using a standard chloroform/phenol method and re-suspended in 1x TE. Illumina
sequencing of the bak1-5 mob1 genome sequence resulted in 43.96 million 90 bp
paired-end reads with a mean insert size of 350 bp; > 98.8% of reads aligned to the
TAIR10 Arabidopsis reference sequence. Average coverage over the nuclear
chromosomes was 49.53. Paired-end reads were aligned to the TAIR10 reference
assembly using BWA v 0.6.1 with default settings (Li and Durbin, 2009). BAM files
were generated using SAMTools v 0.1.8 (Li et al., 2009) and SNPs were called using
the mpileup command. Reads with mapping quality scores less than 20 and
individual bases with sequence quality less than 20 were ignored. Positions
considered for single nucleotide polymorphisms (SNPs) must have had a minimum
coverage of 6 and a maximum of 250. SNPs called at positions where the reference
base was unknown were excluded. Resulting pileup files contained a list of SNPs
and their genomic positions; comparing these to SNPs identified in the bak1-5
control, we were able to identify SNPs unique to bak1-5 mob1. These SNPs were
confirmed in the original bak1-5 mob1 mutant and in backcrossed lines by Sanger
sequencing of PCR amplicons. We then Sanger sequenced the three candidate
genes in the allelic mutant bak1-5 mob2. All primer sequences are listed below.
Molecular cloning
A Gateway-compatible genomic CPK28 fragment containing the entire intergenic
sequence (285 bp) upstream of the translational start codon and including the
endogenous stop codon was amplified from Arabidopsis Col-0 genomic DNA using
Phusion Taq polymerase (New England Biolabs) and cloned into pENTR using the
D/Topo kit (Invitrogen). Additional CPK28 pENTR clones were engineered for
translational fusions using products amplified from Col-0 cDNA containing the
endogenous start codon and with or without the endogenous stop codon. Cauliflower
Mosaic Virus (CaMV) 35S promoter-driven C-terminally tagged yellow fluorescent
protein (YFP) fusions were created after recombination by LR Clonase II (Invitrogen)
into the pXCSG-YFP vector (Matschi et al., 2013). PCR-based site-directed
mutagenesis of CPK28 was achieved though overlapping primers containing the
desired point mutation(s). Primer sequences are listed below. All clones were verified
by Sanger sequencing. BIK1 entry clones were obtained from Jian-Min Zhou,
previously described in (Zhang et al., 2010). 35S promoter-driven BIK1 constructs C-
terminally tagged with 3x hemagglutinin (HA) were generated after recombination by
LR Clonase II (Invitrogen) into the pGWB14 vector (Nakagawa et al., 2007).
35S:FLAG-RBOHD constructs were previously described (Kadota et al., 2014). In-
Fusion (Clontech) compatible fragments were amplified using Phusion Taq
polymerase (New England Biolabs) from entry vectors and cloned into pOPIN-M
(Berrow et al., 2007) to create N-terminal maltose-binding protein (MBP) fusions for
expression in E. coli strain BL21, as previously described (Kadota et al., 2014).
Supplemental plant materials
T-DNA insertion mutants cpk16-1 (Salk_052257), cpk16-2 (Salk_020716), cpk28-1
(Salk_061352) and cpk18-2 (GABI_071G03) were obtained from the European
Arabidopsis Stock Centre in Nottingham, UK (NASC) and genotyped to
homozygosity using allele-specific primers listed below.
Oxidative burst
Eight leaf discs (4 mm diameter) per genotype were collected in 96-well plates and
allowed to recover overnight in sterile water. The water was then removed and
replaced with an eliciting solution containing 17 mg/mL luminol (Sigma Aldrich), 200
µg/mL horseradish peroxidase (Sigma Aldrich), and an appropriate concentration of
the desired PAMP. Luminescence was recorded over a 40 – 60 minute time period
using a charge-coupled device camera (Photek Ltd., East Sussex UK).
Seedling growth inhibition
Seeds were surface-sterilized and sown on 1% MS agar plates. After stratification for
3 d at 4ºC, the plates were transferred to light for 4 d. Seedlings were then
transferred to single wells in 48-well plates containing liquid MS media and an
appropriate concentration of the desired PAMP. 10-12 days later, individual seedlings
were gently blotted dry and weighed using a precision scale (Sartorius).
MAPK activation
Seeds were surface-sterilized and sown on 1% MS agar plates. After stratification for
3 d at 4ºC, the plates were transferred to light for 4 d. Seedlings were then
transferred as two seedlings per well in 24-well plates containing liquid MS media.
10-12 d later, seedlings were elicited over a 30-min time course and ground in liquid
nitrogen. Proteins were extracted in buffer containing 50 mM Tris-HCl (pH 7.5), 10
mM MgCl2, 15 mM EGTA, 100 mM NaCl, 1 mM NaF, 1 mM Na2MoO4, 0.5 mM
activated Na3VO4, 30 mM glycerol 2-phosphate, 0.1% Igepal, 0.5 mM
phenylmethanesulfonyl fluoride, 1x protease inhibitor cocktail (Sigma Aldrich), 100
nM calyculin A (New England Biolabs), and 2 mM dithiothreitol. Samples were
normalized using Bradford Reagent (Biorad), boiled for 5 min in Laemmli sample
buffer and subject to SDS-PAGE and western blot analysis.
Bacterial infections
Pseudomonas syringae pv. tomato DC3000 cultures were grown overnight at 28°C in
liquid LB media containing rifampicin. The cells were collected by gentle
centrifugation and resuspended in 10 mM MgCl2 containing 0.02% Silwet L77 to
OD600 = 0.2 (108 colony forming units per mL). This bacterial suspension was then
sprayed onto 4-5 week old plants until run-off and plants were covered with vented
lids for 3 d. Four microfuge tubes containing three leaf discs (4 mm diameter) from
different plants per genotype were collected in 10 mM MgCl2 and homogenized using
a drill-adapted pestle. Serial dilutions were plated on LB agar containing rifampicin
and colonies were counted manually 2 d later.
Stomatal aperture measurements
Stomatal aperture was measured after treatment with 5 μM flg22 for 1 hr, as
previously described (Macho et al., 2012).
Transient expression in N. benthamiana
Nicotiana benthamiana plants were grown on soil as one plant per pot and used for
transient transformation at 5 weeks post germination. Agrobacterium tumefaciens-
mediated transient transformation of N. benthemiana was performed by infiltrating
leaves with OD600=0.2 of each construct along with the silencing suppressor P19.
Bacteria were prepared in buffer containing 10 mM MgCl2, 10 mM MES and 150 μM
acetosyringone for 3 hours at room temperature prior to infiltration. Samples were
collected 3 d after infiltration.
Western blots
Antibodies used in western blots were as follows: α-GFP (Santa Cruz and/or Roche);
α-mouse-HRP (Sigma Aldrich); α-HA-HRP (Roche); α-rabbit-HRP (Sigma Aldrich), α-
rabbit-TrueBlot-HRP (eBioscience), α-FLAG-HRP (Roche), and α-p42/p44-erk (Cell
Signalling Tech). Polyclonal α-BAK1 and α-FLS2 antibodies were previously
described (Gimenez-Ibanez et al., 2009; Schulze et al., 2010).
Primers used in this study
Primer Name Primer Sequence (5'-3')
Col-0/Ler mapping: flanking bak1-5
IV_16.2_T16L1_F GGGGCAATGTATTTTACAC
IV_16.2_T16L1_R ACTTCCAGCACCAGCTCAC
IV_16.2_F17I5_F CAGCTACACGTTGGCTACA
IV_16.2_F17I5_R GTTTACATCGTCTGCAAATA
Col-0/Ler mapping: flanking mob1
V_23.6_MQJ2_F ATTCTCCGTAGACCACAG V_23.6_MQJ2_R TCAACAGACTCCGCATACT V_26.3_MPA24_F AGGTCAGATCGCTGAGAA V_26.3_MPA24_R TCAAAAATGGCTAATCAAG V_26.9_K9I9_F TGGACTTGAATAGTTAGGCTGTCT
V_26.9_K9I9_R ATTACCAGTACTTAATAAAATGAT
SNP confirmation sequencing
V_2218100_F CATGTTACTTGATTCGGGAA
V_2218100_R GCTGCTTCTCTCTACATGCT
V_26457834-8077_F AGATTCGCCTCTAAAGGCTA
V_26457834-8077_R TTCTCTAACCCACGCATGTG
V_26474069_F CGTTGAGAACTTGATCAATG
V_26474069_R GAGGATCTGTTTCAGTTCGC
V_26540593_F GGTGTTCAGTTTAGTCTCAA
V_26540593_R GCCTTCTTGCCTCAGCTAAC
Sequencing genomic CPK28
CPK28seq_1F CAGTTAAAATTCTCAGAAAT
CPK28seq_1R CTCAACAGCAATAGGAAGAACCAT
CPK28seq_2F GCATCCTCTGCTCTGCTTTGAGGTC
CPK28seq_2R TGATAGCTAGTACTAACCTGGTTTGATA
CPK28seq_3F AGATTCGCCTCTAAAGGCTA
CPK28seq_3R TTCTCTAACCCACGCATGTG
CPK28seq_4F GTATGCTTTCGAGTACTAAAGTTAG
CPK28seq_4R AACATGTAGAGCTGCTGCTACAAACT
CPK28seq_5F TTGTGTACTTGTATCTTTGCT
CPK28seq_5R CTATCGAAGATTCCTGTGAC
Sequencing genomic AtC3H68
AtC3H68seq_1F ATGATGAAGAAAACGAAGAAA
AtC3H68seq_1R CAATGGTGGGCGTTTCCATTTGAT
AtC3H68seq_2F GTAAAGTTGTTTCTTAGTGAT
AtC3H68seq_2R CAGTTTCAAGTGGTTTACCTTTC
AtC3H68seq_3F GCTTCTGCAGCTTTGTCTGC
AtC3H68seq_3R AGGATCTGTTTCAGTTCGCGCTGAC
AtC3H68seq_4F GAGGATTCTTACACAGCT
AtC3H68seq_4R TTACGATCCAAACTTGAGTCTCT
Sequencing genomic MAN7
MAN7seq_1F ATGAAGCTTCTGGCTCTGTTT
MAN7seq_1R TAACGCAAAATCCAAACC
MAN7seq_2F GATCTTGTGGCCAAGTTTTGA
MAN7seq_2R GCAGCCATTTCAGTAATC
MAN7seq_3F GACTCTGTTACTTGTCTAG
MAN7seq_3R CTGGTTTCTTCATTGATTTA
MAN7seq_4F CCAGACTCAAGCGAGCAAT
MAN7seq_4R TCAGTTATTGATTTTGTGACCT
Molecular Cloning and Site-Directed Mutagenesis of CPK28
CPK28_GWY_nopro_F CACCATGGGTGTCTGTTTCTCCGCCA
CPK28_GWY_stop_R CTATCGAAGATTCCTGTGAC
CPK28_GWY_ownpro_F CACCCAGTTAAAATTCTCAGAAAT
CPK28_GWY_nostop_R TCGAAGATTCCTGTGACCTGCAG
CPK28_S245L_F CAGATCAGGGCCTGAATTAGATGTATGGAGCATTGGTGTG
CPK28_S245L_R CACACCAATGCTCCATACATCTAATTCAGGCCCTGATCTG
CPK28_A295V_F GCAACTATAAGTGACAGCGTCAAAGATTTTGTGAAAAAGT
CPK28_A295V_R ACTTTTTCACAAAATCTTTGACGCTGTCACTTATAGTTGC
CPK28_pOPIN_F AAGTTCTGTTTCAGGGCCCGATGGGTGTCTGTTTCTCCGCCA
CPK28_pOPIN_R ATGGTCTAGAAAGCTTTATCGAAGATTCCTGTGACCTGCAG
Genotyping cpk28, cpk16 and cpk18 mutant lines
GABI523B08_F GCGGCGGATTCTTTGACTAA
GABI523B08_R AGTACACAACGGCTCATTATGAA
WiscDSLox264D03_F CAGTTCTATCCCAAAAAGGCC
WiscDSLox264D03_R TCCAGCCCTTACTAGGGTTTC
Salk052257_F ATCAATCGCATCAAACTGGTC
Salk052257_R TATGCGAGGGTGGTGAATTAC
Salk020716_F AATCAACCGAAGAAGATTCGC
Salk020716_F AAGTCCACGAATCCATCTGTG
Salk061352_F TGAATGGCCAACGCTAATAAC
Salk061352_F AGCATTTGTCTCACCACAACC
GABI071G03_F TCCTTCTTTCACCCATGAATG
GABI071G03_F TACAAGCTTTAGGTGGGCATG
Quantitative real-time RT-PCR
FLS2_F ACTCTCCTCCAGGGGCTAAGGAT
FLS2_R AGCTAACAGCTCTCCAGGGATGG
EFR_F CGGATGAAGCAGTACGAGAA
EFR_R CCATTCCTGAGGAGAACTTTG
CERK1_F AAGTGGAGGTTTGGGTGGTGCCG
CERK1_R ACAGCCCCAAAACCACCTTGCCC
BAK1_F GACAACCGCAGTGCGTGGGA
BAK1_R TCGCGAGGCGAGCAAGATCA
BIK1_F TGGGCTCGACCGTACCTCACA
BIK1_R CGGGCGCGACTTGGGTTCAA
RBOHD_F CGAATGGCATCCTTTCTCAATC
RBOHD_R GTCACCGAGAGTGCGGATATG
CPK28_F GGAACTTCGAATGCACACGGGG
CPK28_R GCAGGGCTTGGTGCTCTCTGTG
FRK1_F ATCTTCGCTTGGAGCTTCTC
FRK1_R TGCAGCGCAAGGACTAGAG
At1g51890_F CCAGTTTGTTCTGTAATACTCAGG
At1g51890_R CTAGCCGACTTTGGGCTATC
NHL10_F TTCCTGTCCGTAACCCAAAC
NHL10_R CCCTCGTAGTAGGCATGAGC
ACTIN2_F TCCCTCAGCACATTCCAGCAGAT
ACTIN2_R AACGATTCCTGGACCTGCCTCATC
UBOX_F TGCGCTGCCAGATAATACACTATT
UBOX_R TGCTGCCCAACATCAGGTT
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