The Tomato Calcium Sensor Cbl10 and Its Interacting - IBVF

This article is a Plant Cell Advance Online Publication. The date of its first appearance online is the official date of publication. The article has been
edited and the authors have corrected proofs, but minor changes could be made before the final version is published. Posting this version online
reduces the time to publication by several weeks.
The Tomato Calcium Sensor Cbl10 and Its Interacting Protein
Kinase Cipk6 Define a Signaling Pathway in Plant Immunity
CW
Fernando de la Torre,a Emilio Gutiérrez-Beltrán,a Yolanda Pareja-Jaime,a Suma Chakravarthy,b
Gregory B. Martin,b,c,d and Olga del Pozoa,1
a Instituto
de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas/Universidad de Sevilla, 41092
Seville, Spain
b Boyce Thompson Institute for Plant Research, Ithaca, New York 14853
c Department of Plant Pathology and Plant-Microbe Biology, Cornell University, Ithaca, New York 14853
d Genomics and Biotechnology Section, Department of Biological Sciences, King Abdulaziz University, Jeddah 21589, Saudi Arabia
ORCID IDs: 0000-0001-9852-0431 (O.d.P.); 0000-0002-5143-3709 (F.d.l.T.); 0000-0003-4794-5370 (S.C.); 0000-0003-0044-6830 (G.B.M.).
Ca2+ signaling is an early and necessary event in plant immunity. The tomato (Solanum lycopersicum) kinase Pto triggers
localized programmed cell death (PCD) upon recognition of Pseudomonas syringae effectors AvrPto or AvrPtoB. In a virusinduced gene silencing screen in Nicotiana benthamiana, we independently identified two components of a Ca2+-signaling
system, Cbl10 (for calcineurin B-like protein) and Cipk6 (for calcineurin B-like interacting protein kinase), as their silencing
inhibited Pto/AvrPto-elicited PCD. N. benthamiana Cbl10 and Cipk6 are also required for PCD triggered by other plant
resistance genes and virus, oomycete, and nematode effectors and for host susceptibility to two P. syringae pathogens.
Tomato Cipk6 interacts with Cbl10 and its in vitro kinase activity is enhanced in the presence of Cbl10 and Ca2+, suggesting
that tomato Cbl10 and Cipk6 constitute a Ca 2+ -regulated signaling module. Overexpression of tomato Cipk6 in
N. benthamiana leaves causes accumulation of reactive oxygen species (ROS), which requires the respiratory burst homolog
RbohB. Tomato Cbl10 and Cipk6 interact with RbohB at the plasma membrane. Finally, Cbl10 and Cipk6 contribute to ROS
generated during effector-triggered immunity in the interaction of P. syringae pv tomato DC3000 and N. benthamiana. We
identify a role for the Cbl/Cipk signaling module in PCD, establishing a mechanistic link between Ca2+ and ROS signaling in
plant immunity.
INTRODUCTION
Plants respond to pathogen infection by activating a two-part
immune response, including pattern-triggered immunity (PTI)
and effector-triggered immunity (ETI; Jones and Dangl, 2006).
PTI results from the recognition of microbe- or pathogenassociated molecular patterns (PAMPs), whereas ETI occurs when
cytoplasmic resistance (R) proteins detect specific pathogen effectors. ETI is more robust than PTI and is often accompanied by
a localized programmed cell death (PCD) event known as the
hypersensitive response (HR), which is believed to limit pathogen
establishment and spread by killing both the pathogen and host
cell (Greenberg, 1997).
Both PTI and ETI share early signaling events, including
changes in protein phosphorylation status, cytosolic Ca2+ elevation, production of reactive oxygen species (ROS) in the oxidative burst, and activation mitogen-activated protein kinase
(MAPK) cascades that lead to common downstream defense
responses differing mainly in their kinetics and magnitude (Boller
1 Address
correspondence to [email protected].
The author responsible for distribution of materials integral to the findings
presented in this article in accordance with the policy described in the
Instructions for Authors (www.plantcell.org) is: Olga del Pozo
([email protected]).
C
Some figures in this article are displayed in color online but in black and
white in the print edition.
W
Online version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.113.113530
and Felix, 2009; Tsuda and Katagiri, 2010). Protein phosphorylation and Ca2+ and ROS signaling were described as sequential
events (Blume et al., 2000; Grant et al., 2000; Lecourieux et al.,
2002; Garcia-Brugger et al., 2006) that influence each other differently depending on the pathosystem. For instance, in Nicotiana
benthamiana plants treated with flagellin, Ca2+ influx was required
for the ROS burst, which negatively regulated Ca2+ influx (Segonzac
et al., 2011), whereas in the cryptogein/tobacco cells system,
ROS burst stimulated extracellular Ca2+ influx to the cytosol
(Lecourieux et al., 2002).
The main enzymatic source for the oxidative bursts in Arabidopsis
thaliana, the membrane-bound NADPH oxidases (also known as
respiratory burst homolog [RBOH] proteins), are synergistically
regulated by Ca2+ and phosphorylation (Torres, 2010). RBOHs
possess an N-terminal cytoplasmic regulatory domain, which
contains Ca2+ binding EF hands and phosphorylation sites by
Ca2+-dependent protein kinases (CDPKs or CPKs) that are necessary for RBOH function (Kobayashi et al., 2007; Ogasawara
et al., 2008; Dubiella et al., 2013). Thus, a complex spatio-temporal
Ca2+/ROS crosstalk exists in early immune signaling. To date, it is
still unclear what are the signaling components (proteins and
second messengers) that link initial PAMP or effector recognition
with Ca2+, ROS, and MAPK signaling and how these signaling
events influence each other. A current challenge is to understand
how the cytoplasmic Ca2+ increase is interpreted and turned into
a functional immune response (Segonzac et al., 2011).
The bacterial pathogen Pseudomonas syringae pv tomato (Pst)
causes bacterial speck disease in tomato (Solanum lycopersicum),
The Plant Cell Preview, www.aspb.org ã 2013 American Society of Plant Biologists. All rights reserved.
1 of 17
2 of 17
The Plant Cell
which affects tomato fruit quality and yield resulting in important
economic losses (Pedley and Martin, 2003). Pto, a tomato Ser/Thr
kinase, recognizes Pst effectors AvrPto and AvrPtoB and together
with Prf, a protein containing a nucleotide binding site and a region
of leucine rich repeats elicit an ETI resistance response against
Pst, which includes activation of a complex array of signaling
cascades and involves PCD (Mucyn et al., 2006; Oh and Martin,
2011). AvrPto and AvrPtoB are delivered into the host cell through
a type III secretion system where they suppress PTI and promote
plant disease susceptibility (Anderson et al., 2006; Xiao et al.,
2007; Shan et al., 2008; Hann et al., 2010). Several components
(;25) of the Pto-mediated signaling pathway have been identified
in tomato, tobacco (Nicotiana tabacum), and N. benthamiana using
different loss-of-function approaches (Oh and Martin, 2011). Still,
relevant aspects of early signaling after Pto/Prf activation remain to
be characterized, like the participation of Ca2+ and ROS as second
messengers.
A virus-induced gene silencing (VIGS) screen using randomly
selected cDNA fragments from N. benthamiana identified several genes that caused an alteration in Pto/AvrPto-induced PCD
upon silencing. Among the candidates identified, MAPKKKa,
a positive regulator of PCD, was further characterized and found
to regulate both ETI and disease-associated PCD (del Pozo
et al., 2004). Here, we report the identification and characterization of two components of a Ca2+ signaling module also
identified in this VIGS screen: a calcium sensor (calcineurin
B-like protein [CBL]) and its putative target, a calcineurin B-like
interacting protein kinase (CIPK). In the general context of R
protein/effector signaling, few proteins that sense and relay Ca2+
signals have been described, among them the CDPKs/CPKs,
which have been shown to play a role in defense activation in
different pathosystems (Boudsocq et al., 2010; Kobayashi et al.,
2012; Dubiella et al., 2013). Ca2+-related downstream signaling
events include salicylic acid synthesis, nitric oxide generation,
and transcriptional gene activation (Ma et al., 2008; Du et al.,
2009; Boudsocq et al., 2010; Ma et al., 2012). In Pto-mediated
ETI, APR134, a tomato Calmodulin-like protein, has been the only
Ca2+-signaling component characterized in this pathway (Chiasson
et al., 2005).
Calcium plays an important role as a universal second messenger in adaptation of cells to environmental changes. After
pathogen perception, apoplastic Ca2+ enters the cytoplasm presumably via cyclic nucleotide-gated channels and Glu receptorlike channels (Ma et al., 2009; Kwaaitaal et al., 2011; Tapken et al.,
2013). Specificity is achieved by the stimulus-specific spatial and
temporal Ca2+ influx pattern (calcium signature) and by the local
presence of Ca2+ binding proteins or sensors (CBLs, CaMs,
Calmodulin-like proteins, and CDPKs), which bind Ca2+ with high
affinity through EF hands, a helix-loop-helix structural motif (Harper
et al., 2004). Ca2+ binding changes EF-hand protein conformation,
resulting in activity changes in the sensor itself or modulating the
function of downstream target proteins (sensor relays), transforming Ca2+ changes into cellular responses.
CBLs bind Ca2+ through four EF hands and relay the signal by
interacting specifically with a C-terminal regulatory domain (NAF
or FISL) of CIPKs, Ser/Thr protein kinases belonging to Snf1RELATED KINASE3 family (Suc nonfermenting 1-related kinases, group 3; SnRK3) also known as PKS (protein kinase related
to SOS2) regulating their kinase activity (Ishitani et al., 2000;
Albrecht et al., 2001; Gong et al., 2002; Hrabak et al., 2003).
Thus, it is assumed that CBL/CIPKs modules relay Ca2+ signals
into target protein(s) phosphorylation. To date, few CIPK phosphorylation targets have been described and most of them are
membrane proteins (Quintero et al., 2002; Li et al., 2006; Ho
et al., 2009). Database analysis identified 10 CBLs and at least
26 CIPKs in Arabidopsis (www.arabidopsis.org). Because a specific CBL can interact with several CIPKs and a specific CIPK with
several CBLs, CBL/CIPK module combinatorial possibilities are
vast, thus conferring an extraordinary diversity and versatility of
Ca2+ responsiveness to this signaling system, and their roles are
only beginning to emerge. Most CBLs and CIPKs described so far
are involved in different aspects of abiotic stress signaling and
plant nutrient acquisition (Quintero et al., 2002; Kim et al., 2007;
Quan et al., 2007; Ho et al., 2009; Weinl and Kudla, 2009). Their
involvement in biotic stress signaling has been speculated about
(Kudla et al., 2010), but their role in defense responses remains
unclear.
We report the identification and characterization of two tomato and N. benthamiana participants, Cbl10 and Cipk6, in PCD
events associated with ETI mediated by Pto/AvrPto and by
different R proteins and effectors from oomycetes, fungus, and
virus, thus constituting a convergent signaling node in ETI. We
demonstrate that tomato Cbl10 and Cipk6 interact in planta and
that Cipk6 kinase activity is positively regulated in the presence
of Ca2+ and Cbl10, indicating that Cbl10/Cipk6 constitutes a
functional signaling module regulated by Ca2+. In an attempt to
explore Cipk6 downstream signaling connections, we found that
Cipk6 overexpression led to ROS generation, which was dependent on Cipk6 kinase activity and required respiratory burst
homolog NbRbohB. Importantly, both Cbl10 and Cipk6 interact
with RbohB and the complexes localize at the plasma membrane. Finally, we demonstrate that N. benthamiana Cbl10 and
Cipk6 contribute to the ROS peak generated during ETI in
N. benthamiana in response to Pst infection. Altogether, we present
Cbl10/Cipk6 as participants in ETI PCD, likely integrating Ca2+ and
ROS signaling events via phosphorylation.
RESULTS
N. benthamiana Cbl10 and Cipk6 Play a Role
in Pto-Mediated PCD
In a VIGS screen of randomly selected gene fragments from
N. benthamiana directed at identifying components of Ptomediated PCD, several genes were identified (del Pozo et al., 2004).
N. benthamiana plants silenced with Potato virus X (PVX)–based
constructs cNbME25F4 or cNbME30H4 showed compromised Ptomediated PCD in all of the events examined (for cNbME25F4, four
out of four events showed complete PCD inhibition, whereas for
cNbME30H4, two out of four events showed partial and two no
PCD).
A BLAST search with the cNbME25F4 and cNbME30H4 cDNAs
in the tomato EST databases (www.sgn.cornell.edu and www.tigr.
org) identified two tomato contigs representing partial sequences
for each clone (SGN-U229233 and SGN-E250261 for cNbME25F4;
Role of Cbl10 and Cipk6 in Plant Immunity
Figure 1. Tomato and N. benthamiana Cbl10 and Cipk6 Phylogenetic
Analysis and Protein Structure.
(A) and (B) Phylogenetic trees of CBL amino acid sequences from tomato, Sl-Cbl10; N. benthamiana, Nb-Cbl10; rice, Os-CBL10; Arabidopsis
CBLs; and Calcineurin B protein from Drosophila melanogaster used as
the outgroup (A) as well as CIPK6 amino acid sequences from tomato,
Sl-Cipk6; N. benthamiana, Nb-Cipk6; chickpea, Ca-CIPK6; Arabidopsis,
At-CIPK6; rice, Os-CIPK6; Arabidopsis CIPKs, including At-CIPK8,
At-CIPK24 (SOS2), At-CIPK3, At-CIPK9, and At-CIPK23; tomato SlCipk11 and Sl-Cipk14 (B). At-SnRK2 was used as the outgroup. Phylogenetic trees were done using the neighbor-joining method (ClustalW
program; Thompson et al., 1994) and MEGA 4 software. The scale represents amino acid substitutions, and numbers on the tree represent
bootstrap scores.
3 of 17
SGN-U566705 and SGN-E339676 for cNbME30H4) sharing 95.6
and 92.3% identity, respectively, at the protein level with the
N. benthamiana identified clones. A potato (Solanum tuberosum)
contig, SGN-U271168, spanning the complete coding sequence
for St-Cipk6 was later identified (see Supplemental Figure 1 online).
BLAST performed with both sequences in the Arabidopsis database (www.tair.org) established that cNbME25F4 was a partial
clone of the N. benthamiana ortholog for At-CBL10 (also known as
SOS3-LIKE Ca2+ BINDING PROTEIN8 [SCaBP8]), a CBL protein
(Kim et al., 2007; Quan et al., 2007), and that cNbME30H4
corresponded to a partial N. benthamiana ortholog clone for
At-CIPK6, a CIPK, a target for CBLs (Shi et al., 1999; Tripathi
et al., 2009).
Tomato cDNA clones having the complete open reading frame
were obtained, and we refer to them as Sl-Cbl10 and Sl-Cipk6
(see Supplemental Figure 1 online). Because both N. benthamiana
and tomato displayed a high degree identity at the nucleotide
level (91.2 and 92.4%, with several stretches of more than 21 to
24 nucleotides with 100% identity), either Sl or Nb constructs
were used for effective silencing in both species, and in silencing
experiments we refer to them generally as Cbl10 and Cipk6.
Protein sequences were derived and aligned for all 10 CBLs
present in Arabidopsis, along with Sl-Cbl10, Nb-Cbl10, and a putative rice (Oryza sativa) Os-CBL10 protein (GenBank DQ201203),
and the alignment used to build a phylogenetic tree (Figure 1A; see
Supplemental Data Set 1 and Supplemental Figure 2 online). As
expected, cNbME25F4 appears to be the ortholog of At-CBL10
(Figure 1A). Sl-Cbl10 has four EF hands and an extended
N-terminal hydrophobic domain characteristic of At-CBL10 necessary for membrane association, which is also present in the
putative rice ortholog Os-CBL10 but absent in other CBLs (Quan
et al., 2007). Ser-241 residue in At-CBL10, phosphorylated by
SOS2, is also conserved in Sl-Cbl10 (Ser-237) (Figure 1C; see
Supplemental Figure 2 online) (Lin et al., 2009).
The closest sequence for cNbME30H4 in the Arabidopsis database was At-CIPK6. Protein sequences of putative At-CIPK6
orthologs from tomato, chickpea (Cicer arietinum; GenBank accession number EU492906), and rice (Q6Z9F4) were aligned, together with a representation of other CIPKs with assigned functions
(see Supplemental Data Set 2), and a phylogenetic tree was developed (Figure 1B; see Supplemental Figure 3 online). Indeed,
cNbME30H4 and its putative tomato ortholog were located in the
same clade as At-CIPK6, Ca-CIPK6, and Os-CIPK6 and therefore
were designated as Nb-Cipk6 and Sl-Cipk6, respectively.
Sl-Cipk6 has a typical SnRK3 structure, displaying an N-terminal
kinase catalytic domain and a C-terminal regulatory domain
separated by a junction region (Hrabak et al., 2003). The regulatory
domain contains the NAF/FISL region, which binds CBLs and
(C) Schematic structure of tomato Cbl10 protein representing conserved
Ca2+ binding EF-hands, N-terminal hydrophobic domain, and putative
phosphorylatable Ser-237 residue.
(D) Schematic structure of tomato Cipk6 protein representing N-terminal
catalytic, junction, and C-terminal regulatory domains, protein phosphatase
interaction, NAF/FISL, and activation loop domains. Residues mutagenized
for Cipk6 characterization Lys-43 to Met (K43M) and Thr-172 to Asp
(T172D) are marked.
4 of 17
The Plant Cell
physically overlaps with a protein phosphatase interaction binding
domain (Liu et al., 2000; Albrecht et al., 2001; Ohta et al., 2003). In
the catalytic domain, the conserved ATP binding Lys (Lys-43), and
within the activation loop, a putative trans-phosphorylatable Thr
residue (Thr-172) was observed (Figure 1D).
To confirm and further characterize the phenotype associated
with the loss of function of Cbl10 and Cipk6 in plant immunity, we
cloned cDNA fragments from tomato (SlME25F4 and SlME30H4,
respectively) corresponding to those originally identified as
N. benthamiana VIGS clones into a tobacco rattle virus vector
(TRV; Liu et al., 2002), which causes a more uniform and persistent silencing phenotype than PVX. In order to substantiate the
specificity of their silencing phenotypes, we generated additional
nonoverlapping silencing constructs in TRV for Cbl10 (Sl-Cbl10-59)
and Cipk6 (Sl-Cipk6-5and Sl-Cipk6-39) (see Supplemental Figure 1
online) and used them for VIGS. PCD was monitored visually and
classified according to its extent (Figure 2A). Similar loss of PCD
phenotypes were obtained using either the originally identified
clones or each of the alternative silencing constructs for either
N. benthamiana Cbl10 or Cipk6 gene, indicating that the phenotype is due to specific silencing of Cipk6 or Cbl10 (Figure 2B). Full
Pto/AvrPto-mediated PCD developed in TRV control plants in
80% of the cases, whereas it ranged between 15 and 25% in
TRV-Sl-Cipk6-59, TRV-Sl-Cipk6-39, or TRV-Nb-Cipk6–silenced
N. benthamiana leaves and between 15 and 25% in TRV-SlCbl10-59 or TRV-Nb-Cbl10–silenced N. benthamiana leaves. The
degree of silencing was assessed by RT-PCR, which showed that
N. benthamiana Cbl10 and Cipk6 transcript abundances were reduced ;90% compared with TRV control plants (see Supplemental
Table 1 online).
Silencing specificity was determined by checking possible
changes in transcript abundances of the closest genes found in the
databases (see Supplemental Table 1 online). The tomato genome
sequence was then released and BLAST analysis identified a sequence, SGN-U583600, which shared three stretches of 23, 26,
and 25 nucleotides 100% identical to Cipk6 located toward the 3’
end of the putative gene sequence. Two of them overlapped with
the TRV-Sl-Cipk6-39 silencing construct, whereas none overlapped
with TRV-Sl-Cipk6-59 or TRV-Nb-Cipk6 silencing constructs. RTPCR performed with specific oligonucleotides for SGN-U583600
and cDNA from infected or noninfected tomato and N. benthamiana
leaves did not amplify any band, suggesting that this putative gene
is not transcribed in leaves (see Supplemental Table 1 online). From
these experiments, we conclude that Cbl10 and Cipk6 participate in
Pto/AvrPto-triggered PCD.
N. benthamiana Cbl10 and Cipk6 Contribute to PCD
Triggered by Different Elicitors
Next, we asked whether Cbl10 and Cipk6 expression was also
required in other R/Avr- or general elicitor-triggered PCD. For that
purpose, we used Agrobacterium tumefaciens–mediated transient
transformation of leaves (agroinfiltration) to express NPP-1 (for
necrosis-inducing Phytophthora protein1; Fellbrich et al., 2002),
potato Gpa2/RBP-1 from potato cyst nematode (Sacco et al.,
2009), and potato Rx2/coat protein (CP) from PVX (Bendahmane
et al., 2000) in N. benthamiana leaves silenced for Cbl10, Cipk6,
and TRV control-infected leaves. PCD triggered by NPP-1, Gpa2/
Figure 2. Cbl10 or Cipk6 Silencing in N. benthamiana Compromises
PCD Mediated by Different R Gene/Effector Gene Interactions.
(A) PCD was monitored visually and classified according to its extent: full
PCD, >80%; partial PCD, 20 to 80%; no PCD, <10%.
(B) PCD mediated by Pto is greatly reduced in Cipk6- or Cbl10-silenced
N. benthamiana leaves using the originally identified fragments in the
screen or additional silencing constructions for Cipk6 (TRV-Nb-ME30H4,
TRV-Sl-Cipk6-59, and TRV-Sl-Cipk6-39) and for Cbl10 (TRV-Nb-ME25F4
and TRV-Sl-Cbl10-59) compared with TRV control leaves agroinfiltrated
with Pto or AvrPto. For each silencing construction, we used five plants
with two leaves infiltrated per plant. The experiment was repeated six
times with similar results.
(C) Suppression of PCD elicited by other R genes/elicitors is also observed in Cbl10- or Cipk6-silenced N. benthamiana leaves compared
with TRV control leaves after agroinfiltration of NPP1, RBP-1/Gpa2, and
Rx2/CP. Four plants per line were tested, and PCD was observed
5 d later for NPP1, 7 d later for RBP-1/Gpa2, and 6 d later for Rx2/CP.
TRV-Nb-ME25F4 and TRV-Nb-ME30H4 were used for silencing. Asterisks indicate significant reduction in PCD (Student’s t test; P < 0.05).
[See online article for color version of this figure.]
Role of Cbl10 and Cipk6 in Plant Immunity
5 of 17
Figure 3. Cipk6 and Cbl10 Are Required for Both Immunity and Disease Associated PCD in Tomato after Pst DC3000 Infection.
(A) RG-PtoR–resistant tomato leaves plants were syringe inoculated with Pst DC3000 (0.5 3 107 cfu/mL) and stained for PCD after 18 h with trypan
blue.
(B) and (C) PCD shown in (A) was quantified by measurement of conductivity (B), and growth of Pst DC3000 was assessed 18 h after inoculation (C).
Data presented are means of three to five plants/line, and error bars represent SD. The experiment was repeated three times with similar results.
(D) and (E) TRV control-infected, Cbl10- and/or Cipk6-silenced susceptible RG-Prf3 tomato plants were infected with Pst DC3000 (1 3 104 cfu/mL) (D).
Five days later, leaves were destained to visualize bacterial specks. TRV-Cbl10 or TRV-Cipk6 leaves showed a clear reduction in the number of specks
compared with TRV control leaves as well as reduced growth of Pst DC3000 in TRV-Cipk6 and/or TRV-Cbl10 tomato leaves (RG-Prf3) compared with
TRV control leaves 5 d after Pst DC3000 inoculation (2.5 3 104 cfu/mL) (E). Experiments were performed with an average of five to seven silenced plants
for each silencing construct and repeated at least six times, leading to similar results in five out of seven experiments. Asterisks mark significant
differences from TRV control (Student’s t test; P # 0.025).
[See online article for color version of this figure.]
RBP-1, and Rx2/CP was substantially suppressed in TRV-Cipk6–
and TRV-Cbl10–silenced plants compared with TRV control plants
(Figure 2C). These results demonstrate that Cbl10 and Cipk6 are
required by different R genes/effectors in N. benthamiana, thereby
constituting a convergent signaling node in plant immunity shared
by different pathogen-responsive pathways.
Tomato Cbl10 and Cipk6 Transcript Abundances Increase
upon Pst Infection
Cipk6 and Cbl10 were both identified through VIGS in a leaf PCD
assay in N. benthamiana. Therefore, we wished to confirm their
expression in tomato leaves and other tissues. Tomato Cbl10
and Cipk6 were moderately expressed in all the tissues analyzed
with the exception of stems, where both transcripts were in low
abundance, especially Cipk6 (see Supplemental Figure 4A online). Cbl10 and Cipk6 transcripts were also detectable in roots.
Cbl10 and Cipk6 expression in roots and stems contrasts with
their low abundance reported in roots for Arabidopsis SCABP8/
CBL10 and chickpea CIPK6 and their high level of expression
detected in stems for both Arabidopsis orthologs CIPK6 and
CBL10 (Quan et al., 2007; Kim et al., 2007; Tripathi et al., 2009).
These differences might underlie divergent functions for CBLs
and CIPKs orthologs in different plant species.
We next examined whether transcript abundance of tomato
Cbl10 and Cipk6 changed during ETI and susceptible responses
of tomato to Pst DC3000 (which expresses AvrPto and AvrPtoB)
(see Supplemental Figures 4B and 4C online). Both responses
in tomato are accompanied by PCD, associated with the HR
or with bacterial speck lesion formation, respectively, and are
6 of 17
The Plant Cell
Figure 4. Tomato Cipk6 Is an Active Kinase and Is Regulated by Cbl10 and Ca2+.
(A) Tomato Cipk6 schematic diagram showing kinase and regulatory domains and mutant derivatives. NAF, CBL interaction domain; AD, activation
domain. Mutagenized residues and deleted domains are marked. WT, full-length Cipk6. K43M and T172D, single amino acid substitution of Lys-43 to
Met and Thr-172 to Asp, respectively. DNAF (NAF/FISL amino acids 302 to 321) and DCterm (amino acids 302 to 432) deletions. (T172D)/DNAF and
(T172D)/DCterm combine both mutations within the same protein.
(B) For the phosphorylation assays, Cipk6 and mutant versions were incubated with 1 µg of MBP in the presence [g-32P]ATP and kinase buffer,
electrophoresed on SDS-polyacrylamide gel, and autoradiographed. a.u., arbitrary units.
(C) For the autophosphorylation assays, proteins were incubated with [g-32P]ATP, and samples were processed as in (B).
(D) Equimolecular levels of protein corresponding to Sl-Cipk6 and mutant versions were confirmed by immunoblot using anti-cMyc antibodies.
(E) Cipk6 kinase activity is regulated by Cbl10 in a Ca2+-dependent manner. MBP substrate was incubated in the presence of [g-32P]ATP and Ca2+ or
EGTA in kinase buffer with Cipk6 and with or without GST-Cbl10.
Role of Cbl10 and Cipk6 in Plant Immunity
7 of 17
separated in the timing of their appearance (;16 to 18 h for HR
and 3 to 4 d for specks). For ETI, resistant tomato plants expressing Pto (RG-PtoR) were infiltrated with a high titer of Pst
DC3000 (1 3 108 colony-forming units [cfu]/mL), which generated visible PCD 16 h later, and Cipk6 and Cbl10 mRNA accumulation was determined 0, 4, 8, and 16 h after. Cipk6 transcript
abundance sharply increased 4 h after Pst DC3000 inoculation
and decreased 8 h after. By contrast, Cbl10 transcript abundance was only slightly increased 8 h after inoculation and decreased thereafter.
For the disease interaction, susceptible tomato plants (RG-PtoS)
were inoculated with Pst DC3000 (5 3 105 cfu/mL), and tissue was
collected 0, 1, 2, 3, and 4 d later. Specks were first noticeable 3 d
after infection (DAI). Both Cipk6 and Cbl10 transcript accumulation
increased during the infection process, albeit showing different
patterns. Cipk6 transcript increase could be detected 1 DAI and
continued steadily until it reached its peak accumulation at 3 DAI.
By contrast, Cbl10 transcript accumulation doubled at 3 DAI and
increased at 4 DAI (see Supplemental Figure 4C online).
Tomato Cipk6 and Cbl10 Are Required for Pto/AvrPto ETI
Triggered by Pst DC3000
Next, we examined Cipk6 and Cbl10 function in tomato host responses upon Pst DC3000 infection. To test their role in ETI, we used
VIGS to silence Cbl10 and Cipk6 in resistant tomato plants (RGPtoR). Tomato leaves were syringe infiltrated with a PCD-inducing
titer of Pst DC3000 (0.5 3 107 cfu/mL), along with TRV-Prf and TRV
control-inoculated plants, and PCD was visualized 18 h after infiltration. Infiltrated areas were excised and stained with trypan blue.
PCD did not develop in TRV-Cbl10, TRV-Cipk6 (Figure 3A), or TRVPrf plants (data not shown; see del Pozo et al., 2004), as trypan blue
staining was greatly reduced in comparison to TRV control plants,
which showed confluent PCD with uniform trypan blue staining
covering the entire infiltrated area. These results were supported
by a conductivity assay, which measures ion leakage in leaves
undergoing PCD (Figure 3B). No significant differences were found
in bacterial populations 18 h after infiltration of the RG-PtoR leaves
when comparing TRV control plants with TRV-Cbl10 and TRV-Cipk6
plants (Figure 3C).
N. benthamiana and Tomato Cipk6 and Cbl10 Also
Participate in Disease Lesion Formation Caused by
Two P. syringae Strains
Figure 5. Tomato Cipk6 Expression Results in ROS Generation: Cbl10
and Cipk6 Contribute to ROS Production in Response to Pst DC3000.
(A) Luminol-enhanced chemiluminescent assay in leaf discs agroexpressing Sl-Cipk6, Sl-Cipk6 mutant versions, or control (empty vector)
in pER8 XVE vector after 17b-estradiol treatment. ROS was quantified as
relative light units (RLU) 2 to 3 h after. Data presented are means of 8/10
measures of independent discs. The experiment was repeated five times
with similar results. Asterisks indicate a significant increase compared
with control (Student’s t test; P < 0.05). WT, the wild type.
(B) ROS mediated by Sl-Cipk6(T172D) or Sl-Cipk6(T172D)DCterm was
drastically reduced in TRV-Nb-RbohB plants. Experiment was repeated
twice with similar results, with eight biological replicas and three
P. syringae is a hemibiotrophic pathogen that suppresses PCD
early during infection, but later in the infection process, induces
cell death leading to disease lesions that may contribute to
technical replicates. Single asterisks, significant increase compared with
control (empty vector-agroinfiltrated). Double asterisks, significant decrease compared with TRV control plants (Student’s t test; P < 0.01).
(C) ROS is greatly diminished in TRV-Cbl10 and TRV-Cipk6 and almost
undetectable in TRV-Nb-RbohB Nb plants compared with TRV control
plants after Pst DC3000 (2.5 3 10 8 cfu/mL) inoculation. ROS were
measured for 7 h as in (A). Pst DC3000 DhrcC was employed as a control
for the ETI (2.5 3 108 cfu/mL). The experiment was repeated three times
with similar results.
8 of 17
The Plant Cell
pathogen dissemination. Lesion-associated cell death is considered a PCD that shares molecular mechanisms with ETI PCD,
differing mainly in the timing and number of cells undergoing this
response (Tao et al., 2003; del Pozo et al., 2004). We hypothesized that N. benthamiana Cipk6 and Cbl10 might also play
a role in disease-associated PCD as was observed previously
for MAPKKKa (del Pozo et al., 2004). We analyzed the disease
symptoms caused by 5 3 105 cfu/mL P. syringae pv tabaci infection in TRV-Cipk6, TRV-Cbl10, TRV-Cipk6/TRV-Cbl10–silenced,
and TRV control N. benthamiana leaves (see Supplemental Figure 5
online). Four days after P. syringae pv tabaci infection, fully collapsed
tissue due to successful disease progression was observed in TRV
control plants (see Supplemental Figure 5A online). However, no
disease-associated PCD was apparent in 50 to 60% of the infiltrated areas in single or cosilenced plants for Cipk6 and/or Cbl10
(see Supplemental Figures 5A and 5B online). Bacterial growth
was reduced in TRV-Cbl10, TRV-Cipk6, and to the same extent in
TRV-Cipk6/TRV-Cbl10 plants (see Supplemental Figure 5C online). These observations were substantiated by a conductivity
assay (see Supplemental Figure 5D online).
To characterize Cipk6/Cbl10 role in disease-associated PCD
in tomato caused by Pst DC3000, we silenced Cipk6 and Cbl10
individually or together in susceptible tomato plants (RG-prf3)
and inoculated the resulting plants with 2.5 3 104 cfu/mL of Pst
DC3000. Five days later, mild disease symptoms, consisting of
few and weak specks, could be observed in TRV-Cipk6 and
TRV-Cbl10 plants, whereas in TRV control plants, numerous
well-defined speck lesions were present (Figure 3D). Bacterial
growth was reduced in TRV-Cipk6, TRV-Cbl10, and TRV-Cipk6/
TRV-Cbl10 RG-prf3 plants compared with the TRV control (Figure
3E). TRV-Cipk6/TRV-Cbl10 plants showed similar reduction in
bacterial specks and growth as single silenced TRV-Cipk6 or TRVCbl10, suggesting that Cipk6 and Cbl10 genetically function in the
same pathway. These results were observed in five independent
experiments. Thus, both N. benthamiana and tomato Cbl10 and
Cipk6 are involved in PCD signaling that occurs during both ETI
and disease and contributes to disease susceptibility to two
P. syringae pathovars.
Figure 6. Tomato Cbl10 and Cipk6 Interact in Yeast and in Planta and
with RbohB at the Plasma Membrane.
(A) EGY48 yeast strain cultures containing either Sl-Cipk6, Sl-Cipk6ΔNAF,
Sl-Cipk11, or Sl-Cipk14 in the bait pEG202 vector and Sl-Cbl10 in the prey
vector pJG4-5 were spotted at OD600 = 0.1 and 0.01 on selective medium.
Growth indicates Cipk6/Cbl10 interaction.
(B) Plant protein extracts from leaves agroexpressing Sl-Cipk6-Myc and
Sl-Cbl10-HA proteins were incubated with IgG agarose beads, and
proteins eluted were immunoblotted using anti-cMyc and anti-HA SlCipk6, which did not co-IP with GFP (right panel, lanes derive from same
blots but from no correlative lanes).
(C) Sl-Cipk6-YN and Sl-Cbl10-YC interact with Sl-RbohB in vivo in N.
benthamiana by BiFC. YFP fluorescence was monitored 24 h after agroinfiltration using confocal laser scanning microscopy. Plasma membrane was stained with FM4-64.
Tomato Cipk6 Kinase Activity Is Enhanced by SlCbl10
and Ca2+
Despite the central role attributed to Ca2+ signaling in plant
immunity, little is known about how plants decode Ca2+ signals
into cellular responses leading to plant immunity. Cbl10/Cipk6
participation in immunity-associated PCD prompted us to test if
tomato Cipk6 could participate as a Ca2+ relay through phosphorylation events. We set up to test and characterize Cipk6
kinase activity (Figure 4). As a negative control, a kinase inactive
version was generated, where the conserved catalytic Lys within
the kinase domain was substituted by Met (Sl-Cipk6[K43M]) (Liu
et al., 2000). We also generated different Cipk6 versions mimicking previously described mutations in other CIPKs that conferred enhanced kinase activity (Gong et al., 2002). Initial yeast
two-hybrid analysis followed by structural information has demonstrated that the N-terminal kinase domain and the C-terminal
regulatory domain of SOS2 (Arabidopsis CIPK24) interact intramolecularly through the NAF/FISL domain, inhibiting kinase
Role of Cbl10 and Cipk6 in Plant Immunity
9 of 17
activity possibly by blocking substrate access to the catalytic site
(Albrecht et al., 2001; Sánchez-Barrena et al., 2007; Akaboshi
et al., 2008). We generated a deletion mutant version that
lacked the regulatory domain (Sl-Cipk6DCterm) (Figure 4A). A
Sl-Cipk6DNAF version, lacking the NAF/FISL binding site was
previously generated (see above). It has been also described
that substitution of a conserved Thr within the activation loop
to Asp, mimics phosphorylation (and possibly activation) by
unknown upstream kinases (Guo et al., 2001; Gong et al., 2002).
We generated, through site-directed mutagenesis, a version with
this substitution, Sl-Cipk6(T172D), and obtained double mutants
by combining this point mutation with deletion mutants described
above (Figure 4A).
To determine whether tomato Cipk6 is an active kinase, we
cloned Cipk6 and mutant versions into the pYL436 tandem affinity purification (TAP) vector (Rubio et al., 2005), agroinfiltrated
them into N. benthamiana leaves, and purified the tagged proteins. Cipk6 and mutant versions were observed at the expected
molecular masses, and an in vitro kinase assay was performed
with the recombinant proteins to determine their kinase activity
in the presence of [g-32P]ATP and myelin basic protein (MBP) as
a substrate as well as their autophosphorylation activity (Figures
4B to 4D). Wild type Sl-Cipk6 was observed to have a weak
transphosphorylation and autophosphorylation activity, whereas
Sl-Cipk6(K43M) (kinase inactive) had no activity (Figures 4B and
4C). Altogether, the Cipk6 variants (Sl-Cipk6[T172D], Sl-Cipk6DNAF,
and Sl-Cipk6DCterm) showed noticeably increased kinase and autophosphorylation activity, with Sl-Cipk6(T172D) displaying the
highest kinase activity (approximately threefold above the wild type).
Interestingly, Cipk6 variants resulting from the addition of the
single substitution, T172D, to the deletion mutants (Sl-Cipk6
[T172D]/DNAF and Sl-Cipk6[T172D]/DCterm) increased both kinase activity and most noticeably autophosphorylation activity
with Sl-Cipk6(T172D)/DCterm displaying highest autophosphorylation activity compared with single mutant versions. Kinase and
autophosphorylation assays were performed in parallel with
identical sample loadings, along with an immunoblot to assess
that similar amount of protein was used (Figure 4D). Thus, it
appears that the highest kinase activity is achieved through
transactivation and highest autophosphorylation through release of the catalytic domain of the inhibitory effect of the
regulatory domain.
To test whether tomato Cipk6 kinase activity was modulated by
Cbl10 and Ca2+, in vitro transphosphorylation assays were performed using MBP as substrate. Cipk6 kinase activity increased
significantly ($163) in the presence of both Cbl10 and Ca2+,
therefore demonstrating that Cipk6 kinase activity is regulated by
Cbl10 in a Ca2+-dependent manner (Figure 4E). No Cbl10 phosphorylation by Cipk6 could be observed in the conditions assayed
(Figure 4E). Arabidopsis CBL10 was found previously to be phosphorylated by SOS2 (CIPK24) (Lin et al., 2009).
downstream signaling processes, including ROS production and
PCD (Blume et al., 2000; Grant et al., 2000; Segonzac et al., 2011).
Therefore, given the participation of the Cbl10/Cipk6 module in
ETI-associated PCD, we set out to investigate whether tomato
Cipk6 kinase activity could also affect ROS production.
Tomato Cipk6 and its mutant versions were agroinfiltrated
using an estradiol-inducible system in N. benthamiana leaves,
and production of ROS was quantified in the presence of luminol
in a luminometer (Figure 5A). ROS were detected in samples
expressing Sl-Cipk6 and increased more than two- and fourfold
in samples expressing enhanced kinase activity versions Sl-Cipk6
(T172D) and Sl-Cipk6(T172D)/DCterm, respectively, whereas ROS
was much reduced after expression of the kinase-inactive version
Sl-Cipk6(K43M), indicating that Sl-Cipk6 kinase activity is necessary for ROS generation. The highest ROS accumulation was
detected in Sl-Cipk6(T172D)/DCterm-expressing samples, thus
correlating with highest kinase and autophosphorylation activity
(Figures 4B and 4C) likely as a result of combining both derepression and mimicking-transactivation of kinase activity. Cipk6
and all the mutant versions proteins were expressed similarly
(Figure 5A).
Membrane-bound RBOHs play a key role as ROS-producing
enzymes in plants. N. benthamiana RbohB silenced plants were
susceptible to an avirulent pathogen and showed reduced HR
(Yoshioka et al., 2003). RbohB was also required for ROS accumulation during PTI in response to PAMPs (Segonzac et al., 2011).
In order to determine if RbohB was required for ROS generation
mediated by Cipk6, we expressed Sl-Cipk6(T172D) and Sl-Cipk6
(T172D)/DCterm and measured ROS as described in Figure 5A
in N. benthamiana plants silenced for RbohB and nonsilenced
control plants (Figure 5B). Silencing RbohB completely abolished
Sl-Cipk6(T172D)– and Sl-Cipk6(T172D)/DCterm–mediated ROS
generation. ROS were detected in samples expressing Sl-Cipk6
(T172D) and Sl-Cipk6(T172D)/DCterm in TRV control plants. This
result clearly indicates that N. benthamiana RbohB is required for
tomato Cipk6-mediated ROS generation.
We also determined if tomato Cbl10 and Cipk6 contributed to
ROS generation during the response of N. benthamiana to Pst
DC3000 infection. Using the in vivo luminol-based assay, TRV
control plants infected with Pst DC3000 showed two ROS peaks
approximately at 2.5 and 6 h after infection (Figure 5C). The early
ROS burst is associated with PTI, whereas the later one corresponds to ETI, since TRV control plants infected with Pst DC3000
hrcC (deficient in the type III secretion system and therefore
eliciting PTI but not ETI) showed the first but not the second peak.
Both ROS peaks were substantially reduced in TRV-Cipk6 or
TRV-Cbl10 but not completely abrogated, as observed in TRVRbohB N. benthamiana plants (Figure 5C). Collectively, these
data demonstrate that Cbl10 and Cipk6 contribute to ROS
generation during the immune response to Pst DC3000 in
N. benthamiana.
Tomato Cipk6 Kinase Activity Is Associated with
ROS Production
Cipk6 and Cbl10 Interact in Vivo and Constitute
a Signaling Module
An increase in cytoplasmic Ca2+ is among the earliest responses
upon pathogen recognition and blocking this influx with inhibitors demonstrated that Ca2+-derived signals are necessary for
The fact that plants cosilenced for Cipk6/Cbl10 did not show
an additive loss of PCD or reduced bacterial growth compared
with plants silenced individually for these genes suggested that
10 of 17
The Plant Cell
their proteins might act together as a PCD-inducing signaling
module. To test if these two proteins interact physically, we first
performed a yeast two-hybrid assay. Yeast two-hybrid analysis
and in vitro binding assays have demonstrated that CBLs often
bind a number of CIPKs with different affinity (Albrecht et al.,
2001; Batistic and Kudla, 2009). We found that tomato Cbl10
interacted with Cipk6, whereas, as expected, Sl-Cipk6DNAF, an
altered protein lacking the NAF/FISL domain necessary for CBL
binding, did not interact (Figure 6A). Cbl10 did not interact with
Cipk11 or Cipk14, tomato CIPK proteins with high amino acid
similarity to Cipk6, therefore suggesting that the Cipk6/Cbl10 interaction has a high level of specificity (Figure 6A). Cbl10, Cipk6,
Cipk6DNAF, Cipk11, and Cipk14 were expressed in yeast (see
Supplemental Figure 6 online).
To further examine the Cipk6 and Cbl10 interaction and to test
if the presence of Ca2+ influenced their binding, we attempted
pull-down experiments with tomato Cipk6 and Cbl10 proteins
isolated from Escherichia coli. We successfully expressed Cbl10
fused to glutathione S-transferase (GST), Sl-Cbl10-GST, and
because Cipk6 protein production was limiting, we produced
Sl-Cipk6-MetS35–labeled protein in vitro. Pull-down experiments
demonstrated direct interaction between Cipk6 and Cbl10, which
was not dependent on the presence of Ca2+ in our in vitro assay
(see Supplemental Figure 7 online). The interaction of Arabidopsis CIPK24 (SOS2) and CBL10 (SCaBP8) is also not affected
by Ca2+ (Quan et al., 2007).
In order to examine the likelihood that tomato Cipk6 and
Cbl10 constitute a signaling module in vivo and to further test
the specificity of their interaction, we performed coimmunoprecipitation (co-IP) experiments. We transiently expressed by
agroinfiltration wild-type Sl-Cipk6 or Sl-Cipk6DNAF fused with
a c-Myc tag and Sl-Cbl10 fused with a HA tag in N. benthamiana
leaves (Figure 6B). Sl-Cipk6DNAF accumulated to lower levels;
therefore, we adjusted the input of Sl-Cipk6DNAF to be the
same as Sl-Cipk6 to minimize the effect on the co-IP experiments with Cbl10. Tomato Cbl10 coimmunoprecipitated with
Cipk6, whereas no co-IP was detected with Sl-Cipk6DNAF, as
expected. Also, Cipk6 did not co-IP with another negative control,
green fluorescent protein (GFP) (Figure 6B).
In order to relay Ca2+ signals into target phosphorylation, CBL/
CIPK modules require specific and coordinated subcellular localization with their targets. To examine the likelihood that Cbl10
and Cipk6 could regulate RbohB directly, in vivo interaction between tomato Cbl10 or Cipk6 with RbohB was tested by bimolecular fluorescence complementation (BiFC) experiments in
N. benthamiana leaves (Walter et al., 2004). Fluorescence due to
the reconstitution of the yellow fluorescent protein (YFP) was
observed by confocal microscopy and localized at the periphery
of the cell when the combinations of Sl-Cbl10-CFPC and SlRbohB-YFPN or Sl-Cipk6-CFPC and Sl-RbohB-YFPN were agroinfiltrated into N. benthamiana leaves (Figure 6C). No fluorescence
was detected when SlRbohB-YFPN was agroinfiltrated with empty
vector pXCGW (Figure 6C). Reconstituted YFP signal overlapped
with the plasma membrane stained with the plasma membrane
marker FM4-64 (Figure 6C). These results indicate that tomato
Cbl10 and Cipk6 interact with RbohB at the plasma membrane in
N. benthamiana cells, thus raising the possibility that RbohB is
a phosphorylation target of Cbl10/Cipk6.
DISCUSSION
Several CBL/CIPK complexes have been previously identified as
regulators of different stress responses, including salinity, drought,
cold, and in response to abscisic acid. In this study, we report the
identification of tomato Cbl10 and Cipk6, from a large VIGS fastforward genetic screen (del Pozo et al., 2004), and their functional
characterization as positive regulators of PCD events in ETI mediated by different R genes/effectors from bacteria, oomycete,
nematode, and virus and in susceptible responses to two pathogenic strains of P. syringae. We propose that tomato Cbl10 and
Cipk6 act in a convergent regulatory node in ETI- and diseaseassociated PCD signaling in plant immunity.
Biochemical characterization indicated that tomato Cipk6 is an
active kinase, whose activity is positively regulated by Cbl10 and
Ca2+. Because Cbl10 and Cipk6 interact in planta, we conclude
that Cbl10/Cipk6 constitute a signaling module regulated by Ca2+.
In an attempt to investigate Cipk6 downstream signaling connections, we found that Cipk6 overexpression results in ROS
generation in planta, which required Cipk6 kinase activity and
NADPH oxidase RbohB. Both Cbl10 and Cipk6 interacted in vivo
with RbohB at the plasma membrane, thus suggesting that
RbohB is a target of Cbl10/Cipk6. Importantly, Cbl10 or Cipk6
loss-of-function analysis in N. benthamiana revealed their contribution to ROS bursts occurring during both PTI and ETI in response to Pst DC3000 infection, thus validating their physiological
role in the plant immune response.
Our studies reveal a functional role for a CBL/CIPK module in
ETI PCD and resistance and provide molecular insights into the
mechanistic conversion of Ca2+ signaling into ROS signaling in
plant immunity through still unknown phosphorylation events in
plants. Based on these results, we propose a model where Ca2+
influx after pathogen perception might be detected by Cbl10,
which interacts with Cipk6, thereby derepressing its kinase activity, which in turn phosphorylates RbohB, resulting in ROS
production contributing to PCD and resistance responses.
Ca2+ seems not to be necessary for the Cbl10/Cipk6 interaction, at least in our in vitro assay. However, Cipk6 kinase
activity increased notably in the presence of Cbl10 and Ca2+,
demonstrating the relevance of Ca2+ regulation in the system.
Similarly, SOS2/SOS3 and Arabidopsis CBL2/CIPK14 displayed
Ca2+-independent interaction and Ca2+-dependent kinase activity (Halfter et al., 2000; Akaboshi et al., 2008). Among the
single mutants, Sl-Cipk6(T172D) displayed the greatest kinase
and autophosphorylation activity. Thr-172 is a phosphorylatable
residue located in the activation loop and is conserved at the
same position in all SnRK3s. Substitution of this residue to Asp,
to mimic Thr phosphorylation, gives CIPKs enhanced kinase
activity (Guo et al., 2001; Gong et al., 2002). Because this activated state was observed in several cases in which Ser/Thr was
mutated to an Asp, it has been proposed that CIPKs could also
be activated by a phosphorylation cascade (Harper et al., 2004).
Changes in the phosphorylation state of proteins are among
the earliest events in plant immunity (Dietrich et al., 1990; Felix
et al., 1991). In the future, identification of a kinase(s) that could
regulate Cipk6 activity could fill in knowledge gaps in early immune signaling in plants between early phosphorylation events
and Ca2+-mediated responses. One possible candidate is Pto.
Role of Cbl10 and Cipk6 in Plant Immunity
However, the participation of N. benthamiana Cipk6 in diverse R
gene/effector-elicited PCD events would argue for the involvement
of a convergent kinase playing this role.
In plants, cell death induced by pathogen infection is associated with both disease resistance and susceptibility. In the
tomato/Pst DC3000 interaction, both ETI and susceptibility are
associated with PCD events, which seem to share some common
regulators and physiological changes but their development occurs at different timing and involves different number of cells (del
Pozo et al., 2004; López-Solanilla et al., 2004; Tao et al., 2003).
In contrast with HR PCD, the molecular basis of diseaseassociated PCD that takes place during compatible interactions is largely unknown. In this work, we observed that
tomato and N. benthamiana Cbl10 and Cipk6 positively regulate
both immunity-associated PCD and disease-associated PCD and
contribute to disease susceptibility in a fashion similar as described previously for MAPKKKa (del Pozo et al., 2004). This
raises the possibility that Cbl10/Cipk6 and MAPKKKa act in the
same pathway or that crosstalk between them might exist. We did
not detect any interaction between MAPKKKa (full-length or kinase domain) and tomato Cipk6 (wild-type or constitutive active
derivatives) by yeast two-hybrid assays (O. del Pozo, unpublished
data). MAPKKKa overexpression results in PCD, whereas overexpression of tomato Cipk6 (wild-type or enhanced kinase activity
versions) in the presence or absence of Cbl10 did not result in
PCD (O. del Pozo, unpublished data). Possibly, an increase of
cytosolic Ca2+ is required for full Cipk6 kinase activation. Alternatively, Cipk6 kinase activity and ROS generation might be
necessary but not sufficient for PCD execution. In this regard, it is
known that H2O2 can diffuse and activate PCD; however, it must
be accompanied by suppression of ROS detoxification mechanisms and by a balanced concentration of nitric oxide (Mittler
et al., 1999; Delledonne et al., 2001). Because Cbl10 and Cipk6
loss-of-function phenotypes are reminiscent of those previously
described for MAPKKKa (del Pozo et al., 2004), it will be important to explore in the future possible crosstalk between
Cipk6 and MAPKKKa or other MAPK cascades in PCD regulation. However, as we have stated previously for MAPKKKa
(del Pozo et al., 2004), we cannot rule out that Cbl10/Cipk6
might regulate other susceptibility-related pathways in addition to controlling cell death. Perturbation of these pathways
could also lead to inhibition of bacterial growth as there is no
direct link between lesion formation and virulence or ETI PCD
and increased resistance as shown in Pst DC3000 mutant strains
that affect both forms of PCD (López-Solanilla et al., 2004;
Gassmann, 2005).
We discovered evidence of a direct and positive connection
between tomato Cipk6 kinase activity and ROS production in
planta; more importantly, we provide genetic and physiological
in vivo evidence that Cbl10 and Cipk6 contribute to ROS generated during both PTI and ETI peaks in the N. benthamiana/Pst
DC3000 interaction. These results indicate that ROS generation
in ETI and PTI might be mechanistically linked at the Cbl10/
Cipk6 signaling node. Because Cbl10 and Cipk6 are interacting
partners in yeast and in planta (using co-IP) and both interact
directly with RbohB in N. benthamiana plasma membrane (by
BiFC), we propose that RbohB is a target of the Cbl10/Cipk6
module, thus providing a mechanistic link between Ca2+-derived
11 of 17
signals, phosphorylation, and ROS signaling during the plant
immune response to bacterial pathogens.
Work performed in elicitor-treated suspension cultures indicated
that phosphorylation and Ca2+ signaling are required for ROS
generation (Blume et al., 2000; Grant et al., 2000, Kimura et al.,
2012), whereas other studies demonstrated that ROS production
was necessary for Ca2+ influx or played a positive feedback role
(Van Breusegem et al., 2008). These studies revealed a specific
and complex crosstalk between Ca2+ and ROS signaling in early
immune responses in different plant–pathogen interactions. An
oxidative burst is a hallmark in nearly all plant responses to
pathogen attack and plays an important signaling role in PCD
(Torres, 2010). Different enzymatic sources of ROS have been
described (Daudi et al., 2012; Ishiga et al., 2012; Rojas et al.,
2012); however, the membrane-bound RBOH complex seems to
play a central role in generating apoplastic ROS in PTI and ETI
and possibly in amplifying ROS generated in different cellular
compartments (Torres, 2010). The role of specific RBOH enzymes
in pathogen-elicited PCD and resistance is complex, and they
play distinct roles depending on the pathogen and the plant
species. For instance, Arabidopsis rbohF plants are more resistant to Peronospora parasitica, displaying enhanced HR, but
are impaired in resistance to compatible bacteria, whereas
N. benthamiana plants silenced for RbohB (Arabidopsis RBOHF
ortholog) are more susceptible to Phytophtora infestans and
present reduced HR, but appear not to contribute to bacterial
resistance (Yoshioka et al., 2003; Segonzac et al., 2011; Chaouch
et al., 2012). A role for Arabidopsis RBOHs in signaling has been
proposed, as they regulate the spread of salicylic acid–mediated
cell death and the synthesis of salicylic acid and defense-related
metabolites (Torres et al., 2005; Chaouch et al., 2012). Recently,
a model for CPK5 and RBOHD in a self-activating circuit that
activates distal plant defense has been proposed (Dubiella et al.,
2013). In the future, it will be interesting to determine if Cipk6
regulation of RbohB contributes to defense-related metabolite
production and if it affects the outcome of defense responses
regulation in both tomato and N. benthamiana.
Plant RBOH proteins are regulated by Ca2+ at different levels.
They contain EF hands that can bind Ca2+ (Keller et al., 1998)
and are phosphorylated during pathogen responses by CDPKs
or CPKs (Kobayashi et al., 2007; Dubiella et al., 2013). Additional
events of RBOH phosphorylation had been described in proteomic analysis performed in Arabidopsis cells after elicitor treatment
(Benschop et al., 2007; Nühse et al., 2007). These results highlight
the importance of Ca2+ and phosphoregulation of RBOHs in early
immune signaling, and we propose that CIPKs are candidates for
such phosphorylation events in plant responses to pathogen attack, in addition to CDPKs or CPKs.
Recent reports indicate that at least in vitro, Arabidopsis RBOH
proteins might be also regulated by different Ca2+-regulated kinases that play a role in abiotic stress responses (Sirichandra et al.,
2009; Drerup et al., 2013). Arabidopsis CIPK6, CBL10, and orthologs have been shown to participate in different processes like salt
stress tolerance, development, regulation of potassium channel
AKT2 activity or modulation, or abscisic acid signaling (Kim et al.,
2007; Quan et al., 2007; Tripathi et al., 2009; Held et al., 2011; Chen
et al., 2012; Ren et al., 2013). We did not observe any abnormal
growth or development in N. benthamiana or tomato plants
12 of 17
The Plant Cell
silenced for either Cbl10, Cipk6, or both, growing under standard
greenhouse conditions. Because N. benthamiana and tomato
plants become fully silenced when they are over 1 month old, such
early development-associated phenotypes might not have been
apparent in our silenced plants. Interestingly, ROS are also generated during salt stress and during development (Borsani et al.,
2001; Hernández et al., 2001), and RBOHs participate in initial
phases of salt stress (Jiang et al., 2012). It will be interesting to
determine whether Arabidopsis CIPK6 also mediates ROS production and its possible contribution to immunity, salt tolerance, or
development.
Earlier, a connection between CIPKs and reactive oxygen
signaling was suggested, as different SOS2-interacting proteins
(NUCLEOSIDE DIPHOSPHATE KINASE2 and two catalases
[CAT2 and CAT3]) involved in ROS signaling and decomposition,
respectively, were identified (Verslues et al., 2007). SOS1, a Na+/
H+ antiporter, which is a target of the SOS3/SOS2 complex, has
been reported to be posttranscriptionally regulated by ROS and
to interact with oxidative responses regulators (Katiyar-Agarwal
et al., 2006; Chung et al., 2008). In rice cultured cells treated with
PAMPs, CIPK14/15 appear to mediate ROS production (Kurusu
et al., 2010), and, more recently, Arabidopsis CIPK26 was shown
to interact with RBOHF in planta and positively and negatively
regulate its ROS-producing activity in human cultured cells
(HEK293T) (Kimura et al., 2012; Drerup et al., 2013). In planta
validation of such results in the context of a specific physiological response will consolidate these promising results.
CBLs and CIPKs have been described to interact specifically
with protein phosphatases, the chaperone-like protein DNAJ and
a 14-3-3 protein (Fuglsang et al., 2007; Yang et al., 2010). Understanding the regulation of the formation of the Cbl10/Cipk6
complex, identification of additional regulatory components,
together with transcriptional regulation of their genes under developmental-, tissue-, and cell-specific and environmental or biotic
stress conditions will be important for understanding how downstream Cbl10/Cipk6 signaling specificity is achieved from upstream Ca2+-derived signals in abiotic and biotic stress signaling.
Our results and other recent reports that suggest the participation of rice CIPK14/15 in regulation of defense gene
expression and phytoalexin production in rice cultured cells
and the possible regulation of NPR1 by PKS5 (Arabidopsis
CIPK24) phosphorylation (Kurusu et al., 2010; Xie et al., 2010)
support a role for CBL/CIPK participation in different aspects
of plant defense. Thus, CBL/CIPK complexes are important
players in modulating outputs during biotic interactions. In
the future, we will investigate the molecular basis of tomato
Cbl10/Cipk6 regulation of RbohB ROS production. For that
purpose, we will determine if RbohB is a Cipk6 phosphorylation substrate, identify the putative residues phosphorylated,
and analyze the contribution of these putative phosphorylation events to ROS generation, PCD, and resistance in the
plant immune responses to pathogenic bacteria through functional analysis. Alternatively, phosphorylation-independent regulation of RbohB by Cipk6 also could be plausible, in a similar
fashion to SOS2 (CIPK24) regulation of the H+/Ca2+ antiporter
CAX1 (Cheng et al., 2004). Thus, characterization of RbohB
regulation by Cipk6 and identification and characterization of
additional Cipk6 phosphorylation targets involved in plant
immunity will be the next important steps to gain understanding
about the cellular processes regulated by this key regulator of
PCD.
METHODS
Plant Material and Bacterial Strains
Resistant tomato (Solanum lycopersicum) line Rio Grande-PtoR (RGPtoR; Pto/Pto and Prf/Prf), susceptible lines Rio Grande-PtoS (RG-PtoS;
ptp/pto and Prf/Prf), and Rio Grande-prf3 (RG-prf3; Pto/Pto and prf/prf)
and Nicotiana benthamiana were used for VIGS. Agrobacterium tumefaciens strains GV2260 and GV3101 were used for VIGS in N. benthamiana
and tomato, respectively, and C58C1 for Agrobacterium-mediated transient
expression in N. benthamiana. Pseudomonas syringae pv tomato strain
DC3000 and P. syringae pv tomato strain DC3000 DhrcC- (Pst DC3000
DhrcC) were used in tomato and N. benthamiana infection analysis, and
P. syringae pv tabaci strain 11528R was used for pathogen assays in
N. benthamiana.
cDNA Cloning for Tomato Cipk6, Cbl10, and RbohB
Tomato Cipk6, Cbl10, and RbohB cDNAs containing the complete open
reading frames were obtained by RT-PCR using as a template a tomato
cDNA library obtained from Pst DC3000-infected leaves using Gatewayadapted oligonucleotides OPS-13/OPS-14 for Cipk6, OPS-56/OPS-64 for
Cbl10, and OPS-637/OPS-638 for RbohB and cloned into the pDONR207
vector (Invitrogen; Supplemental Table 2).
Site-Directed Mutagenesis and Deletion Mutants
PCR-based site mutagenesis was performed to generate the substitutions
K43M and T172D in Sl-Cipk6 using the QuickChange kit (Stratagene) using
the following oligonucleotides: OPS-40/OPS-41 for Sl-Cipk6(K43M) and
OPS-42/OPS-43 for Sl-Cipk6(T172D). For Sl-Cipk6DNAF and Sl-Cipk6DCterm, OPS-44/OPS-45 and OPS-13/OPS-98 were used, respectively
(Supplemental Table 2).
Electrolyte Leakage Assay and PCD Staining
Trypan blue staining in tomato was performed as described by del Pozo
et al. (2004). Ion leakage assays were performed with three discs per plant
(1 cm diameter) floated in 3 mL of MilliQ water for 2 h at room temperature
with gentle shaking, and conductivity was measured with an Acorn Con 6
meter (Oakton Instruments).
VIGS
PVX clones containing N. benthamiana partial cDNAs corresponding to
Cipk6 (cNbME30H4) and Cbl10 (cNbME35F4) were identified previously
(del Pozo et al., 2004). Tomato identical clones were amplified and
transferred into the TRV silencing vector pYL215 (Liu et al., 2002) to obtain
TRV-Cipk6 and TRV-Cbl10. TRV-Cipk6-59, a 403-bp Cipk6 fragment,
Cipk6-3, a 463-bp fragment, and TRV-Cbl10-5, a 205-bp fragment, were
PCR amplified using Gateway-adapted oligonucleotides OPS-15/OPS16, OPS-19/OPS-20, and OPS-22/OPS-24, respectively. PCR products
were gel purified, inserted into the TRV vector by the Gateway reaction
(Invitrogen), sequenced, and transformed into Agrobacterium strain
GV2260. TRV-Nb-RbohB silencing construct was kindly provided by
Cecile Segonzac (Segonzac et al., 2011). For silencing in tomato and
N. benthamiana, seedlings with two emerging leaflets and 14-d-old plants,
respectively, were syringe infiltrated with cultures containing pYL215 derived
constructs and mixed with pTRV1, both with an OD600 of 0.075, according to
procedures described by Liu et al. (2002).
Role of Cbl10 and Cipk6 in Plant Immunity
Agrobacterium-Mediated Transient Transformation Assay
Agrobacterium-mediated transient expression of proteins in N. benthamiana
leaves was performed as described by He et al. (2004). Agrobacterium
GV2260 cultures carrying Pto, avrPto, Cf9, avr9, PtoY207D, and Bax were
infiltrated at OD600 = 0.2; for cultures carrying RBP1 or Gpa2, OD600 = 0.1;
and for cultures carrying NPP1, RX2, or CP, OD600 = 0.04 was used. OD600
was adjusted for each culture to synchronize PCD development in
N. benthamiana and allow parallel scoring. Tomato Cipk6 and mutant
derivatives were cloned into the estradiol-inducible pER8-XVE vector (Zuo
et al., 2000), with a GFP fusion tag at the C terminus (oligonucleotides
OPS-51, OPS-178, and OPS-404), and into the pYL436 TAP-tag vector
(Rubio et al., 2005) (oligonucleotides OPS-189, OPS-190, and OPS-191).
Agrobacterium strain C58C1 carrying XVE, pYL436 TAP-tag, or the tomato bushy stunt virus p19 gene constructs was adjusted to an OD600 =
0.4 (Supplemental Table 2).
Yeast Two-Hybrid Assay
Tomato Cbl10 tagged with an HA epitope was cloned into pJG4-5 vector
under control of the Gal-inducible promoter and tomato Cipk6 gene and
derivatives (fused to the LexA DNA binding domain) into pEG202 vector
and transformed in the yeast strain EGY48 (ura3, his3, trp1, lexAPo-leu2)
following the LiAc/PEG method (Yeast Protocols Handbook, Clontech).
Two-hybrid tests were performed on indicator plates supplemented with
2% Gal and 1% raffinose to induce prey fusion protein expression. Activation of the chromosomal LEU2 reporter was tested by scoring growth on
minimal medium lacking uracil, His, Trp, and Leu (Golemis and Khazak,
1997). Expression of fusion proteins was verified by immunoblot using
monoclonal antibodies mouse anti-LexA SC-7544 (Santa Cruz Biotechnology) or rat anti-HA 3F10 (Roche Applied Science).
Preparation and Purification of Tomato Cipk6 Recombinant Proteins
Tomato Cipk6 and derivatives were PCR amplified with LIC-compatible
primer OPS-189 (forward) and OPS-190 or OPS-191 (reverse) oligonucleotides for full-length or Cipk6DCterm, respectively, and cloned as
described by Popescu et al. (2007 Supplemental Table 2). Six-week-old
N. benthamiana leaves were coinfiltrated with a mix of Agrobacterium
C58C1 cultures carrying pYL436-Cipk6 or derivatives and p19. Three
days later, leaves were collected, frozen in liquid nitrogen, ground, and
mixed 1:2 (v/v) with extraction buffer (100 mM Tris-HCl, pH 7.5, 150 mM
NaCl, 5 mM EDTA, 10% glycerol, 0.1% Triton X-100, 13 Complete
protease inhibitors [Roche], and 1 mM phenylmethylsulfonyl fluoride) and
centrifuged for 20 min at 14,000g. Supernatants were collected and filtered through four layers of Miracloth (Calbiochem). Ten milliliters was
incubated with 50 mL IgG beads (Amersham Biosciences) for 2 h at 4°C
with gentle rotation. After centrifugation at 150g for 3 min at 4°C, the IgG
beads were recovered and washed three times with 10 mL of washing
buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, and 0.1%
Triton X-100) and once with 10 mL of cleavage buffer (50 mM Tris-HCl, pH
7.5, 150 mM NaCl, 10% glycerol, 0.1% Triton X-100, and 1 mM DTT) at
4°C. Elution from the IgG beads was performed by incubation with 10 mL
(20 units) of Prescission protease (Amersham Biosciences) in 1 mL of
cleavage buffer at 4°C with gentle rotation.
Preparation and Purification of Tomato Cbl10 Recombinant Protein
To produce GST-Cbl10 recombinant protein, GST coding region was cloned
in frame in the NdeI and NcoI sites of pET30a expression vector. Then, Cbl10
was cloned in frame into the EcoRI site, sequenced, and transformed into
Escherichia coli strain BL21-codonPlus-(DE3)-RIL (Stratagene). Cells were
grown at 37°C with shaking in Luria Bertani broth containing 50 µg/mL
kanamycin and 34 µg/mL chloramphenicol until OD600 = 0.6; 1 mM isopropyl-
13 of 17
b-D-thiogalactoside was then added and incubated for 14 h at 10°C with
shaking. Cells were pelleted by centrifugation at 4000g, resuspended in PBS
buffer, pH 8.0, lysed by sonication, and centrifuged at 22,000g, and the
recombinant proteins in soluble phase were purified by affinity chromatography using GST MicroSpin columns (Amersham).
Kinase Assays
For autophosphorylation assays, purified tomato Cipk6 and mutant version
proteins were incubated in kinase buffer (20 mM Tris, pH 7.5, 15 mM MgCl2,
1 mM DTT, 50 µM ATP, and 10 µCi of [g-32P]ATP [Amersham; 3000 Ci/mmol,
1 Ci = 37 GBq]) in a final volume of 30 mL and incubated 45 min at
30°C; 1 mM CaCl2 or 10 mM EGTA was included in the mixture or in the
kinase buffer, respectively. Reactions were terminated by adding 10 mL of
43 Laemmli loading buffer, and samples were separated on 12% SDSPAGE gels. Gels were stained with Coomassie Brilliant Blue (Bio-Rad) and
visualized by autoradiography. Cipk6 kinase transactivity was determined in
the presence or absence of 100 ng of GST-Cbl10 fusion protein, and 3 µg of
MBP (Roche), 5 µCi of [g-32P]ATP, and 50 µM ATP were added to the kinase
reaction mixture and incubated at 30°C for 45 min. Samples were separated
on 15% SDS-PAGE gels. Kinase activity was visualized by autoradiography.
ROS Detection and Measurement
N. benthamiana leaf discs (0.28 cm2) were floated on 100 mL of distilled
water in a 96-well white-bottom plate for 8 to 12 h at room temperature at
dark. Water was replaced with 100 mL solution containing 100 µM luminol
(Sigma-Aldrich) and 1 µg of horseradish peroxidase. Expression was
induced by adding 5 nM 17b-estradiol and 0.1% Silwet L-77. For ROS
detection after bacterial infection, 2.5 3 108 cfu/mL Pst DC3000 or Pst
DC3000 DhrcC was added to the wells, and ROS were measured in vivo
as luminescence using a Varioskan Flash Multimode Reader (Thermo
Scientific) every 30 s up to 8 h.
BiFC Assay
Tomato Cipk6 and Cbl10 were Gateway cloned into the pXCGW vector,
using oligonucleotides OPS13/OPS63 and OPS56/OPS64, respectively;
tomato RbohB was cloned into the pXNGW vector (OPS-637/OPS-638).
For the BiFC assay, Agrobacterium C58C1 cultures (OD600 = 0.5) carrying
pXCGW-Sl-Cipk6 or pXCGW-Sl-Cbl10, pXNGW-Nb-RbohB and tomato
bushy stunt virus p19, were mixed at 1:1:1 v/v/v ratio, were coinfiltrated
in N. benthamiana plants and kept in the greenhouse for 48 h. YFP reconstitution was visualized by confocal microscopy. FM4-64 was infiltrated (20 µM) and observed immediately by confocal microscopy. See
Supplemental Table 2.
Accession Numbers
Sequence data from this article can be found in the GenBank/EMBL data
libraries under the following accession numbers: cNbME25F4 (JF974260),
cNbME30H4 (JF974261), Nb-Cipk6 (EB449468), Sl-Cbl3 (SGN-U585014),
Sl-Cbl4 (SGN-U587071), Sl-Cbl10 (JF831199; Solyc08g065330), Sl-Cipk3
(SGN-U580739), Sl-Cipk6 (JF831200; Solyc12g010130), Sl-Cipk11 (JF831201;
Solyc06g082440), Sl-Cipk14 (JF831202; Solyc10g085450), Sl-RbohB
(Solyc03g117980), Os-Cbl10 (DQ201204), Os-Cipk6 (Q6Z9F4), Ca-Cipk6
(ACC96114), and Dm-CalciB (NM_080002). From Arabidopsis, they are
as follows: At-CIPK6 (AT4g30960), At-CIPK8 (AT4g24400), At-CIPK24
(SOS2, AT5g35410), At-CIPK3 (AT2g26980), At-CIPK9 (AT1g01140),
At-CIPK23 (AT1g30270), At-SnRK2 (At1g10940), At-CBL1 (AT4G17615),
At-CBL2 (AT5G55990), At-CBL3 (AT4G26570), At-CBL4 (AT5G24270),
At-CBL5 (AT4G01420), At-CBL6 (AT4G16350), At-CBL7 (AT4G26560),
At-CBL8 (AT1G64480), At-CBL9 (AT5G47100), and At-CBL10 (AT4g33000).
14 of 17
The Plant Cell
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. ORF Cloning of Sl-Cbl10 and Sl-Cipk6 and
Derivative VIGS Constructs.
Supplemental Figure 2. Cbl10 Protein Sequence Alignment.
Supplemental Figure 3. SlCipk6 Protein Sequence Alignment.
Supplemental Figure 4. Cipk6 and Cbl10 Genes Are Expressed in All
Tissues Analyzed and Their Expression Is Induced after Pathogen Infection.
Supplemental Figure 5. Reduction of Disease Symptoms in N.
benthamiana Plants Silenced for Cipk6 or Cbl10.
Supplemental Figure 6. Tomato Cipk and Cbl10 Proteins Were
Expressed in Yeast.
Supplemental Figure 7. Cbl10/Cipk6 Interaction Is Not Calcium
Dependent in in Vitro Pull-Down Assays.
Supplemental Table 1. TRV-Cbl10 and TRV-Cipk6 Constructs Specifically Target Their Corresponding mRNAs in Tomato.
Supplemental Table 2. Oligonucleotides Used in This Work.
Supplemental Data Set 1. Text File of Alignment Corresponding to
Phylogenetic Analysis in Figure 1A.
Supplemental Data Set 2. Text File of Alignment Corresponding to
Phylogenetic Analysis in Figure 1B.
ACKNOWLEDGMENTS
We thank Peter Moffett for providing Rx2/CP and Gpa2/RBP-1 constructs and the co-IP protocol, Cecile Segonzac for TRV-Nb-RbohB
construct and helping with the ROS assay, and Mary Beth Mudgett for
Gateway-compatible BiFC vectors. We thank Jose Manuel Pardo for
critically reading the article and for suggesting fruitful experiments. This
work was funded in part by the European Regional Development Fund
through the Ministerio de Ciencia e Innovación (BIO2005-02136 and
BIO2009-08648), Junta de Andalucía (P07-CVI-03171), Marie Curie Programme through the International Reintegration grants (MIRG-CT2005-031174) (O.D.); National Science Foundation IOS-0841807 and
(IOS-1025642) (G.B.M.). O.D. was supported in part by the Junta de Andalucía
(Programa de Retorno de Investigadores), F.D. by a Juan de la Cierva contract
(Ministerio de Ciencia e Innovación, Spain), and Y.P.-J. by Grant BIO200908648. E.G.-B. was a recipient of an Formación de Personal Investigador
fellowship (Ministerio de Educación, Spain).
AUTHOR CONTRIBUTIONS
F.D. and O.D. designed experiments and analyzed the data. O.D. wrote
most of the article, and F.D. and G.B.M. helped with the writing. F.D.
performed most of experiments. O.D. performed initial VIGS assays. E.G.-B.
performed yeast two-hybrid and RT-PCR analyses. Y.P.-J. performed tomato
RbohB cloning and BiFC. S.C. performed in planta experiments using
oomycete, viral, and nematode effectors. G.B.M. assisted with data analysis.
Received May 13, 2013; revised July 1, 2013; accepted July 12, 2013;
published July 31, 2013.
REFERENCES
Akaboshi, M., Hashimoto, H., Ishida, H., Saijo, S., Koizumi, N.,
Sato, M., and Shimizu, T. (2008). The crystal structure of plant-
specific calcium-binding protein AtCBL2 in complex with the regulatory
domain of AtCIPK14. J. Mol. Biol. 377: 246–257.
Albrecht, V., Ritz, O., Linder, S., Harter, K., and Kudla, J. (2001).
The NAF domain defines a novel protein-protein interaction module
conserved in Ca2+-regulated kinases. EMBO J. 20: 1051–1063.
Anderson, J.C., Pascuzzi, P.E., Xiao, F., Sessa, G., and Martin, G.B. (2006).
Host-mediated phosphorylation of type III effector AvrPto promotes
Pseudomonas virulence and avirulence in tomato. Plant Cell 18: 502–514.
Batistic, O., and Kudla, J. (2009). Plant calcineurin B-like proteins and
their interacting protein kinases. Biochim. Biophys. Acta 1793: 985–992.
Bendahmane, A., Querci, M., Kanyuka, K., and Baulcombe, D.C.
(2000). Agrobacterium transient expression system as a tool for the
isolation of disease resistance genes: application to the Rx2 locus
in potato. Plant J. 21: 73–81.
Benschop, J.J., Mohammed, S., O’Flaherty, M., Heck, A.J., Slijper, M.,
and Menke, F.L. (2007). Quantitative phosphoproteomics of early
elicitor signaling in Arabidopsis. Mol. Cell. Proteomics 6: 1198–1214.
Blume, B., Nürnberger, T., Nass, N., and Scheel, D. (2000). Receptormediated increase in cytoplasmic free calcium required for activation of
pathogen defense in parsley. Plant Cell 12: 1425–1440.
Boller, T., and Felix, G. (2009). A renaissance of elicitors: Perception
of microbe-associated molecular patterns and danger signals by
pattern-recognition receptors. Annu. Rev. Plant Biol. 60: 379–406.
Borsani, O., Valpuesta, V., and Botella, M.A. (2001). Evidence for a role of
salicylic acid in the oxidative damage generated by NaCl and osmotic
stress in Arabidopsis seedlings. Plant Physiol. 126: 1024–1030.
Boudsocq, M., Willmann, M.R., McCormack, M., Lee, H., Shan, L.,
He, P., Bush, J., Cheng, S.H., and Sheen, J. (2010). Differential
innate immune signalling via Ca(2+) sensor protein kinases. Nature
464: 418–422.
Chaouch, S., Queval, G., and Noctor, G. (2012). AtRbohF is a crucial
modulator of defence-associated metabolism and a key actor in the
interplay between intracellular oxidative stress and pathogenesis
responses in Arabidopsis. Plant J. 69: 613–627.
Chen, L., Ren, F., Zhou, L., Wang, Q.Q., Zhong, H., and Li, X.B. (2012).
The Brassica napus calcineurin B-Like 1/CBL-interacting protein kinase
6 (CBL1/CIPK6) component is involved in the plant response to abiotic
stress and ABA signalling. J. Exp. Bot. 63: 6211–6222.
Cheng, N.H., Pittman, J.K., Zhu, J.-K., and Hirschi, K.D. (2004). The protein
kinase SOS2 activates the Arabidopsis H(+)/Ca(2+) antiporter CAX1 to
integrate calcium transport and salt tolerance. J. Biol. Chem. 279: 2922–2926.
Chiasson, D., Ekengren, S.K., Martin, G.B., Dobney, S.L., and
Snedden, W.A. (2005). Calmodulin-like proteins from Arabidopsis
and tomato are involved in host defense against Pseudomonas syringae
pv. tomato. Plant Mol. Biol. 58: 887–897.
Chung, J.S., Zhu, J.K., Bressan, R.A., Hasegawa, P.M., and Shi, H.
(2008). Reactive oxygen species mediate Na+-induced SOS1 mRNA
stability in Arabidopsis. Plant J. 53: 554–565.
Daudi, A., Cheng, Z., O’Brien, J.A., Mammarella, N., Khan, S.,
Ausubel, F.M., and Bolwell, G.P. (2012). The apoplastic oxidative
burst peroxidase in Arabidopsis is a major component of patterntriggered immunity. Plant Cell 24: 275–287.
Delledonne, M., Zeier, J., Marocco, A., and Lamb, C. (2001). Signal
interactions between nitric oxide and reactive oxygen intermediates
in the plant hypersensitive disease resistance response. Proc. Natl.
Acad. Sci. USA 98: 13454–13459.
del Pozo, O., Pedley, K.F., and Martin, G.B. (2004). MAPKKKalpha is
a positive regulator of cell death associated with both plant immunity
and disease. EMBO J. 23: 3072–3082.
Dietrich, A., Mayer, J.E., and Hahlbrock, K. (1990). Fungal elicitor
triggers rapid, transient, and specific protein phosphorylation in parsley
cell suspension cultures. J. Biol. Chem. 265: 6360–6368.
Role of Cbl10 and Cipk6 in Plant Immunity
Drerup, M.M., Schlücking, K., Hashimoto, K., Manishankar, P.,
Steinhorst, L., Kuchitsu, K., and Kudla, J. (2013). The Calcineurin
B-like calcium sensors CBL1 and CBL9 together with their interacting
protein kinase CIPK26 regulate the Arabidopsis NADPH oxidase RBOHF.
Mol. Plant 6: 559–569.
Du, L., Ali, G.S., Simons, K.A., Hou, J., Yang, T., Reddy, A.S., and
Poovaiah, B.W. (2009). Ca(2+)/calmodulin regulates salicylic-acidmediated plant immunity. Nature 457: 1154–1158.
Dubiella, U., Seybold, H., Durian, G., Komander, E., Lassig, R., Witte,
C.P., Schulze, W.X., and Romeis, T. (2013). Calcium-dependent protein
kinase/NADPH oxidase activation circuit is required for rapid defense
signal propagation. Proc. Natl. Acad. Sci. USA 110: 8744–8749.
Felix, G., Grosskopf, D.G., Regenass, M., and Boller, T. (1991). Rapid
changes of protein phosphorylation are involved in transduction of the
elicitor signal in plant cells. Proc. Natl. Acad. Sci. USA 88: 8831–8834.
Fellbrich, G., Romanski, A., Varet, A., Blume, B., Brunner, F.,
Engelhardt, S., Felix, G., Kemmerling, B., Krzymowska, M., and
Nürnberger, T. (2002). NPP1, a Phytophthora-associated trigger of
plant defense in parsley and Arabidopsis. Plant J. 32: 375–390.
Fuglsang, A.T., Guo, Y., Cuin, T.A., Qiu, Q., Song, C., Kristiansen,
K.A., Bych, K., Schulz, A., Shabala, S., Schumaker, K.S.,
Palmgren, M.G., and Zhu, J.K. (2007). Arabidopsis protein kinase
PKS5 inhibits the plasma membrane H+-ATPase by preventing
interaction with 14-3-3 protein. Plant Cell 19: 1617–1634.
Garcia-Brugger, A., Lamotte, O., Vandelle, E., Bourque, S.,
Lecourieux, D., Poinssot, B., Wendehenne, D., and Pugin, A.
(2006). Early signaling events induced by elicitors of plant defenses.
Mol. Plant Microbe Interact. 19: 711–724.
Gassmann, W. (2005). Natural variation in the Arabidopsis response to the
avirulence gene hopPsyA uncouples the hypersensitive response from
disease resistance. Mol. Plant Microbe Interact. 18: 1054–1060.
Golemis, E.A., and Khazak, V. (1997). Alternative yeast two-hybrid
systems. The interaction trap and interaction mating. Methods Mol.
Biol. 63: 197–218.
Gong, D., Guo, Y., Jagendorf, A.T., and Zhu, J.K. (2002).
Biochemical characterization of the Arabidopsis protein kinase
SOS2 that functions in salt tolerance. Plant Physiol. 130: 256–264.
Grant, M., Brown, I., Adams, S., Knight, M., Ainslie, A., and Mansfield,
J. (2000). The RPM1 plant disease resistance gene facilitates a rapid and
sustained increase in cytosolic calcium that is necessary for the
oxidative burst and hypersensitive cell death. Plant J. 23: 441–450.
Greenberg, J.T. (1997). Programmed cell death in plant–pathogen
interactions. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48: 525–545.
Guo, Y., Halfter, U., Ishitani, M., and Zhu, J.K. (2001). Molecular
characterization of functional domains in the protein kinase SOS2
that is required for plant salt tolerance. Plant Cell 13: 1383–1400.
Halfter, U., Ishitani, M., and Zhu, J.K. (2000). The Arabidopsis SOS2
protein kinase physically interacts with and is activated by the calciumbinding protein SOS3. Proc. Natl. Acad. Sci. USA 97: 3735–3740.
Hann, D.R., Gimenez-Ibanez, S., and Rathjen, J.P. (2010). Bacterial
virulence effectors and their activities. Curr. Opin. Plant Biol. 13:
388–393.
Harper, J.F., Breton, G., and Harmon, A. (2004). Decoding Ca(2+) signals
through plant protein kinases. Annu. Rev. Plant Biol. 55: 263–288.
He, X., Anderson, J.C., del Pozo, O., Gu, Y.Q., Tang, X., and Martin,
G.B. (2004). Silencing of subfamily I of protein phosphatase 2A
catalytic subunits results in activation of plant defense responses
and localized cell death. Plant J. 38: 563–577.
Held, K., Pascaud, F., Eckert, C., Gajdanowicz, P., Hashimoto, K.,
Corratgé-Faillie, C., Offenborn, J.N., Lacombe, B., Dreyer, I., Thibaud,
J.B., and Kudla, J. (2011). Calcium-dependent modulation and plasma
membrane targeting of the AKT2 potassium channel by the CBL4/CIPK6
calcium sensor/protein kinase complex. Cell Res. 21: 1116–1130.
15 of 17
Hernández, J.A., Ferrer, M.A., Jiménez, A., Barceló, A.R., and Sevilla, F.
(2001). Antioxidant systems and O(2)(.-)/H(2)O(2) production in the
apoplast of pea leaves. Its relation with salt-induced necrotic lesions in
minor veins. Plant Physiol. 127: 817–831.
Hrabak, E.M., et al. (2003). The Arabidopsis CDPK-SnRK superfamily
of protein kinases. Plant Physiol. 132: 666–680.
Ho, C.H., Lin, S.H., Hu, H.C., and Tsay, Y.F. (2009). CHL1 functions
as a nitrate sensor in plants. Cell 138: 1184–1194.
Ishiga, Y., Ishiga, T., Wangdi, T., Mysore, K.S., and Uppalapati, S.R.
(2012). NTRC and chloroplast-generated reactive oxygen species regulate
Pseudomonas syringae pv. tomato disease development in tomato and
Arabidopsis. Mol. Plant Microbe Interact. 25: 294–306.
Ishitani, M., Liu, J., Halfter, U., Kim, C.S., Shi, W., and Zhu, J.K. (2000).
SOS3 function in plant salt tolerance requires N-myristoylation and
calcium binding. Plant Cell 12: 1667–1678.
Jiang, C., Belfield, E.J., Mithani, A., Visscher, A., Ragoussis, J.,
Mott, R., Smith, J.A., and Harberd, N.P. (2012). ROS-mediated
vascular homeostatic control of root-to-shoot soil Na delivery in
Arabidopsis. EMBO J. 31: 4359–4370.
Jones, J.D., and Dangl, J.L. (2006). The plant immune system. Nature
444: 323–329.
Katiyar-Agarwal, S., Zhu, J., Kim, K., Agarwal, M., Fu, X., Huang, A.,
and Zhu, J.K. (2006). The plasma membrane Na+/H+ antiporter SOS1
interacts with RCD1 and functions in oxidative stress tolerance in
Arabidopsis. Proc. Natl. Acad. Sci. USA 103: 18816–18821.
Keller, T., Damude, H.G., Werner, D., Doerner, P., Dixon, R.A., and
Lamb, C. (1998). A plant homolog of the neutrophil NADPH oxidase
gp91phox subunit gene encodes a plasma membrane protein with
Ca2+ binding motifs. Plant Cell 10: 255–266.
Kim, B.G., Waadt, R., Cheong, Y.H., Pandey, G.K., DominguezSolis, J.R., Schültke, S., Lee, S.C., Kudla, J., and Luan, S. (2007).
The calcium sensor CBL10 mediates salt tolerance by regulating ion
homeostasis in Arabidopsis. Plant J. 52: 473–484.
Kimura, S., Kaya, H., Kawarazaki, T., Hiraoka, G., Senzaki, E.,
Michikawa, M., and Kuchitsu, K. (2012). Protein phosphorylation
is a prerequisite for the Ca2+-dependent activation of Arabidopsis
NADPH oxidases and may function as a trigger for the positive
feedback regulation of Ca2+ and reactive oxygen species. Biochim.
Biophys. Acta 1823: 398–405.
Kobayashi, M., Ohura, I., Kawakita, K., Yokota, N., Fujiwara, M.,
Shimamoto, K., Doke, N., and Yoshioka, H. (2007). Calcium-dependent
protein kinases regulate the production of reactive oxygen species by
potato NADPH oxidase. Plant Cell 19: 1065–1080.
Kobayashi, M., Yoshioka, M., Asai, S., Nomura, H., Kuchimura, K.,
Mori, H., Doke, N., and Yoshioka, H. (2012). StCDPK5 confers
resistance to late blight pathogen but increases susceptibility to
early blight pathogen in potato via reactive oxygen species burst.
New Phytol. 196: 223–237.
Kudla, J., Batistic, O., and Hashimoto, K. (2010). Calcium signals:
The lead currency of plant information processing. Plant Cell 22:
541–563.
Kurusu, T., Hamada, J., Hamada, H., Hanamata, S., and Kuchitsu, K.
(2010). Roles of calcineurin B-like protein-interacting protein kinases in
innate immunity in rice. Plant Signal. Behav. 5: 1045–1047.
Kwaaitaal, M., Huisman, R., Maintz, J., Reinstädler, A., and Panstruga,
R. (2011). Ionotropic glutamate receptor (iGluR)-like channels mediate
MAMP-induced calcium influx in Arabidopsis thaliana. Biochem. J. 440:
355–365.
Lecourieux, D., Mazars, C., Pauly, N., Ranjeva, R., and Pugin, A.
(2002). Analysis and effects of cytosolic free calcium increases in
response to elicitors in Nicotiana plumbaginifolia cells. Plant Cell 14:
2627–2641.
16 of 17
The Plant Cell
Li, L., Kim, B.G., Cheong, Y.H., Pandey, G.K., and Luan, S. (2006). A
Ca(2)+ signaling pathway regulates a K(+) channel for low-K response in
Arabidopsis. Proc. Natl. Acad. Sci. USA 103: 12625–12630.
Lin, H., Yang, Y., Quan, R., Mendoza, I., Wu, Y., Du, W., Zhao, S.,
Schumaker, K.S., Pardo, J.M., and Guo, Y. (2009). Phosphorylation of
SOS3-LIKE CALCIUM BINDING PROTEIN8 by SOS2 protein kinase
stabilizes their protein complex and regulates salt tolerance in Arabidopsis.
Plant Cell 21: 1607–1619.
Liu, J., Ishitani, M., Halfter, U., Kim, C.S., and Zhu, J.K. (2000). The
Arabidopsis thaliana SOS2 gene encodes a protein kinase that is required
for salt tolerance. Proc. Natl. Acad. Sci. USA 97: 3730–3734.
Liu, Y., Schiff, M., and Dinesh-Kumar, S.P. (2002). Virus-induced
gene silencing in tomato. Plant J. 31: 777–786.
López-Solanilla, E., Bronstein, P.A., Schneider, A.R., and Collmer, A.
(2004). HopPtoN is a Pseudomonas syringae Hrp (type III secretion system)
cysteine protease effector that suppresses pathogen-induced necrosis
associated with both compatible and incompatible plant interactions. Mol.
Microbiol. 54: 353–365.
Ma, W., Smigel, A., Tsai, Y.C., Braam, J., and Berkowitz, G.A. (2008). Innate
immunity signaling: Cytosolic Ca2+ elevation is linked to downstream nitric
oxide generation through the action of calmodulin or a calmodulin-like
protein. Plant Physiol. 148: 818–828.
Ma, W., Smigel, A., Verma, R., and Berkowitz, G.A. (2009). Cyclic
nucleotide gated channels and related signaling components in
plant innate immunity. Plant Signal. Behav. 4: 277–282.
Ma, Y., Walker, R.K., Zhao, Y., and Berkowitz, G.A. (2012). Linking ligand
perception by PEPR pattern recognition receptors to cytosolic Ca2+
elevation and downstream immune signaling in plants. Proc. Natl. Acad.
Sci. USA 109: 19852–19857.
Mittler, R., Herr, E.H., Orvar, B.L., van Camp, W., Willekens, H.,
Inzé, D., and Ellis, B.E. (1999). Transgenic tobacco plants with
reduced capability to detoxify reactive oxygen intermediates are
hyperresponsive to pathogen infection. Proc. Natl. Acad. Sci. USA
96: 14165–14170.
Mucyn, T.S., Clemente, A., Andriotis, V.M., Balmuth, A.L., Oldroyd,
G.E., Staskawicz, B.J., and Rathjen, J.P. (2006). The tomato
NBARC-LRR protein Prf interacts with Pto kinase in vivo to regulate
specific plant immunity. Plant Cell 18: 2792–2806.
Nühse, T.S., Bottrill, A.R., Jones, A.M., and Peck, S.C. (2007).
Quantitative phosphoproteomic analysis of plasma membrane proteins
reveals regulatory mechanisms of plant innate immune responses. Plant
J. 51: 931–940.
Ogasawara, Y., et al. (2008). Synergistic activation of the Arabidopsis
NADPH oxidase AtrbohD by Ca2+ and phosphorylation. J. Biol.
Chem. 283: 8885–8892.
Oh, C.S., and Martin, G.B. (2011). Effector-triggered immunity
mediated by the Pto kinase. Trends Plant Sci. 16: 132–140.
Ohta, M., Guo, Y., Halfter, U., and Zhu, J.K. (2003). A novel domain in the
protein kinase SOS2 mediates interaction with the protein phosphatase
2C ABI2. Proc. Natl. Acad. Sci. USA 100: 11771–11776.
Pedley, K.F., and Martin, G.B. (2003). Molecular basis of Pto-mediated
resistance to bacterial speck disease in tomato. Annu. Rev. Phytopathol.
41: 215–243.
Popescu, S.C., Popescu, G.V., Bachan, S., Zhang, Z., Seay, M.,
Gerstein, M., Snyder, M., and Dinesh-Kumar, S.P. (2007).
Differential binding of calmodulin-related proteins to their targets
revealed through high-density Arabidopsis protein microarrays.
Proc. Natl. Acad. Sci. USA 104: 4730–4735.
Quan, R., Lin, H., Mendoza, I., Zhang, Y., Cao, W., Yang, Y., Shang, M.,
Chen, S., Pardo, J.M., and Guo, Y. (2007). SCABP8/CBL10, a putative
calcium sensor, interacts with the protein kinase SOS2 to protect
Arabidopsis shoots from salt stress. Plant Cell 19: 1415–1431.
Quintero, F.J., Ohta, M., Shi, H., Zhu, J.K., and Pardo, J.M. (2002).
Reconstitution in yeast of the Arabidopsis SOS signaling pathway
for Na+ homeostasis. Proc. Natl. Acad. Sci. USA 99: 9061–9066.
Ren, X.L., Qi, G.N., Feng, H.Q., Zhao, S., Zhao, S.S., Wang, Y., and Wu,
W.H. (2013). Calcineurin B-like protein CBL10 directly interacts with AKT1
and modulates K+ homeostasis in Arabidopsis. Plant J. 74: 258–266.
Rojas, C.M., Senthil-Kumar, M., Wang, K., Ryu, C.M., Kaundal, A., and
Mysore, K.S. (2012). Glycolate oxidase modulates reactive oxygen speciesmediated signal transduction during nonhost resistance in Nicotiana
benthamiana and Arabidopsis. Plant Cell 24: 336–352.
Rubio, V., Shen, Y., Saijo, Y., Liu, Y., Gusmaroli, G., Dinesh-Kumar, S.P.,
and Deng, X.W. (2005). An alternative tandem affinity purification strategy
applied to Arabidopsis protein complex isolation. Plant J. 41: 767–778.
Sacco, M.A., Koropacka, K., Grenier, E., Jaubert, M.J., Blanchard,
A., Goverse, A., Smant, G., and Moffett, P. (2009). The cyst
nematode SPRYSEC protein RBP-1 elicits Gpa2- and RanGAP2dependent plant cell death. PLoS Pathog. 5: e1000564.
Sánchez-Barrena, M.J., Fujii, H., Angulo, I., Martínez-Ripoll, M.,
Zhu, J.K., and Albert, A. (2007). The structure of the C-terminal
domain of the protein kinase AtSOS2 bound to the calcium sensor
AtSOS3. Mol. Cell 26: 427–435.
Segonzac, C., Feike, D., Gimenez-Ibanez, S., Hann, D.R., Zipfel, C., and
Rathjen, J.P. (2011). Hierarchy and roles of pathogen-associated
molecular pattern-induced responses in Nicotiana benthamiana. Plant
Physiol. 156: 687–699.
Shan, L., He, P., Li, J., Heese, A., Peck, S.C., Nürnberger, T., Martin,
G.B., and Sheen, J. (2008). Bacterial effectors target the common
signaling partner BAK1 to disrupt multiple MAMP receptor-signaling
complexes and impede plant immunity. Cell Host Microbe 4: 17–27.
Shi, J., Kim, K.N., Ritz, O., Albrecht, V., Gupta, R., Harter, K., Luan, S.,
and Kudla, J. (1999). Novel protein kinases associated with calcineurin
B-like calcium sensors in Arabidopsis. Plant Cell 11: 2393–2405.
Sirichandra, C., Gu, D., Hu, H.C., Davanture, M., Lee, S., Djaoui, M.,
Valot, B., Zivy, M., Leung, J., Merlot, S., and Kwak, J.M. (2009).
Phosphorylation of the Arabidopsis AtrbohF NADPH oxidase by
OST1 protein kinase. FEBS Lett. 583: 2982–2986.
Tao, Y., Xie, Z., Chen, W., Glazebrook, J., Chang, H.S., Han, B., Zhu, T.,
Zou, G., and Katagiri, F. (2003). Quantitative nature of Arabidopsis
responses during compatible and incompatible interactions with the
bacterial pathogen Pseudomonas syringae. Plant Cell 15: 317–330.
Tapken, D., Anschütz, U., Liu, L.H., Huelsken, T., Seebohm, G.,
Becker, D., and Hollmann, M. (2013). A plant homolog of animal
glutamate receptors is an ion channel gated by multiple hydrophobic
amino acids. Sci. Signal. 6: ra47.
Thompson, J.D., Higgins, D.G., and Gibson, T.J. (1994). CLUSTAL W:
Improving the sensitivity of progressive multiple sequence alignment
through sequence weighting, position-specific gap penalties and weight
matrix choice. Nucleic Acids Res. 22: 4673–4680.
Torres, M.A. (2010). ROS in biotic interactions. Physiol. Plant. 138:
414–429.
Torres, M.A., Jones, J.D., and Dangl, J.L. (2005). Pathogen-induced,
NADPH oxidase-derived reactive oxygen intermediates suppress spread
of cell death in Arabidopsis thaliana. Nat. Genet. 37: 1130–1134.
Tripathi, V., Parasuraman, B., Laxmi, A., and Chattopadhyay, D.
(2009). CIPK6, a CBL-interacting protein kinase is required for
development and salt tolerance in plants. Plant J. 58: 778–790.
Tsuda, K., and Katagiri, F. (2010). Comparing signaling mechanisms
engaged in pattern-triggered and effector-triggered immunity. Curr.
Opin. Plant Biol. 13: 459–465.
Van Breusegem, F., Bailey-Serres, J., and Mittler, R. (2008).
Unraveling the tapestry of networks involving reactive oxygen species in
plants. Plant Physiol. 147: 978–984.
Role of Cbl10 and Cipk6 in Plant Immunity
Verslues, P.E., Batelli, G., Grillo, S., Agius, F., Kim, Y.S., Zhu, J.,
Agarwal, M., Katiyar-Agarwal, S., and Zhu, J.K. (2007). Interaction
of SOS2 with nucleoside diphosphate kinase 2 and catalases reveals
a point of connection between salt stress and H2O2 signaling in
Arabidopsis thaliana. Mol. Cell. Biol. 27: 7771–7780.
Walter, M., Chaban, C., Schütze, K., Batistic, O., Weckermann, K.,
Näke, C., Blazevic, D., Grefen, C., Schumacher, K., Oecking, C.,
Harter, K., and Kudla, J. (2004). Visualization of protein interactions in
living plant cells using bimolecular fluorescence complementation. Plant
J. 40: 428–438.
Weinl, S., and Kudla, J. (2009). The CBL-CIPK Ca(2+)-decoding signaling
network: Function and perspectives. New Phytol. 184: 517–528.
Xiao, F., He, P., Abramovitch, R.B., Dawson, J.E., Nicholson, L.K.,
Sheen, J., and Martin, G.B. (2007). The N-terminal region of
Pseudomonas type III effector AvrPtoB elicits Pto-dependent immunity
and has two distinct virulence determinants. Plant J. 52: 595–614.
17 of 17
Xie, C., Zhou, X., Deng, X., and Guo, Y. (2010). PKS5, a SNF1-related kinase,
interacts with and phosphorylates NPR1, and modulates expression of
WRKY38 and WRKY62. J. Genet. Genomics 37: 359–369.
Yang, Y., Qin, Y., Xie, C., Zhao, F., Zhao, J., Liu, D., Chen, S.,
Fuglsang, A.T., Palmgren, M.G., Schumaker, K.S., Deng, X.W.,
and Guo, Y. (2010). The Arabidopsis chaperone J3 regulates the
plasma membrane H+-ATPase through interaction with the PKS5
kinase. Plant Cell 22: 1313–1332.
Yoshioka, H., Numata, N., Nakajima, K., Katou, S., Kawakita, K.,
Rowland, O., Jones, J.D., and Doke, N. (2003). Nicotiana
benthamiana gp91phox homologs NbrbohA and NbrbohB participate in
H2O2 accumulation and resistance to Phytophthora infestans. Plant Cell
15: 706–718.
Zuo, J., Niu, Q.W., and Chua, N.H. (2000). Technical advance: An
estrogen receptor-based transactivator XVE mediates highly inducible
gene expression in transgenic plants. Plant J. 24: 265–273.