Download Mapping out the roles of MAP kinases in plant defense

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Protein-kinase recognition sequence motifs.
Trends. Biochem. Sci.15, 342–346
Patrick J. Hussey*
Timothy J. Hawkins
Dept of Biological Sciences, University of
Durham, South Road, Durham, UK DH1 3LE.
*e-mail: [email protected]
Mapping out the roles of MAP kinases in plant defense
Roger W. Innes
Arabidopsis contains 20 MAP kinase genes,
but their roles in plant physiology have
remained largely unknown because of a
lack of mutants. Recent papers from two
groups have shed new light on the function
of two different MAP kinases. The
Arabidopsis MPK4 gene appears to
negatively regulate salicylic acid-mediated
defense responses and positively regulate
jasmonic acid-induced responses. The
tobacco SIPK gene (orthologous to
Arabidopsis MPK6 ) appears to positively
regulate programmed cell death.
one has shown a complete
MAPKKK–MAPKK–MAPK assembly
other than by heterologous assays in
yeast4,5. Equally significant, no one has
identified a direct target of a plant MAPK,
or determined their biological functions.
The two papers I will discuss here
represent important advances in our
understanding of plant MAPKs in that
they provide our first insights into the
biological functions of two distinct MAPK
family members, Arabidopsis MPK4 and
tobacco SIPK.
Mitogen-activated protein kinases
(MAPKs) are signal transduction
proteins and are found in all eukaryotes
analyzed to date. They are typically
involved in transducing extracellular
signals1. Although MAPKs have been
studied in great detail in animal systems
and in yeast, little is known about their
functions in plants. In animal and yeast
systems, MAPKs are activated by a
kinase relay consisting of an MAPKK
and an MAPKKK (Ref. 1). This also
appears to be true in plants2,3 (Fig. 1).
Activation of MAPKKKs in animals and
yeast is typically controlled by
transmembrane receptors, although
numerous other proteins are known to
regulate MAPKKK activity1.
Piecing together a complete pathway
from a receptor to a MAPK target has not
been accomplished in plants. Indeed, no
First MAPK mutant in plants
http://plants.trends.com
The Arabidopsis genome contains
20 MAPK-like genes6, but until recently
no mutations had been reported. That
has changed with the publication of a
transposon insertion mutation in the
Arabidopsis MPK4 gene by Morten
Petersen et al.7 The mpk4 mutant was
identified in a standard forward-genetic
screen as a severe dwarf. It was only after
the disrupted gene was identified that the
researchers knew that they had found an
MAPK mutant.
Dwarfism in plants can be caused by
many different things; thus the challenge
for Petersen et al. was to determine why
disruption of MPK4 resulted in growth
defects. Previous work had indicated that
MPK4 is activated by multiple abiotic
stresses, including cold, low humidity,
hyper-osmolarity, touch and wounding,
suggesting that its primary role might
be in regulating adaptation to
environmental stress8. Surprisingly, the
mpk4 mutant behaved like wild-type
plants in response to these stresses7.
This forced Petersen et al. to scrap the
abiotic stress connection and think more
broadly about the possible causes of the
dwarfed phenotype.
‘…the mpk4 mutation causes a greatly
elevated level of salicylic acid and…the
majority of the mpk4 mutant phenotypes…
can be alleviated simply by reducing the
endogenous levels of salicylic acid via the
expression of a bacterial salicylate
hydroxylase gene…’
One well known cause of dwarfism in
plants is constitutive expression of
defense responses associated with
elevated levels of salicylic acid9,10.
A quick check of defense gene mRNA
levels in the mpk4 mutant revealed
constitutively high levels, thus
suggesting that MPK4 might directly or
indirectly regulate salicylic acidmediated defenses. This led Petersen
et al. into a thorough genetic and
physiological analysis of defense
response pathways in the mpk4 mutant.
This work yielded several important
conclusions. Foremost among these was
that the mpk4 mutation causes a greatly
elevated level of salicylic acid, and that
1360-1385/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(01)02058-1
Research Update
the majority of the mpk4 mutant
phenotypes (e.g. enhanced resistance to
bacterial and oomycete pathogens,
enhanced defense gene expression) can
be alleviated simply by reducing the
endogenous levels of salicylic acid via the
expression of a bacterial salicylate
hydroxylase gene (NahG).
Significantly, NahG only partially
suppressed the dwarf phenotype of the
mpk4 mutant. Furthermore, induction of
the defense genes PDF1.2 and THI1 by
jasmonic acid is blocked in the mpk4
mutant, even in the NahG-containing
line7. Thus, MPK4 must regulate more
than just salicylic acid levels. It is not
clear whether these three phenotypes
(dwarfism, jasmonic acid insensitivity
and high salicylic acid levels) are
independent effects of the mpk4
mutation, or whether the defect in
jasmonic acid signaling leads indirectly to
elevated levels of salicylic acid and
dwarfism. In support of the hypothesis
the three phenotypes are independent
effects of the mpk4 mutation, there are
precedents from yeast and animal
systems for single MAPKs functioning in
two or more independent signal
transduction pathways, presumably via
independent protein complexes1,11.
Additionally, a single MAPK pathway can
have multiple targets1, thus the multiple
effects of the mpk4 mutation are not
particularly surprising.
Although mpk4 mutant plants are
dwarfed, there appears to be no gross
disruption of cellular homeostasis. This
is seen in the lack of lesions in mpk4
plants and by the relatively small
number of genes whose expression is
altered7. In addition, expression of a
kinase inactive form of the MPK4 protein
in the mpk4 mutant failed to rescue any
of the mutant phenotypes, which argues
against the possibility of ‘promiscuous’
cross-talk occurring among MAPK
pathways in the absence of MPK4
protein12. These observations indicate
that MPK4 plays a specific role in the
regulation of defense responses, and
that the mpk4 mutant phenotype is
not simply the result of pleiotropic
effects caused by gross disruption of
cellular functions.
To fully understand the role of the
MPK4 protein, the upstream and
downstream components of this MAPK
pathway need to be identified. There are
already yeast two-hybrid and in vitro
http://plants.trends.com
TRENDS in Plant Science Vol.6 No.9 September 2001
393
Environmental
signal
H2O2, SA, cold,
drought or wounding
?
Ethylene
Receptor
?
?
ETR1
MAPKKK
ANP1, 2 or 3
AtMEKK1
CTR1
MAPKK
AtMKK4
MEK1
?
MAPK
MPK6
MPK4
?
Targets
?
?
?
ERF1?
EIN3?
Effects
Cell
death?
Auxin
responses
SA
PDF1.2
Triple
response
TRENDS in Plant Science
Fig. 1. Representative examples from Arabidopsis of the three general classes of proposed plant mitogen-activated
protein kinase (MAPK) cascades. Blue arrows indicate positive regulation and red Ts indicate negative regulation.
Abbreviations: CTR1, constitutive triple response factor; EIN3, ethylene insensitive 3 protein; ERF1, ethylene
response factor 1; ETR1, ethylene receptor; PDF1.2, plant defensin protein; SA, salicylic acid.
data that point to the MAPKK and
MAPKKK components that probably
regulate MPK4 (Fig. 1)3–5, but the
upstream receptor(s) and downstream
targets are unknown. Likely candidates
for the downstream targets would be
transcription factors that bind to the
PDF1.2 promoter. Although these have
not been identified, the fact that PDF1.2
induction requires the ethylene signal
transduction pathway13 points to the
EIN3 and ERF transcription factors.
These two proteins are known to
constitute a transcription factor cascade
leading to the induction of ethylene
inducible genes14. However, it is worth
noting in this context that mpk4 mutants
do not display developmental defects
associated with the ein3 mutation, such
as lack of a triple response, nor does
mpk4 suppress the constitutive triple
response of ctr1 mutants (J. Mundy, pers.
commun.), which seems to rule out EIN3
as a likely target, but leave open the
possibility that ERF1 and/or related
transcription factors are targets.
There is even less to go on as far as
identifying upstream receptors that
regulate MPK4, or even the upstream
signals that activate it. Somewhat
unexpected is that neither jasmonic
acid nor ABA activate MPK4 (J. Mundy,
pers. commun.); thus, MPK4 is
currently an MAPK in search of an
activator and a target. However, it is the
only plant MAPK for which we have a
mutant phenotype.
Potential role for tobacco SIPK
In contrast with MPK4, the tobacco SIPK
protein has many known activators,
including avirulent pathogens15.
Unfortunately, no one has been able to
identify an insertional mutation in the
Arabidopsis ortholog, MPK6, in spite of
efforts by multiple laboratories. SIPK was
first identified because it is strongly
activated by wounding and by salicylic
acid, and can be easily detected by in-gel
protein kinase assays16. Using a transient
expression assay in Arabidopsis
protoplasts, Jen Sheen and colleagues
recently identified a family of closely
related MAPKKKs (ANP1, ANP2 and
ANP3) that appear to function at the top
of the SIPK/MPK6 pathway17 (Fig. 1).
Perhaps significantly, these MAPKKKs
also appear to regulate auxin responses
and cell division17–19. This observation
suggests that the failure to isolate
394
Research Update
mutations in MPK6 might be because
it is an essential regulator of the plant
cell cycle.
This possibility is important to keep in
mind when interpreting the recent paper
from Shuqun Zhang and colleagues, who
propose that SIPK can participate
directly in the induction of programmed
cell death in tobacco leaves in response
to pathogen elicitors20. In this work,
a constitutively activated mutant version
of the MAPKK protein, NtMEK2, was
shown to activate SIPK in vitro and in
vivo. Activation of SIPK in tobacco leaves
preceded induction of several known
defense genes and induction of cell death.
This cell death response was apparently
not dependent on salicylic acid because it
was not suppressed by expression of the
NahG transgene20. Although it is
possible that SIPK is directly responsible
for induction of cell death during a
pathogen-induced hypersensitive
response (HR), one must be cautious in
interpreting the above data. Sustained
activation of SIPK by the mutant
MAPKK could be causing pleiotropic
effects (e.g. activation of inappropriate
cell division), which then cause
activation of programmed cell death
via a pathway independent of the
pathogen-induced HR.
However, even with this caution, this
work is significant because it convincingly
shows that NtMEK2 activates SIPK
in vivo. Given the data mentioned above
from Sheen and colleagues17,18, one would
predict that NPK1 (tobacco ortholog
of the Arabidopsis ANP genes) functions
as the MAPKKK that activates
NtMEK2 (Fig. 1). Thus, it should now
be possible to assemble the full
MAPKKK–MAPKK–MAPK cascade for
SIPK/MPK6, which would be a first for
any MAPK pathway in plants. Based on
two-hybrid and complementation
analyses performed in yeast4,5, the full
triad for MPK4 can also be predicted
(Fig. 1). Significantly, the MAPKKKs
predicted to regulate MPK4 and MPK6
belong to distinct subclasses17.
Three general classes of MAPK cascades
in plants
Three subclasses of MAPKKKs have
been defined in plants, the MEKK1 class,
the ANP class and the Raf class17.
Members of all three subclasses have
now been implicated in the regulation of
disease resistance because the Raf-like
http://plants.trends.com
TRENDS in Plant Science Vol.6 No.9 September 2001
MAPKKK gene EDR1 has recently been
shown to negatively regulate plant
defense responses, including
programmed cell death21. In addition, a
second Raf-like gene CTR1 functions to
negatively regulate ethylene inducible
genes22, including the PDF1.2 gene. Thus
the picture that emerges is one of
competing MAPK pathways that
positively and negatively regulate plant
defenses (Fig. 1).
Acknowledgements
I thank John Mundy and his laboratory
members for permission to discuss
unpublished results. Work discussed from
my laboratory was supported by National
Institutes of Health grant R01-GM46451
and a grant from Novartis Agricultural
Biotechnology Research, Inc.
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Roger W. Innes
Dept of Biology, Indiana University,
Bloomington, IN 47405-3700, USA.
e-mail: [email protected]
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