NPR1 in Plant Defense: It's Not over 'til It's Turned over

Abstract

NPR1 is a key transcriptional coregulator in plant defense responses. In this issue, Spoel et al. (2009) demonstrate that proteasome-mediated degradation of NPR1 in the nucleus promotes efficient expression of defense response genes following infection and prevents spurious activation of defensive responses in the absence of infection.

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804 Cell 137, May 29, 2009 ©2009 Elsevier Inc.
Corl et al. provide a nal piece of evi-
dence for the specicity of the EGFR/
ERK pathway in regulating ethanol
resistance. They found t wo subsets of
cells in the y brain that are responsi-
ble for the increase in ethanol sensitiv-
ity caused by overexpression of EGFR.
Overexpression of EGFR in either dop-
aminergic neurons or insulin-producing
cells (IPCs) in th e  y b rain is suf cient to
increase ethanol resistance. The hppy
gene is broadly expressed, so it will be
critical to demonstrate that the site of
action for hppy is also in dopaminergic
neurons and IPCs. This would demon-
strate the necessity of these two neu-
ronal foci for ethanol resistance and
would corroborate the observation
that expression of a dominant-negative
EGFR in IPCs is sufcient to increase
e th a n o l s e n si t i vi t y . S e ve r a l r e g i on s o f t h e
y br ain ha ve be en im pli cated in ethan ol
resistance (Scholz, 2009). Therefore, it
will be impor tant to determine whether
ethanol has broad targets in the brain
with the ERK pathway mediating a sub-
set of the behavioral responses to etha-
nol, or whether several redundant path-
ways are at work in ethanol resistance
with the observed specicity due to the
expression of an ERK pathway-interact-
ing molecule that is unique to IPCs and
dopaminergic neurons.
The new study by Corl et al. boosts our
understanding of alcohol resistance. Yet,
potential targets still abound. Signaling
pathways using cAMP are contenders
for targets of ethanol (Moore et al., 1998).
Ligand-gated ion channels, including the
GABA, acetylcholine, glycine, and NMDA
receptors, as well as various potassium
channels have also been implicated as
ethanol targets (Harris et al., 2008). At
least 100 different knockout mice exhibit
alterations in ethanol sensitivity (Crabbe
et al., 2006). So far, however, remarkably
few of these putative targets have been
shown to bind directly to ethanol (Harris
et al., 2008). Thus, it will be important to
test whether Happyhour itself or a reg-
ulator of Happyhour is a direct ethanol
target. Hopefully, with further genetic
screens and careful validation of tar-
gets we will eventually be able to distill
a cohesive model for how ethanol alters
behavior.
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Systemic acquired resistance (SAR) is an
inducible form of plant defense confer-
ring broad-spectrum immunity to sec-
ondary infection of plant tissues above
the initial infection site. SAR is triggered
by systemic increases in salicylic acid
(SA) levels following local infection by
certain phytopathogens (Durrant and
Dong, 2004) and results in the transcrip-
tional activation of ~10% of the genes in
the Arabidopsis genome. NPR1 (nonex-
pressor of pathogenesis-related genes
1) is a key SAR regulator. NPR1 contains
a BTB/POZ (broad-complex, tramtrac,
bric-à-brac/poxvirus, zinc nger) domain
and an ankyrin-repeat domain. In the
absence of infection, NPR1 is predomi-
nantly oligomeric and sequestered in the
cytoplasm. Upon pathogen challenge,
NPR1 is reduced to a monomeric state
and translocates to the nucleus (Mou
NPR1 in Plant Defense:
It’s Not over ’til It’s Turned over
M. Shahid Mukhtar,1 Marc T. Nishimura,1 and Jeff Dangl1,*
1Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
*Correspondence: dangl@email.unc.edu
DOI 10.1016/j.cell.2009.05.010
NPR1 is a key transcriptional coregulator in plant defense responses. In this issue, Spoel et al.
(2009) demonstrate that proteasome-mediated degradation of NPR1 in the nucleus promotes effi-
cient expression of defense response genes following infection and prevents spurious activation
of defensive responses in the absence of infection.

Cell 137, May 29, 2009 ©2009 Elsevier Inc. 805
et al., 2003). Within the nucleus, NPR1
physically interacts with TGA-bZIP tran-
scription factors, inducing expression
of defense response genes via a largely
unknown mechanism to activate SAR.
Spoel et al. (2009) now show that pro-
teasome-mediated turnover of nuclear
NPR1 regulates SAR. They nd that block-
ing NPR1 degradation by use of protea-
some inhibitors or by genetic knockdown
of Cullin3 (CUL3; a component of cullin-
RING ubiquitin ligases) activates expres-
sion of NPR1 target genes in otherwise
uninduced cells, though to a lesser
extent than salicylic acid treatment.
Spoel et al. observe continual degrada-
tion of nuclear NPR1 in the absence of
inducer, which they suggest is likely to
restrict the ability of NPR1 to serve as
a transcriptional coactivator. Thus, the
authors reasoned that NPR1 degrada-
tion is vital to limiting transcriptional
activation of SAR, thereby avoiding the
tness consequences associated with
a constitutive defense response in the
absence of infection. However, it is still
unclear why NPR1 enters the nucleus
in the absence of inducer or infection.
One plausible explanation is that before
NPR1 is targeted for degradation it may
regulate additional genes in a manner
independent of salicylic acid. NPR1 is
recruited to a cis-regulator y element in
the promoter of the PR1 (pathogenesis-
related 1) gene via an unknown protein(s),
independent of the transcription factor
TGA2 and salicylic acid. Yet, in this case,
the PR1 gene is not activated (Figure 1;
Rochon et al., 2006).
A key observation made by Spoel and
colleagues is that salicylic acid treat-
ment or pathogen-dependent activation
of SAR do not prevent NPR1 degrada-
tion. These unexpected results ques-
tion whether nuclear NPR1 turnover is
required for activation of target genes
and disease resistance. The authors use
a combination of genetic and biochemi-
cal approaches to block NPR1 turnover.
They convincingly demonstrate that
these transcriptional responses are com-
promised in (1) plants with diminished
expression of the E3 ligases CUL3A
and CUL3B, (2) plants that express an
Figure 1. NPR1 Degradation and Plant Defense Responses
Depicted is a model for proteasome-mediated regulation of the transcriptional activity of NPR1.
(A) In uninduced cells, a small amount of monomeric NPR1 (nonexpressor of pathogenesis-related genes 1) is constantly translocating from cytoplasm to the
nucleus. NPR1 is recruited to the PR1 (pathogenesis-related gene 1) promoter through an unknown protein, but NPR1 and TGA transcription factors do not
interact with each other and PR1 is not activated. Interaction of monomeric NPR1 with the CUL3-based E3 ligase protein complex is mediated by an unknown
substrate adaptor protein (Adp-A) before recruitment of NPR1 to other target gene promoters. Ubiquitin (Ub)-dependent NPR1 degradation via a nuclear protea-
some pathway prevents activation of NPR1 target genes.
(B) In cells in which systemic acquired resistance is induced, a large amount of monomeric NPR1 translocates to the nucleus. A pool of NPR1 is phosphorylated
before target gene expression. Both unphosphorylated and phosphorylated NPR1 may interact with TGA transcription factors. NPR1 is also likely to change
partners from unknown protein to TGA transcription factors (Rochon et al., 2006). PR1 is activated following the interaction of NPR1 with a TGA transcription
factor. Unphosphorylated NPR1 is also recruited to target gene promoters by unknown transcription factors, leading to the assembly of the RNA polymerase
II (Pol II) initiation complex and subsequent activation of target gene transcription. This pool of NPR1 may be phosphorylated by a kinase attached to Pol II. A
high-afnity interaction of phosphorylated NPR1 and the CUL3-based E3 ligase protein complex is mediated by a different proposed substrate adaptor protein
(Adp-B). Degradation of NPR1 following target gene activation allows fresh NPR1 to be recruited for the next round of transcription initiation. WRKY proteins
regulate NPR1 transcript levels. Oligomerization of NPR1 occurs through intermolecular disulde bonds. S-nitrosothiol (SNO)-facilitated NPR1 oligomerization
and thioredoxin (TRX)-based monomerization are shown.

806 Cell 137, May 29, 2009 ©2009 Elsevier Inc.
NPR1 protein with phosphorylation
site mutations, and (3) wild-type plants
treated with a proteasome inhibitor.
They observe that the pattern of NPR1
degradation upon pathogen infection is
biphasic. This led the authors to hypoth-
esize that NPR1 is rapidly degraded after
initial activation of target gene transcrip-
tion in preparation for a new round of
transcription initiation following recruit-
ment of fresh NPR1 and other cofactors.
Given that NPR1 degradation occurs
constantly, it remains unclear how the
cell maintains a proper homeostasis
between NPR1 oligomers and mono-
mers. Tada et al. (2008) recently showed
that NPR1 is sequentially oxidized and
reduced leading to NPR1 oligomeriza-
tion and monomerization, respectively,
following infection. Additionally, basal
and salicylic acid-induced expression
of NPR1 appears to be controlled by yet
unidentied WRKY transcription factors
(Figure 1; Eulgem and Somssich, 2007).
This suggests the existence of a feed-
back loop that maintains the oligomeric
form of NPR1 at a particular concentra-
tion in the cytoplasm. This may explain
not only the onset of ef cient SAR by
transcriptional regulation coupled with
proteolysis but also the inactivation of
SAR once cellular salicylic acid concen-
trations decrease to basal levels.
Cullin3-based E3 ligases target BTB
domain-containing proteins for ubiquitin-
dependent degradation, making NPR1 a
potential target of this pathway. Spoel
and coworkers demonstrate that NPR1
associates with CUL3 and other compo-
nents of the COP9 signalosome, which
controls proteasomal degradation. The
authors further support their results with
genetic data, showing that NPR1 protein
stability is enhanced in plants decient in
COP9 or in plants decient in both CUL3A
and CUL3B. As NPR1 does not physi-
cally interact with CUL3, this interaction
is likely to be mediated via an unidenti-
ed BTB domain-containing adaptor
protein. Arabidopsis contains 77 BTB
domain proteins, including ve NPR1
paralogs (Stogios et al., 2005). Zhang
et al. (2006) have shown that NPR3 and
NPR4, like NPR1, can interact with TGA
transcription factors. Surprisingly, plants
lacking both NPR3 and NPR4 display ele-
vated disease resistance and PR1 gene
expression, suggesting that these para-
logs are negative regulators of defense
gene transcription. These phenotypes
are partially dependent on NPR1. Thus,
it is possible that NPR1 paralogs may
facilitate the NPR1-CUL3 interaction. It is
also possible that NPR1 family members
might interact with each other. To gain
further understanding of CUL3 function
in NPR1 degradation, the NPR1-CUL3
interaction should be investigated in
plants lacking functional NPR3, NPR4,
or TGA transcription factors. Moreover,
the NPR1-CUL3 interaction should also
be tested in plants expressing nonfunc-
tional NPR1 alleles to conrm the speci-
city of this interaction.
Proteasome-mediated degradation
is often regulated by posttranslational
modications including phosphoryla-
tion. Spoel and colleagues demonstrate
that NPR1 turnover is promoted by
phosphor ylation of key residues (Ser11/
Ser15) present in an IkB-like phospho-
degron motif. Distinct mechanisms lead
to NPR1 degradation in uninduced and
SAR-induced nuclei: NPR1 phosphory-
lation is not required for degradation in
the former, whereas it is indispensable in
the latter. It remains unclear how CUL3
differentiates between unphosphory-
lated and phosphorylated forms of NPR1
under different physiological conditions.
Compared to wild-type NPR1, the NPR1
protein with phosphomimetic site muta-
tions (NPR1S11D/S15D) exhibits increased
degradation. In comparison, the NPR1
protein lacking these key phosphoryla-
tion sites (NPR1S11A/S15A) displays both
reduced polyubiquitination and reduced
interaction with CUL3. This led to the
proposition that phosphorylation may
create or stabilize a binding site for the
CUL3-based ubiquitin ligase, thereby
regulating degradation. However, other
possible scenarios include additional
modications of NPR1 in response to an
inducer of SAR or replacement of sub-
strate adaptor protein(s) that may facili-
tate interaction between different forms
of NPR1 and CUL3 (Figure 1).
In the model suggested by Spoel et
al., promoter-targeted NPR1 is phospho-
rylated by a kinase associated with RNA
polymerase II following transcription initi-
ation. It is thereby marked as “exhausted,”
becomes rapidly ubiquitinated, and is
then degraded. Notably, NPR1S11D/S15D
can still interact with TGA transcription
factors and efciently induces transcrip-
tion. Therefore NPR1 phosphorylation
could be independent of this turnover
cycle. Alternatively, phosphorylation of
a non-chromatin-bound pool of NPR1
could be mediated by a different kinase.
A similar mechanism is shown for the
yeast transcriptional activator Gcn4,
in which two different kinases, an RNA
polymerase II-associated Srb10 and a
non-chromatin-bound Pho85, contribute
to Gcn4 degradation either by targeting
different pools of Gcn4 or by respond-
ing to different cellular signals (Chi et al.,
2001). Further phosphorylation-depen-
dent modications are also plausible,
including ubiquitination of NPR1 to regu-
late its functional lifetime, as has been
shown for human SRC-3 coactivator (Wu
et al., 2007).
Spoel et al. (2009) provide deep
insights into understanding the opposing
roles of proteasome-mediated degrada-
tion in SAR. Future research will explore
how salicylic acid and phosphorylation
regulate the dynamic formation and dis-
ruption of NPR1-chromatin complexes.
ACKNOWLEDGMENTS
This work was supported by NIH grant
R01GM066025 to J.L.D.
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