SRFR1 Negatively Regulates Plant NB-LRR Resistance Protein Accumulation to Prevent Autoimmunity
et al. (2010) SRFR1 Negatively Regulates Plant NB-LRR Resistance Protein Accumulation to Prevent Autoimmunity. PLoS
Pathog 6(9): e1001111. doi:10.1371/journal.ppat.1001111
SRFR1 Negatively Regulates Plant NB-LRR Resistance Protein Accumulation to Prevent Autoimmunity
Yingzhong Li 0
Shuxin Li 0
Dongling Bi 0
Yu Ti Cheng 0
Xin Li 0
Yuelin Zhang 0
Ken Shirasu, RIKEN Plant Science, Japan
0 1 National Institute of Biological Sciences , Zhongguancun Life Science Park, Beijing , People's Republic of China, 2 College of Life Sciences, Peking University , Beijing, People's Republic of China, 3 Michael Smith Laboratories , University of British Columbia , Vancouver, British Columbia , Canada
Plant defense responses need to be tightly regulated to prevent auto-immunity, which is detrimental to growth and development. To identify negative regulators of Resistance (R) protein-mediated resistance, we screened for mutants with constitutive defense responses in the npr1-1 background. Map-based cloning revealed that one of the mutant genes encodes a conserved TPR domain-containing protein previously known as SRFR1 (SUPPRESSOR OF rps4-RLD). The constitutive defense responses in the srfr1 mutants in Col-0 background are suppressed by mutations in SNC1, which encodes a TIR-NB-LRR (Toll Interleukin1 Receptor-Nucleotide Binding-Leu-Rich Repeat) R protein. Yeast two-hybrid screens identified SGT1a and SGT1b as interacting proteins of SRFR1. The interactions between SGT1 and SRFR1 were further confirmed by co-immunoprecipitation analysis. In srfr1 mutants, levels of multiple NB-LRR R proteins including SNC1, RPS2 and RPS4 are increased. Increased accumulation of SNC1 is also observed in the sgt1b mutant. Our data suggest that SRFR1 functions together with SGT1 to negatively regulate R protein accumulation, which is required for preventing autoactivation of plant immunity.
To protect themselves from infections by microbial pathogens,
plants have evolved a large number of immune receptors to sense
pathogen-derived molecules and trigger defense responses .
Resistance (R) proteins with nucleotide-binding (NB) and
Leucinerich repeat (LRR) domains constitute the main type of intracellular
plant immune receptors. In animals, similar nucleotide-binding
domain and LRR-containing (NLR) proteins also function as
intracellular immune receptors . In plants, activation of
NBLRR R proteins often results in localized programmed cell death
known as hypersensitive response (HR), accumulation of defense
hormone salicylic acid (SA), and high expression of resistance
marker genes termed Pathogenesis-Related (PR) genes .
Among the components that are required for R protein
triggered immune responses, RAR1, HSP90 and SGT1 are three
conserved proteins that function in correct folding and
stabilization of NLR R proteins . Loss of RAR1 function leads to
compromised resistance mediated by multiple R proteins
[5,6,7,8,9]. Accumulation of barley MLA proteins, potato Rx,
and Arabidopsis RPM1 and RPS5 was reduced when RAR1
function was compromised [7,10,11]. Compromising the activity
of HSP90 also caused reduced accumulation of several R proteins
including RPM1, RPS5 and Rx [11,12,13]. The functions of
SGT1 appear to be more complex. Silencing of SGT1 in Nicotiana
benthamiana resulted in reduced accumulation of Rx, suggesting
that similar to RAR1 and HSP90, SGT1 is required for
maintaining the protein level of Rx . On the other hand,
reduced accumulation of RPS5, but not RPM1 or RPS2, in the
rar1 mutant background can be suppressed by the sgt1b
loss-offunction mutation. It was suggested that SGT1b antagonize RAR1
in regulating the accumulation of certain R proteins .
SGT1 contains three domains including the TPR
(tetratricopeptide repeat) domain, the CS (present in CHP and SGT1
proteins) domain and the SGS (SGT1 specific) domain . RAR1
contains two conserved cysteine and histidine rich domains named
CHORD-I and CHORD-II . Both SGT1 and RAR1 function
as cochaperones of HSP90 [15,16,17]. The CS domain of SGT1
and CHORD-I domain of RAR1 bind to HSP90. The CHORDII
domain of RAR1 binds to SGT1. In Arabidopsis genome, there
are two copies of SGT1 genes, SGT1a and SGT1b. Loss of the
function of both genes lead to lethality . Both plant and animal
NLR proteins are substrates of the HSP90-RAR1-SGT1
chaperone complex . Binding of SGT1 to these substrates is probably
through the SGS domain in SGT1 and LRRs in NLRs .
Arabidopsis SNC1 encodes a TIR-NB-LRR type of R protein
. In the snc1 mutant, a gain-of-function mutation located in the
region between NB and LRR constitutively activates downstream
defense responses. snc1 mutant plants exhibit dwarf morphology,
accumulate high levels of salicylic acid (SA), and constitutively
express pathogenesis-related (PR) genes and resistance to pathogens
. Overexpression of SNC1 also results in constitutive activation
of defense responses . A recent report showed that the
expression of SNC1 is regulated at chromatin level by MOS1,
which encodes a large protein with a conserved BAT2 domain
The nucleotide-binding domain and leucine-rich
repeatscontaining (NLR) proteins are structurally conserved
immune receptors found in both animals and plants.
Correct folding of NLR proteins requires two conserved
proteins, SGT1 and HSP90. We showed that another
evolutionarily conserved protein, SRFR1, interacts with
SGT1 in both yeast two-hybrid assays and
co-immunoprecipitation analysis. Loss-of-function mutations in SRFR1
result in constitutive activation of immune responses. The
constitutive activation of immune responses in the srfr1
mutants is dependent on the NLR Resistance (R) protein
SNC1. In srfr1 mutant plants, levels of multiple R proteins
including SNC1, RPS2 and RPS4 are elevated. Consistent
with previous findings that SGT1b is involved in the
negative regulation of protein levels of certain NLR R
proteins, increased accumulation of SNC1 is also observed
in the sgt1b mutant. Our data suggest that SRFR1
functions together with SGT1 to negatively regulate NLR
R protein accumulation to prevent autoimmunity in plants.
Because autoimmunity is detrimental to plant growth and
development, R protein mediated immunity is subjected to tight
control. Since overexpression of R genes often leads to constitutive
activation of defense responses [20,22], transcription of R genes
need to be controlled properly to keep R protein levels below a
threshold to avoid constitutive activation of R protein-mediated
immune responses. At protein level, without the presence of the
microbial pathogens, R proteins are kept in an auto-inhibited
conformation through intramolecular interactions . Here we
report that an SGT1-interacting protein negatively regulates R
protein accumulation to prevent auto-activation of immune
Identification and characterization of the snc5-1 npr1-1
In Arabidopsis, NPR1 (Nonexpresser of PR genes 1) is an essential
signaling component downstream of SA . To search for
negative regulators of defense responses independent of NPR1, an
npr1-1 suppressor screen was previously conducted . A mutant
named snc5-1 npr1-1 was found to constitutively express the BGL2
(PR2) Promoter-GUS reporter gene in the npr1-1 mutant background
(Figure S1). snc5-1 npr1-1 exhibited a dwarf morphology
(Figure 1A) similar to snc1, an auto-activated TIR-NB-LRR R
gene mutant identified in an independent npr1-1 suppressor screen
. Plants heterozygous for snc5-1 npr1-1 displayed wild type
morphology, indicating that the snc5-1 mutation is recessive.
In snc5-1 npr1-1 mutant plants, both PR1 and PR2 were
constitutively expressed (Figure 1B and 1C). To test whether snc5-1
npr1-1 over-accumulates SA, SA levels in snc5-1 npr1-1 and wild
type plants were measured with high-performance liquid
chromatography (HPLC). As shown in Figure 1D, both free and total SA
(free SA plus glucose-conjugated SA) levels in snc5-1 npr1-1 plants
were much higher than in wild type controls.
Since the defense marker PR genes were activated in snc5-1
npr1-1, we tested whether snc5-1 npr1-1 has enhanced pathogen
resistance. snc5-1 npr1-1 seedlings were challenged with
Hyaloperonospora arabidopsidis Noco2 (H. a. Noco2), an oomycete downy
mildew pathogen virulent on Arabidopsis Col-0 ecotype. As shown
in Figure 1E, sporulation of H. a. Noco2 on snc5-1 npr1-1 plants
was much less than on wild type plants, indicating that defense
responses are constitutively activated in snc5-1 npr1-1.
Map-based cloning of snc5-1
To map the snc5-1 mutation, snc5-1 npr1-1 (in the Col-0 ecotype)
was crossed with the wild type Ler ecotype to generate a
segregating F2 population. In the F2 progeny, plants homozygous
at the snc5-1 locus were identified based on the dwarf morphology
of snc5-1. Interestingly, the percentage of plants with dwarf
morphology in the F2 population was less than one quarter,
suggesting that there may be a natural modifier of snc5-1 in Ler.
Crude mapping using 24 dwarf plants suggested that two loci are
required for the mutant phenotype: one is closely linked to the
lower arm of chromosome 4 (marker F19F18 at 17.7 MB) and the
other is linked to the middle of chromosome 4 (marker FCA5, at 9
For fine mapping of the locus on the lower arm of chromosome
4, we first identified F2 plants homozygous for the Col-0
sequence at marker FCA5 and heterozygous at marker F19F18.
About 500 F3 plants from these F2 lines were genotyped with the
markers T16L1 and F19F18. The snc5-1 mutation was further
mapped to a 92 kb region between markers F6G17 and F19F18
after analyzing the recombinants between T16L1 and F19F18.
Sequence analysis of the genes in this region identified a single G
to A mutation in At4G37460, which introduces an early stop
codon in the middle of the protein (Figure 2A). At4G37460 was
predicted to encode a TPR domain-containing protein. Analysis
of At4G37460 expression using the microarray database at The
Arabidopsis Information Resource found that it is expressed in all
To confirm the mutation in At4G37460 causes the activation of
defense responses, we analyzed two additional T-DNA knockout
alleles of At4G37460, snc5-2 (SAIL_412_E08) and snc5-3
(SAIL_216_F11), both carrying T-DNA insertions in exons of
At4g37460 (Figure 2A). These two mutants showed similar dwarf
morphology as snc5-1 npr1-1 (Figure 2B). RT-PCR analysis
showed that full length At4G37460 was no longer expressed in
the two T-DNA mutants (Figure S2). Both mutants accumulated
high levels of SA (Figure 2C). Consistent with previous reports that
NPR1 functions in negative feedback regulation of SA
accumulation [19,26], the snc5-1 npr1-1 double mutant accumulated
higher levels of SA than the snc5-2 and snc5-3 single mutants. Like
snc5-1 npr1-1, snc5-2 and snc5-3 also constitutively expressed PR1
(Figure 2D) and PR2 (Figure 2E) and exhibited enhanced
resistance to H. a. Noco2 (Figure 2F), suggesting that the mutations
in At4G37460 cause the activation of defense responses. It also
indicates that the locus in the middle of chromosome 4 is probably
a natural modifier of snc5-1.
Recently it was reported that mutants of At4g37460 named srfr1
(suppressors of rps4-RLD) in the RLD ecotype background exhibited
enhanced resistance against Pseudomonas syringae pv. tomato DC3000
expressing avrRps4 . Unlike the snc5 mutants in Col
background, defense responses are not constitutively activated in
the srfr1 mutants identified in RLD ecotype and these mutants
remain fully susceptible to the virulent P.s.t. DC3000 strain
without avrRps4 .
To be consistent with the literature, we renamed snc5-1, snc5-2
and snc5-3 in the Columbia background as srfr1-3, srfr1-4 and
srfr15, respectively. The protein encoded by At4g37460 is referred to as
SRFR1. Sequence analysis revealed that SRFR1 is conserved in
plants and vertebrates (Figure S3), but not present in yeast and
invertebrates such as C. elegans and D. melanogaster. The biochemical
function of the protein is unknown.
Figure 1. Defense responses are constitutively activated in snc5-1 npr1-1. (A) Morphology of wild type (Col-0), npr1-1 and snc5-1 npr1-1
plants grown on soil. The picture was taken when the plants were four weeks old. (BC) Expression levels of PR1 (B) and PR2 (C) in wild type, npr1-1
and snc5-1 npr1-1 compared to Actin 1. Error bars represent standard deviation from three measurements. (D) Free and total SA levels in wild type
(Col-0), npr1-1 and snc5-1 npr1-1. Error bars represent standard deviation from four measurements. (E) Growth of H. a. Noco2 on wild type (Col-0),
npr1-1 and snc5-1 npr1-1. Error bars represent standard deviation from three measurements.
snc5-1/srfr1-3 activates SNC1-mediated resistance
To further map the modifying locus affecting srfr1-3 npr1-1
mutant morphology, we identified F2 plants that are homozygous
for Col-0 at marker F19F18 (close to SRFR1) and heterozygous at
marker FCA5 (close to the modifier). About 500 F3 plants from
these lines were genotyped using the markers FCA5 and F1N20.
The modifier was further mapped to the region between marker
FCA6 and FCA8 after analyzing the recombinants between FCA5
and F1N20. This region contains the RPP4 R-gene cluster (Parker
et al. 1997), which SNC1 is a member of.
To identify the modifier required for the mutant phenotypes of
srfr1-3 npr1-1, we mutagenized srfr1-3 npr1-1 with EMS and looked
for suppressors of srfr1-3 npr1-1. Because the SNC1 locus is highly
polymorphic in different ecotypes , we hypothesized that it
may be the natural modifier. When we sequenced the SNC1 locus
in four of the suppressor mutants, we found that two of them
contained mutations in SNC1 (Figure S4). To confirm that SNC1 is
indeed the modifier of srfr1-3 npr1-1, we crossed snc1-r1, a known
null mutant allele of SNC1 containing a deletion of 8 bp in the first
exon , into srfr1-3 npr1-1. We found that the snc1-r1 srfr1-3
npr1-1 mutant plants displayed wild type morphology (Figure 3A),
a stronger suppression compared to mutant alleles with point
mutations identified from the srfr1-3 suppressor screen. Further
analysis of the triple mutant showed that the elevated SA levels
(Figure 3B), constitutive expression of PR genes (Figure 3C3D)
and resistance to H. a. Noco2 (Figure 3E) in srfr1-3 npr1-1 were
also blocked by the snc1-r1 mutation, suggesting that srfr1-3
activates SNC1-mediated resistance pathways.
To test whether activation of defense responses in srfr1-3 npr1-1
was caused by overexpression of SNC1 at transcription level, the
expression level of SNC1 was determined by real-time RT-PCR.
As shown in Figure 3F, SNC1 expression in srfr1-3 npr1-1 is only
slightly higher than that in wild type and npr1-1 plants. The small
increase in SNC1 transcript level probably is not the cause of the
dramatic phenotypes observed in srfr1-3 npr1-1.
Interestingly, the snc1-r1 srfr1-3 npr1-1 triple mutant is less
susceptible to H. a. Noco2 than the snc1-r1 npr1-1 double mutant
(Figure 3E), suggesting that srfr1-3 may also affect
SNC1independent resistance responses. To test whether srfr1-3 affects
resistance specified by additional R genes, we analyzed resistance
mediated by RPP4, RPS2 and RPS4 in snc1-r1 srfr1-3 npr1-1. As
shown in Figure S5A, the snc1-r1 srfr1-3 npr1-1 triple mutant
displayed enhanced resistance to H. a. Emwa1 comparing to the
snc1-r1 npr1-1 double mutant, suggesting that the srfr1-3 mutation
enhances RPP4-mediated resistance. In addition, snc1-r1 srfr1-3
npr1-1 exhibited enhanced resistance to P.s.t. DC3000 carrying
avrRpt2 or avrRps4 comparing to npr1-1 (Figure S5B and S5C),
indicating that resistance mediated by RPS2 and RPS4 is also
enhanced by the srfr1-3 mutation.
SRFR1/SNC5 interacts with SGT1a and SGT1b
SRFR1 contains a TPR domain at its N-terminal half and a
conserved C-terminal domain with unknown function. Since TPR
domains are often involved in protein-protein interactions, SRFR1
probably functions through association with other proteins. To
identify interacting partners with SRFR1, we performed a yeast
two-hybrid screen using the full-length SRFR1 as bait. Seven
positive cDNA clones were identified on synthetic dropout plates
lacking Histidine (data not shown). Sequence analysis showed that
one clone contained SGT1a (encoding amino acid 1-351) and
another contained SGT1b (encoding amino acid 6-358) cDNA. To
confirm the interactions between SRFR1 and SGT1a/b, the
cDNA clones were recovered from yeast and used for additional
assays. As shown in Figure 4A, both SGT1a and SGT1b interact
with SRFR1 but not the empty vectors in the yeast two-hybrid
assays. b-Gal assays were also performed to confirm the
interactions (data not shown).
To determine which parts of SRFR1 and SGT1a/1b interact
with each other, we created a series of deletion constructs of
SRFR1 and SGT1a/1b (Figure 4B). As shown in Figure 4C and
4D, the N-terminal TPR domain but not the C-terminal half of
SRFR1 interacted with SGT1a and SGT1b, suggesting that
SRFR1 interacts with SGT1 through its TPR domain. When the
TPR domain of SRFR1 was expressed together with the truncated
SGT1a/1b proteins, it was found to interact with the TPR
domains of SGT1a and SGT1b (Figure 4E), but not with the CS
plus SGS domains in the yeast two-hybrid assay (Figure 4F). These
interactions were further confirmed by b-Gal assays (data not
shown). We also tested whether the TPR domain of SRFR1
selfassociates in the yeast two-hybrid assays. As shown in Figure S6,
the TPR domain of SRFR1 interacts with the TPR domain of
SGT1b but not itself.
SRFR1/SNC5 associates with SGT1 in planta
To test whether SRFR1 and SGT1 associate with each other in
planta, we conducted co-immunoprecipitation (co-IP) analysis.
First, we generated a polyclonal antibody against SRFR1, which
has a predicted size of 118 kD. The anti-SRFR1 antiserum
detected a protein around 120 kD present in wild type but not the
srfr1-3 npr1-1 or srfr1-4 mutant plants (Figure S7), indicating that
the antibody specifically detects SRFR1. Next we performed IP
experiments using an anti-SGT1 antibody that can detect both
SGT1a and SGT1b. As a control, we also performed IP using an
anti-MPK4 antibody. Both SRFR1 and MPK4 were localized to
Figure 3. Loss of SNC1 function suppresses constitutive defense responses in snc5-1/srfr1-3. (A) Morphology of wild type (Col-0), snc1-r1
npr1-1, srfr1-3 npr1-1 and snc1-r1 srfr1-3 npr1-1 plants grown on soil. The picture was taken when the plants were four weeks old. (B) Free and total SA
in wild type (Col-0), snc1-r1 npr1-1, srfr1-3 npr1-1 and snc1-r1 srfr1-3 npr1-1. Error bars represent standard deviation from four measurements. (CD)
Expression levels of PR1 (C) and PR2 (D) in wild type (Col-0), snc1-r1 npr1-1, srfr1-3 npr1-1 and snc1-r1 srfr1-3 npr1-1 compared to Actin1. Error bars
represent standard deviation from three measurements. (E) Growth of H. a. Noco2 on wild type (Col-0), snc1-r1 npr1-1, srfr1-3 npr1-1 and snc1-r1
srfr13 npr1-1. Error bars represent standard deviation from three measurements. (F) Expression levels of SNC1 in wild type (Col-0), npr1-1 and srfr1-3 npr1-1
determined by q-RT-PCR. Error bars represent standard deviation from three measurements.
cytosol and nucleus (Figure S8). Proteins that were
immunoprecipitated by the antibodies were subsequently detected by western
blot analysis using the SGT1, MPK4 or SRFR1 antibodies. As
shown in Figure 5, SRFR1 co-immunoprecipitates with SGT1,
but not with MPK4, indicating that SRFR1 and SGT1 associate
with each other in planta.
The SNC1 protein level is elevated in snc5/srfr1 mutants
Since SRFR1/SNC5 interacts with SGT1 and SGT1 has been
shown to regulate R protein stability through its association with
RAR1 and HSP90, we tested whether the accumulation of SNC1
is affected in the srfr1 mutants. We generated a SNC1-specific
antibody against a peptide unique in the SNC1 protein. SNC1 has
a predicted size of 147 kD. The anti-SNC1 antibody detected a
protein around 150 kD in the wild type, but not in the snc1-r1
deletion mutant (Figure 6A), indicating that the antibody is specific
against SNC1. In the srfr1-3 npr1-1, srfr1-4 and srfr1-5 mutant
plants, SNC1 protein levels are much higher than that in the wild
type plants, suggesting that loss of the function of SRFR1 results in
over-accumulation of SNC1. To test whether mutations in SGT1b
and SGT1a affect the accumulation of SNC1, we also analyzed the
SNC1 protein levels in the sgt1b deletion allele edm1-1  and
sgt1a-3, a T-DNA knockout allele of sgt1a. Real-time RT-PCR
showed that the expression of SGT1a was dramatically decreased
in sgt1a-3 (Figure S9). We observed increased accumulation of
SNC1 protein in edm1-1, but not in sgt1a-3 (Figure 6B). Taken
together, both SRFR1 and SGT1b contribute to the negative
regulation of SNC1 stability.
RPS2 and RPS4 protein levels are elevated in snc1-r1
srfr1-3 and snc1-r1 srfr1-3 npr1-1
To test whether srfr1 mutations affect the accumulation of RPS2
and RPS4 proteins, we crossed RPS2-HA or RPS4-HA transgenic
lines, expressed under their native promoters [31,32], into snc1-r1
Figure 4. SRFR1 interacts with SGT1a and SGT1b in yeast two-hybrid analysis. (A) Interactions between full-length SRFR1 with SGT1a and
SGT1b. (B) A diagram of the truncated proteins of SRFR1, SGT1a and SGT1b expressed in yeast two-hybrid vectors. (CD) Interactions between the
TPR domain (C) and C-terminal part (D) of SRFR1 with SGT1a and SGT1b. (EF) Interactions between the TPR domain of SRFR1 with the TPR domains
(E) or CS-SGS domains of SGT1a and SGT1b.
srfr1-3 and snc1-r1 srfr1-3 npr1-1 backgrounds. The snc1-r1
mutation was included in the analysis to avoid the effect of
constitutive activation of defense responses on the accumulation of
the R proteins. In the snc1-r1 srfr1-3 plants, the transcript level of
RPS2 was similar to that in wild type plants whereas the transcript
of RPS4 was about twice as much as that in wild type plants
(Figure 7A and 7B). As shown in Figure 7C and 7D, both
RPS2HA and RPS4-HA accumulated to higher levels in snc1-r1 srfr1-3
than in wild type plants.
In the snc1-r1 srfr1-3 npr1-1 triple mutant, the transcript levels
of RPS2 and RPS4 were similar to those in wild type plants
(Figure 7E and 7F). As shown in Figure 7G, RPS2-HA
accumulated to a higher level in snc1-r1 srfr1-3 npr1-1 than in
wild type. Accumulation of RPS4-HA was also increased in the
triple mutant (Figure 7H), but the increase was not as dramatic as
that observed in the snc1-r1 srfr1-3 double mutant, suggesting that
the increased RPS4-HA protein level in snc1-r1 srfr1-3 was partly
due to increased transcription of RPS4. These data suggest that
In a suppressor screen of npr1-1 to search for negative
regulators of immune responses, we identified snc5-1/srfr1-3 that
constitutively expresses PR genes and pathogen resistance. Since
loss-of-function mutations in SNC1 block activation of defense
responses in srfr1-3 npr1-1, the resistance activated by srfr1-3 is
mediated by the R protein SNC1. In addition, SNC1 protein
over-accumulated in srfr1 mutants, suggesting that SRFR1
regulates the stability of SNC1 and over-accumulation of SNC1
caused the activation of immune responses. A previous study
showed that srfr1 mutants in the RLD ecotype background do not
activate constitutive defense responses . The lack of
constitutive defense responses in the srfr1 mutants is probably due to
the absence of a functional SNC1 gene in the RLD background,
whereas the enhanced resistance to DC3000 with avrRps4 may be
caused by increased accumulation of an unidentified R protein
that recognizes AvrRPS4.
From a yeast two-hybrid screen, we found that SRFR1 interacts
with SGT1a and SGT1b. In planta interactions between SGT1 and
SRFR1 were confirmed by co-IP experiments. Like in srfr1
mutants, elevated SNC1 protein level was also observed in edm1-1,
the deletion mutant allele of sgt1b. This is consistent with SRFR1
and SGT1 function together to regulate the stability of SNC1.
Interestingly, the over-accumulation of SNC1 in sgt1b mutant
plants does not cause constitutive activation of defense responses,
suggesting that SNC1 protein over-accumulated in sgt1b mutant
may have reduced activity. Since SGT1 may have dual functions
in negative regulation of R protein accumulation as well as positive
regulation of R protein folding , it is likely that the
overaccumulated R proteins in sgt1b mutant are not folded correctly
without the assistance of SGT1b, thus not able to trigger immune
SGT1 contains three domains, the N-terminal TPR domain,
the central CS and C-terminal SGS domain. The CS domain
interacts with both RAR1 and HSP90 while the SGS domain may
form contacts with the LRRs of R proteins [10,16]. The function
of the TPR domain is unclear. Interestingly, the TPR domain of
SGT1 is missing in some non-plant species such as C. elegans ,
suggesting that the TPR domain may have a specialized function.
Our study showed that the TPR domain of SGT1 interacts with
SRFR1, suggesting that this domain may function in negative
regulation of R protein accumulation, which is consistent with the
association of SGT1 with components of the SCF (SKP1, Cullin,
F-box protein) ubiquitin ligase complex [33,34] and SGT1 is
required for SCF-mediated auxin responses . The TPR
domain of SGT1b has previously been shown to be dispensable for
the function of SGT1b in regulating R protein mediated resistance
as well as auxin signaling when it was overexpressed . It
remains to be determined whether a truncated SGT1b without the
TPR domain under its own promoter is able to complement the
phenotypes of sgt1b as well as the embryo lethality phenotype in
the sgt1a sgt1b double mutant.
Analysis of SGT1 functions in Arabidopsis has been
complicated by the presence of two closely related SGT1 proteins with
overlapping functions [14,35]. STG1a is expressed at a lower level
than SGT1b, but it has intrinsic activity to complement the sgt1b
mutant when its expression is increased to a certain level. Thus the
mutant phenotypes of sgt1b are probably results of partial loss of
SGT1 functions. While SGT1b has been shown to be required for
the function of a number of R proteins (reviewed by Shirasu ),
RPS5 function is not affected in sgt1b. The SGT1a activity may be
sufficient for proper folding of RPS5. An unexpected result is that
a loss-of-function mutation in SGT1b suppresses the reduced
accumulation of RPS5 and loss of RPS5 function in rar1,
implicating that SGT1b may also play a role in the negative
regulation of R protein accumulation . Our data support the
model proposed by Azevedo et al.  that SGT1 has dual
functions in regulating R protein-mediated immune responses. In
addition to its function as a co-chaperone of HSP90 in positively
regulating R protein folding, it may also be involved in the
negative regulation of R protein stability by association with
SRFR1 through its N-terminal TPR domain.
In addition to SNC1, SRFR1 may regulate the accumulation of
other R proteins. In the srfr1-3 mutant plants, both RPS2-HA and
RPS4-HA fusion proteins accumulate to higher levels than in wild
type plants. Because knockout of SNC1 is sufficient to block the
constitutive defense responses in the srfr1-3 mutant, the increased
accumulation of other R proteins such as RPS2 and PRS4
probably has not reached the threshold levels that would cause
activation of these R proteins. Since SRFR1 and SGT1 are both
conserved in plants and animals and SGT1 is required for the
functions of animal NLR proteins such as NOD1, NOD2 and
NLRP3 [36,37], it will be interesting to test whether the homologs
of SRFR1 in animals also function as negative regulators of NLR
protein-mediated immune responses.
Materials and Methods
as previously described . snc5-2/srfr1-4 (SAIL_412_E08),
snc53/srfr1-5 (SAIL_216_F11) and sgt1a-3 (SALK_122139C) were
obtained from the Arabidopsis Biological Resource Center
(ABRC). Homozygous plants for snc5-2/srfr1-4 were identified
by PCR using primers 59-tcatcactaattccgcaacg-39 and
59-cgacttatgtaacggatcag-39. Homozygous plants for snc5-3/srfr1-5 were
identified by PCR using primers 59-ctatggttctactgagctcg-39 and
59tgctcatggtttagttagcc-39. The RPS2-HA and RPS4-HA transgenic
lines were described previously [31,32].
snc1-r1 npr1-1 is a deletion mutant of snc1 described previously
. The snc1-r1 srfr1-3 npr1-1 triple mutant was generated by
crossing snc1-r1 npr1-1 with srfr1-3 npr1-1 and genotyping the F2
population. srfr1-3 mutation were identified by PCR using primers
59-caattttcctgtcttgaccagggttcg-39 followed by digestion with TaqI. Plants homozygous
for snc1-r1 were identified by PCR using primers 10C-WT-F
(59cctggtgcctgaatgaattg-39) and 10C-R (59-atcatccgatggtgtcatag-39).
Plant material and growth conditions
All plants were grown under 16 hour light at 23uC and 8 hour
dark at 20uC. srfr1-3 npr1-1 was identified from an
EMSmutagenized mutant population in the npr1-1 mutant background
Infection of H. a. Noco2 was carried out on two-week-old
seedlings by spraying with spore suspensions at a concentration of
50,000 spores per ml of water. The plants were kept at 18uC in 12
h light/12 h dark cycles with 95% humidity. Infections were
scored seven days post inoculation by counting the number of
spores with a hemocytometer.
RNA was extracted from the 12-day-old seedlings grown on MS
plates using the RNAiso reagent (Takara). Reverse transcription
(RT) was performed using the M-MLV reverse transcriptase from
Takara. For gene expression analysis, real-time PCR was carried
out using the Perfect Real Time kit (Takara). The sequences of
primers used for amplification of PR-1, PR-2 and Actin1 were
described previously . SA was extracted as previously
described and measured using HPLC .
Map-based cloning of snc5-1/srfr1-3
Markers used for mapping were designed based on the
Monsanto Arabidopsis polymorphisms and Landsberg sequence
collections . The primer sequences for AP20 are
59-gacgacatattgcacattttcatattg39. Primers for F6G17 are 59-cacttccctggtgcgtccaa-39 and
59ggacagaagatacaggtgag-39. The primer sequences for F19F18 are
59-aatcaatgattctatatacacatg-39 and 59-gacgaagattgcttggtgag-39.
The primer sequences for FCA5 are 59-aatgcggtgttacccatggc-39
and 59-actcttccgataaacttcctc-39. The primer sequences for FCA8
are 59-gtcttcctctgccatttcac-39 and 59-gttgcgaaaagcagagattg-39. All
the markers are based on Indel polymorphisms.
Co-immunoprecipitation and antibodies
About 0.9 g of 12-day-old seedlings were ground in liquid
nitrogen to fine powder and 0.9 ml of grinding buffer with 50 mM
Tris-HCl (pH 7.5), 10 mM MgCl2, 150 mM NaCl, 0.1% NP40, 1
mM PMSF, and 1 x Protease Inhibitor Coctail (Roche,
11873580001) was added to the powder. The sample was
resuspended, transferred to 1.5 ml tubes and spun at 21,000 g
for 10 min at 4uC. The supernatant was transferred to a tube
containing 20 ml Protein A agarose beads (GE Healthcare,
171279-03) for pre-cleaning. After rotating for 25 minutes, the
sample was spun at 21,000 g for 5 min at 4uC. 40 ml of the
supernatant was saved as input. Antibody was added to the rest of
the supernatant and the sample was kept at 4uC with continuous
rotation for 23 hours. 20 ml of Protein A agrose beads was
subsequently added to the sample and kept at 4uC with continuous
rotation for 1 hour. The beads were spun down at 4,000 rpm for
30 sec at 4uC. The beads were washed with 1 ml of grinding buffer
for three times before immunoprecipitated proteins were eluted
with 40 ml 2 x SDS loading buffer.
SGT1b and the TPR domain of SRFR1 (a. a. 1-567) were
expressed in E. coli and used to generate the anti-SGT1b and
antiSRFR1 antibodies in rabbit. The Anti-SNC1 antibody was
generated against an SNC1-specific peptide (KAKSEDEKQS).
The anti-MPK4 antibody was from Sigma (A6979). The anti-HA
antibody was from Roche (REF#11867423001). Nuclei-depleted
(DN) and nuclear (N) protein extracts of wild type plants were
prepared as previously described .
Yeast two-hybrid screen
To create the SRFR1 bait plasmid, SRFR1 cDNA was amplified
by primers 59-aaaactgcagggcccatgaggcctcaatcgttgtaagtgctaag-39
and 59-cgcggatccggccgtcaaggccaatggcgacggcgacggcgaca-39 and
cloned into pGBKT7 (Clontech). The plasmid was sequenced
and transformed into the yeast strain Y1348. The Arabidopsis
prey library in pGADT7 was kindly provided by Dr. Qi Xie.
40 mg of the library DNA were transformed to yeast strain
containing the bait plasmid. The transformed yeast cells were
plated on the SD-Leu-Trp-His containing 3 mM 3AT. DNA
inserts from the positive clones were amplified by PCR using
primers T7 and AD-seq-R (59-agatggtgcacgatgcacag-39). The
DNA fragments from PCR were digested with HinfI to group
the positive clones into different classes. DNAs from representative
clones were sequenced. The plasmids from selected positive clones
were extracted and transformed into E. coli to amplify the DNA for
further analysis. For yeast growth assays, overnight yeast cultures
were diluted to different concentration and plated on SD-Leu-Trp
and SD-Leu-Trp-His dropout plates.
To make the bait plasmid containing the N-terminal half of
SRFR1 (a.a 1-567), the cDNA fragment was amplified using
59-aaaactgcaggcccatgaggcctcatgcatcaagttccacgtcaa-39. To make the bait
plasmid containing the C-terminal half of SRFR1 (a.a. 568-1052),
the cDNA fragment was amplified using
59-aaaactgcagggcccatgaggcctcaatcgttgtaagtgctaag-39. The DNA fragments were cloned
into the pGBKT7.
SGT1 fragments were amplified by PCR and cloned into the
prey vector pGADT7. SGT1a-TPR1-120 was amplified using
59-gccgaattctcgagtcattctgtgattagaaaattgc-39. SGT1b1-120 was amplified using
59-gccgaattctcgagtcattcttctgcaatacgaagat-39. SGT1a121-351 was amplified using
59-ccggaattcgaagagaaagatttggttca-39 and 59-cgcggatcctcagatctcccatttcttga-39.
SGT1b121-358 was amplified using
59-ccggaattcgagaaagatttggttcagcc-39 and 59-cgcggatcctcaatactcccacttcttga-39. The bait and
prey vectors expressing the SRFR1 and SGT1 fragments were
cotransformed into the Y1348 strain for yeast two-hybrid assays.
Figure S1 GUS staining of npr1-1 and snc5-1 npr1-1.
Two-weekold seedlings grown on MS media were stained for GUS activity.
Both npr1-1 and snc5-1 npr1-1 contain the BGL2 (PR2)
PromoterGUS reporter gene.
Found at: doi:10.1371/journal.ppat.1001111.s001 (0.22 MB PDF)
Figure S2 Location of the T-DNA insertions (A) and
semiquantitative RT-PCR analysis of SNC5 expression in the T-DNA
knockout mutants snc5-2 and snc5-3 (B). Primers F1 and R1 were
used in PCR amplification of snc5-2. Primers F2 and R2 were used
in PCR amplification of snc5-3. The locations of the primers are
indicated in (A).
Found at: doi:10.1371/journal.ppat.1001111.s002 (0.26 MB PDF)
Figure S3 Alignment of SRFR1 and its homologs in rice, human
and mouse. AtSRFR1, OsSRFR1, MmSRFR1, and HsSRFR1
represent NP_195462, NP_001058749, NP_663582, and
NP_078801 respectively. The sequences were retrieved from
NCBI and aligned by the ClustalX2. The aligned data were
further analyzed by the BOXSHADE 3.21 (http://www.ch.
Found at: doi:10.1371/journal.ppat.1001111.s003 (0.84 MB PDF)
Figure S4 Two suppressor mutants of srfr1-3 npr1-1 carrying
mutations in SNC1. (A) Morphology of snc1-12 srfr1-3 npr1-1 and
snc1-13 srfr1-3 npr1-1. (B) Molecular lesions in SNC1 identified
from snc1-12 and snc1-13.
Found at: doi:10.1371/journal.ppat.1001111.s004 (0.40 MB PDF)
Figure S5 Immunity mediated by RPP4, RPS2 and RPS4 is
enhanced in snc1-r1 srfr1-3 npr1-1. (A) Growth of H. a. Emwa1 on
WT (Col-0), snc1-r1 npr1-1, snc1-r1 srfr1-3 npr1-1, and eds1-2 (Col).
Two-week-old seedlings were sprayed with H. a. Emwa1 at a
concentration of 50,000 spores per ml water. Infection was scored
7 days after inoculation by counting the number of spores per
gram of tissue. Error bars represent standard deviations from three
measurements. (B-C) Growth of P.s.t. DC3000 avrRpt2 (B) and
P.s.t. DC3000 avrRps4 (C) on WT (Col-0), snc1-r1, npr1-1 and
snc1r1 srfr1-3 npr1-1. Leaves of five-week old plants were infiltrated
with P.s.t. DC3000 carrying avrRpt2 or avrRps4 (OD600 = 0.001).
Bacterial growth was determined at Day 0 and Day 3. The values
presented are averages of six replicates 6 standard deviations
(SD). *, P,0.001, significant difference from npr1-1.
Found at: doi:10.1371/journal.ppat.1001111.s005 (0.25 MB PDF)
Figure S6 Yeast two-hybrid analysis of self-association of the
TPR domain of SRFR1.
Found at: doi:10.1371/journal.ppat.1001111.s006 (0.28 MB PDF)
Figure S7 Western blot analysis of the SRFR1 protein in wild
type and snc5 mutants using the anti-SRFR1 antibody.
Found at: doi:10.1371/journal.ppat.1001111.s007 (0.17 MB PDF)
Figure S8 Localization of SRFR1 and MPK4. Immunoblot
analysis of SRFR1 and MPK4 in nuclei-depleted (DN) and nuclear
(N) protein extracts of wild type plants. Equal proportions of
nuclei-depleted and nuclear protein extracts were loaded.
AntiPEPC was used as a cytosolic marker, and anti-Histone H3 was
used as a nuclear marker.
Found at: doi:10.1371/journal.ppat.1001111.s008 (0.47 MB PDF)
Figure S9 Location of the T-DNA insertion in sgt1a-3 (A) and
real-time RT-PCR analysis of SGT1a expression in wild type
(Col0) and sgt1a-3 (B). Primers used for the PCR analysis are indicated
in (A). Error bars represent standard deviation from three
Found at: doi:10.1371/journal.ppat.1001111.s009 (0.23 MB PDF)
We thank Dr. Jianmin Zhou for the anti-SGT1 antiserum, Dr. Qi Xie for
the yeast two-hybrid library, Dr. Zuhua He for the sgt1a-3 mutant seeds,
Dr. Jijie Chai for the bacterial strain expressing the SGT1b protein, Dr.
Jane Parker for the transgenic line expressing the RPS4-HA protein and
Dr. Brain Staskawicz for the transgenic line expressing the RPS2-HA
protein. We thank Jacqueline Monaghan for careful reading of the
manuscript and Yan Li for her help with making the figures. We also thank
the NIBS Antibody Center for making the antibodies used in the study.
Conceived and designed the experiments: SL DB XL YZ. Performed the
experiments: YL SL DB YTC. Analyzed the data: YL SL DB YTC XL
YZ. Wrote the paper: YL XL YZ.
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