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A Haplotype-Specific Resistance Gene Regulated by BONZAI1
Mediates Temperature-Dependent Growth Control
in Arabidopsis
Shuhua Yang1 and Jian Hua1,2
Department of Plant Biology, Cornell University, Ithaca, New York 14853
Plant growth homeostasis and defense responses are regulated by BONZAI1 (BON1), an evolutionarily conserved gene.
Here, we show that growth regulation by BON1 is mediated through defense responses. BON1 is a negative regulator of
a haplotype-specific resistance (R) gene SNC1. The bon1-1 loss-of-function mutation activates SNC1, leading to
constitutive defense responses and, consequently, reduced cell growth. In addition, a feedback amplification of the
SNC1 gene involving salicylic acid is subject to temperature control, accounting for the regulation of growth and defense by
temperature in bon1-1 and many other mutants. Thus, plant growth homeostasis involves the regulation of an R gene by
BON1 and the intricate interplay between defense responses and temperature responses.
INTRODUCTION
Plant growth is controlled by internal programs and external
factors. Internally, the basic cellular growth machinery is
programmed in a developmental context to accumulate cell
mass and increase cell number to a certain size. Positional cues
are sent from neighboring cells, and hormones produced locally
or distantly execute global growth regulation on individual cells.
External signals, both biotic and abiotic, also greatly influence
plant growth. Temperature, with its daily fluctuation and
seasonal change, is one of the major environmental factors that
regulate plant growth, distribution, and survival (Long and
Woodward, 1988). One of the most prominent plant responses
to extreme temperatures is cold acclimation, where plants
tolerate freezing better if treated with low temperatures before
being exposed to freezing conditions. Another response is
vernalization, where a long period of cold treatment can accelerate flowering in many plants. The molecular mechanisms of
these responses to extreme temperatures have been revealed
recently through intensive studies (Sheldon et al., 2000;
Viswanathan and Zhu, 2002). Ambient temperatures also affect
various processes of growth and development. Flowering time
and leaf initiation rate are modulated by thermal time (accumulative heat), with plants at higher temperature growing faster and
flowering earlier (Poethig, 2003). Ambient temperature also
influences the ability of plants to respond to many other hormonal, abiotic, and biotic signals. For instance, auxin-mediated
hypocotyl growth, flowering time control by phytochrome, and
some resistance (R) gene–mediated disease resistances are
temperature dependent (Whitham et al., 1994; Gray et al., 1998;
Halliday et al., 2003).
1 These
authors contributed equally to this paper.
whom correspondence should be addressed. E-mail jh299@
cornell.edu; fax 607-255-5407.
Article, publication date, and citation information can be found at
www.plantcell.org/cgi/doi/10.1105/tpc.020479.
2 To
To investigate the molecular mechanisms of growth responses
to ambient temperatures, we are genetically dissecting the
control of growth homeostasis at varying temperatures in
Arabidopsis (Arabidopsis thaliana). Wild-type Arabidopsis plants
achieve a similar size when grown at temperatures ranging from
16 to 308C. The genetic control of this growth homeostasis was
revealed by mutants (such as acaulis1, acaulis 3, acaulis4, and
bonzai1 [bon1]) that cannot maintain constant size at different
temperatures (Akamatsu et al., 1999; Hua et al., 2001). The lossof-function mutant bon1-1 has a dwarf phenotype mostly
because of reduced cell size at 228C, but it resembles the wild
type at 288C, indicating an essential role of BON1 in growth
homeostasis (Hua et al., 2001). BON1 encodes a member of the
copine gene family that is highly conserved among protozoa,
plants, nematodes, and mammals (Creutz et al., 1998). The
deduced BON1 protein, like other copine proteins, has at its N
terminus two calcium-dependent phospholipid binding C2 domains that are mostly found in signal transduction or membrane
trafficking molecules (Rizo and Sudhof, 1998). BON1 binds to
phospholipids in a calcium-dependent manner in vitro, and it is
localized to the plasma membrane in plants (Hua et al., 2001).
The C-terminal region of BON1 shows homology to the A domain
of integrins (Williams et al., 1999) and is proposed to mediate
protein–protein interaction, to possess an intrinsic protein kinase
activity, or both (Caudell et al., 2000). The A domain in BON1
mediates the interaction of BON1 with its putative functional
partner BAP1 that also contains a C2 domain (Hua et al., 2001).
The presence of C2 domains in both BON1 and BAP1 suggests
that they are involved in a biological process regulated by the
membrane system, calcium state, or by both factors.
In addition to regulating growth homeostasis, BON1 is also
shown to modulate defense responses. A bon1 mutant allele
cpn1-1 (which we will refer to as bon1-4) exhibits precocious
cell death and enhanced disease resistance under low humidity or low temperature conditions (Jambunathan et al., 2001;
Jambunathan and McNellis, 2003). It is intriguing that both
growth and defense are affected by bon1 mutations in a
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The Plant Cell
temperature-dependent manner. Emerging molecular data
suggest that these two processes are intricately intertwined.
Mutations or transgenes that perturb cellular metabolism and
plant growth cause lesion mimic phenotypes and defense
responses characteristic of systemic acquired resistance (SAR)
(Mittler et al., 1995; Molina et al., 1999; Clough et al., 2000).
Compromised cell growth is found in some mutants with
constitutive pathogen responses, such as cpr1 and mpk4
(Bowling et al., 1994; Petersen et al., 2000). Furthermore, genes
functioning in the signaling pathway of defense responses, such
as TIP49a, RAR1, and SGT1, play important roles in growth and
development (Austin et al., 2002; Azevedo et al., 2002; Holt et al.,
2002; Liu et al., 2002). Thus, defense responses, once thought to
be specific for pathogen invasions, have extensive interplay with
growth regulation. Moreover, it is becoming evermore evident
that defense responses are intimately connected with abiotic
responses. Light, humidity, and temperature have all been
demonstrated to influence certain defense responses (Dietrich
et al., 1994; Yoshioka et al., 2001; Xiao et al., 2003). The
molecular mechanisms of the interaction between growth,
defense, and abiotic responses are yet to be revealed.
We report in this study an investigation of the molecular
mechanisms of growth and defense regulated by BON1 and
modulated by temperature. We found that BON1 negatively
regulates a haplotype-specific R gene, SNC1. The bon1 lossof-function mutation activates defense responses that lead to
compromised cell growth in a SNC1-containing accession but
not in accessions without a functional SNC1. Furthermore, we
found that SNC1 is under a positive feedback regulation involving
salicylic acid (SA) and that this regulatory loop is subject to
temperature modulation. Thus, plant growth homeostasis
controlled by BON1 involves repression of an R gene and
modulation of defense responses by temperature.
Figure 1. bon1-2 Has Wild-Type Morphology.
RESULTS
Identification of a Natural Modifier of bon1
To better understand the molecular mechanism by which BON1
controls growth homeostasis, we took advantage of a naturally
occurring bon1 modifier. The bon1 mutant alleles bon1-1 and
bon1-2 confer drastically different phenotypes. At 228C, bon1-1
mutants show greatly reduced plant size and have twisted leaves
and short inflorescence stems, whereas bon1-2 mutants resemble the wild type (Figure 1). The phenotypic differences
between the two mutants are unlikely caused by differences in
allele strength because both appear to be loss-of-function
mutations. Both bon1-1 and bon1-2 contain T-DNA insertions
in exons at positions corresponding to amino acids 394 and 61,
respectively, in the BON1 predicted protein. These insertions
would result in truncated proteins. In addition, no wild-type
BON1 transcripts were detected in either of the mutants by RNA
gel blot analysis (data not shown), indicating that both are lossof-function mutants.
Given that both bon1-1 and bon1-2 appear to be loss-offunction mutations, we considered the possibility that the
phenotypic difference was because of differences in accession
Col, bon1-1, Ws, and bon1-2 plants grown at 228C before (A) and after
(B) bolting. bon1-1 is dwarf with dark-green and twisted leaves and short
inflorescence stems. bon1-2 resembles wild-type Ws.
(ecotype) background. bon1-1 was isolated from the Columbia
(Col) accession, whereas bon1-2 was isolated from the
Wassilewskija (Ws) accession. F2 progeny of bon1-1 backcrossed to Col segregated one-quarter of dwarf plants, whereas
F2 progeny of bon1-1 crossed to Ws segregated one-sixteenth
of bon1-1–like dwarf plants (Table 1), suggesting the presence
of a bon1 suppressor in Ws. Consistent with this view, F2 progeny of bon1-2 crossed to the Col wild type segregated
approximately one-sixteenth of bon1-1–like dwarf plants,
whereas F2 progeny of bon1-2 backcrossed to Ws did not
segregate any dwarf plants (Table 1). These data confirm that
the phenotypic differences are attributable to accession background and suggest that the modifier of the bon1 phenotype is
conferred by differences in a single nuclear gene. The latter point
was further supported when a quarter of the F2 progeny from
a cross between bon1-1 and bon1-2 exhibited a dwarf phenotype (Table 1). Natural variations at this locus modify the bon1
phenotype, and we named this locus MOB (modifier of bon1).
BON1 and Temperature Regulate an R gene
Table 1. Analysis of the Genetic Basis of the Phenotypic Differences
between bon1-1 and bon1-2
F2 Progeny of
Wild Type
Mutant
Ratio Expected
P Value
bon1-1
bon1-1
bon1-2
bon1-2
bon1-1
74
100
>400
117
314
23
7
0
8
115
3:1
15:1
ND
15:1
3:1
0.80
0.90
ND
0.94
0.39
3
3
3
3
3
Col
Ws
Ws
Col
bon1-2
P value was calculated based on x2.
The Col variant of MOB is required for the dwarf phenotype of
bon1-1, whereas the Ws variant of MOB dominantly suppresses
this dwarf phenotype.
Cloning of the MOB Gene
We employed a map-based cloning approach to identify the
MOB gene. bon1-1 (in Col) was crossed to bon1-2 (in Ws), and
the dwarf plants segregated in the F2 progeny were chosen for
mapping. Codominant cleaved amplified polymorphic sequence
(CAPS) markers (Konieczny and Ausubel, 1993) and simple
sequence length polymorphism (SSLP) markers (Bell and Ecker,
1994) were used to locate the MOB gene on chromosome IV
between markers SC5 and AG (http://www.arabidopsis.org/
index.jsp) (Figure 2A). New SSLP and CAPS markers between
SC5 and AG were generated between Col and Ws. Further
mapping using 2500 plants refined the position to a 120-kb
region between markers VRN2 and TGCAPS2 (Gendall et al.,
2001) (Figure 2A). The recombination rate of this region is
extremely low compared with the average of 200 to 250 kb/
centimorgan in Arabidopsis, which is likely because of the
presence of the RPP5 complex locus in this region. RPP5 is an R
gene conferring resistance to the oomycete pathogen Peronospora parasitica Noco2 strain in Arabidopsis accession Landsberg erecta (Ler) (Parker et al., 1997). The locus consists of
multiple copies of functional and apparently nonfunctional RPP5
homologs (Noel et al., 1999). In Col, the locus contains eight
homologs of RPP5 (named Col-A to Col-H) covering a region of
80 kb (Figure 2C), whereas in Ler, this locus contains 10 homologs of RPP5 (named La-A to La-J) covering a region of >95 kb
(Noel et al., 1999). There is a pronounced divergence and lack of
synteny among the homologs in this region between Col and Ler,
which may account for the significant recombination suppression noted in previous map-based cloning in this region (Gendall
et al., 2001; Stokes et al., 2002). Although the RPP5 complex
locus in Ws has not been sequenced, the strong recombination
suppression in this region between Col and Ws suggests the
presence of a divergent complex locus in Ws as well.
We reasoned that one of the RPP5 homologs differing
between Col and Ws in this cluster could be the MOB gene
because bon1-1 resembles the gain-of-function mutants of the
second gene in the RPP5 cluster Col-B (At4g16890), also known
as SNC1 and BAL (we will refer to this gene as SNC1). snc1-1
contains a missense mutation, which likely renders the mutant
protein constitutively active (Zhang et al., 2003). bal is an
epigenetic allele of SNC1 (we will refer to it as snc1-2), which
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causes overexpression of SNC1 without an alteration in the
nucleotide sequences (Stokes et al., 2002). Both snc1-1 and
snc1-2 mutations activate an SA-dependent defense response
pathway that leads to enhanced defense responses. In addition,
they both exhibit a dwarf phenotype similarly to bon1-1 (Li et al.,
2001; Stokes et al., 2002). It is thus possible that MOB is one of
the RPP5 homologs in the complex locus and that the activation
of this homolog by bon1-1 leads to the observed growth defects.
If so, the loss of function of this homolog should suppress the
bon1-1 phenotype.
To determine whether mutations in any of the RPP5 homologs
in the cluster can suppress the bon1-1 phenotype, we isolated
mutants of these genes from the Salk T-DNA insertion line
collections (http://signal.salk.edu/cgi-bin/tdnaexpress). Mutant
lines identified for this cluster are shown in Figure 2B underneath
the corresponding genes, and they are all in the Col accession
background. T-DNA insertions are found in exons of SNC1, Col-C,
Col-D, Col-E, Col-G, Col-F, and Col-H, presumably resulting
in nonfunctional proteins even if the genes are transcribed. For
Col-A (also known as RPP4), only mutants with T-DNA inserted
into the introns were found, but they are likely to be loss-offunction mutants because no wild-type transcripts were detected by RNA gel blot analysis (data not shown). Each of these
mutants was crossed to bon1-1, and their F2 progeny were
analyzed. The bon1-1–like dwarf phenotype was observed in
about one-quarter of the F2 progeny from each cross, with the
exception of the crosses between bon1-1 and two snc1 mutants
(data not shown). Both of the two mutants, SALK_047058 and
SALK_052814, have T-DNA insertions in the first exon, and we
named them snc1-11 and snc1-12, respectively. We confirmed
the loss-of-function nature of snc1-11 by RNA gel blot analysis,
revealing a transcript much smaller than that of the wild type in
snc1-11 (Figure 2C). The F2 progeny of the crosses between
bon1-1 and either snc1-11 or snc1-12 segregated the bon1-1–
like phenotype in about one-sixteenth of plants, indicating that
snc1-11 and snc1-12 suppress bon1-1 in a dominant manner.
Furthermore, when mutants of the RPP5 cluster were crossed
to bon1-2, all segregated bon1-1–like phenotypes in the F2
progeny except for crosses between bon1-2 and snc1-11 or
snc1-12, confirming that SNC1 is the MOB gene that mediates
the dwarf phenotype in bon1. bon1-1 snc1-11 double mutants
were identified, and they are indistinguishable from wild-type Col
(Figure 2D).
SNC1 Is Specific to the Col Accession
That MOB (SNC1) is functional in Col indicates that Ws does not
have a functional SNC1 gene. To determine whether this is
because of the absence of an SNC1 ortholog or because of
a mutated SNC1 gene in Ws, we attempted to isolate SNC1-like
genes from Ws. Because genes in the RPP5 cluster from Col
share high sequence homology, primers specific to SNC1, but
not to any of the other RPP5 homologs, were used to amplify
a putative SNC1 variant from Ws using RT-PCR.
This approach produced a cDNA from a gene tightly linked to
the VRN2 marker, making it a likely member of the RPP5 cluster
in Ws (data not shown). Furthermore, the gene has intron splice
sites consistent with those identified in other members of the
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Figure 2. The Col-Specific SNC1 Gene Mediates the bon1-1 Phenotype.
(A) A genetic map of the region carrying the MOB locus. The corresponding nucleotide positions (in Col) are numbered in kilobases below the line. MOB
was positioned between markers VRN2 and TGCAPS2. The number of recombinants confirmed is indicated underneath the markers.
(B) A molecular map of the Col RPP5 complex locus. Predicted genes are shown by arrows indicating the direction of transcription. The locus name, the
complex locus name (Noel et al., 1999), and the genetic name (if known) are given for each gene. T-DNA insertion lines from the Salk Institute are
indicated below the corresponding genes.
(C) SNC1 RNA expression in bon1-1 and bon1-1 snc1-11. SNC1 transcript is 4.5 kb in the wild type and <1 kb in snc1-11.
(D) Suppression of bon1-1 growth phenotype by snc1-11. Col, bon1-1, and bon1-1 snc1-11 grown at 228C are shown. bon1-1 snc1-11 resembles wildtype Col.
RPP5 gene family (Meyers et al., 2003). We have named this gene
SNW for SNC1-like in Ws. The very 39 end of the SNW (possibly
300 bp of the coding sequence) was not obtained, as estimated
by the SNW transcript size and its sequence alignment with other
members. This deduced SNW protein exhibits high homology to
SNC1 only at the N terminus in the Toll and interleukin-1
receptors (TIR) region (Figure 3). Its nucleotide binding (NB) and
Leu-rich repeat (LRR) regions are not more similar to SNC1 than
to RPP4 encoded by the first gene in the Col RPP5 cluster (Figure
3). It thus appears that there is no closely related ortholog of
SNC1 in Ws. In fact, sequence comparison between Col and Ler
accessions indicates that genes in the RPP5 cluster have
evolved rapidly (Noel et al., 1999), and orthologous relationships
may not exist for homologs in different accessions.
To test the hypothesis that SNC1 has rapidly evolved and is
likely unique to Col, we crossed bon1-1 to two other accessions:
Ler and Nossen-0. F2 progeny of these two crosses segregated
dwarf plants at a ratio far less than one-quarter as found in the
cross to Col (data not shown), suggesting that these two
accessions may not have SNC1 orthologs. The SNC1 gene is
likely Col haplotype specific, and a functional SNC1 is required
for the exhibition of growth defect in bon1-1.
SNC1 Mediates the Disease Resistance Phenotype
of bon1
Based on our results, BON1 appears to be a negative regulator of
the R gene SNC1. In this model, SNC1 would be activated in
bon1-1. Activation of an R gene by the recognition of a specific
pathogen avirulence (Avr) gene usually induces a local hypersensitive response, which also triggers a secondary defense
response (SAR) through the signal SA (Glazebrook, 2001).
Constitutive activation of SAR by the activation of SNC1 could
therefore account for the enhanced disease resistance phenotype observed in bon1-4 mutants in the Col background.
To determine whether SNC1 mediates enhanced defense
responses in bon1 mutants, we analyzed and compared the
disease resistance phenotypes of bon1-1, bon1-1 snc1-11, and
bon1-2. Similar to previous observations with bon1-4, bon1-1
exhibited a strong resistance to the virulent pathogen
BON1 and Temperature Regulate an R gene
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Figure 3. Sequence Comparison among SNC1, SNW, and RPP4.
TIR domain and NB-Apaf-1, R proteins, and Ced4 domain are labeled. Identical amino acids are indicated by white letters with black background. High
sequence identity between SNC1 and SNW is found mostly in the TIR and NB-Apaf-1, R proteins, and Ced4 domains. The C-terminal LRR region of
SNW is not more similar to that of SNC1 than to that of RPP4.
P. parasitica at 228C. Under these conditions, wild-type Col is
sensitive, as shown by the formation of sporangiophores on most
leaves several days after exposure to P. parasitica spores
(Figures 4A and 4B). No sporangiophores were detected on any
of the leaves of bon1-1, indicating a strong resistance to this
pathogen (Figures 4A and 4B). Furthermore, the defense
response marker gene PR1 (Pathogenesis Related1) is upregulated in bon1-1 grown at 228C compared with that in the Col wild
type (Figure 4C), suggesting that defense responses involving
SAR are upregulated in bon1-1. By contrast, bon1-2, without
a functional SNC1, does not exhibit an enhanced resistance to
virulent pathogen P. parasitica Emwa1 strain. Sporangiophore
formation is similar on bon1-2 leaves to that on the wild-type
Ws leaves at 228C (Figure 4A), and PR1 gene upregulation is
usually not observed in bon1-2 (data not shown). That the
heightened disease resistance in bon1-1 is mediated by SNC1
is further supported by the suppression of the resistance
phenotype in the loss-of-function mutant snc1-11. bon1-1
snc1-11 is as sensitive to P. parasitica as wild-type Col, as
demonstrated by the similar extent of sporangiophore formation
between the double mutant and Col (Figures 4A and 4B). The
upregulation of PR1 in bon1-1 is greatly suppressed by snc1-11
(Figure 4C), indicating that the enhanced disease resistance in
bon1-1 is largely mediated by SNC1. These data all support
a role for BON1 as a negative regulator of SNC1 in Col wild-type
plants.
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The Plant Cell
The Growth Defect of bon1-1 Is a Result of Constitutive
Defense Responses
Because constitutive defense responses could lead to compromised plant growth in some mutants (Heil and Baldwin, 2002), we
tested whether the dwarf phenotype of bon1-1 is a consequence
of the activation of defense responses. SA is known to be a signal
for local defense and for SAR (Glazebrook, 2001), we therefore
introduced into bon1-1 a nahG transgene encoding an enzyme
that degrades SA (Bowling et al., 1994). bon1-1 nahG plants, in
great contrast with bon1-1, have close to wild-type morphology
(Figure 4C). They also exhibit drastically reduced resistance to P.
parasitica and reduced PR1 expression compared with bon1-1
(Figures 4B and 4C). Because the effect of nahG is more
pleotropic than lowering SA level (Heck et al., 2003), we also
generated a double mutant between bon1-1 and the sid2-1
mutant that is defective in SA biosynthesis (Wildermuth et al.,
2001). The bon1-1 sid2-1 double mutant has a wild-type
phenotype (data not shown), further indicating an involvement
of SA accumulation in the growth defect of bon1-1.
SNC1 belongs to the TIR-NB-LRR type of R genes (Meyers
et al., 2003), and several members of this type of R genes are
shown to require the function of EDS1 and PAD4 (Aarts et al.,
1998; Glazebrook, 2001). We found that both eds1 and pad4 can
suppress the disease resistance phenotype as well as the growth
defects of bon1-1. EDS1 exists as tandem repeats in Col, and
both genes (At3g48090 and At3g48080) appear to encode
functional proteins. A loss-of-function mutant of At3g48090,
SALK_071051, was isolated and named eds1-22. Virulent P.
parasitica sporulated on bon1-1 eds1-22, but to a less extent
than on Col, indicating a partial suppression of bon1-1 resistance
by eds1-22 (Figure 4B). PR1 expression is greatly reduced in
bon1-1 eds1-22 compared with that in bon1-1 (Figure 4C). Furthermore, bon1-1 eds1-22 has a nearly wild-type growth phenotype (Figure 4D). The residual resistance and growth defect in
bon1-1 eds1-22 is likely attributable to the presence of another
functional EDS1 gene, At3g48080, a view supported by the wildtype phenotype of a double mutant generated between bon1-1
and a null eds1-1 mutant from Ws (data not shown). pad4-1
totally suppressed the bon1-1 phenotype. bon1-1 pad4-1 is
as sensitive to P. parasitica as the wild-type Col (Figure 4B),
and PR1 expression is absent in bon1-1 pad4-1 (Figure 4C). In
Figure 4. The bon1-1 Growth Defect Is Induced by Constitutive Defense
Responses.
(A) Disease resistance phenotype of bon1-1, bon1-2, and bon1-1snc111. No growth of P. parasitica is observed in bon1-1, whereas a similar
amount of growth is found between Col and bon1-1 snc1-11
and between the Ws wild type and bon1-2. The experiment was
repeated twice, and similar trends were observed.
(B) Resistance to virulent P. parasitica is suppressed by nahG, eds1-22,
pad4-1, and snc1-11. Sporulations on representative leaves are shown.
(C) Suppression of PR1 gene upregulation in bon1-1 by snc1-11,eds122, and pad4-1 mutations and by the transgene nahG. rRNAs detected
by ethidium bromide staining were used as a control.
(D) Suppression of the dwarf phenotype of bon1-1 by the transgene
nahG and mutations of eds1-22 and pad4-1.
BON1 and Temperature Regulate an R gene
addition, bon1-1 pad4-1 is indistinguishable from the wild type.
By contrast, a mutation in NDR1 required for the function of
several coiled coil (CC)-NB-LRR type of R genes (Aarts et al.,
1998; Glazebrook, 2001) could not suppress the bon1-1
phenotype (data not shown). Thus, the activation of defense
responses mediated by a TIR-NB-LRR type of R gene and SA is
responsible for the growth defect in bon1-1.
SNC1 Is Subject to a Positive Feedback Regulation
We next investigated how SNC1 is regulated by BON1 and found
that the SNC1 transcript level is altered in bon1-1. RNA gel blot
analysis shows that bon1-1 grown at 228C has at least five times
more SNC1 transcript compared with the wild type (Figure 5A).
Overexpression of SNC1 by the strong 35S promoter of
Cauliflower mosaic virus or by an epigenetic mechanism has
been shown to induce constitutive defense responses (Stokes
et al., 2002), making it possible to attribute the activation of SNC1
by bon1-1 to the regulation of SNC1 transcript abundance.
Further study shows that this regulation may be indirect
because of the existence of positive feedback regulation of
SNC1. We found that SNC1 is transcriptionally upregulated by
SA. Wild-type plants treated with SA have drastically increased
amounts of SNC1 transcript compared with control plants
treated with water (Figure 5B). It is thus possible that the bon1
mutation causes activation of the SNC1 protein, which leads to
SA accumulation and a subsequent elevation of the SNC1
transcript. Consistent with this idea, we observed an increase in
SNC1 transcript abundance in snc1-1 (Figure 5A), although
snc1-1 is caused by a missense mutation likely affecting the
SNC1 protein activity (Zhang et al., 2003). To test the hypothesis
that the upregulation of the SNC1 transcript is a secondary
effect, we used pad4 and nahG to block this feedback
regulation. In bon1-1 pad4 and bon1-1 nahG mutants, the
induction of SNC1 by bon1-1 is eliminated (Figure 5C), indicating
that BON1 likely acts on the SNC1 transcript abundance through
feedback regulation.
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The SNC1 Pathway Is Temperature Regulated
BON1 regulates plant growth in a temperature-dependent
manner as indicated by the fact that at a higher temperature
(288C) the growth defects that bon1-1 exhibits at 228C are not
observed. The enhanced disease resistance phenotype of bon14 regulation on defense responses is also temperature dependent (Jambunathan and McNellis, 2003). PR1 expression is
upregulated at 228C but is not detected at 288C in bon1-1 (Figure
6A). We asked whether this temperature regulation could act
upon the SNC1 pathway by analyzing the temperature responses of the snc1 gain-of-function mutants (snc1-1 and snc12) and an SNC1-related mutant, cpr1. The cpr1 mutation has not
been molecularly identified. However, the CPR1 gene was
mapped close to the SNC1 locus and is proposed to be an
epigenetic allele of SNC1 (Stokes and Richards, 2002). All these
mutants have constitutive expression of SAR and exhibit dwarf
phenotypes resembling bon1-1 at 228C (Figure 6B). When these
mutants were grown at 288C, they all had a wild-type morphology
(Figure 6B), indicating that temperature could regulate SNC1 or
its downstream components independently of BON1. Thus,
temperature regulation of the bon1-1 phenotype could be
attributed to its modulation of the SNC1 pathway.
We found that temperature regulates the level of components
in the feedback regulation loop consisting of SNC1, EDS1/PAD4,
and SA. As in bon1-1, the SNC1 RNA transcript at 228C is more
abundant in snc1-1, snc1-2, and cpr1 than in the wild type (Figure
6C). The SNC1 RNA transcript is not detectable at 288C by RNA
gel blot analysis in any of these mutants (Figure 6C), indicating
that temperature regulates the RNA transcript abundance of
SNC1 in the mutants. Transcripts of EDS1 genes are also
upregulated in bon1-1, and this upregulation is abolished by high
temperature (Figure 6D). Similar regulation is found for PAD4
transcript level and SA amount in bon1-1 (data not shown). Thus,
in bon1-1, all components in the regulatory loop tested have
a higher level at 228C than at 288C. Interestingly, EDS1
transcripts appear to be regulated by temperature in wild-type
Col as well. We consistently see more EDS1 transcripts at 228C
Figure 5. SNC1 Is under a Positive Feedback Regulation.
(A) Upregulation of the SNC1 RNA transcripts in bon1-1 and snc1-1 compared with Col.
(B) Induction of the SNC1 transcript by SA application. RNAs were extracted 2 d after plants were sprayed with 0, 0.5, and 1 mM of SA.
(C) Suppression of SNC1 upregulation in bon1-1 by pad4-1 and nahG. Wild-type level of the SNC1 transcripts is observed in bon1-1 pad4-1 and bon1-1
nahG.
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a haplotype-specific R gene. We propose that BON1 is a negative
regulator of the haplotype-specific R gene SNC1 (Figure 7).
Activation of SNC1 by bon1-1 leads to constitutive defense
responses, and through this effect, plant growth is compromised
(Figure 7). Furthermore, SNC1 is under positive feedback
regulation involving EDS1, PAD4, and SA. Temperature primarily modulates one or several of the components in this feedback regulation, conferring a downregulation of all components
in the pathway by higher temperatures (Figure 7). This provides a mechanism for the temperature regulation of the bon1
phenotype.
Interaction of Growth and Defense
Figure 6. Temperature Regulation of the bon1-1 Phenotype.
(A) PR1 expression in bon1-1 at 228C and 288C. The PR1 gene is
upregulated in bon1-1 at 228C but not at 288C.
(B) Phenotypes of Col, bon1-1, cpr1-1, snc1-1, and snc1-2 grown at
228C and 288C. Higher temperature (288C) suppresses the dwarf
phenotype of these mutants exhibited at 228C.
(C) SNC1 RNA expression in the wild type, bon1-1, cpr1-1, and snc1-2
grown at 228C and 288C. Induction of the SNC1 transcript in these
mutants at 228C is abolished at 288C.
(D) EDS1 RNA expression in the wild type and bon1-1 at 228C and 288C.
The EDS1 transcript level is higher at 228C than at 288C in both the wild
type and bon1-1.
Our dissection of growth homeostasis regulated by BON1
underlines the importance of regulating defense responses in
plant growth control. BON1 normally represses the function of
the R gene SNC1. The loss of BON1 function activates SNC1,
leading to constitutive defense responses and subsequently
compromising cell growth. It has long been recognized that
disease resistance has a fitness cost. Compromised cell growth
is sometimes found in mutants with constitutive pathogen
responses, possibly because of resource reallocation from
growth to defense, the metabolic burden of defense responses
on plants, or both (Heil and Baldwin, 2002). There can be a fitness
cost even with the acquisition of a resistance trait such as an R
gene RPM1 (Tian et al., 2003). Thus, defense responses need to
be repressed under most conditions and only need to be induced
in the presence of pathogen invasion.
Creation of new R genes is beneficial for plants because each
new gene can enable the plant to recognize new varieties of
pathogens. Plants appear to drive R gene duplication through
complex R gene loci like that of RPP5, in which duplicated genes
have evolved to confer resistance to distinct pathogens (Parker
et al., 1997; Noel et al., 1999; van der Biezen et al., 2002). The
advantage of multiple R genes, however, is counterbalanced by
the fitness cost of R gene misexpression, making it critical that
each gene be expressed only when needed. A negative
regulation of the R gene activity by endogenous plant genes
like BON1 can confer tight control over the activation of defense
responses and may be prevalent among the R genes.
than at 288C (Figure 6D), suggesting that some components in
the SNC1 pathway might be modulated by temperature even
when the autoregulatory loop is not activated.
Figure 7. Model for Growth Homeostasis Controlled by BON1.
DISCUSSION
In this study, we further investigated the molecular mechanisms of
growth homeostasis and defense responses regulated by BON1.
We identified a natural modifier of bon1, SNC1, which is
BON1 is a negative regulator of the R gene SNC1. SNC1, when activated,
can induce SA accumulation and defense responses that in turn inhibit
cell growth. A positive feedback regulation exists between SNC1 and SA,
and high temperature suppresses this feedback amplification or components in the feedback regulation.
BON1 and Temperature Regulate an R gene
Regulation of SNC1 by BON1
The activation of SNC1 by the bon1-1 mutation demonstrates
that BON1 is a negative regulator of SNC1. This is one of the few
recently uncovered examples where an R gene is regulated by
a plant gene. The plant R gene was proposed to specifically
recognize the pathogen Avr gene in the gene-for-gene model
(Flor, 1971) whose simplest biochemical interpretation is a
receptor-ligand model where the Avr protein directly interacts
and activates the R protein (Van der Biezen and Jones, 1998).
The limited repertoire of R proteins compared with the varieties
of pathogens led to the guard hypothesis. Under the guard
hypothesis, non-R plant proteins are guarded by R proteins. The
non-R plant proteins sense the pathogen Avr proteins and
mediate the activation of the guard R proteins (Dangl and Jones,
2001; Martin et al., 2003). One of the examples is the Arabidopsis
RIN4 protein, a bacterial virulence target guarded by two R
proteins, RPM1 and RPS2. Phosphorylation or elimination of the
RIN4 protein by the Avr proteins activates RPM1 and RPS2,
respectively, and triggers defense responses (Mackey et al.,
2002, 2003; Axtell and Staskawicz, 2003).
The interaction between SNC1 and BON1 supports the guard
hypothesis that R genes are regulated by plant genes and that
there is an indirect interaction between Avr and R proteins. The R
protein SNC1 appears to be normally repressed by a negative
regulator as suggested by the gain-of-function snc1-1 mutation
(Zhang et al., 2003). Whether BON1 is the negative regulator of
SNC1 protein and guarded by SNC1 in analogy to RIN4 by RPS2
is yet to be determined. The pathogen effector Avr for SNC1, if
any exists, is not known. It is therefore currently not possible to
test whether an Avr protein targets BON1 for modification or
elimination, an event that would then be recognized by SNC1.
BON1 is a protein evolutionarily conserved from paramecium to
human, and it likely has a cellular function in an essential process
such as membrane trafficking or signal transduction. This cellular
function also could contribute to defense responses (by facilitating PR protein secretion or by transducing calcium signals)
and thus may be targeted by pathogen Avr proteins. Plants
may counteract this targeting by using SNC1 to monitor the
BON1 protein or other molecules in the BON1 pathway to
recognize pathogen invasion.
The molecular mechanism of how BON1 negatively regulates
SNC1 is not known. SNC1 transcript levels are regulated by
BON1, but this regulation could be indirect and through feedback
regulation of SNC1. We have shown that SA upregulates SNC1
transcripts and that the elevated level of SNC1 could be because
of an accumulation of SA by an activated SNC1 protein. Similar
positive feedback regulation between an R gene and SA has
been observed in plants carrying a mutant form of the R gene
SSI4. Though SSI4 transcript is upregulated in the mutant, the
gain-of-function missense mutation, but not the overexpression
of SSI4, results in an enhanced disease resistance phenotype
(Shirano et al., 2002). In addition, SA is demonstrated to induce
some R genes of the TIR-NB-LRR type but not the CC-NB-LRR
type (Shirano et al., 2002). Thus, defense responses in bon1-1
could be amplified through a positive autoregulation of SNC1
and the upregulation of other R genes by SA. This view would
suggest that the increase in SNC1 transcription in bon1-1
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mutants is indirect and may not be indicative of the primary
mechanism by which BON1 acts in pathogen response.
It is possible that BON1 regulates the protein activity of SNC1
more directly and BON1 might be guarded by SNC1. BON1 and
SNC1 proteins could potentially have direct physical interaction.
BON1 is localized to the plasma membrane (Hua et al., 2001).
The subcellular localization of SNC1 is not known, but it could be
plasma membrane associated as shown for the CC-NB-LRR
type of R protein, RPM1 (Boyes et al., 1998). The association of
BON1 with SNC1 could inhibit the activity of SNC1, and the
elimination of BON1 could therefore activate the SNC1 activity.
Alternatively, there is no physical interaction between BON1 and
SNC1, and the direct regulation of SNC1 is performed by
downstream components of BON1. The regulation of SNC1 by
BON1 could be through other mechanisms, including transcriptional control. For instance, a slight upregulation of SNC1
transcription in bon1-1 (beyond our detection sensitivity) could
be amplified by the positive feedback regulation to induce full
activation of SNC1.
It is intriguing that the loss-of-function snc1 mutation is
dominant over wild-type SNC1 in suppressing bon1-1 phenotype. This phenomenon could be caused by haploid insufficiency. A threshold of SNC1 activity might be required for it to
activate defense responses, and one copy of SNC1 is below the
threshold. A feedback regulation could amplify the activity above
the threshold, conferring a strong activation of defense
responses with two copies of wild-type SNC1 but no apparent
activation with one copy of SNC1.
Though defense responses regulated by BON1 are mostly
mediated by SNC1, an SNC1-independent defense-related
pathway also appears to be regulated by BON1. In bon1-1
snc1-11 double mutants, we consistently observed residual PR1
expression, although at a much reduced level than in bon1-1. We
also occasionally observed PR1 upregulation in bon1-2 but not in
the Ws wild type. Thus, there seems to be a weak upregulation of
defense responses by bon1 mutations in the absence of SNC1.
This could be because of a weak activation of other SNC1related R genes. Alternatively, it could be because of some basal
activation of defense responses by the bon1 mutation, and SNC1
acts as a specific amplifier of the basal resistance.
Interaction of Disease Resistance and Temperature
Responses
Disease resistance is known to be modulated by abiotic factors,
such as light, temperature, and humidity, but the molecular basis
for this modulation is largely unexplored except for a few cases.
In one case, temperature has been shown to modulate RNA
silencing–mediated defense (Szittya et al., 2003). At a low
temperature, both virus and transgene-triggered RNA silencing
are inhibited, and the amount of small interfering RNAs are
gradually reduced from higher to lower temperatures (27 to
158C). In another case, temperature is shown to regulate the
synthesis of SA (Malamy et al., 1992). High temperature prevents
induction of PR genes and disease resistance mediated by the
R gene N, and this is correlated with an inhibition of SA
accumulation, although SA does not appear to be the sole signal
regulated by temperature.
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In this study, we found that the amount of components in the
feedback regulatory loop (including SNC1, EDS1, PAD4, and SA)
is greatly reduced at high temperatures in bon1-1 mutants. This
likely accounts for the modulation of bon1 phenotype by
temperature. It is conceivable that common signaling molecules
and similar feedback regulation exist in defense responses
induced by some other R genes. Temperature regulation of the
level of individual components provides a mechanism for the
modulation of defense responses by environmental signals in
many other cases.
Because of the feedback regulation among SNC1, PAD4/
EDS1, and SA, the regulation of these components by temperature may not be independent. Temperature may primarily
regulate one component in the pathway and subsequently
regulate all components in the pathway through the feedback
regulation. The primary target of temperature regulation is yet
to be determined. It is interesting to note that the EDS1 genes
have a higher expression at 228C than at 288C even in the wild
type, suggesting that temperature regulation could exist independently of feedback amplification. Further study of the
growth homeostasis controlled by BON1 and temperature will
shed more light on the interplay between growth, defense, and
temperature response as well as the evolution and function of R
genes.
METHODS
Plant Growth Conditions and Treatments
Plants were grown at 228C or 288C under constant light with 30 to 70%
relative humidity unless stated otherwise. For SA treatment, 3-week-old
Col plants were sprayed with SA in 0.005% Silwet L-77 or only 0.005%
Silwet L-77 (for control), and tissues were collected 2 d later for RNA
extraction. Pathogen resistance test on P. parasitica was as described
with modifications (Kim and Delaney, 2002). Briefly, 2-week-old plants
were sprayed with strains Noco2 (for plants in Col background) or Emwa1
(for plants in Ws background) and kept at 228C under high humidity
(covered with plastic dome). The number of sporangiophores on the
leaves was counted a week later. Approximately 70 leaves were counted
for each genotype.
larger population of mutant plants was surveyed with SC5 and AG, and
recombinants between MOB and SC5 or MOB and AG were selected.
Information on the existing markers can be found at The Arabidopsis
Information Resource (http://www.arabidopsis.org/index.jsp). New
markers we identified as polymorphic between Col and Ws are as
follows. SC5, AccI digest, Col (0.55 kb) and Ws (0.3 kb and 0.25 kb);
VRN2, Col (1.5 kb) and Ws (0.8 kb); and TGCAP2, DdeI digest, Col (0.5 kb
and smaller fragments) and Ws (0.35 kb and smaller fragments).
Isolation of the SNW Gene
SNC1-specific primers ColB-1 (59-ATATGGAGATAGCTTCTTCTTC
TG-39) and ColB-8 (59-CTTGGAGAGAGGTTCAACCAC-39) were used to
amplify close to full length of the SNW gene from the Ws genomic DNA.
Oligo(dT) (18-mer) was used for cDNA synthesis by reverse transcription
reaction on RNA extracted from Ws. Primers ColB-1 and WsColB2
(59-CCTTGATGACACCTCTACAGTC-39) were subsequently used to
amplify the cDNA of SNW from the cDNA pools.
DNA Analysis and Genotyping
Standard molecular techniques were used (Sambrook et al., 1989).
Genomic DNAs were prepared from a small piece of leaves for PCR
genotyping as described (Konieczny and Ausubel, 1993). Thirty-five
cycles were used to amplify the genomic DNA, and each cycle consists of
948C for 30 s, 528C for 30 s, and 728C for 2 min, and the extension time
varied according to the length of the PCR products.
RNA Analysis
Total RNAs were extracted using Tri Reagent (Molecular Research,
Cincinnati, OH) from 3-week-old plants unless stated otherwise. Ten or
twenty micrograms of total RNAs per sample were used for RNA gel blot
analysis. PR1, EDS1, and PAD4 genes were amplified from the genomic
DNA using PCR. SNC1-specific gene fragment was amplified from exon 1
or exons 1 and 2 of SNC1. The PCR products were then used for singlestranded DNA probe synthesis by 30 cycles of Taq polymerase reaction
with only the antisense primers. Standard hybridization procedure was
followed (Sambrook et al., 1989).
Accession Number
The GenBank accession number for partial SNW genomic sequence is
AY510018.
Plant Materials
bon1-2 was isolated from the Wisconsin Knockout Facility by PCR-based
screening following the procedures instructed by the facility (http://
www.biotech.wisc.edu/Arabidopsis/). The T-DNA insertion site in bon1-2
was identified by sequencing the PCR product amplified with a T-DNA
border primer and a BON1-specific primer. T-DNA insertion lines for
genes in the RPP5 cluster and for EDS1 were isolated from the Salk
T-DNA line collection (http://signal.salk.edu/cgi-bin/tdnaexpress) available through the ABRC. PCR-based genotyping was used to identify
homozygous single mutants and double mutants.
Map-Based Cloning of MOB
The mapping population was created by crossing bon1-1 to bon1-2, and
dwarf plants from the F2 progeny were chosen for mapping. Bulk
segregation analysis was performed on pools of 50 plants with SSLP and
CAPS markers available for Col and Ws (Lukowitz et al., 2000), and the
MOB gene was located on the lower arm of chromosome IV. Subsequently, the MOB gene was placed between markers SC5 and AG. A
Sequence data from this article have been deposited with the EMBL/
GenBank data libraries under accession number AY510018.
ACKNOWLEDGMENTS
We thank G. Fink for discussions; P. Grisafi, H. Garnsey, and M.
Callahan for technical assistance; K. Roberg-Perez for critical reading of
the manuscript; and X. Li, X. Dong, E. Richards, B. Staskawicz, J.
Dewdney, and the ABRC for mutant seeds.
Received December 23, 2003; accepted February 11, 2004.
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