Nonsense-Mediated mRNA Decay Factors, UPF1 and UPF3,
Contribute to Plant Defense
Hee-Jeong Jeong1,4, Young Jin Kim1,4, Sang Hyon Kim2, Yoon-Ha Kim3, In-Jung Lee3,
Yoon Ki Kim1 and Jeong Sheop Shin1,*
School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea
Division of Bioscience and Bioinformatics, Myongji University, Yongin 449-728, Korea
3
Division of Plant Bioscience, Kyungpook National University, Daegu 702-701, Korea
4
These authors contributed equally to this work.
*Corresponding author: E-mail, [email protected]; Fax, +82-2-927-9028.
(Received August 5, 2011; Accepted October 18, 2011)
2
In Arabidopsis, the NMD-defective mutants upf1-5 and
upf3-1 are characterized by dwarfism, curly leaves and late
flowering. These phenotypes are similar to those of mutants
showing constitutive pathogenesis-related (PR) gene expression, salicylic acid (SA) accumulation and, subsequently, resistance to pathogens. The disease symptoms of upf1-5 and
upf3-1 mutants were observed following infection with the
virulent pathogen Pst DC3000 with the aim of determining
whether the loss of nonsense-mediated mRNA decay (NMD)
is involved in disease resistance. These mutant plants
showed not only enhanced resistance to Pst DC3000, but
also elevated levels of endogenous SA, PR gene transcripts
and WRKY transcripts. UPF1 and UPF3 expression was
down-regulated in Pst DC3000-infected Arabidopsis plants,
but the expression of various NMD target genes was
up-regulated. The expression of 10 defense-related genes
was elevated in cycloheximide (CHX)-treated plants. The
transcriptional ratios of eight of these 10 defense-related
genes in CHX-treated to non-treated plants were lower in
NMD-defective mutants than in the wild-type plants. These
eight defense-related genes are possibly regulated by the
NMD mechanism, and it is clear that an alternatively spliced
transcript of WRKY62, which contains a premature termination codon, was regulated by this mechanism. Taken together, our results suggest that UPF1 and UPF3, which are
key NMD factors, may act as defense-related regulators associated with plant immunity.
Keywords: Arabidopsis Enhanced disease resistance Nonsense-mediated mRNA decay (NMD) Premature termination codon (PTC) NMD-defective mutants Salicylic
acid (SA).
Abbreviations: c.f.u., colony-forming units; CHX, cycloheximide; dpi, days post-inoculation; ICS, isochorismate synthase;
JA, jasmonic acid; NMD, nonsense-mediated mRNA decay;
PAMP, pathogen-associated molecular pattern; PR gene,
pathogenesis-related gene; Pst DC3000, Pseudomonas syringae
pv. tomato DC3000; PTC, premature termination codon; RT–
PCR, reverse transcription–PCR; SA, salicylic acid; UPF,
UP-FRAMESHIFT; UTR, untranslated region.
Regular Paper
1
Introduction
Nonsense-mediated mRNA decay (NMD) is an mRNA quality
control mechanism in eukaryotes which controls the expression of ‘normal’ genes—approximately 1–10% of yeast, mammalian or plant transcriptome is altered in NMD-defective
cells—and eliminates mRNAs bearing nonsense mutations
by degrading ‘faulty’ transcripts encoding potentially dominant
negative proteins (Rehwinkel et al. 2006, Kurihara et al.
2009, Rebbapragada and Lykke-Andersen 2009). The
UP-FRAMESHIFT(UPF) proteins are known as the conserved
core of the NMD machinery. Three UPF proteins (UPF1, UPF2
and UPF3) were first identified in Saccharomyces cerevisiae, and
four orthologs in human (hUPF1, hUPF2, hUPF3a and hUPF3b)
have also identified (Conti and Izaurralde 2005). The
Arabidopsis genome also contains three UPF genes (van Hoof
and Green 2006). Arabidopsis UPF1 and UPF3 mutants were
analyzed to identify several mRNA substrates for NMD using
microarrays (Hori and Watanabe 2005, Yoine et al. 2006). UPF1
is required for the rapid degradation of mRNAs containing both
spliced and unspliced premature termination codons (PTCs).
In Arabidopsis, UPF3 has been found to suppress aberrantly
spliced mRNAs containing PTCs.
The mRNA surveillance pathway is also known to be
implicated in several essential pathological processes
in human genetic diseases and cancers. There is evidence that
carriers of hereditary diseases and disorders are protected
by the NMD-mediated process, while the clinical manifestations of other genetic disease are exacerbated by NMD
Plant Cell Physiol. 52(12): 2147–2156 (2011) doi:10.1093/pcp/pcr144, available online at www.pcp.oxfordjournals.org
! The Author 2011. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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(Holbrook et al. 2004). However, there is no direct evidence
that NMD is involved in the regulation of pathological processes in plant diseases in a manner similar to its regulation
of pathological processes in human genetic diseases. Indirect
evidence of this possibility was reported by Yi and Richards
(2007) who observed that PTC-containing alternatively spliced
transcripts of SURPRESSOR OF NPR1 (SNC1), which functions in
disease resistance against bacterial pathogens (Stokes et al.
2002), were accumulated in upf1-5 and upf3-1 mutants,
suggesting that these transcripts might be direct NMD targets.
However, Arabidopsis upf1 and upf3 mutants show vegetative
and floral abnormalities, such as jagged leaves, late flowering
and fused flowers (Arciga-Reyes et al. 2006). Mutants showing
disease resistance via constitutive pathogenesis-related (PR)
gene expression as well as salicylic acid (SA) accumulation
also display vegetative and floral abnormalities (Heil and
Baldwin 2002). The similarity of these abnormalities suggests
that loss of NMD may be involved in disease resistance mechanisms against pathogens.
To identify any possible change in infectivity in NMDdefective mutants upon bacterial challenges, we compared
the phenotypes of the insertional mutants upf1-5 and upf3-1
with those of wild-type plants following bacterial infection and
analyzed the expression levels of a subset of genes related to
plant defense. We also examined the expression levels of genes
regulated in the pathological pathway in cycloheximide (CHX)treated Arabidopsis to ascertain whether or not these genes are
bona fide targets for NMD.
Results
and UPF3 are involved in basal resistance rather than
R-gene-mediated resistance.
The PR genes PR1, PR2 and PR5 were more highly
expressed in NMD-defective mutants than in wild-type
plants, while the expression of PR4 decreased dramatically
and that of PDF1.2 decreased slightly in these mutants
(Fig. 2A). Moreover, after bacterial infection, PR1, PR2 and
PR5 were also more highly expressed in NMD-defective mutants than in wild-type plants (Fig. 2B). These results suggest
that the expression of UPF1 and UPF3 suppresses resistance to
the virulent pathogen and is involved in SA-responsive defense
mechanisms.
The SA content and ICS1 expression are higher in
NMD-defective mutants than in wild-type plants
Total SA content in NMD-impaired mutants and the expression levels of ISO-CHORISMATE SYNTHASE 1 (ICS1) encoding
isochorismate synthase, which produces SA from chorismate
(Wildermuth et al. 2001), were examined to determine whether
A
B
NMD-defective mutant plants show enhanced
basal resistance to Pst DC3000
The phenotypes of NMD-defective mutants were similar to
those of mutants showing disease resistance via constitutive
PR gene expression as well as SA accumulation. We hypothesized that loss of NMD is involved in plant disease resistance. To
test our hypothesis, we inoculated wild-type, SA-deficient
NahG transgenic and upf1-5 and upf3-1 insertional mutant
plants with Pst DC3000. The inoculated leaves of wild-type
and NahG transgenic plants showed progressive chlorotic or
necrotic symptoms, whereas similar symptoms were not visible on the leaves of upf1-5 and upf3-1 (NMD-defective mutants) at 6 days post-inoculation (dpi) (Fig. 1A), indicating
that NMD-defective mutant plants exhibited enhanced resistance to Pst DC3000 infection. Consistent with the visual
observation, the numbers of colony-forming units (c.f.u.) per
1 cm2 of leaf tissue of NMD-defective mutants were about
35- and 100-fold less than those of the wild-type and NahG
plants, respectively, during the first 2–3 dpi (Fig. 1B).
However, slightly reduced growth of avirulent bacteria, Pst
DC3000 (avrRpm1), was observed in the NMD-defective mutants (data not shown). These findings suggest that UPF1
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Fig. 1 Responses of Arabidopsis thaliana (ecotype Col-0) wild-type,
upf1-5, upf3-1 and NahG plants to inoculation with Pst DC3000.
(A) Phenotypes of 5-week-old wild-type, upf1-5, upf3-1 and NahG
plants at 6 dpi. (B) Growth rate of Pst DC3000 in wild-type (Col-0),
upf1-5, upf3-1 and NahG plants. Four-week-old plants were
vacuum-infiltrated with 1 104 (A) and 1 105 (B) c.f.u. ml1 of virulent Pst DC3000. Data are presented as the mean ± SD (n = 3).
Different letters denote significant differences between values calculated by Student’s t-test (P < 0.05, n = 3).
Plant Cell Physiol. 52(12): 2147–2156 (2011) doi:10.1093/pcp/pcr144 ! The Author 2011.
NMD factors contribute to plant defense
A
B
Fig. 2 Relative transcript levels of defense marker genes. (A) Relative
transcript levels of SA-dependent defense marker genes (PR1, PR2 and
PR5) and JA-dependent defense marker genes (PR4 and PDF1.2) in
4-week-old wild-type (Col-0), upf1-5 and upf3-1 plants. (B) Relative
transcript levels of SA-dependent defense marker genes (PR1, PR2 and
PR5) in 1 107 Pst DC3000-treated 4-week-old wild-type (Col-0),
upf1-5 and upf3-1 plants 24 h post-inoculation. The expression level
of the wild-type plants was set to 1.0 after normalization relative to
EF1 for the quantitative (q) PCR analysis. Error bars represent the SD
of three replicates. Different letters denote significant differences between values calculated by Student’s t-test (P < 0.05, n = 3).
the enhanced resistance of NMD-defective mutants is involved
in SA-mediated defense. The level of total SA in the upf1-5 and
upf3-1 mutants was 2-fold higher than that in the wild-type
plants (Fig. 3A), and the relative transcript level of ICS1 was
about 3- and 2-fold higher, respectively, in non-infected and Pst
DC3000-infected plants of these mutants (Fig. 3B). SA
is synthesized either from phenylalanine by phenylalanine
ammonia lyase or from chorismate by ICS (Shah 2003). These
results demonstrate that increased ICS1 expression induced an
elevated accumulation of SA in these NMD-defective mutants,
subsequently enhancing resistance to Pst DC3000.
NPR1, EDS1, PAD4, EDS5 and WRKY genes are
more highly expressed in NMD-defective
mutants than in wild-type plants
To confirm whether the enhanced resistance of NMD-defective
mutants is related to the SA-dependent defense pathway, we
analyzed the genes induced by elevated levels of SA by real-time
PCR. NONEXPRESSOR OF PR GENES 1 (NPR1) is an essential
regulator of SA-induced PR gene expression and systemic
acquired resistance (SAR) (Pieterse and Van Loon 2004).
Increased SA induces NPR1 transcription and converts NPR1
from an oligomer to a monomeric form (Mou et al. 2003). The
monomeric NPR1 can interact with TGA factors and in turn
stimulates the binding of TGA factors to the TGA box in the
promoter of PR1 (Johnson et al. 2003). In our experiments,
NPR1 expression levels in the upf1-5 and upf3-1 mutants were
2-fold higher than those in wild-type plants, and, after bacterial
infection, the levels in upf1-5 and upf3-1 mutants were 4- and
2-fold higher than those in wild-type plants, respectively
(Fig. 4A). WRKY18, -53 and -70 are direct targets of NPR1,
and both WRKY18 and WRKY53 positively regulate PR gene
expression (Wang et al. 2006). WRKY18, WRKY53 and WRKY70
were up-regulated in both non-inoculated and Pst
DC3000-inoculated NMD-defective mutants (Fig. 4B). These
results demonstrate that higher expression of NPR1 leads to
induction of these genes. Navarro et al. (2004) reported that
WRKY28 binds to the promoter of ICS1 and up-regulates its
expression. van Verk et al. (2009) proposed that WRKY28 may
be a linker protein between pathogen-associated molecular
pattern (PAMP) signaling and the biosynthesis of SA. In our
experiments, the expression of WRKY28 was higher in upf3-1
mutant plants than in wild-type plants (Fig. 4B) and the
expression of ICS was also elevated in this mutant (Fig. 3B),
implying that higher expression of WRKY28 leads to induction
of ICS1 expression in upf3-1. It is well known that WRKY70 is an
essential requirement for SA-mediated defense against Erysiphe
cichoracearum and Pst DC3000 (Li et al. 2006). WRKY62
is regulated by cytosolic NPR1 (Mao et al. 2007). In our experiments, both WRKY62 and WRKY70 were more highly expressed
in bacteria-infected as well as non-infected upf1-5 and upf3-1
mutants than in the wild-type plants (Fig. 4B). These results
demonstrate that up-regulated expression of WRKY70 induces
SA-dependent defense and that higher expression of NPR1
leads to induction of WRKY62 in these NMD-defective
mutants.
Both ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) and
PHYTOALEXIN DEFICIENT 4 (PAD4) are essential for SA accumulation, and EDS1 is also required for pathogen-induced PAD4
mRNA accumulation. The EDS5 protein regulated by PAD4 is
an essential component of SA-dependent signaling for disease
resistance in Arabidopsis (Nawrath et al. 2002). As shown in
Fig. 4C, the expression levels of PAD4, EDS1 and EDS5 were all
higher in both non-infected and bacteria-infected upf1-5 and
upf3-1 mutants than in the wild-type plants. These results indicate that the up-regulated expression of essential components for SA-dependent defense leads to SA accumulation in
NMD-defective mutants.
Down-regulation of UPF1 and UPF3 affects the
accumulation of NMD target transcripts
We next checked the changes in the expression of UPF1, UPF3
and NMD direct target genes in virulent bacteria-inoculated
Arabidopsis. The expression levels of UPF1 and UPF3 were
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A
B
C
Fig. 3 Total SA contents and relative transcript levels of ICS1. (A) Total SA contents in the wild-type (Col-0), upf1-5 and upf3-1 plants. Data are
presented as the mean ± SD (n = 3). Different letters denote significant differences between values calculated by Student’s t-test (P < 0.05, n = 3).
(B) Relative transcript levels of ICS1 in wild-type, upf1-5 and upf3-1 leaves by real-time quantitative (q) PCR analysis. The expression level of the
wild-type plants was set to 1.0 after normalization relative to EF1 for qPCR analysis. Data are presented as the mean ± SD (n = 3). Different
letters denote significant differences between values calculated by Student’s t-test (P < 0.05, n = 3).
lower in the Pst DC3000-infected plants than in the mock
control but, in contrast, the known NMD target transcripts
At5g40920, AGL88 and MPG accumulated in the infected
plants (Fig. 5A). These results suggest that when plants are
challenged by virulent bacteria, the expression of both UPF1
and UPF3 is down-regulated, which in turn positively affects the
accumulation of NMD target transcripts.
Eight defense-related genes are considered to be
bona fide targets for NMD
We also examined the expression levels of genes regulated in
the pathological pathway in CHX-treated Arabidopsis to
ascertain whether or not these genes are bona fide targets for
NMD. CHX is a translational inhibitor which interferes with the
peptidyl transferase activity of the 60S ribosomal subunit.
As NMD is a translation-dependent process, treatment with
CHX would be expected to inhibit NMD. The transcripts
whose expression was increased in the CHX-treated plants
can be considered to be bona fide targets for NMD
(Arciga-Reyes et al. 2006). As shown above, the expression
levels of 13 defense-related genes showed relatively higher
expression levels in upf1-5 and upf3-1 mutants than in the
wild-type plants (Figs. 2A, 3B, 4). In 10 of 13 genes—PAD4,
EDS1, EDS5, WRKY18, WRKY53, WRKY70, WRKY62, WRKY28,
PR1 and PR2—expression was higher in the CHX-treated plants
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than in the mock-treated control (Fig. 5B). To confirm if the
higher expression of these 10 genes was caused by an
NMD-defective effect, we compared the ratios of the expression
levels in CHX-treated plants with those in non-treated plants of
wild-type, upf1-5 and upf3-1. As shown in Fig. 6A, the ratios of
PAD4, EDS5, WRKY18, WRKY53, WRKY62, WRKY28, PR1 and
PR2 expression levels in wild-type plants were higher than
those in NMD-defective mutants. Thus, only these eight
genes can be considered to be bona fide targets for NMD.
To identify if any are PTC-containing aberrant transcripts
via alternative splicing, we amplified transcripts from the first to
last exons by reverse transcription–PCR (RT–PCR) using the primers (named ‘-ORF’) that are listed in Supplementary Table
S1. We found that only WRKY62 produced two alternative
transcripts (Fig. 6B). One of these two transcripts contained
a PTC but the other did not. Within the detection limit of our
analysis, we could not detect more splicing variants. The
expression levels of PTC-containing WRKY62 transcripts,
WRKY62-2 (e+b) and WRKY62-2 (f+b) that were amplified in
the same transcript with different primer sets, and the normal
transcript (WRKY62-1) were 3.5-fold and 57-fold higher,
respectively, in CHX-treated plants than those in non-treated
plants (Fig. 6D, left). The expression ratios of these transcripts
in CHX-treated to non-treated plants were higher in wild-type
plants than in NMD-defective mutants (Fig. 6D, right). Taken
all together, we concluded that these eight defense-related
Plant Cell Physiol. 52(12): 2147–2156 (2011) doi:10.1093/pcp/pcr144 ! The Author 2011.
NMD factors contribute to plant defense
A
genes, especially two WRKY62 transcripts, may be targets for
NMD.
Discussion
NMD-defective mutants show enhanced resistance
to Pst DC3000 via an SA-dependent pathway
B
C
Fig. 4 The expression of NPR1, WRKY genes and genes regulating ICS1
expression in wild-type, upf1-5, upf3-1 and NahG leaves by real-time
quantitative (q) PCR analysis. (A) Relative transcript levels of NPR1 in
wild-type, upf1-5, upf3-1 and NahG leaves by real-time qPCR.
(B) Relative transcript levels of WRKY genes in wild-type, upf1-5,
upf3-1 and NahG leaves by real-time qPCR. (C) Expression pattern
of genes regulating ICS1 expression (PAD4, EDS1 and EDS5) in
wild-type (Col-0), upf1-5, upf3-1 and NahG plants. The expression
level of the wild type was set to 1.0 after normalization relative to
EF1 for qPCR analysis. Data are presented as the mean ± SD (n = 3).
Different letters denote significant differences between values calculated by Student’s t-test (P < 0.05, n = 3).
Our findings of enhanced resistance against bacteria (Fig. 1)
and up-regulated expression of SA-dependent marker genes,
PR1, PR2 and PR5, in NMD-defective mutants (Fig. 2) indicate
that the expression of UPF1 and UPF3 suppresses resistance to
the virulent pathogen via SA-responsive defense mechanisms.
Elevated SA contents in NMD-defective mutants without bacterial infection and up-regulated ICS1 transcriptional expression with or without infection (Fig. 3) also support this
conclusion. However, SA contents in NMD-defective mutants
at 24 h post-inoculation were not higher than those in
wild-type plants (data not shown). Therefore, we concluded
that the higher SA contents in NMD-defective mutants
induce dwarfism, curly leaves, late flowering, enhanced basal
resistance and up-regulated expression of the essential components of SA-dependent signaling for disease resistance in these
mutants, but levels of SA accumulation induced by pathogen
infection were not very different between NMD-defective mutants and the wild-type plant.
In our experiments, NPR1 expression levels in the upf1-5 and
upf3-1 mutants were 2-fold higher than those in wild-type
plants (Fig. 4A) and PR1 expression was simultaneously
up-regulated (Fig. 2A, B). These results indicate that elevated
levels of SA induce NPR1 transcription, which in turn leads to
increased levels of NPR1 in the monomeric form, thereby activating PR1 gene expression in NMD-defective mutants.
WRKY18, WRKY53 and WRKY70 are direct targets of NPR1,
and both WRKY18 and WRKY53 positively regulate PR gene
expression (Wang et al. 2006). As shown in Figs. 3B and 4B,
ICS1 was highly expressed in both upf1-5 and upf3-1, while
WRKY28 was highly expressed in upf1-5, but not in upf3-1,
compared with the wild-type. The increased expression of
WRKY28 and ICS1 in upf3-1 may infer that its resistance
is derived from PAMP signaling and the biosynthesis of
SA, whereas the increased expression of ICS1 but not
WRKY28 in upf1-5 infers its enhanced resistance by SA accumulation. In turn, WRKY70 functions to ‘fine-tune’ the SA and
jasmonic acid (JA) defense pathways by inducing SA-dependent
responses and repressing JA-dependent responses (Li et al.
2004, Li et al. 2006). WRKY62, which is regulated by cytosolic
NPR1, negatively modulates JA-responsive gene expression
(Mao et al. 2007). In our experiments, both WRKY62 and
WRKY70 were more highly expressed in upf1-5 and upf3-1 mutants than in the wild-type plants (Fig. 4B), and JA-dependent
defense marker genes, PR4 and PDF1.2, were down-regulated in
these mutants (Fig. 2A), indicating that up-regulated expression of WRKY62 and WRKY70 may down-regulate the expression of JA-dependent defense genes in NMD-defective mutants.
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A
B
Fig. 5 Expression pattern of NMD key factors and NMD target genes in Pst DC3000-treated Arabidopsis and expression levels of defense-related
genes in CHX-treated Arabidopsis. (A) mRNA expression analysis of NMD key factors (UPF1 and UPF3) and NMD target genes in Pst
DC3000-treated Arabidopsis. Mock, 10 mM MgCl2; infected, vacuum-infiltrated with 1 105 Pst DC3000. Expression of the mock (10 mM
MgCl2-only) wild-type (Col-0) plant was set to 1.0 after normalization relative to EF1 for the quantitative (q) PCR analysis analysis. Error
bars represent the SD of three replicates. (B) Increased expression of PAD4, EDS1, EDS5, WRKY18, WRKY53, WRKY70, WRKY62, WRKY28, PR1 and
PR2 genes in CHX-treated plants. Mock, buffer only; CHX, 20 mM CHX. Expression of the mock (buffer-only) wild-type (Col-0) plant was set to 1.0
after normalization relative to EF1 for qPCR analysis. Error bars represent the SD of three replicates.
As shown in Fig. 4C, the expression levels of PAD4, EDS1 and
EDS5 were all higher in both non-infected and bacteria-infected
upf1-5 and upf3-1 mutants than in the wild-type plants, revealing that up-regulated PAD4, EDS1, EDS5 and ICS1 induce SA
accumulation in NMD-defective mutants. All these results suggest that elevated expression of PAD4, EDS1 and EDS5 in
NMD-defective mutants can induce the expression of ICS1
and in turn accumulate SA, maintaining the higher expression
of NPR1, WRKY18, WRKY53, WRKY70, PR1, PR2 and PR5 in
these mutants.
Regulation of defense-related genes via the
NMD mechanism may enhance pathogen
resistance in NMD-defective mutants
We observed that the expression levels of UPF1 and UPF3 were
lower in the Pst DC3000-infected plants than in the mock control, but, in contrast, the known NMD target transcripts
At5g40920, AGL88 and MPG accumulated in the infected
plants (Fig. 5A), and PAD4, EDS1, EDS5, WRKY18, WRKY53,
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WRKY70, WRKY62, WRKY28, PR1 and PR2 expression was
higher in the CHX-treated plants than in the mock-treated
control (Fig. 5B). We also observed that the ratios of PAD4,
EDS5, WRKY18, WRKY53, WRKY62, WRKY28, PR1 and PR2
expression levels in CHX-treated wild-type plants to those
in non-treated wild-type plants were higher than in NMDdefective mutants (Fig. 6A) and that one of two alternatively
spliced transcripts of WRKY62 was a PTC-containing aberrant
transcript (Fig. 6B). These results suggest that the NMD mechanism regulates the expression of several defense-related genes
and subsequently can control plant defense.
AtUPF1 is involved in not only NMD but also in RNA interference (Arciga-Reyes et al. 2006) and the sugar signaling pathway (Yoine et al. 2006). AtUPF3 is also known to suppress
aberrant spliced PTC-containing mRNA in Arabidopsis
(Hori and Watanabe 2005) and to be enriched in the nucleolus
(Kim et al. 2009). Because AtUPF1 and AtUPF3 can affect not
only NMD but also other mechanisms, the up-regulated
disease-related genes in the NMD-defective mutants, upf1-5
and upf3-1, can also be affected by other mechanisms. Thus,
Plant Cell Physiol. 52(12): 2147–2156 (2011) doi:10.1093/pcp/pcr144 ! The Author 2011.
NMD factors contribute to plant defense
A
B
C
D
Fig. 6 The expression of defense-related genes in CHX-treated wild-type plants and NMD-defective mutants. (A) The ratios of the expression
levels of those genes in CHX-treated plants to those in non-treated plants of the wild type, upf1-5 and upf3-1. (B) Detection of possible alternative
transcripts of PAD4, EDS5, WRKY18, WRKY53, WRKY62, WRKY28, PR1 and PR2 genes in CHX-treated plants. The defense-related genes
(continued)
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the expression levels of those 13 disease-related genes induced
in NMD mutants were identified in CHX-treated plants. As
expected, the expression levels of ICS1, WRKY60 and PR5 in
CHX-treated plants were not higher than those in the wild
type (Fig. 5B).
Both normal termination codons and PTCs can elicit NMD,
and PTC recognition is not a significant step in the NMD mechanism. Moreover, the criterion of NMD substrates remains unclear in mammals as well as plants (Rebbapragada and
Lykke-Andersen 2009). It is known that mRNA which was
more highly expressed in NMD-defective cells than in the
wild type and accumulated in cells treated by a translational
repressor, such as CHX, can be considered as a substrate for
NMD (Rebbapragada and Lykke-Andersen 2009). Generally, the
expression levels of NMD targets in CHX-treated plants are
10-fold higher than in non-treated plants, whereas in our
experiments the expression levels of defense-related genes
which were up-regulated in NMD-defective mutants were 11to 79-fold higher. Because CHX can induce effects other rather
than an NMD-inhibiting effect, we compared the ratios of gene
expression in CHX-treated to those in non-treated wild-type
plants with those ratios in NMD-defective mutants in order to
exclude other effects of CHX treatment. Because the NMD
system is already destroyed in these mutants, the NMDinhibiting effect of CHX would be decreased compared with
the wild-type plants. If the expression ratio in a gene is lower in
NMD-defective mutants, this gene can be a bona fide target for
NMD. In our experiments, the ratios of PAD4, EDS5, WRKY53,
WRKY62, WRKY28, PR1 and PR2 expression levels in
CHX-treated upf1-5 mutants to those in non-treated upf1-5
were lower compared with the wild-type, and the ratios of
EDS5, WRKY18, WRKY62, PR1 and PR2 expression levels in
CHX-treated upf3-1 to those in non-treated upf3-1 were also
lower compared with the wild-type. Thus, we could conclude
that UPF1, not UPF3, regulates PAD4 and WRKY53 expression,
UPF3, not UPF1, regulates WRKY18 and WRKY28, and both
UPF1 and UPF3 control the expression of EDS5, WRKY62, PR1
and PR2. Therefore, these eight defense-related genes can be
bona fide targets for NMD, and regulation of defense-related
genes via the NMD mechanism may enhance resistance to
pathogens in NMD-defective mutants.
To identify which transcripts of these eight genes bear nonsense mutations, we surveyed whether or not these eight genes
contain a long 30 untranslated region (UTR), introns in the
30 UTR and an upstream open reading frame (uORF).
However, we could not find them in these eight genes. Thus,
we amplified from the first to the last exon by RT–PCR to
determine whether or not any genes produce alternatively
spliced transcripts because alternative splicing can make a
PTC via frameshift. We found that only WRKY62 produced
two alternative transcripts (Fig. 6B) and that one of the two
transcripts of WRKY62 was a PTC-containing aberrant transcript, while the other transcript appeared to be normal.
In addition, the remaining genes (PAD4, EDS5, WRKY18,
WRKY53, WRKY28, PR1 and PR2) did not produce any alternative forms of transcripts. Taken all together, we therefore conclude that the NMD factors UPF1 and UPF3 do play a role in the
host defense pathway, possibly via controlling PTC-containing
abberant and normal transcripts of several defense-related
genes in Arabidopsis.
Materials and Methods
Plant materials and growth conditions
Arabidopsis thaliana (ecotype Col-0), T-DNA insertion lines
[SALK_112922 (upf1-5) in UPF1 and SALK_025175 (upf3-1) in
UPF3] and the SA-deficient NahG transgenic plant, which
served as a susceptible control, were grown in soil (Sunshine
Mix #5; Sun Gro Horticulture) at 22 C, 70–80% relative humidity under 12/12 h light/dark conditions.
Pathogen infection
Pst DC3000 were cultured in fresh King’s B medium and then
resuspended in 10 mM MgCl2 to a final concentration of
104–107 c.f.u. ml1 with 0.005% Silwet L-77 (Lehle Seeds).
Leaves of 4-week-old Arabidopsis plants were then vacuum
infiltrated with bacteria and rinsed with tap water to remove
excessive bacteria on the surface of leaves. Pst DC3000 were
recovered on King’s B media containing rifampicin (Katagiri
et al. 2002).
CHX treatment
Two-week-old Arabidopsis seedlings were vacuum infiltrated
with 20 mM CHX (BioVision) in 0.1 MS medium [0.44 g l1
Murashige and Skoog Basal Salt Mixture (Duchefa), 3 g l1 sucrose, pH 5.8] for 5 min. The seedlings were kept at room temperature for 4 h and used for total RNA extraction.
Fig. 6 Continued
accumulated in CHX-treated Arabidopsis were amplified from the first to the last exons by RT–PCR. EF1 was used as an internal control. Mock, buffer
only; CHX, 20 mM CHX. (C) The structures of pre-mRNA and two alternatively spliced WRKY62 variants. The first diagram shows the structure of
pre-mRNA and the positions of primers used for RT–PCR and real-time quantitative (q) PCR analysis. Descriptions of these primers are given in
Supplementary Table S1. WRKY62-1 and WRKY62-2 are alternatively spliced WRKY62 variants. White boxes, black boxes, lines and arrows indicate,
respectively, UTRs, exons, introns and primers. a, WRKY62_F; b, WRKY62_R; c, WRKY62_ORF_F; d, WRKY62_ORF_R; e, WRKY62_intron_F; f,
WRKY62_intron_1_F. (D) Relative expression levels of WRKY62 transcripts in CHX-treated Arabidopsis by real-time qPCR. Mock, buffer only; CHX,
20 mM CHX. Expression of the mock (buffer-only) wild-type (Col-0) plant was set to 1.0 after normalization relative to EF1 for qPCR analysis. Error bars
represent the SD of three replicates (left). The ratios of expression levels of those genes in CHX-treated plants to those in non-treated plants of wild-type,
upf1-5 and upf3-1 (right).
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Plant Cell Physiol. 52(12): 2147–2156 (2011) doi:10.1093/pcp/pcr144 ! The Author 2011.
NMD factors contribute to plant defense
RNA extraction and quantitative PCR analysis
Õ
Total RNA was isolated using a NucleoSpin RNA Plant kit
(Macherey-Nage). Total RNA (5 mg) was reverse transcribed
with a Transcriptor First Strand cDNA Synthesis kit (Roche
Applied Science) using anchored oligo(dT)18 primers.
For the real-time PCR, the LightCyclerÕ 480II and
LightCyclerÕ 480 SYBR Green I Master Mix (Roche
Diagnostics) were used according to the manufacturer’s
instructions. The real-time PCR cycling program consisted of
an initial polymerase activation at 95 C for 10 min, followed by
45 cycles of 95 C for 10 s, 58 C for 10 s, and 72 C for 20 s.
Following the amplification phase, a melting curve analysis
was conducted from 65 to 97 C, with a cooling step at 40 C
for 10 s (ramp rate of 2.0 C s1). The fluorescence was monitored using the Mono Hydrolysis Probe setting (483–533 nm)
following the 72 C extension phase of each cycle. The second
derivative maximum method in the LightCyclerÕ 480 quantification software (Roche Diagnostics) was used to analyze the
data. The reproducibility parameters were calculated from
these data in the Microsoft Excel software program. Each
cDNA sample was amplified in triplicate, and the EF1 gene
was used as the internal control.
Total SA extraction and quantification
SA was extracted and quantified as described by Seskar et al.
(1998). Leaf tissues from 2-week-old Arabidopsis plants were
ground to powder, and independent samples (0.4–0.7 g FW)
were analyzed in triplicate by HPLC equipped with a Shimadzu
RF-10AXL fluorescence detector (Shimadzu Co.) fitted with a
C18 reverse-phase HPLC column (Waters). The flow rate was
1.0 ml min1. o-Anisic acid (Sigma-Aldrich) was used as an internal standard.
Supplementary data
Supplementary data are available at PCP online.
Funding
This research was supported by the Ministry for Food,
Agriculture, Forestry and Fisheries [a grant from the
Technology Development Program for Agriculture and
Forestry]; the Rural Development Administration, Republic of
Korea [grants from the Next-Generation BioGreen21 Program
(PJ008198; PJ008160)].
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