Rice OsPAD4 functions differently from Arabidopsis AtPAD4 in

The Plant Journal (2014) 78, 619–631
doi: 10.1111/tpj.12500
Rice OsPAD4 functions differently from Arabidopsis AtPAD4
in host-pathogen interactions
Yinggen Ke, Hongbo Liu, Xianghua Li, Jinghua Xiao and Shiping Wang*
National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China
Received 11 January 2014; revised 15 February 2014; accepted 19 February 2014; published online 11 March 2014.
*For correspondence (e-mail [email protected]).
SUMMARY
The extensively studied Arabidopsis phytoalexin deficient 4 (AtPAD4) gene plays an important role in
Arabidopsis disease resistance; however, the function of its sequence ortholog in rice is unknown. Here, we
show that rice OsPAD4 appears not to be the functional ortholog of AtPAD4 in host-pathogen interactions,
and that the OsPAD4 encodes a plasma membrane protein but that AtPAD4 encodes a cytoplasmic and
nuclear protein. Suppression of OsPAD4 by RNA interference (RNAi) increased rice susceptibility to the biotrophic pathogen Xanthomonas oryzae pv. oryzae (Xoo), which causes bacteria blight disease in local tissue.
OsPAD4-RNAi plants also show compromised wound-induced systemic resistance to Xoo. The increased
susceptibility to Xoo was associated with reduced accumulation of jasmonic acid (JA) and phytoalexin
momilactone A (MOA). Exogenous application of JA complemented the phenotype of OsPAD4-RNAi plants
in response to Xoo. The following results suggest that OsPAD4 functions differently than AtPAD4 in
response to pathogen infection. First, OsPAD4 plays an important role in wound-induced systemic resistance, whereas AtPAD4 mediates systemic acquired resistance. Second, OsPAD4-involved defense signaling
against Xoo is JA-dependent, but AtPAD4-involved defense signaling against biotrophic pathogens is salicylic acid-dependent. Finally, OsPAD4 is required for the accumulation of terpenoid-type phytoalexin MOA
in rice-bacterium interactions, but AtPAD4-mediated resistance is associated with the accumulation of
indole-type phytoalexin camalexin.
Keywords: bacterial blight, bacterial streak, jasmonate, systemic resistance, Oryza sativa, Xanthomonas
oryzae.
INTRODUCTION
Plants live in a complex environment. To defeat attempted
pathogen invasion, plants use effective defense responses
at the infection site and in systemic tissues. At the infection
site, plants use the following two-tiered innate immune
system to protect themselves: (i) pathogen-associated
molecular pattern-triggered immunity (PTI), microbe-associated molecular pattern-triggered immunity or basal
resistance; and (ii) effector-triggered immunity (ETI), genefor-gene or race-specific resistance (Jones and Dangl,
2006; Thomma et al., 2011). PTI is initiated by plasma
membrane-localized plant pattern recognition receptors;
ETI is activated by cytoplasm-localized resistance (R) proteins (Jones and Dangl, 2006). As emerging data suggest
that both plant pattern recognition receptors and R proteins can mediate a race-specific high level of resistance,
or qualitative resistance, we named the gene conferring
qualitative resistance the ‘major resistance’ (MR) gene
(Thomma et al., 2011; Zhang and Wang, 2013).
© 2014 The Authors
The Plant Journal © 2014 John Wiley & Sons Ltd
Plants use induced immunity to protect themselves from
subsequent pathogen invasion in distal or systemic tissues
after the first local infection; this type of immunity is separated into two classes: systemic acquired resistance (SAR)
and induced systemic resistance (ISR) (Grant and Lamb,
2006). The phytohormones salicylic acid (SA), jasmonic
acid (JA) and ethylene have been implicated as signals
associated with systemic defense responses (Shah, 2009).
The SAR pathway appears to partially overlap with PTI and
ETI (Thomma et al., 2011). The accumulation of SA in distal tissue is the key for establishing SAR (An and Mou,
2011; Fu and Dong, 2013); however, JA as the systemic
signal for SAR is controversial (Fu and Dong, 2013). Both
JA-dependent and JA-independent SAR have been
reported (Truman et al., 2007; Chaturvedi et al., 2008; Attaran et al., 2009). ISR frequently depends on the JA/ethylene signal pathway in both monocots and dicots (Shoresh
et al., 2010; Balmer et al., 2013). It is controversial whether
619
620 Yinggen Ke et al.
SA can also function as the systemic signal for ISR. Barley
ISR against biotrophic pathogens is SA dependent (Molitor
et al., 2011), whereas Pseudomonas fluorescens WCS374rinduced Oryza sativa (rice) ISR against the hemibiotrophic
Magnaporthe oryzae is dependent on the JA/ethylenemodulated signal, but is independent of SA signaling (De
Vleesschauwer et al., 2008).
The SA-dependent and JA/ethylene-dependent pathways frequently interact either synergistically or antagonistically in plant-pathogen interactions (Durrant and
Dong, 2004). Arabidopsis phytoalexin deficient 4
(AtPAD4), a pathogen and SA-induced defense-responsive
gene, encodes a lipase-like protein and is required for the
expression of multiple defense responses (Glazebrook
et al., 1997; Zhou et al., 1998; Jirage et al., 1999). AtPAD4
functions in SA-dependent defense signaling by interacting with another lipase-like protein, enhanced disease
susceptibility 1 (AtEDS1), in a positive feedback loop to
promote SA biosynthesis, and it is required in some MR
gene-mediated resistance to the biotrophic parasite Peronospora parasitica (Glazebrook et al., 1997; Falk et al.,
1999; Feys et al., 2001; Wiermer et al., 2005). Induced
expression of the pathogenesis-related 1 (PR1) gene is a
marker of SAR in Arabidopsis (Uknes et al., 1992; Bowling
et al., 1994). Pathogen-induced PR1 expression is AtPAD4
dependent, but exogenous SA-induced PR1 expression is
AtPAD4 independent (Zhou et al., 1998). Disease resistance signaling involving AtPAD4, downstream of
AtMPK4, may require the suppression of JA/ethylene signaling, however. The Arabidopsis pad4 mutant shows an
increased susceptibility to the biotrophic pathogen Pseudomonas syringae pv. tomato DC3000, which is accompanied by induced expression of the marker gene PDF1.2 of
JA/ethylene signaling (Jirage et al., 2001; Brodersen et al.,
2006). Furthermore, the Arabidopsis HRT protein confers
resistance to turnip crinkle virus, and SA-induced HRT
expression is AtPAD4 dependent (Zhu et al., 2011); however, AtPAD4-mediated resistance to Myzus persicae
(green peach aphid) is SA independent (Louis et al.,
2012).
Bacterial blight caused by biotrophic Xanthomonas
oryzae pv. oryzae (Xoo) is one of the most devastating
diseases of rice worldwide (Kou and Wang, 2013). SA, JA
and ethylene appear to have complex roles in the resistance of rice to Xoo. Previous studies have reported that
the induction of defense-responsive genes involved in the
resistance of rice to Xoo is accompanied by: an increased
accumulation of SA and a reduced level of JA (Qiu et al.,
2007; Xiao et al., 2009; Shen et al., 2010); an increased
accumulation of JA, but not of SA (Tao et al., 2009); an
increased accumulation of both SA and JA, but with a
reduced level of ethylene (Tao et al., 2009; Shen et al.,
2010, 2011); or reduced levels of SA and JA (Ding et al.,
2008; Fu et al., 2011). The exact roles of SA and JA in the
response of rice to Xoo infection remain to be elucidated,
however.
Pathogen infection frequently influences the expression
of rice OsPAD4, the sequence homolog of AtPAD4.
OsPAD4 shows differential expression in resistant and susceptible plants during Xoo infection, suggesting the putative involvement of OsPAD4 in the response of rice to Xoo
(Qiu et al., 2007; Ding et al., 2008; Tao et al., 2009; Xiao
et al., 2009). To examine this inference, we generated
OsPAD4-suppressing plants and analyzed the role of
OsPAD4 in rice-Xoo interactions. These analyses confirmed
that OsPAD4 regulates rice defense responses against Xoo
and Xanthomonas oryzae pv. oryzicola (Xoc), which cause
bacterial streak disease; however, OsPAD4 plays a different
role in host-pathogen interactions compared with AtPAD4.
RESULTS
OsPAD4 encodes a plasma membrane protein
Sequence analysis showed that the sequence homolog of
Arabidopsis AtPAD4 is the protein encoded by gene locus
LOC_Os11g09010 in the rice genome. Thus, this gene is
the sequence ortholog of AtPAD4, and we refer to it as
OsPAD4 in this study. OsPAD4 (accession number:
AK243523) is a single-copy gene in the rice genome. Its
encoding protein and AtPAD4 share 26% sequence identity
and 37% sequence similarity. The most similar regions
between OsPAD4 and AtPAD4 are the predicted lipase
regions, which cover approximately 215 amino acids, have
31% sequence identity and 38% sequence similarity, and
harbor the catalytic triad of lipase, the conserved serine
aspartate and histine residues (Brady et al., 1990) (Figure
S1).
Sequence analysis showed that PAD4 orthologs are conserved in at least 15 monocots and dicots (Figure S1); however, their predicted encoding proteins have different sizes
ranging from 484 to 670 amino acids. According to amino
acid sequence alignment, the partial lengths of the 15
PAD4 orthologs that continuously cover approximately 415
amino acids and harbor lipase regions and flanking
sequences of the lipase regions were used to analyze the
evolutionary relationship. The 15 PAD4 orthologs were
classified into three major groups according to the phylogenetic tree (Figure 1a). OsPAD4 and its orthologs from
other monocots were classified as group II, AtPAD4 was
classified as group III and the PAD4 orthologs from other
dicots were classified as group I. The results of bioinformatic analyses suggest that most of these PAD4 orthologs
from different species are membrane proteins. The exceptions are AtPAD4 and wine grape PAD4, which are predicted to be soluble proteins and do not have putative
transmembrane helixes. This prediction is support by the
evidence that AtPAD4 localizes in the cytoplasm and
nucleus (Feys et al., 2005).
© 2014 The Authors
The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 78, 619–631
Rice OsPAD4 in defense responses 621
(b)
76
44
57
94
99
99
99
100
53
93
Cassava
Castor bean
Poplar
I
Wine grape
Soybean
Papaya
Cucumber
Spotted monkey flower
Sorghum
Maize
II
Foxtail millet
Rice
Brachypodium distachyon
Barley
Arabidopsis
III
GFP
PAD4-GFP
67
99
0.05
Bright
Merged
a
b
c
d
e
f
GFP
(a)
Figure 1. Phylogenetic analysis of the evolution of PAD4-type proteins and subcellular localization of OsPAD4.
(a) PAD4-type proteins are evolutionarily conserved in different plant species. Sequences were analyzed by the unweighted pair group method using the arithmetic method, with genetic distance calculated by MEGA 5.2 (http://www.megasoftware.net). The numbers for interior branches indicate the bootstrap values (%)
for 1000 replications. The scale bar represents 0.5 amino acid substitutions per site. The accession numbers of some PAD4-like proteins in the NCBI protein database (http://www.ncbi.nlm.nih.gov) are NP_001067424 (Oryza sativa, rice), NP_190811 (Arabidopsis), XP_002449170 (Sorghum), ACN26767 (Zea mays, maize),
XP_003577748 (Brachypodium distachyon), XP_002522923 (Ricinus communis, castor bean), XP_002307051 (Pupulus, poplar), NP_001242860/XP_003532051
(Glycine max, soybean), and CBI21592 (Vitis vinifera, wine grape). The accession numbers of other PAD4-like proteins are evm.model.supercontig_37.13 (Carica
papaya, papaya), Cucsa.353810.1 (Cucumis sativus, cucumber), BAJ89353 (Hordeum vulgare, barley), cassava4.1_004098m (Manihot esculenta, cassava),
mgv1a004394m (Mimulus guttatus, spotted monkey flower), and Si026081m (Setaria italica, foxtail millet) in PlantGDB (http://www.plantgdb.org/prj/GenomeBrowser).
(b) OsPAD4-GFP fusion protein localized in the plasma membrane of rice protoplasts. The P35S:OsPAD4-GFP was transiently expressed in stem rice protoplasts
isolated from japonica rice variety Zhonghua 11. Bars represent 10 lm.
To determine its subcellular localization, P35S:OsPAD4GFP was transiently expressed in rice protoplasts, with the
fusion protein localized in the plasma membrane
(Figure 1b). Transgenic rice plants carrying PUbi:OsPAD4GFP were also generated. OsPAD4-GFP also localized in
the plasma membrane of rice stem protoplasts isolated
from these transgenic plants (Figure S2). These results
confirm that OsPAD4 is a plasma membrane protein.
Suppressing OsPAD4 increased rice susceptibility to Xoo
To examine the role of OsPAD4 in rice-Xoo interactions,
we suppressed its expression in susceptible rice variety
Mudanjiang 8 by using an RNA interference (RNAi) strategy. Eight independent transgenic plants, D146RM1–
D146RM8, were obtained. These T0 plants showed no obvious phenotypic changes during the developmental stage.
All these T0 transgenic plants were inoculated with Xoo
strain PXO61 at the booting (panicle development) stage.
Seven of the eight plants showed an increased susceptibility to PXO61, with the disease area ranging from 61 to
85%, versus 46% for wild-type Mudanjiang 8 (Figure 2a).
The increased susceptibility was associated with suppressed expression of OsPAD4 in these transgenic plants;
the correlation between disease area and OsPAD4 expression level in the T0 transgenic plants was 0.8833 (significant at a = 0.01; n = 8). Bacterial growth analysis showed
that the growth rate of PXO61 on transgenic plants was
significantly higher (P < 0.05) than the growth rate on wildtype plants at 8–12 days after infection (Figure 2b). These
results suggest that the increased susceptibility of the
transgenic plants may be attributable to the suppression of
OsPAD4.
To examine this hypothesis, T1 plants of two T1 families generated from two susceptible T0 plants (D146RM6
and D146RM7) were further analyzed individually at the
booting stage for their response to the Xoo strain
PXO99 and the OsPAD4 transcript level. The increased
susceptibility of T1 plants co-segregated with reduced
OsPAD4 transcripts (Figure 2c). The correlations between
disease area and OsPAD4 transcripts were 0.5282 (significant at a = 0.05; n = 15) and 0.5724 (significant at
a = 0.05; n = 15) for D146RM6 and D146RM7 T1 families,
respectively. T2 plants of two T2 families from the two
T1 families were also analyzed individually at the booting
stage for their response to the Xoo strain PXO61 and
OsPAD4 expression. The increased susceptibility of these
T2 plants negatively correlated with OsPAD4 transcript
levels (D146RM6-7 T2 family, r = 0.8312; significant at
a = 0.01; n = 14; D146RM7-3 T2 family, r = 0.5464;
a = 0.05; n = 15; Figure S3). These results confirm that
the increased susceptibility of transgenic plants is associated with the suppression of OsPAD4: OsPAD4 protein
© 2014 The Authors
The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 78, 619–631
622 Yinggen Ke et al.
100
Xoo infection b a b a b
80
b
b
60
40
20
0
1.6
OsPAD4 expression
1.2
0.8
b
a
0.4
0.0
a
WT 1
2
a
a
Lesion area (%)
Relative
expression level
80
b
10
D146RM6_7
D146RM7_3
8
6
a
4
3 4 5 6 7
D146RM (T0 plants)
0
b a
Xoo infection
b
b
100
80
60
60
40
40
20
20
0
1.6
0
1.2
OsPAD4 expression
0.9
1.2
0.8
b
b b b b b
4
8
Time after inoculation (day)
8
a a
b b b
Xoo growth
Mudanjiang 8
a
100
(c)
12
(b)
Bacterial growth
(log [cfu leaf–1])
Relative
expression level
Lesion
area (%)
(a)
b
b
a
a
a
a
a
a b
b
b
b
a
0.3
Xoo infection
a
a a a
a a
OsPAD4 expression
0.6
0.4
a
12
a
a
a a
a
a
a a
a
a
a
0.0
0.0
WT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
D146RM6 T1 family
WT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
D146RM7 T1 family
Figure 2. OsPAD4-transgenic rice plants (D146RM) had increased susceptibility to Xoo. Plants were inoculated with Xoo at the booting stage. Data represent
means (three replicates from one plant for gene expression, between two and five replicates from one plant for disease area, and three replicates from each type
of plant for bacterial growth) standard deviations. The ‘a’ and ‘b’ indicate a significant difference was detected between transgenic plants and wild-type (WT;
Mudanjiang 8) plants (P < 0.01 or P < 0.05, respectively).
(a) Transgenic T0 plants with suppressed OsPAD4 expression showed increased susceptibility to Xoo strain PXO61. D146RM8 is a negative transgenic plant.
(b) Growth of Xoo strain PXO61 in the leaves of OsPAD4-suppressing T2 (D146RM6-7 and D146RM7-3) plants; cfu, colony-forming units.
(c) Increased susceptibility of OsPAD4-suppressing T1 plants to Xoo strain PXO99 was associated with suppressed OsPAD4 expression.
is a positive regulator in the resistance of rice to Xoo.
We refer to these transgenic plants as OsPAD4-RNAi
plants.
Suppressing OsPAD4 did not influence gene-for-gene
resistance to Xoo
AtPAD4 is required for RPS4-mediated and RPP5-mediated
gene-for-gene resistance to Pseudomonas syringae and
Phytophthora parasitica in Arabidopsis (Glazebrook et al.,
1997; Parker et al., 2000). RPS4 and RPP5 encode cytoplasmic nucleotide-binding leucine-rich repeat (LRR) proteins
(Leister et al., 1996; Parker et al., 1997). To ascertain
whether suppressing OsPAD4 influenced MR gene-mediated race-specific resistance, we crossed an OsPAD4-RNAi
plant (D146RM7) with transgenic rice line Rb49, which carries MR gene Xa3/Xa26 and has the genetic background of
Mudanjiang 8. Xa3/Xa26, encoding an LRR receptor kinaselike protein, confers race-specific resistance to Xoo, and
rice plants carrying Xa3/Xa26 are resistant to Xoo strain
PXO61 but are susceptible to Xoo strain PXO99 (Sun et al.,
2004; Cao et al., 2007). The F2 plants (Rb49/OsPAD4-RNAi)
carrying both OsPAD4-RNAi construct and Xa3/Xa26 generated from this cross were analyzed. These F2 plants
showed a level of resistance to PXO61 similar to that
against Rb49, but they were more susceptible to PXO99
than to Rb49 (Figure 3a). These results suggest that
OsPAD4 may not function in Xa3/Xa26-initiated defense
signaling.
© 2014 The Authors
The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 78, 619–631
Rice OsPAD4 in defense responses 623
F2 plants (OsPAD4-RNAi7/WRKY13-oe) from this cross
were analyzed. The F2 plants carrying both OsPAD4RNAi and WRKY13-oe constructs showed increased
resistance to Xoo compared with the F2 plants carrying
only the OsPAD4-RNAi construct and the OsPAD4-RNAi
line, but they showed increased susceptibility to Xoo
compared with the F2 plants carrying only the WRKY13oe construct and the WRKY13-oe line (Figure 3b). These
results suggest that OsPAD4 and WRKY13 do not function in the same defense signaling pathway as in the
rice-Xoo interaction, further supporting the inference that
OsPAD4 is not involved in Xa3/Xa26-mediated resistance.
To further examine the inference that OsPAD4 does
not function downstream of Xa3/Xa26 in defense signaling, we crossed the OsPAD4-RNAi line (D146RM7) with
the WRKY13-overexpressing (WRKY13-oe) transgenic rice
line (D11UM7-2), which has the genetic background of
Mudanjiang 8 (Qiu et al., 2007). WRKY13 is a transcriptional repressor that positively regulates the quantitative
resistance of rice to both Xoo and M. oryzae, and negatively regulates the responses of rice to abiotic stresses.
Suppressing WRKY13 compromises Xa3/Xa26-mediated
resistance against Xoo, indicating that WRKY13 functions
in the defense signaling pathway downstream of Xa3/
Xa26 (Qiu et al., 2007, 2008, 2009; Xiao et al., 2013). The
(b)
1.2
1.0
*
0.8
0.6
**
**
**
**
0.4
Disease area (%)
80
OsPAD4
0.2
75
*
50
25
**
0
WT Rb49 F2
M7 WT Rb49
PXO99
0
5
4
F2
M7
WRKY13
120
90
60
30
0
1.5
OsPAD4
1.2
0.9
0.6
0.3
**
*a
*
a
0.0
**
a
a
a**
b
**
a
WT
1 2
3 4
5
6
7
8
9 10
F2 plants
Negative sibling from the F2 population
D49OM6 (Xa21;
resistant reaction)
3
**
a
b
2
1
**
PXO61
WT (susceptible reaction)
a
Rb49 (Xa3/Xa26;
*a
resistant reaction)
*
b
a
2
1
20
WRKY13-oe
3
40
OsPAD4-RNAi
5
4
Relative expression level
**
Xoo infection
Relative expression level
**
**
(c)
Xoo (PXO61) infection
60
0
150
0.0
100
Disease area (%)
Relative expression level
(a)
*
b
**
b
ck
0.5
*a
F2 Plant carrying only WRKY13-oe construct
b
**
a
F2 carrying only OsPAD4-RNAi construct
*a
b
a
a
24
48
F2 Plant carrying both OsPAD4-RNAi and
WRKY13-oe constructs
0
1
4
8
12
Time after inoculation of PXO61 (h)
Figure 3. Analyses of the relationship of OsPAD4 and MR gene-mediated resistance to Xoo. Rice variety Mudanjiang 8 is a wild type (WT) and is susceptible to
Xoo strains PXO61 and PXO99. Rb49 and D49OM6 are transgenic lines carrying transgenic MR genes Xa3/Xa26 and Xa21, respectively, driven by their native
promoters and with the genetic background of Mudanjiang 8; D146RM7 (M7), OsPAD4-RNAi transgenic line with the genetic background of Mudanjiang 8;
WRKY13-oe, WRKY13-overexpressing line D11UM7-2 with the genetic background of Mudanjiang 8. Plants were inoculated with Xoo strain PXO61 or PXO99 at
the booting stage. Gene expression was analyzed by qRT-PCR. Bars represent means (three replicates for gene expression and 10–25 leaves from between two
and five plants for disease area) standard deviation. The ‘a’ and ‘b’ indicate that a significant difference was detected between inoculated plants and non-inoculated control (ck) plants (P < 0.01 or P < 0.05, respectively). Asterisks indicate that a significant difference was detected between wild-type plants and other
plants after the same treatment (**P < 0.01 and *P < 0.05).
(a) OsPAD4 was not involved in Xa3/Xa26-mediated resistance. F2 plants carrying both OsPAD4-RNAi construct and Xa3/Xa26 were generated from a cross
between Rb49 and D146RM7.
(b) OsPAD4 and WRKY13 did not function in the same defense signaling pathway during the rice-Xoo interaction. F2 plants were generated from the cross
between D146RM7 and WRKY13-oe lines.
(c) OsPAD4 showed differential expression in resistant and susceptible reactions after infection with Xoo.
© 2014 The Authors
The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 78, 619–631
624 Yinggen Ke et al.
AtPAD4 is required for SAR establishment (Jing et al.,
2011; Shah and Zeier, 2013). To determine whether
OsPAD4 is involved in rice SAR, we first inoculated the
leaves of rice plants that developed early with Xoc strain
RH3, and then inoculated the leaves on the same tiller
that developed later with Xoo strain PXO99 at 1, 2 or
3 days after the first inoculation. OsPAD4-RNAi plants
were more susceptible to RH3 compared with wild-type
plants (Figure 4a). After the first inoculation with RH3,
OsPAD4-RNAi and wild-type plants, as well as the negative siblings (OsPAD4-RNAi) from OsPAD4-RNAi segregating populations, showed less susceptibility to Xoo
compared with the plants without the first inoculation
(Figure 4b); however, suppressing OsPAD4 impaired the
induced systemic resistance. In the systemic leaves, the
disease area of wild-type/OsPAD4-RNAi plants caused
by Xoo infection was reduced by more than 50%, whereas
the disease area of OsPAD4-RNAi plants caused by Xoo
was only reduced by approximately 20%. Mock inoculation with phosphate-buffered saline (PBS) also reduced
the susceptibility of OsPAD4-RNAi and wild-type/OsPAD4RNAi plants to Xoo in systemic leaves (Figures 4b and
S4). This reduced susceptibility was also impaired after
suppressing OsPAD4. The OsPAD4-RNAi plants had fewer
reduced disease areas and higher bacterial growth rates
compared with those of wild-type/OsPAD4-RNAi plants
in systemic tissue after mock inoculation (Figures 4b and
S4). In addition, inoculation and mock inoculation resulted
in a similar level of reduced susceptibility to Xoo in systemic leaves of both OsPAD4-RNAi and wild-type plants.
Inoculation with Xoc and mock inoculation caused
Lesion length (cm)
(a)
2.5
2.0
Xoc infection
b
a a
b
b b
b
1.0
0.5
0.0
WT
6+
6–
7+
OsPAD4-RNAi
7–
None-PXO99
RH3-3d PXO99
RH3-2d PXO99
Mock-3d PXO99
RH3-1d PXO99
100 Xoo infection in systemic tissue
b*
a a* * *
80
a a a*
aaa
**
a
60
40
20
0
(c)
RH3-15d
RH3-16d
RH3-17d
1.5
(b)
Disease area (%)
Wound-induced systemic resistance was impaired in
OsPAD4-RNAi plants
wounds. These results suggest that OsPAD4 may be
required for wound-induced systemic resistance instead
of SAR.
Relative expression level
These results are consistent with differential expression
patterns of OsPAD4 in resistant and susceptible reactions
(Figure 3c). Rice variety Mudanjiang 8, which is susceptible to PXO61, is the wild type for transgenic rice lines
Rb49 and D49OM6 (Sun et al., 2004; Cao et al., 2007;
Zhao et al., 2009). D49OM6 carries the MR gene Xa21,
which encodes LRR receptor kinase protein and also
mediates race-specific resistance to Xoo (Zhao et al.,
2009). OsPAD4 expression in Rb49 and D49OM6 showed
a similar pattern in comparison with OsPAD4 expression
in wild-type plants. OsPAD4 expression was induced to a
maximum level at 8 h after infection in both resistant
and susceptible reactions (Figure 3c); however, the
OsPAD4 transcript levels were significantly higher in susceptible wild-type plants compared with the resistant
Rb49 and D49OM6 plants before infection, and during
most time points after infection. These results suggest
that OsPAD4 may not be specific for Xa21-mediated
resistance either, although it is involved in the regulation
of the rice-Xoo interaction.
*
**
** ****
WT
***** *
* **
6–
7+
OsPAD4-RNAi
6+
6
7–
a
OsPAD4 in wounded
local tissue
a
4
a
a
b
2
b
0
10
a
OsPAD4 in unwounded
systemic tissue
a
8
6
a
4
b
2
0
a
ck
1
3
6
12
a
a
a
24
48
72
Time after wounding (h)
Figure 4. The responses of OsPAD4-RNAi plants to bacterial infection and
wounding; WT, wild-type Mudanjiang 8; 6+ and 7+, OsPAD4-RNAi T2 lines
D146RM6 and D146RM7, respectively; 6 and 7, negative siblings from
D146RM6 and D146RM7 segregating populations, respectively. Bars represent means (between five and 12 leaves from between two and four plants
for lesion length, 10–25 leaves from between two and five plants for disease
area and three replicates for gene expression) standard deviations. The
‘a’ and ‘b’ indicate a significant difference was detected between wild-type
and OsPAD4-RNAi plants or between treated and untreated control (ck)
plants (P < 0.01 or P < 0.05, respectively; a and c).
(a) Suppressing OsPAD4 increased rice susceptibility to Xoc strain RH3 as
shown at 15–17 days after inoculation.
(b) Suppressing OsPAD4 compromised induced resistance to Xoo in rice.
Plants were first inoculated in the early-developing leaves with Xoc strain
RH3 or PBS (mock inoculation), or without inoculation (none), and were
then inoculated in the late-developing leaves with Xoo strain PXO61 at
1–3 days after the first inoculation. Asterisks indicate that a significant difference was detected between no first treatment (none) and treatment
within the same plant material (**P < 0.01 and *P < 0.05).
(c) Wounding induced OsPAD4 in both local and unwounded systemic
leaves in rice variety Mudanjiang 8, as analyzed by qRT-PCR.
© 2014 The Authors
The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 78, 619–631
Rice OsPAD4 in defense responses 625
enzyme in the JA biosynthetic pathway in rice (Mei et al.,
2006). Rice JAZ8, encoding the jasmonate ZIM-domain
(JAZ) protein, is induced by pathogens and JA, and functions in JA-dependent signaling (Figure S5; Liu et al., 2012;
Yamada et al., 2012). The expression of JAZ8, PR1a and
AOS2 was induced in infected rice leaves after inoculation
with Xoo and in unwounded systemic leaves after wounding in wild-type and OsPAD4-RNAi plants (Figure 5). The
transcript levels of the three genes in OsPAD4-RNAi plants
were significantly lower than those in wild-type plants
without infection, however, and after Xoo infection. Consistent with the expression patterns of these genes, the JA
content was increased in infected rice leaves and
in unwounded systemic leaves, but the JA content in
OsPAD4-RNAi plants was significantly lower than that in
wild-type plants (Figure 5).
Arabidopsis AtPAD4-mediated defense responses are SA
dependent (Zhou et al., 1998). To ascertain whether
OsPAD4-mediated bacterial resistance was associated with
SA, we analyzed the SA content in the same samples used
for JA quantification and JA-related gene expression
analyses (Figure 5). The SA content was reduced but there
To further determine whether OsPAD4 was essential for
wound-induced systemic resistance, we analyzed OsPAD4
expression after wounding by mock inoculation with PBS.
OsPAD4 expression was rapidly induced in local (early)
leaves at 1 h, and reached the highest level at 12 h after
wounding (Figure 4c). Its expression was also rapidly
induced in late-developing unwounded leaves at 1 h, and
reached the highest level at 3 h after wounding the earlydeveloping leaves (Figure 4c). This result suggests that
OsPAD4 may play a role in wound-induced bacterial resistance.
Impaired disease resistance was associated with
repressed JA signaling in OsPAD4-RNAi plants
Jasmonic acid (JA) is a wound-responsive hormone, and
wounding can increase the accumulation of JA (Mousavi
et al., 2013). To determine whether OsPAD4-mediated
resistance was JA dependent, we examined the expression
of JA-responsive genes. Rice PR1a expression was markedly induced by wounding, JA and SA, but JA and SA only
weakly influenced OsPAD4 expression (Figure S5; Agrawal
et al., 2000). AOS2 encodes allene oxide synthase, a key
Infected leaves
120
JAZ8
60
a
a ** a
b
****
72
**
a **
a
48
24
0
90
72
** *
*
b b
**
a
AOS2
12
6
0
20
4
JA
270
180
0
50
40
a
a
90
****
**
a
**
a
**
a
**
a **
a
a
****
bb
**
a
SA
20
b ** b
b
10
0
750
600
b
a
b
**
b b **
b
a
450
*
300
0
b
**
**
*
ck
6
**
a **
12
b
* **
a
24
**
a
48
Time after Xoo infection (h)
© 2014 The Authors
The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 78, 619–631
b
b
a
*
a
**
**
a a
**
a **
****
a
**
**
a b
*a *a
a
AOS2
a
**
a
*a
**
****
JA
a a **
a
**
a
**
a
100
*a
a
50
16
*
a aa
**
a
a
a
150
****
**
a
**
b
**
b
*
b*
a ** a*
*
24
72
*
b b **
a
SA
b
12
8
4
0
400
a
MOA
150
200
0
20
30
**
b
Concentration
(ng g–1 FW)
0
250
a
Concentration
(µg g–1 FW)
360
**
a
****
**
a
a
PR1a
3
8
*
*a
9
18
a a **
a
a
12
a
a *a **
**
**
a a
* *
0
15
16
a
Concentration
(ng g–1 FW)
Concentration
(ng g–1 FW)
*
WT
OsPAD4-RNAi 6
OsPAD4-RNAi 7
a
2
36
0
450
Concentration
(µg g–1 FW)
**
a b
a
54
Concentration
(ng g–1 FW)
**
a a
a **
a
PR1a
JAZ8
4
**
**
a a
**
a
30
96
6
**
a
90
0
120
Unwounded systemic leaves
8
a
Relative expression level
150
Relative expression level
Figure 5. Suppressing
OsPAD4
markedly
reduced the accumulation of jasmonic acid (JA)
and the phytoalexin momilactone A (MOA), and
the expression of JA-responsive genes and the
JA synthesis-related gene. Gene expression
was analyzed by qRT-PCR. Bars represent
means (from three replicates) standard deviations. The ‘a’ and ‘b’ indicate a significant difference was detected between treated and
untreated control (ck) plants (P < 0.01 or
P < 0.05, respectively). Asterisks indicate that a
significant difference was detected between
wild-type plants and OsPAD4-RNAi plants after
the same treatment (**P < 0.01 and *P < 0.05).
FW, fresh weight.
MOA
a
300
200
100
0
****
ck
** **
a
b
3
12
Time after wounding (h)
a*
626 Yinggen Ke et al.
tion of SA, we treated rice plants with either 200 lM or
5 mM of SA. Both treatments could not induce rice resistance to Xoo (Figure S6), although exogenous application
of SA (200 lM) was able to induce the expression of PR1a
(Figure S5). These results further support the inference that
OsPAD4 functions in the JA-dependent pathway in the
interaction between rice and Xoo.
was an increased accumulation of JA in infected rice
leaves of wild-type and OsPAD4-RNAi plants. There was
no obvious difference in SA content between wild-type
and OsPAD4-RNAi plants during the early infection stage.
At 48 h after Xoo infection, the OsPAD4-RNAi plants had a
lower level of SA than did the wild-type plants (Figure 5).
Moreover, similar SA levels were detected in unwounded
systemic leaves after wounding (Figure 5). These results
suggest that OsPAD4-involved disease resistance may be
associated with JA signaling.
To further determine whether OsPAD4-mediated bacterial resistance was JA dependent, we analyzed the
responses of OsPAD4-RNAi plants to the exogenous application of JA (150 lM). Previous studies have shown that
exogenous application of JA enhances the resistance of
rice to Xoo (Yamada et al., 2012). Consistent with previous
reports, exogenous application of JA reduced rice susceptibility to Xoo in wild-type and OsPAD4-RNAi plants, as
shown by disease area and Xoo growth rate compared
with mock-treated plants (Figure 6a). JA treatment complemented the phenotype of OsPAD4-RNAi plants: these
plants showed disease areas and bacterial growth rates
similar to those of mock-treated wild-type plants (P > 0.05;
Figures 6a, b). Suppression of OsPAD4 impaired the
enhanced resistance to Xoo conferred by exogenous JA,
however: the OsPAD4-RNAi plants had fewer reduced disease areas and higher bacterial growth rates compared
with those of JA-treated wild-type plants.
Exogenous application of SA can induce rice resistance
to both Xoo and the fungal pathogen M. oryza (Song et al.,
2001; Iwai et al., 2007; Xu et al., 2013). To ascertain
whether the increased susceptibility of OsPAD4-RNAi
plants could be complemented by the exogenous applica-
(a)
(b)
WT
OsPAD4-RANi7
**
a
30
a
20
a
10
0
DISCUSSION
PAD4 is a putative triacylglycerol lipase. The role of Arabidopsis AtPAD4 in pathogen-induced defense responses has
WT JA
OsPAD4-RANi6 JA
OsPAD4-RANi7 JA
10
Bacteria [log (cfu leaf–1)]
Disease area (%)
40
Xoo
**
** infection
Arabidopsis AtPAD4 positively regulated the pathogeninduced accumulation of indole-type phytoalexin camalexin (Zhou et al., 1998). To test whether Xoo induced rice
phytoalexin accumulation dependent on OsPAD4, we
quantified the known rice phytoalexins, the terpenoid-type
phytoalexin momilactone A (MOA) and the flavonoid-type
phytoalexin sakuranetin, in OsPAD4-RNAi plants after Xoo
infection. Xoo infection induced MOA accumulation in
wild-type plants, and MOA also rapidly accumulated in
unwounded systemic leaves of wild-type plants after
wounding (Figure 5). However, the MOA content in the
local infected leaves and systemic leaves after wounding
of OsPAD4-RNAi plants was significantly lower than that in
wild-type plants (Figure 5). In contrast, the sakuranetin
content was very low (fresh weight approximately 0.005–
0.5 ng g1) in wild-type and OsPAD4-RNAi plants. These
results suggest that the increased susceptibility of
OsPAD4-RNAi plants may also be associated with the
reduced accumulation of MOA.
WT mock
OsPAD4-RANi6 mock
OsPAD4-RANi7 mock
OsPAD4-RANi6
50
Accumulation of phytoalexins was impaired in OsPAD4RNAi plants
Xoo growth
8
6
4
Mock
JA
Treatment
0
8
12
Time after inoculation (day)
Figure 6. Exogenous application of jasmonic acid (JA) influenced Oryza sativa (rice) response to Xoo invasion in wild-type (WT) and OsPAD4-RNAi plants.
Plants were sprayed with 150 lM JA or a solution not containing JA (mock) during the tillering to booting stages; 2 days after treatment, plants were inoculated
with Xoo strain PXO61. Bars represent means (17–44 replicates from between eight and 16 plants for disease area and three replicates for bacterial growth) standard deviations. The ‘a’ indicates a significant difference was detected between the same plants with JA treatment and mock treatment (P < 0.01). Asterisks
(**) indicate that a significant difference was detected between WT plants and OsPAD4-RNAi plants after the same treatment (P < 0.01).
(a) Exogenous application of JA reduced rice susceptibility to Xoo.
(b) Exogenous application of JA reduced the growth of bacterium; 0 day, 1 h after inoculation of Xoo; cfu, colony-forming units.
© 2014 The Authors
The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 78, 619–631
Rice OsPAD4 in defense responses 627
been extensively studied. AtPAD4 functions as a positive
regulator in the SA-dependent signaling pathway, and as a
negative regulator in the JA-dependent signaling pathway,
in disease resistance of Arabidopsis (Zhou et al., 1998;
Brodersen et al., 2006). Rice OsPAD4 is the sequence ortholog of AtPAD4. The present results suggest that
OsPAD4 positively regulates rice defense responses to bacterial pathogens: suppressing OsPAD4 compromises the
resistance of rice to Xoo and Xoc. The following evidence
suggests that OsPAD4 may not be the functional ortholog
of AtPAD4 in host-pathogen interactions, however, considering that OsPAD4 is a plasma membrane protein and that
AtPAD4 is a cytoplasmic and nuclear protein. OsPAD4 and
AtPAD4 appear to play different roles in host resistance
against pathogens.
OsPAD4 is required for wound-induced systemic
resistance
Induced immunity is an important method used by plants
to defeat pathogen attacks. Wounding allows pathogens,
including Xoo and Xoc, to invade plants (Li and Wang,
2013). Plants can also be wounded by the invasion of some
pathogens (Balmer et al., 2013; Kobayashi and Kobayashi,
2013). Wound-induced resistance is a strategy used by
plants to defend themselves from pathogen challenges.
Wounding can induce disease resistance in dicots and
monocots (Schweizer et al., 1998; Walters et al., 2006).
AtPAD4 mediates SA-dependent SAR to biotrophic pathogens, but not wound-induced systemic resistance in
Arabidopsis (Jing et al., 2011; Rietz et al., 2011). The present results show that OsPAD4 is involved in induced resistance to Xoo. Suppressing OsPAD4 impaired induced
disease resistance; however, this gene is required for
wound-induced systemic resistance and may not be essential for SAR, and is supported by the following evidence. A
first infection and a mock (wound) infection induced the
same level of impaired resistance to Xoo in systemic tissues of OsPAD4-RNAi plants (Figure 4b). Wounding stimulates JA biosynthesis (Chung et al., 2008; Glauser et al.,
2008; Koo et al., 2009). The increased susceptibility of
OsPAD4-RNAi plants to Xoo was associated with reduced
JA content in local tissues, and the wounding-induced
accumulation of JA in systemic tissues was compromised
in OsPAD4-RNAi plants (Figure 5). Finally, OsPAD4 expression was rapidly induced not only in wounded tissues but
also in unwounded systemic tissues (Figure 4c). These
results suggest that OsPAD4 and AtPAD4 play different
roles in induced resistance: OsPAD4 is required for woundinduced resistance and AtPAD4 is required for SAR.
OsPAD4-involved defense signaling is JA dependent
In general, resistance against biotrophic and hemibiotrophic pathogens is usually regulated by the SA-dependent
pathway, whereas resistance against necrotrophic patho-
gens is frequently controlled by the JA/ethylene-dependent
pathway (Bari and Jones, 2009). AtPAD4 as a positive regulator modulates Arabidopsis resistance to biotrophic
pathogens in an SA-dependent manner, but as a negative
regulator it mediates Arabidopsis resistance to necrotrophic
pathogen in a JA-dependent pathway (Glazebrook et al.,
1997; Zhou et al., 1998; Brodersen et al., 2006). Xoo is a
biotrophic bacterium (Kou and Wang, 2013). These results
show that suppressing OsPAD4 compromised Xoo-induced
JA accumulation and JAZ8 expression, which is a marker
of JA-dependent signaling (Yamada et al., 2012), in local
tissues and wound-induced JA accumulation in
unwounded systemic tissues (Figure 5). Exogenous application of JA complemented the phenotype of OsPAD4RNAi plants in response to Xoo infection, however
(Figure 6a). These results suggest that unlike AtPAD4,
OsPAD4 plays a role in JA-dependent signaling in rice
resistance against biotrophic pathogens.
Accumulation of MOA may contribute to the OsPAD4mediated defense response
One of the plant defense responses against pathogens is
producing secondary metabolites, such as phytoalexins, to
kill pathogens (Ahuja et al., 2012). Until now, only two
types of phytoalexins, camalexin and rapalexin A, have
been detected in Arabidopsis (Ahuja et al., 2012). AtPAD4
functions in the SA-dependent pathway and positively regulates pathogen-induced phytoalexin camalexin accumulation in Arabidopsis-pathogen interactions (Zhou et al.,
1998). The indole-type camalexin, which is synthesized
from tryptophan by a cytosolic pathway, is the major phytoalexin form in Arabidopsis (Glawischnig, 2007; Geu-Flores et al., 2011). It is suggested that JA signaling controls
camalexin synthesis (Rowe et al., 2010); however, SA signaling-independent camalexin synthesis and signalingdependent camalexin synthesis have also been proposed
by different studies (Nawrath and Metraux, 1999; Roetschi
et al., 2001; Denby et al., 2005).
The synthesis of camalexin in rice has not yet been
reported; however, rice can synthesize the terpenoid
phytoalexin MOA, which has not been reported in Arabidopsis. MOA accumulation was induced by M. oryzae in rice
(Hasegawa et al., 2010; Riemann et al., 2013). MOA can
inhibit the growth of the rice fungal pathogen M. oryzae
(Dillon et al., 1997; Grayer and Kokubun, 2001; Hasegawa
et al., 2010). In addition, Xoo infection also induced MOA
accumulation in rice (Liu et al., 2012). Exogenous JA
induced MOA accumulation (Rakwal et al., 1996), and
M. oryzae-induced MOA accumulation was suppressed in
the JA-deficient mutant (Riemann et al., 2013). These
results suggest that MOA accumulation is dependent on
JA signaling. Our present results also show that Xoo infection induces the accumulation of MOA in wild-type plants,
and that reduced MOA content is associated with reduced
© 2014 The Authors
The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 78, 619–631
628 Yinggen Ke et al.
JA levels in OsPAD4-RNAi plants, indicating that the
impaired resistance of OsPAD4-RNAi plants may be at least
partly attributable to the reduced accumulation of MOA.
Thus, OsPAD4 functions differently from AtPAD4 by promoting MOA synthesis in a JA-dependent pathway.
In conclusion, OsPAD4 functions differently than AtPAD4
in response to pathogen infection. This difference may
arise because OsPAD4 is a plasma membrane-associated
protein and AtPAD4 is a cytoplasmic and nuclear protein.
Future studies with the subcellular localization domain
switch may provide insight on this perspective.
EXPERIMENTAL PROCEDURES
Bioinformatics
using calli derived from mature embryos of japonica rice varieties
Mudanjiang 8 or Zhonghua 11 (Lin and Zhang, 2005).
Wound treatment
The leaves of rice plants at the booting (panicle development)
stage were penetrated with 500 ll of 10 mM PBS using a syringe
(Schaad et al., 1996).
Pathogen inoculation
To evaluate bacterial blight disease, transgenic and wild-type
plants were inoculated with Philippine Xoo strains PXO61 and
PXO99 by the leaf-clipping method during the booting stage
(Chen et al., 2002). The extent of disease was scored by measuring the percentage of disease area [(lesion length/leaf
length) 9 100] at approximately 10 days after inoculation. The
bacterial growth rate in rice leaves was measured by counting the
colony-forming units (Sun et al., 2004).
For analysis of systemic acquired resistance, the early-developing leaves of rice plants were infected with Chinese Xoc strain
RH3 by the penetration method using a syringe, and then the latedeveloping leaves on the same tiller were inoculated with PXO99
at 1–3 days after the first inoculation. The Xoc inoculum was prepared using 10 mM of PBS.
For analysis of wound-induced systemic resistance, the earlydeveloping leaves of rice plants were wounded and then the latedeveloping leaves on the same tiller were inoculated with PXO99
at 3 days after wounding.
The amino acid sequence of Arabidopsis AtPAD4 (accession number NP_190811 in the protein database of the National Center for
Biotechnology Information, NCBI; http://www.ncbi.nlm.nih.gov)
was used to screen the rice genome database (http://blast.ncbi.nlm.nih.gov/Blast) using the BLASTP program (Altschul et al.,
1997). The rice amino acid sequence NP_001067424 (gene locus
LOC_Os11g09010) showed the highest sequence similarity to
AtPAD4. The genomic sequence of LOC_Os11g09010 was used
to search the Knowledge-based Oryza Molecular Biological Encyclopedia database (KOME; http://cdna01.dna.affrc.go.jp/cDNA/
Wblast2.html), and a full-length cDNA AK234523 (cDNA clone
J100075L24) corresponding to LOC_Os11g09010 from japonica
rice (O. sativa ssp. japonica) variety Niponbare was identified.
This cDNA clone was kindly provided by the RIKEN Yokohama
Institute (Suzuki et al., 1997).
Web-based topology prediction programs TMHMM (http://
www.cbs.dtu.dk/services/TMHMM/), HMMTOP (http://www.enzim.hu/
hmmtop) and PHOBIUS (http://phobius.sbc.su.se/) were used to
predict the orientation of the transmembrane regions of PAD4
protein.
Rice plants growing in a glasshouse until the booting stage were
sprayed with 150 lM JA, or with 200 lM or 5 mM SA, in 0.1% (v/v)
methanol and 0.015% (v/v) Tween 20, or were mock-sprayed with
0.1% methanol and 0.015% Tween 20 until uniformly wet. The
sprayed plants were kept sealed in plastic shade for 2 days. After
hormone treatment, the plants were inoculated with Xoo strain
PXO61.
Subcellular localization of OsPAD4
Gene expression analysis and quantification of SA and JA
To determine the subcellular localization of the OsPAD4 protein,
the OsPAD4 gene was fused with the green fluorescent protein
(GFP) gene at the 30 end. The coding region of OsPAD4, amplified
from cDNA clone J100075L24 using gene-specific primers (Table
S1), was cloned into vector pU1391 for the stable expression of
OsPAD4-GFP. The pU1391 vector was constructed by inserting
fragments containing the maize ubiquitin promoter (PUbi) and GFP
into vector pCOMBIA1391 (Shen et al., 2010). For transient expression of OsPAD4-GFP, the coding region of OsPAD4 was cloned
into the pM999-GFP vector to produce the OsPAD4-GFP fusion
construct driven by a cauliflower mosaic virus 35S promoter
(P35S). The construct was transiently expressed in rice stem protoplasts, as described by Zhang et al. (2011). Green fluorescent
signal was examined using a confocal microscope (TCS SP2;
Leica, http://www.leica.com).
The 3-cm leaf fragments next to the bacterial infection sites were
used for RNA isolation and phytohormone quantification. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was
conducted using gene-specific primers (Table S2), as described
previously (Qiu et al., 2007). The expression level of the rice actin
gene was used to standardize the RNA sample for each qRT-PCR.
The expression level relative to that of controls was presented.
The same samples used for gene expression analysis were used
for phytohormone quantification. Samples were prepared and JA
and SA were quantified using an ultrafast liquid chromatograph/
electrospray ionization/tandem mass spectrometry system, as
described previously (Liu et al., 2012).
Rice transformation
To construct an RNAi vector of OsPAD4, a 566-bp DNA fragment
amplified from OsPAD4 cDNA using gene-specific primers (Table
S1) was inserted into transformation vector pDS1301 (Yuan et al.,
2007). The OsPAD4-RNAi and OsPAD4-GFP vectors were transformed into Agrobacterium tumefaciens strain EHA105 by electroporation. Agrobacterium-mediated transformation was performed
Hormone treatment
Statistical analyses
The significant differences between control samples and treated
samples were analyzed by pairwise Student’s t-tests in EXCEL
(Microsoft, http://www.microsoftstore.com). The correlation analysis between gene transcript level and disease level was performed
using CORREL analysis in EXCEL.
ACKNOWLEDGEMENTS
This work was supported by grants from the National Natural
Science Foundation of China (31330062), the National Program on
© 2014 The Authors
The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 78, 619–631
Rice OsPAD4 in defense responses 629
the Development of Basic Research in China (2012CB114005) and
the National Program of High Technology Development of China
(2012AA10A303).
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online version of this article.
Figure S1. Alignment of the lipase regions of PAD4 homologs
from different species.
Figure S2. OsPAD4-GFP fusion protein localized in the plasma
membrane of rice protoplasts.
Figure S3. An increased susceptibility of OsPAD4-suppressing T2
plants to Xoo strain PXO61 was associated with suppressed
OsPAD4 expression.
Figure S4. Suppressing OsPAD4 compromised induced resistance
to Xoo in rice.
Figure S5. Exogenous application of JA and SA markedly influenced the expression of PR1a and JAZ8 analyzed by qRT-PCR.
Figure S6. Exogenous application of SA did not influence the rice
response to Xoo in both wild-type (WT) and OsPAD4-RNAi plants.
Table S1. PCR primers used for the construction of vectors, detection of positive transgenic plants and sequencing.
Table S2. Primers used for quantitative PCR in gene expression
analysis.
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