Experimental Procedures
Plant materials and greenhouse conditions
The rice transformants used in this study were in the genetic background of Oryza
sativa L. cv. Nipponbare (NB). The rice cultivars Sensho, Chubu32, Koganenishiki, and
Mutsuhikari were used for comparisons of blast resistance {Fukuoka, 2001
#769;Sugiura, 2002 #726}. Unless otherwise noted, plants were grown in soil (Bonsol
No.2; Sumitomo Chemical Co. Ltd., Osaka, Japan) in a greenhouse under the following
conditions: 30/25C (day/night) and 50% relative humidity.
Microarray analysis
Microarray and data analyses were carried out essentially as described previously
{Shimono, 2007 #748}. The fourth leaves of NB rice at the 4-leaf stage were harvested
from four plants and pooled. Total RNA was extracted from two replicate sets of rice
plants using an RNeasy Plant Mini Kit (Qiagen, www.qiagen.com). The extracted RNA
was labeled with Cy3 and then hybridized to an Agilent Rice Oligo Microarray (Agilent
Technologies, Palo Alto, CA, USA; www.agilent.com).
Construction and plant transformation
We generated the plasmid pZH1:WRKY45:NT:2 by excising a fragment encoding
WRKY45 from pZH1:Ubi:WRKY45:NT {Shimono, 2007 #748} with SfiI and PacI and
inserting it into the HindIII site of pZH1 {Kuroda, 2010 #502} together with a
double-strand linker (5′-AGC TTG GCC AAA T-3′ and 5′-TGG CCA-3′). Subsequently,
a double-stranded linker consisting of 5′-AGC TGG CGC GCC ATT TAA ATA-3′ and
5′-AGC TTA TTT AAA TGG CGC GCC-3′ was inserted into the HindIII site to
generate AscI and SwaI sites.
Next, we generated the constructs for constitutive expression of WRKY45 in
rice driven by the promoters of various genes (ChaC, SADP, AAT, CBS, NONE, LTP,
eEF1α, OsUbi7, ACT8, OsUbi2, OsUbi3, OsUbi5, GAPDH1, GAPDH3, and ACT7).
We amplified 2-kb upstream sequences (from initiation codons) of these genes by PCR
with
PrimeSTAR
Max
DNA
Polymerase
(Takara
Bio,
Otsu,
Japan;
www.takara-bio.co.jp) using rice genomic DNA as the template and the primers listed
in Table S4. The DNA sequences were inserted into the HindIII site of
pZH1:WRKY45:NT:2 using an In-Fusion HD Cloning Kit (Takara Bio). The OsUbi1
promoter was excised from AP:UTP, which contains the OsUbi1 promoter and
terminator in pUC198AP {Kuroda, 2010 #502}, with HindIII and BglII. The excised
OsUbi1 promoter was then inserted into the HindIII site of pZH1:WRKY45:NT:2 with a
double-stranded linker (5′-GAT CTC TAG-3′ and 5′-AGC TCT AGA-3′). To remove a
heat-shock element (HSE) from the OsUbi1 promoter, the sequences flanking both sides
of the HSE were amplified by PCR. Then, the two fragments were inserted between the
EcoNI and HincII sites of AP:UTP to replace the HSE sequence in the OsUbi1
promoter using an In Fusion HD Cloning Kit (Clontech Laboratories, Mountain View,
CA, USA). Subsequently, a fragment containing the OsUbi1 promoter without the HSE
(POsUbi1∆HSE) was inserted into the HindIII site of pZH1:WRKY45:NT:2 with a
double-stranded linker (5′-GAT CTC TAG-3′ and 5′-AGC TCT AGA-3′).
The OsUbi7:GUS construct was generated by amplifying the GUS coding
sequence by PCR using pSMAHdN632L-M2GUS {Hakata, 2010 #724} as the template.
The GUS coding sequence was then inserted between the HindIII and BamHI sites of
pZH1:OsUbi7:WRKY45:NT to yield pZH1:OsUbi7:GUS:NT.
Rice was transformed with these plasmids using an Agrobacterium-mediated
method {Toki, 2006 #175}. Transformant lines harboring a single copy of the transgene
were selected by screening T0 plants by qPCR with HPT and OsUbi1 primers (Table
S4). The intactness of the transgenes was analyzed by PCR.
Disease resistance tests
For the M. oryzae resistance test, fungal spores of a compatible race (007.0, 003.0,
031.1 or 037.1) {Kiyosawa, 1984 #817} were suspended in 0.02% Tween20 at a density
of 1–3×105/ml and then sprayed onto rice plants at the 5-leaf stage. Then, the plants
were incubated in a chamber for 20 h in the dark at 23C with 100% relative humidity.
After 1 week in a greenhouse, disease symptoms were evaluated by counting lesions
(number of lesions per 10-cm leaf area) and by determining the proportion of the
diseased area by scanning the leaves and determining the number of pixels in lesion
areas. Panicle blast resistance testing was performed as described previously in a
greenhouse using M. oryzae race 003.0 {Hayashi, 2010 #368}.
For the Xoo resistance test, bacteria of compatible races of Japanese origin
(T7174, T7147, and T7133) and those isolated in Sri Lanka (SL6-1) {Ochiai, 2000
#812}, China (CN1-1) and Myanmar (MY1) (Ochiai et al., unpublished) were
suspended in distilled water at a density of OD600=0.03 and inoculated onto the
youngest fully expanded leaves of plants at the 9–10 leaf stage using the scissors-dip
method {Kauffman, 1973 #770}. Disease symptoms were evaluated by measuring the
length of lesions after 2 weeks.
Gene expression analysis
Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA;
www.invitrogen.com/) from the fifth leaves of plants at the 5-leaf stage (T2) or the
youngest fully expanded leaves of plants grown for 1.5 month after transplanting (T0
and T1) from plates to soil. Reverse-transcription was carried out using ReverTra Ace
qPCR
RT
Master
Mix
with
gDNA
Remover
(TOYOBO,
Osaka,
Japan;
http://www.toyobo-global.com). Quantitative reverse-transcription polymerase chain
reactions (qRT-PCR) were run on a Thermal Cycler Dice TP800 system (Takara Bio)
using SYBR Premix ExTaq I (Takara Bio). The primers used for qRT-PCR are listed in
Table S4.
Immunoblot analysis
Total protein was extracted from the fifth leaves of plants at the 5-leaf stage in buffer (8
M Urea, 10% SDS, 20 mM Tris-HCl, pH 6.8). Proteins were separated on 10% SDS–
polyacrylamide gels (10 µg/lane) and immunoblotting was carried out using
anti-WRKY45 antibody, which was raised against the C-terminal peptide of WRKY45,
as described previously {Matsushita, 2013 #752;Shimono, 2012 #747}.
Histochemical analysis of GUS activity
Whole plants at the 4.5–5-leaf stage were rinsed with acetone to remove surface wax
and then stained for 20 h to detect GUS activity. The GUS staining solution consisted of
1 mM X-glucuronide, 100 mM phosphate buffer pH 7.0, 10 mM EDTA, 0.5 mM
potassium ferricyanide, 0.5 mM potassium ferrocyanide, 20% methanol, and 0.05%
Silwet L-77. After GUS staining, the samples were immersed in 70% ethanol overnight
to remove chlorophylls.
Evaluation of agronomic traits in a greenhouse and confined fields
To evaluate the agronomic traits of rice transformants grown in a greenhouse, the seeds
of WRKY45-expressing transformant lines and NB were sown on MS plates and grown
for 14 d. To select homozygotes, genomic DNAs were extracted from the transformants
and transgene copy numbers were determined by qPCR using HPT and OsUbi1 primers
(Table S4). The seedlings of homozygotes were transplanted into soil and grown in a
greenhouse. The temperature and humidity were changed according to a preset program
or outside conditions, and the positions of the pots were rotated in the greenhouse each
week. The plants were harvested after seed maturation and agronomic traits (plant
length, number of panicles and grains) were measured.
For the field trial in Korea, the seedlings of T6 (PZmUbi) and T2 (POsUbi7)
homozygous transgenic lines and NB control lines at the 4-leaf stage were transplanted
into a confined paddy field at Jinju (35°1'N, 128°1'E; annual rainfall, 1512.8 mm; 38 m
asl; annual average temperature, 13.1°C). The field had been dressed with basal
fertilizer (in kg/ha, 92:45:36 N:P:K). The plants had a spacing of 15 cm × 30 cm and
were grown from May to October, 2011. The plants were harvested at maturity and the
plant length, panicle number, grain weight, filling rate, 1000-grain weight, and grain
number were recorded for 16–20 plants in each line. Plants located at the borders were
excluded from data scoring.
In Colombia, T7 (PZmUbi) and T3 (POsUbi7) homozygous transgenic lines along
with the NB control were transplanted into a confined paddy field at the International
Center for Tropical Agriculture (CIAT), Palmira (3°30′N, 76°21′W; annual rainfall,
1,000 mm; 965 m asl; annual average temperature, 26°C). The field trials were
conducted during two growing seasons; September–December, 2011, and March–July,
2013). We used a randomized block design with six replications. Each plot was 2 m × 2
m and contained four rows. In each plot, 20 seedlings per line were randomly
transplanted with a 10 × 20 cm spacing 20–25 d after sowing. Fertilizer (in kg/ha,
180:140:60 N:P:K) was applied according to the recommendations for lowland rice in
Colombia. Nitrogen fertilizer was supplied in three equal portions: 60 kg ha−1 N as
basal at 2 days after transplanting (DAT), 60 kg ha−1 N at 10 DAT, and 60 kg ha−1 N at
30 DAT. The following morpho-agronomic traits were scored at plant maturity: plant
length, days to heading, number of panicles (48 plants per line), biomass and grain yield
(16–20 plants per line). Plants located at the borders were excluded from analyses.
Abiotic stress treatments
For the cold treatment, rice plants were grown on a 1:1 mixture of Bonsol No. 2 and
fertilizer-free soil in a greenhouse. When they reached the 5.5–6-leaf stage, they were
transferred to a growth chamber and kept under the following conditions for 7 d: 8C,
60% humidity, 14-h light/10-h dark photoperiod. Then, the plants were returned to the
greenhouse and allowed to recover for 7 d.
For the high-salinity treatment, rice plants were grown in a 1:1 mixture of
Bonsol No. 2 and fertilizer-free soil in the greenhouse. When they reached the 4.5–
5-leaf stage, they were irrigated with 250 mM NaCl for 10 d, and then with water for 7
d.