Suppelmentary Data

Fig.S1. The gene structure of OsNRT2.3a/b.

Fig.S2. Detection of the T-DNA single copy insertion lines of OsNRT2.3a-RNAi by Southern Blot and TAIL PCR analyses.

Fig.S3. The mRNA and protein levels of OsNRT2.3a in the roots of osnrt2.3 knockdown lines (r1 and r2) and WT plants.

Fig.S4. The protein expression of OsNRT2s in roots of osnrt2.3a knockdown mutants (r1 and r2) and WT plants

Fig.S5. Comparison of growth and nitrate concentration of osnrt2.3a knockdown mutants and WT plants under 5 mM nitrate supply.

Fig.S6. Nitrate contents of different individual leaves of osnrt2.3a mutants and wild type.

Fig.S7. The relative mRNA expression of OsNRT2.3b and OsNRT2.4 in the shoots of osnrt2.3a knockdown mutants (r1 and r2) and WT plants.

Table S1. The primers for semi-quantitative RT-PCR of OsNRT2s and OsNAR2.1 genes

Table S2. Gene-specific PCR primers used for real-time RT-PCR

Table S3. The Primers and Procedures for TAIL-PCR

Fig.S1. The gene structure of OsNRT2.3a/b.

The gene structure of OsNRT2.3a/b and target sequence for OsNRT2.3a RNA interference (F and R: the forward and reverse primer, respectively).

Fig.S2. Detection of the T-DNA single copy insertion lines of OsNRT2.3a-RNAi by Southern Blot and TAIL PCR analyses.

(A) Southern blot to identify the single copy insertion lines. P: Probe; M:Maker; The genomic DNA of WT, r1 and r2 were all digested with BamⅠand HindⅢ for 6 hr. (B) The T-DNA insertion sites in the genome of r1 and r2 lines of OsNRT2.3a-RNA interference mutation.


Fig.S3. The mRNA and protein levels of OsNRT2.3a in the roots of osnrt2.3 knockdown lines (r1 and r2) and WT plants.

(A) Total RNA isolated from roots of RNAi lines (r1 and r2) and WT plants grown in IRRI solution was analyzed by real time quantitative RT-PCR using gene-specific primers. The value of OsNRT2.3a was normalized to the rice OsActin control; (B) Western blot analysis of OsNRT2.3a expression in the rice roots. Total proteins from osnrt2.3a knockdown lines and WT were separated by SDS-PAGE, transferred to a polyvinylidene fluoride membrane (Whatman), and hybridized with an OsNRT2.3a-specific antibody (dilution 1:500) and an OsActin-specific antibody (dilution: 1:5000). Each lane was loaded with equal quantity of protein (50 µg).


Fig.S4. The protein expression of OsNRT2s in roots of osnrt2.3a knockdown mutants (r1 and r2) and WT plants

Total proteins from osnrt2.3a knockdown mutants and WT were separated by SDS-PAGE, transferred to a polyvinylidene fluoride membrane (Whatman), and hybridized with an OsNAR2.1 (dilution 1:2000), OsNRT2.1/2.2 (1:2000), OsNRT2.3b-specific antibody (1:500), OsNRT2.4 (1:1000) and an OsActin- specific antibody (1:2000). Each lane was loaded with equal quantity of protein (50 g). overnight at 4℃. The membrane was then incubated with the appropriate secondary antibody (1:20000, Pierce), followed by chemiluminescence detection and exposed to x-ray Film.

Fig.S5. Comparison of growth and nitrate concentration of osnrt2.3a knockdown mutants and WT plants under 5 mM nitrate supply.

Rice seedlings of WT and mutants were grown in IRRI solution containing 1 mM (NH4)2SO4 for 4 weeks and then N starved for 3 days, and were then grown with 5 mM nitrate for one further week. (A) Appearance of the seedlings treated with 5 mM NO3-; (B) The dry weight of shoots and roots of the knockdown mutants and WT treated with 5 mM NO3-; (C) Nitrate concentration in the shoots and roots of mutants (r1 and r2) and WT treated with 5 mM NO3-. Error bar are means ±SE of three replicates. Significant differences between WT and mutants are indicated with different letters (P<0.05, one-way ANOVA).Bar = 3 cm.


Fig.S6. Nitrate contents of different individual leaves of osnrt2.3a mutants and wild type.

Individual 4 leaves were collected in the order counted from the top of rice and nitrate content was measured. The rice plants were grown in IRRI solution containing 1 mM NH4NO3 for 1 week under 16-h photoperiod.


Fig.S7. The relative mRNA expression of OsNRT2.3b and OsNRT2.4 in the shoots of osnrt2.3a knockdown mutants (r1 and r2) and WT plants.

Total RNA isolated from roots of mutants (r1 and r2) and WT plants grown in IRRI solution containing 0.5 mm NO3- was analyzed by semi-quantitative RT-PCR using gene-specific primers. The mRNA of rice OsActin was used as control. The number of PCR cycles was 35 for amplification of OsNRT2.3b and OsNRT2.4 and 24 for amplification of OsActin.

Supplementary Table S1.

Name / Primers / Length（bp） / Tm(℃)
OsNRT2.1
(AB008519) / F:5'- CACGGTGCAAGTCTCAAG -3'
R:5'- GGTATAAATGCCTCTCCC -3 / 316 / 50
OsNRT2.2
(AK109733) / F:5'- TGGAACATTTGGATCCTCC -3'
R:5'-CCATGACGACATACTCTAG -3’ / 638 / 53
OsNRT2.3a
(AK109776) / F:5'- GCTCATCCGCGACACCCT -3'
R:5'- GTCGAAGCGGTCGTAGAA -3 / 673 / 55
OsNRT2.3b
(AK072215) / F:5'- CGTTCGCCGTGTT -3'
R:5'- TCGAAGCGGTCGTAGAAG -3 / 607 / 55
OsNAR2.1
(AP004023.2) / F: 5'- CAGTCGGTTTGGTTTGTCAG -3'
R:5'- TGAGGGAGGCGTGGATGC -3' / 557 / 55
OsNRT2.4
(LOC_Os01g3672) / F:5’-GCTCGCCTTCCCCTACGACCTC-3’
R:5’- CAGACGAAGGGAACGATGC-3’ / 501 / 55
OsNia1
(AK102178) / F：GAGCTCCTGATCAAGATATAC
R：TTCAGAAGACGAGGCAGGAC / 586 / 55
OsACT
(LOC_Os03g50890) / F:5’-CCTCGTCTCGACCTTGCTGGG-3’
R:5’-GAGAACAAGCAGGAGGACGGC-3’ / 666 / 55

The primers for semi-quantitative RT-PCR of OsNRT2s and OsNAR2.1 genes


Supplementary Table S2.

Gene-specific PCR primers used for real-time RT-PCR

Name / Primers
qOsNRT2.3a-F / 5’-CGCTGCTGCCGCTCATCCG-3’
qOsNRT2.3a-R / 5’-CCGTGCCCATGGCCAGAC-3’
qOsNRT2.1-F / 5’-CTTGTTGCAAACGGTGATGA-3’
qOsNRT2.1-R / 5’-GCCTCTCCCTTATTATACCTCCG-3’
qOsNRT2.2-F / 5’-CGGAGCACGCCTAATTAAGAG-3’
qOsNRT2.2-R / 5’-CTCCATGACGACATACTCTAGATA-3’
qOsActin-F / 5’-TTATGGTTGGGATGGGACA-3’
qOsActin-R / 5’-AGCACGGCTTGAATAGCG-3’


Supplementary Table S3

The Primers and Procedures for TAIL-PCR

Name / primers
SP1 / 5’-GCATGACGTTATTTATGAGATGGG-3’
SP2 / 5’-AATATAGCGCGCAAACTAGG-3’
SP3 / 5’-GCGGTGTCATCTATGTTAC-3’
AD1 / 5’-TGWGNAGWANCASAGA-3’
AD2 / 5’-STTGNTASTNCTNTGC-3’
AD3 / 5’-WAGTGNAGWANCANAGA-3’
AD4 / 5’-WCAGNTGWTNGTNCTG-3’
TAIL 1° REACTION PROGRAM:
Control Method: CALCULATED
1=4° for 2 min.
2=92° for 2 min.
3=95° for 1 min.
4=94° for 30 sec.
5=66° for 1 min.
6=72° for 2 min.
7=Go to step 4 for 5 more cycles
8=94° for 30 sec.
9=30° for 3 min.
10=Ramp for 72° at 0.2°/sec, 72° for 2 min. 30 sec.
11=94° for 5 sec.
12=66° for 1 min.
13=72° for 2 min.
14=94° for 5 sec.
15=66° for 1 min.
16=72° for 2 min.
17=94° for 5 sec.
18=44° for 1 min.
19=72° for 2 min.
20=Go to step 12, for 15 more cycles
21=72° for 5 min.
22=4° forever
23=END / TAIL 2° REACTION PROGRAM:
Control Method: CALCULATED
1=95° for 2 min.
2=94° for 10 sec.
3=66° for 1 min.
4=72° for 2 min.
5=94° for 10 sec.
6=66° for 1 min.
7=72° for 2 min.
8=94° for 10 sec.
9=44° for 1 min.
10=72° for 2 min.
11=Go to step 2, for 12 more cycles
12=72° for 5 min.
13=4° forever
14=END / TAIL 3° REACTION PROGRAM:
Control Method: CALCULATED
1=95° for 2 min.
2=94° for 10 sec.
3=60° for 1 min.
4=72° for 2 min.
5=Go to step 2, for 19 more cycles
6=72° for 5 min.
7=4° forever
8=END