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SUPPLEMENTAY FIGURES
Phosphate starvation induced OsPHR4 mediates the PiSignaling and Homeostasis in Rice
Wenyuan Ruan1§, Meina Guo1§, Ping Wu2, Keke Yi1 *
Author affiliation:
1Key Laboratory of Plant Nutrition and Fertilizer, Ministry of Agriculture,
Institute of Agricultural Resources and Regional Planning, Chinese
2 College of Life Science, Zhejiang University.
§Wenyuan Ruan and Meina Guo contributed equally to this work.
*To whom correspondence should be addressed: [email protected]
E-mail of all authors:
Wenyuan Ruan ([email protected])
Meina Guo ([email protected])
Ping Wu ([email protected])
Keke Yi ([email protected])
Fig. S1 Amino acid sequence comparison of OsPHR1, OsPHR2, OsPHR3, and
OsPHR4
The PHR1-subfamily members in rice have conserve MYB DNA binding domain
(indicated by red box) and colied-coli (CC) (indicated by green box) domain. The two
conserved domain are located on the protein C-term.
1.5
Root
Shoot
1.0
0.5
+P
-P
PHR1
PHR2
PHR3
PHR4
PHR1
PHR2
PHR3
PHR4
0.0
PHR1
PHR2
PHR3
PHR4
PHR1
PHR2
PHR3
PHR4
Relative expression level
(PHR2, 3 and 4 vs PHR1)
2.0
+P
-P
Figure S2 Related mRNA levels of OsPHR1-4 genes (OsPHR1 set as 1)
Two-week-old plants grown in Pi-sufficient (200 μM Pi) solution were
transferred to Pi-sufficient or Pi-lacking solution for other 7d; RNA from roots
and shoots were isolated separately. qRT-PCR was performed using specific
primers (Supplemental Table S1) for OsPHR1, 2, 3 and 4. Values represent
means ±SD of three replicates.
Fig. S3 Nucleotide aliment of OsPHR1, OsPHR2, OsPHR3, and OsPHR4
DNA homology analysis indicated that there is no homology on the 5’ term for the four
OsPHRs . Therefore the sequence from 4bp to 207bp of OsPHR4 was chosen as the target
for RNA interfering design.
Relative mRNA expression
30
WT
25
PHR4-Ri-5
20
PHR4-Ri-11
PHR4-OE-9
15
PHR4-OE-14
10
2.0
1.5
1.0
0.5
Sh
o
R ot
oo
Sh t
o
R ot
oo
Sh t
o
R ot
oo
Sh t
o
R ot
oo
Sh t
o
R ot
oo
t
0.0
Figure S4 The relative expression levels of OsPHR4 in RNAi and overexpressors
The plants of WT, PHR4-Ri and PHR4-OE grown in Pi-sufficient (200 μM Pi) solution for
three-week before RNA isolation. qRT-PCR was performed using specific primers
(Supplemental Table S1) for OsPHR4. Values represent means ±SD of three
replicates.
Relative mRNA expression level
1.5
WT
PHR4-Ri-5
PHR4-Ri-11
1.0
0.5
0.0
OsPHR1
OsPHR2
OsPHR3
Fig S5 Knock down of OsPHR4 does not affect the expression of OsPHR1-3
The plants of WT, PHR4-Ri-5 and PHR4-Ri-11 grown in Pi-sufficient (200 μM
Pi) solution for three-week before RNA isolation. qRT-PCR was performed
using specific primers (Supplemental Table S1) for OsPHR1, 2, and 3. Values
represent means ±SD of three replicates.
Table S1 Primers used in this study
Usage
qRT-PCR primers
Over expression
primers
GUS fusion primers
Primers (enzyme site)
Sequences ( 5’ to 3’)
OsACTIN-RT-F
OsACTIN-RT-R
OsIPS1-RT-F
OsIPS1-RT-R
OsmiR827-RT-F
OsmiR827-RT-R
OsPT2-RT-F
OsPT2-RT-R
OsPHR1-RT-F
ATCCTGACGGAGCGTGGTT
CCAGGGCGATGTAGGAAAGC
TTGGCAATTATTCGGTGGAT
ACCATTTCACCATCCTCTTTATG
GCCCTGGATATGTTTTAGTAC
GGGTGAAAATGTATTGTCGAT
CACAAACTTCCTCGGTATGCT
GAAACCCCACAAATCCACAAC
CCAAGAGCCAGTGTTGTCAGA
OsPHR1-RT-R
OsPHR2-RT-F
GAGAAATAAAGGGAGCAGCCAT
CGCTTTGTAGATGCTGTCAATC
OsPHR2-RT-R
OsPHR3-RT-F
OsPHR3-RT-R
OsPT10-RT-F
OsPT10-RT-R
OsIPS2-RT-F
AGACCCTCATCACATCCTCATTATC
GCAGAAACCTCTTCAGCACCTT
ATGATTATTCGTCATGCACCATT
GAGCTCGCACCTCAGCAT
GAGTTCACTCACACGGAGACC
CCT TCTTCTGGATTCCTCTC
OsIPS2-RT-R
OsPHR4-RT-F
OsPHR4-RT-R
OsSPX1-RT-F
OsSPX1-RT-R
AGTTCACCACAAAAGATACAGTAG
TACCATTCCTGCCTCACCCT
GCCAATATCACCATCACCACC
GAAGTTTGGGAAGAGCCTGAGT
TGTAGTTGAGGGCGCTGTAGTT
OsPHO2-RT-F
Os PHO2-RT-R
ACAATGCTACAAGTCCTGGTCTCAGTTC
GCAGCTCTTGTCGCCCTCATC
OsPHR4-Ov-F (KpnI)
CGGGGTACCATGAGCTCCCAGAGTGTTGT
OsPHR4-Ov-R (XhoI)
CCGCTCGAGCTATGAACACTGGTGCTCATC
OsPHR4-P-GUS-F(BamHI) CGCGGATCCTTTGTAGCCTGCTGAACTAATC
OsPHR4-P-GUS-R(KpnI)
CGGGGTACCGCTGGTTCAGTTGATTGATGG
Yeast-one or two
hybrids fusion to
pB42AD primers
OsPHR4-y1-AD-F(EcoRI)
CCGGAATTCATGAGCTCCCAGAGTGTTGT
OsPHR4-y1-AD-R(XhoI)
CCGCTCGAGCTATGAACACTGGTGCTCATC
Yeast-one hybrids
fusion to pLacZ2u
primers
OsPHR4-P-F(KpnI)
CGGGGTACCGAGGGACCTTAACTCTCTCCT
OsPHR4-P -R(XhoI)
CCGCTCGAGGCTGGTTCAGTTGATTGATGG
OsPHR4 fusion to
pGX4T-1 primers
OsPHR4-GST-F(EcoRI)
CCGGAATTCATGAGCTCCCAGAGTGTTGT
OsPHR3-GST-R(XhoI)
OsPHR4-probe-F
OsPHR4-probe-R
CCGCTCGAGCTATGAACACTGGTGCTCATC
GAATATCTGATGCTGTGAAT
TTTTGGAGAAATTTCCCGTC
EMSA probe
primers