Supplemental Figure 1. Detection of the AvrPiz- t:Myc protein expression in transgenic rice by immunoblot analysis. The transgene AvrPiz-t:Myc was transformed into Nipponbare (NPB) via the agro-transformation method. 2-1, 7-1 and 9-2 are three independently transformed lines. The anti- Myc antibody was used to detect the AvrPiz-t- Myc fusion protein. IB:Į-Myc AvrPiz-t:Myc-NPB 2-2 7-1 9-2 Ponceau S stain NPB Supplemental Data. Park et al. (2012). Plant Cell 10.1105/tpc.111.105429
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Supplemental Data. Park et al. (2012). Plant Cell 10.1105 ......NPB Supplemental Data. Park et al. (2012). Plant Cell 10.1105/tpc.111.105429. ... 170 130 70 40 100 55 35 KDa RING Finger
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Supplemental Figure 1. Detection of the AvrPiz-t:Myc protein expression in transgenic rice by immunoblot analysis. The transgene AvrPiz-t:Mycwas transformed into Nipponbare (NPB) via the agro-transformation method. 2-1, 7-1 and 9-2 are three independently transformed lines. The anti-Myc antibody was used to detect the AvrPiz-t-Myc fusion protein.
IB:Į-Myc
AvrPiz-t:Myc-NPB
2-2 7-1 9-2
Ponceau S stain
NPB
Supplemental Data. Park et al. (2012). Plant Cell 10.1105/tpc.111.105429
Supplemental Figure 2. Disease reaction of 3-week old AvrPiz-t transgenic plants to M. oryzae isolates RB22 (virulent) and C9240 (avirulent). Photos were taken 7 d after spray inoculation.
RB22 (virulent) C9240 (avirulent)
NPBAvrPiz-t-
NPB NPBAvrPiz-t-
NPB
Supplemental Data. Park et al. (2012). Plant Cell 10.1105/tpc.111.105429
Supplemental Figure 3. Expression of the FLAG:APIP6 and FLAG:APIP6 H58Y proteins in N. benthamiana and E. coli.(A) Expression of FLAG:APIP6 and FLAG:APIP6 H58Y in N. benthamiana. Agrobacterium carrying the FLAG:APIP6 construct was agroinfected, and the treated tissues were harvested 1 to 3 DAI as indicated. MG132 (50 µM) was infiltrated 2 DAI, and the treated tissues were harvested 3 DAI. The TAP tag protein was expressed as an internal control and was detected by immunoblot with peroxidase anti-peroxidase (PAP). The transcriptional level of each gene was determined by RT-PCR.(B) Expression of FLAG:APIP6 and FLAG:APIP6 in E. coli. FLAG:APIP6 and FLAG:APIP6 H58Y was expressed in E. coli as fusion proteins either with MBP (left) or GST (right). Proteins were detected by immunoblot with anti-MBP, anti-GST and anti-FLAG antibodies to ensure that FLAG and APIP6 are in-frame. Arrow indicates the position of the FLAG:APIP6 or FLAG:APIP6 H58Y fusion protein either with MBP (left) or GST (right).
APIP6
IB:Į-FLAG
IB:PAP
1 D
AI
2 D
AI
3 D
AI
MG
132
FLAG:APIP6 H58Y
1 D
AI
2 D
AI
3 D
AI
MG
132
FLAG:APIP6A
B
70100
55
40
35
25
7055
40
35
25
100
IB:Į-GST
IB:Į-FLAGG
ST
GST
:FLA
G:A
PIP6
GST
:FLA
G:A
PIP6
H58
Y
IB:Į-FLAG
130
70100
55
40
7055
40
130100
IB:Į-MBP
B
MB
P
MB
P:FL
AG
:API
P6
MB
P:FL
AG
:API
P6 H
58Y
Supplemental Data. Park et al. (2012). Plant Cell 10.1105/tpc.111.105429
Supplemental Figure 4. BiFC assay for the APIP6 H58Y and AvrPiz-t interaction in N. benthamiana using Agrobacterium-mediated transient expression system. APIP6 H58Y was fused to the N-terminal fragment of YFP (nYFP) and AvrPiz-t was fused to the C-terminal fragment of YFP (cYFP). Images were captured by fluorescence confocal microscopy. (Objective=20X)(A) Co-expression of APIP6 H58Y:nYFP with AvrPiz-t:cYFP reconstituted BiFC. (B) Co-expression of nYFP with AvrPiz-t:cYFP (C) Co-expression of cYFP with APIP6 H58Y:nYFP.(D) Co-expression of APIP6 H58Y:nYFP with AvrPiz-t:cYFP along with RFP:SPIN1. Re-constitution of BiFC was predominantly observed in nucleus as it co-localized with the nuclear protein RFP:SPIN1 (Objective=40X)
B
C
A
GFP Merged
GFP RFP
GFP+RFP Merged
D
Supplemental Data. Park et al. (2012). Plant Cell 10.1105/tpc.111.105429
170130
70
40
100
55
35
KDa
RING Finger
35 80
70
40
100
55
KDa
IB:Į-Ub
IB:Į-MBP
A
B
C
Supplemental Figure 5. Structure and E3 ubiquitin ligase activity of APIP6.(A) Structure of APIP6.(B) Scheme of APIP6 RING finger composition and the location of the H58Y mutation in the RING finger domain. (C) APIP6 E3 ligase activity assay. MBP:APIP6 and its RING finger domain mutant, MBP:APIP6 H58Y, were assayed for E3 ligase activity in the presence of wheat (Triticum aestivum) E1, E2 (UBC10, At5g53300), and ubiquitin. MBP (lane 5) was used as a negative control. Immunoblot was performed with the anti-ubiquitin antibody to detect polyubiquitin bands. Immunoblot with the anti-MBP antibody was used to determine the amount of MBP:APIP6, MBP:APIP6 H58Y, and MBP protein loading in each lane.
E2E1
MBP:APIP6MBP:APIP6 H58Y
- + + ++ - + ++ + - +- - - -
++-+
+++-
MBPUb
- - - -+ + + -
++
-+
++-+-+
Silencing target region
Supplemental Data. Park et al. (2012). Plant Cell 10.1105/tpc.111.105429
IB:Į-Ub
IB:Į-MBP
KDa
70
170130
70100
55
40
70
4055
MBP:APIP6GST:AvrPiz-t:HA
+-
++
GST:AvrPi-ta:HA - -
+-
+
H58Y
+
-
IB:Į-HA
Supplemental Figure 6. Ubiquitination of AvrPiz-t by APIP6 and suppression of APIP6 E3 ligase activity by AvrPiz-t in vitro.(A) In vitro ubiquitination assay of GST:AvrPiz-t:HA by the MBP:APIP6 fusion protein. (B) Suppression of APIP6 E3 ligase activity by AvrPiz-t. E3 ligase activity of APIP6 in the absence of GST:AvrPiz-t:HA and in the presence of GST:Avr-Pita:HA were used as controls. Relative E3 ligase activity was calculated by comparison to the control (lane 1) using ImageJ software.(C) Immunoblot with the anti-MBP antibody to quantify the MBP:APIP6 or MBP:APIP6 H58Y protein in each lane.
A
B
C1.0 0.8 1.1
Supplemental Data. Park et al. (2012). Plant Cell 10.1105/tpc.111.105429
80
58
46
175
KDa
IB:Į-MBP
MBP:AvrPiz-t
80
58
46
175
KDa
Supplemental Figure 7. Suppression of APIP6 E3 ligase activity by AvrPiz-t (without the signal peptide sequence) and ubiquitination of AvrPiz-t by APIP6. The conditions for the E3 ligase activity assay were the same as described in Supplemental Figure 5C. (A) Immunoblot with the anti-ubiquitin antibody to detect polyubiquitin bands. Relative E3 ligase activity was calculated by comparison to the control (lane 1) using ImageJ software.(B) Immunoblot with the anti-MBP antibody to detect the MBP:AvrPiz-t protein
+-MBP:AvrPiz-t
A
B
IB:Į-Ub
0.71.0
Supplemental Data. Park et al. (2012). Plant Cell 10.1105/tpc.111.105429
A
B
Supplemental Figure 8. BlastN results of APIP6 silencing fragment.(A)100% identity to Apip6 (Os05g06270), (B) 80% identity to Os01g0350900
Supplemental Data. Park et al. (2012). Plant Cell 10.1105/tpc.111.105429
Supplemental Figure 9. Specificity of APIP6 silencing.APIP6 silencing fragment used to make APIP6RNAi lines specifically targets APIP6 and there is no off-target effect on Os01g0350900. Relative gene expression of APIP6 andOs01g0350900 was measured by qRT-PCR in leaves of wild-type Toride (TRD) and APIP6RNAi lines. Values were means of three independent transgenic APIP6RNAi lines. Error bars represent the s.e.m . (*P value <0.05)
APIP6 Os01g0350900
Relative�Expressio
n
*
Supplemental Data. Park et al. (2012). Plant Cell 10.1105/tpc.111.105429
Supplemental Data. Park et al. (2012). Plant Cell 10.1105/tpc.111.105429
Supplemental Table 1. The list of APIPs identified by Y2H
Name Putative function APIP1 Hypothetical protein APIP2 C3HC4-type Ring finger E3 ubiquitin ligase APIP3 Hypothetical protein APIP4 Bowman-Birk proteinase inhibitor APIP5 bZip transcription factor bzip121 APIP6 C3HC4-type Ring finger E3 ubiquitin ligase APIP7 AKT1-like potassium channel APIP8 UFD1 like protein APIP9 Oticosapeptide/Phox/Bem1p APIP10 C3HC4-type Ring finger E3 ubiquitin ligase APIP11 3-methylcrotonyl-CoA carboxylase APIP12 Nucleoporin2 containing protein
Supplemental Data. Park et al. (2012). Plant Cell 10.1105/tpc.111.105429
Supplemental Table 2. Window pane analysis for APIP6 RNAi target region specificity.
Query number Sequence of 21 nt fragment Gene ID with 100%
Cloning of AvrPiz-t into pXUN-HA and RT-PCR AvrPiz-tR 5’GTCGACCTATTGGCGCTGAGCCTGAG3’
AvPiz-HAF1 5’CCCGGATCCGAATTCATGAGCTTCGTACAATGC3’
Cloning of AvrPiz-t:HA into pGDG and pGex-6p-1 AvPiz-HAR2 5’AGGCTCGAGACTAGTTAAGCGTAAT3’
APIP6-mutF 5’CGAGTCGACAAGCTTCTACATCCTTGGGGTGTGC3’
Mutation of the APIP6 RING figure domain
APIP6-mutR 5’AGGCTGCAGTGTGGCTACGAGTTCCACC3’TAP-RTF 5’CCTCCATCTCCCAAACCT3’ RT-PCR of TAP tag TAP-RTR 5’GCTCTTCCATCTGCTGCTCT3’ AP6-RTF 5’TTAGTGAACAGTACCAGCAGA3’ qRT-PCR of APIP6 AP6-RTR 5’AACTCCACCAGTTTGTCTCA3’NAC4-RT-F 5’TCCTGCCACCATTCTGAGATG3’ qRT-PCR of NAC4 NAC4-RT-R 5’TTGCAGAATCATGCTTGCCAG3’KS4-RT-F 5’TCGCATTGCGTGTGCAA3’ qRT-PCR of KS4 KS4-RT-R 5’TTGGAACTTCCGACATCGAAA3’ OsUG_F 5’TTCTGGTCCTTCCACTTTCAG 3’ qRT-PCR of rice
genomic ubiquitin OsUG_R 5’ACGATTGATTTAACCAGTCCATGA 3’ MoPot2_F 5’ACGACCCGTCTTTACTTATTTGG 3’ qRT-PCR of M. oryzae