2 5 7 57 9 11 13 Most normal plants found 61 64 17 65 68 72 76 … · 2016-05-19 · ago1-3/+...
5
ago1-3/+ FLAG-AGO1 WT (T1) WT phenotype Morphological defects 2 9 11 5 7 13 17 32 30 37 Most normal plants found Morphological defects Most normal plants found Morphological defects Most normal plants found Morphological defects ago1-3/+ FLAG-AGO1 H988F (T1) ago1-3/+ FLAG-AGO1 D762A (T1) ago1-3/+ FLAG-AGO1 D848A (T1) 57 64 65 68 72 76 82 87 61 90 102 104 106 151 153 156 157 159 160 163 171 177 182 168 136 Figure S1. Dominant-negative effect of slicer-deficient AGO1 on wild type plants (A) Morphological phenotypes of independent transgenic lines transformed with FLAG-AGO1 WT, H988F, D762A, or D848A in an ago1-3/+ background. For each construct, 28 T1 plants were selected among those with wild type appearance (in FLAG-AGO1WT), or the closest to wild type that could be found (in slicer-deficient constructs) (upper trays) for further analyses and line selection. 14 plants with morphological defects representa- tive of the variety found in the population were also selected for analysis (lower half trays). Compared to the other slicer-deficient mutants, very few T1 plants expressing FLAG-AGO1 D848A were relatively normal and fertile. (B) Western blot analyses of flowers of the T1 plants highlighted in (A). While plants with morphological defects expressing FLAG-AGO1 WT (serrations on the leaves margins) showed low expression of the transgene (lines 30, 32, 37), the more severe phenotypic abnormalities in plants expressing slicer-deficient FLAG-AGO1 did not correlate with low expression of the transgene (lines 82, 87, 90, 136, 160, 168, 171, 182). ago1-3/+ FLAG-AGO1 FLAG Coomassie WT H988F D762A D848A 2 5 7 13 17 32 57 64 90 102 136 151 153 160 168 106 104 WT 5 7 9 11 13 17 30 32 37 ago1-3/+ FLAG-AGO1 FLAG H988F 61 64 65 68 72 76 82 87 90 ago1-3/+ FLAG-AGO1 FLAG D848A 156 157 159 160 163 168 171 177 182 ago1-3/+ FLAG-AGO1 FLAG A B
Transcript of 2 5 7 57 9 11 13 Most normal plants found 61 64 17 65 68 72 76 … · 2016-05-19 · ago1-3/+...
ago1-3/+ FLAG-AGO1 WT (T1)
WT
phen
otyp
eM
orph
olog
ical
de
fect
s
2
9 11
5 7
13
17
3230
37
Mos
t nor
mal
pl
ants
foun
dM
orph
olog
ical
de
fect
s
Mos
t nor
mal
plan
ts fo
und
Mor
phol
ogic
al
defe
cts
Mos
t nor
mal
plan
ts fo
und
Mor
phol
ogic
al
defe
cts
ago1-3/+ FLAG-AGO1 H988F (T1)
ago1-3/+ FLAG-AGO1 D762A (T1) ago1-3/+ FLAG-AGO1 D848A (T1)
57
64
65 68
72 76
82
87
61
90
102 104 106 151 153 156 157
159 160 163
171
177
182
168
136
Figure S1. Dominant-negative effect of slicer-deficient AGO1 on wild type plants(A) Morphological phenotypes of independent transgenic lines transformed with FLAG-AGO1 WT, H988F, D762A, or D848A in an ago1-3/+ background. For each construct, 28 T1 plants were selected among those with wild type appearance (in FLAG-AGO1WT), or the closest to wild type that could be found (in slicer-deficient constructs) (upper trays) for further analyses and line selection. 14 plants with morphological defects representa-tive of the variety found in the population were also selected for analysis (lower half trays). Compared to the other slicer-deficient mutants, very few T1 plants expressing FLAG-AGO1D848A were relatively normal and fertile.(B) Western blot analyses of flowers of the T1 plants highlighted in (A). While plants with morphological defects expressing FLAG-AGO1WT (serrations on the leaves margins) showed low expression of the transgene (lines 30, 32, 37), the more severe phenotypic abnormalities in plants expressing slicer-deficient FLAG-AGO1 did not correlate with low expression of the transgene (lines 82, 87, 90, 136, 160, 168, 171, 182).
ago1-3/+ FLAG-AGO1
FLAG
Coomassie
WT H988F D762A D848A
2 5 7 13 17 32 57 64 90 102
136
151
153
160
168
106
104
WT
5 7 9 11 13 17 30 32 37
ago1-3/+ FLAG-AGO1
FLAG
H988F
61 64 65 68 72 76 82 87 90ago1-3/+ FLAG-AGO1
FLAG
D848A
156
157
159
160
163
168
171
177
182
ago1-3/+ FLAG-AGO1
FLAG
A
B
0
10
20
30
-6 -4 -2 0 2 4 6
miRNA targets - KO vs WT-13d-L
og10
(p-a
dj)
Log2 (Fold Change)
0
10
20
30
-8 -6 -4 -2 0 2 4 6 8
miRNA targets - KO vs WT-8d
Log2 (Fold Change)
0
10
20
30
-6 -4 -2 0 2 4 6
miRNA targets - SD vs WT-13d
Log2 (Fold Change)
0
10
20
30
40
-6 -4 -2 0 2 4 6
miRNA targets - SD vs WT-8d
Log2 (Fold Change)
Figure S2. miRNA target regulation in ago1 mutants vs. different wild type controlsVolcano plots of differentially regulated miRNA targets in 13-day-old seedlings of KO (ago1-3) or slicer-deficient AGO1 (average expression among the three catalytic mutants FLAG-AGO1 D762A, E803A, and H988F in an ago1-3 null background)), compared to two different FLAG-AGO1 wild type controls, also in an ago1-3 null background, harvested either 13 days (same age) or 8 days (same developmental stage) after germination.
Col-0
TIR1
35S:
TIR1
35S:
TIR1
mut
*
Figure S3. Test of TIR1 antibodyWestern blot of total protein extracts from Col-0 and from transgenic lines expressing either a wild type TIR1 mRNA (35S-TIR1) or a TIR1 mRNA with a mutated miR393 binding site (35S-TIR1mut). Vertical dashed line indicates presence of a number of lanes on the original gel with samples not relevant for the present study.
Table S8 - Primers and probes
Cloning primers LA2-ago1Pro/HA.F ATGTACCCAUACGATGTTCCTGAC
LA3-HA/ago1.R ACAGCGUAATCTGGAACGTCATATG LA34.ago1Pro.F GGCTTAAUCTATCCAAATTCCAAACCATACG LA35.ago1Pr/HA.R ATGGGTACAUGATGATTCCTGTGAAAATAACAC LA36.HA/ago1.F ACGCTGUGAGAAAGAGAAGAACGGATG LA37.ago1Ter.R GGTTTAAUGATTCTGTCGATTGCTTTGCTGG
USER Cassette USER-AvrII-PacI.F 5‘Phos-CTAGGGCTGAGGCTTAATTAAACCTCAGCC-3'
USER-AvrII-PacI.R 5‘Phos-CTAGGGCTGAGGTTTAATTAAGCCTCAGCC-3'
Primers for site-directed mutagenesis LA32.pX6-QC/AvrII.F CATCCCCTCGACCTAGGTACTGTACATATAACC LA33.pX6-QC/AvrII.R GGTTATATGTACAGTACCTAGGTCGAGGGGATG
Primers for genotyping LA319.ago10-1.LP ACTCGGTGAAGGGTTAGAGTC
LA320.ago10-1.RP AACTTGGAGACATGGCAAATGC LBb1.3 (SALK) ATTTTGCCGATTTCGGAAC
Probes U6 AGGGGCCATGCTAATCTTCTC
miR159 TAGAGCTCCCTTCAATCCAAA miR160 TGGCATACAGGGAGCCAGGCA miR166 GGGGAATGAAGCCTGGTCCGA miR172 ATGCAGCATCATCAAGATTCT miR398 AAGGGGTGACCTGAGAACACA miR5026 ACGTGTCACGATCTTATGAGT miR160c* CATGCTTGACTCCTTGTACGC miR173* TTTCGCTTACACAGAGAATCA
Table S11: Antibodies
Antibody Animal Dilution for WB Gene Theoretica
l MW Apparent
MW Immunogen Description Origin
FLAG mouse 1:1000 - - - Synthetic peptide: DYKDDDDK
Monoclonal ANTI-FLAG M2 Antibody (HRP conjugated) Sigma (F3165)
HA mouse 1:1000 - - - Synthetic peptide: YPYDVPDYA Monoclonal anti-HA Antibody Abnova (12CA5)
AGO1 rabbit 1:3000 AT1G48410 116.5 KDa ~ 130 KDa Synthetic N-terminal
11 aa peptide (VRKRRTDAPSE)
Affinity purified by Eurogenetec Eurogentec SA
AGO2 rabbit 1:2000 AT1G31280 113.5 KDa ≲ 130 KDa Schott et al., 2012 Gift from O. Voinnet (ETH Zürich, Switzerland)
CSD1 rabbit 1:5000 AT1G08830 15.1 KDa ≲ 15 KDa Gift from Dan Kliebenstein (University of California, Davis, CA)
CSD2 rabbit 1:5000 AT2G28190 22.2 KDa ≲ 20 KDa Recombinant CSD2 Polyclonal Agrisera (AS06 170)
TIR1 rabbit 1:1500 AT3G62980 66.8 KDa ≳ 70 KDa See Supplemental Figure 3 Gift from L. Navarro (Ecole Normale Supérieure de Paris, France)
