Supplemental Figure 1 - Plant Cell fileThe amount of DYT1-EYFP fluorescence in the tapetal cytoplasm...
Transcript of Supplemental Figure 1 - Plant Cell fileThe amount of DYT1-EYFP fluorescence in the tapetal cytoplasm...
Supplemental Figure 1. Phenotypic analyses of the DYT1-GR transgenic plants and anthers before and after DEX induction. (A) Pro:DYT1-GR/dyt1-3 plants without DEX treatment All the siliques were short, and after several days of treatment, the newly formed siliques began to elongate and produce seeds. The red arrow indicates the newly elongated silique. (B) The flowers and anthers of Pro:DYT1-GR transgenic plants in the dyt1-3 background, before and after treatment with DEX. Scale bar for the flower: 500 µm, and for the anther: 20 µm.
BPro:DYT1-GR/dyt1-3�
−DEX +DEX
A −DEX +DEX
Supplemental Figure 1!
Pro:DYT1-GR/dyt1-3�
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E-stage!5! L-stage!5! stage!6! stage!7!
I-Total-Cyto/I-Total-Cell!
I-Total-Nu/I-Total-Cell!
Supplemental Figure 2!
Supplemental Figure 2. Statistical analyses of the subcellular localization pattern of DYT1-EYFP in tapetal cells at anther stage 5-7. (A) The amount of DYT1-EYFP fluorescence in the tapetal cytoplasm (I-Cyto) or nucleus (I-Nu) relative to the total amount in the tapetal cell (I-Total-Cell), and the ratio between these two Values are the mean ± SD, n≥30. (B) The percentage of DYT1-EYFP amount in the cytoplasm or nucleus of tapetal cells during anther stage 5-7. (C) The tapetal cell numbers analyzed in each anther stage.
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Cell number Cyto-only Cyto & Nu Nu-only DYT1-E-stage 5 110 0 110 0
DYT1-L-stage 5 68 0 68 0
DYT1-stage 6 166 0 15 151
DYT1-stage 7 174 0 0 174
DYT1� BIF –stage 6 106 94 12 0
DYT1� BIF –stage 7 118 101 17 0
mDYT1F139DL141D-stage 6 92 74 18 0
mDYT1F139DL141D-stage 7 104 83 21 0
mDYT1I143DI144D-stage 6 84 84 0 0
mDYT1I143DI144D-stage 7 115 115 0 0
DYT1N-bHLH010BIF –stage 6 108 0 0 108
DYT1N-bHLH010BIF –stage 7 120 0 0 120
C
anther stage I-Cyto/I-Total-Cell I-Nu/I-Total-Cell Nu/Cyto
E-stage 5 0.57±0.07 0.43±0.08 0.77±0.25
L-stage 5 0.30±0.09 0.70±0.09 2.60±1.13
stage 6 0.22±0.09 0.78±0.10 3.64±1.12
stage 7 0.10±0.05 0.90±0.06 11.49±6.62
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YT1-
EYFP
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I-Cyto/I-Total-Cell
I-Nu/I-Total-Cell
Supplemental Figure 3. Functional enrichment of the 143 DYT1-ineraction candidates from screens using the yeast two-hybrid system. Pies with different colors suggest different functional clades, numbers on the pies indicate gene number in each clade.
Supplemental Figure 3!
transcription factors
protein synthesis and degeneration-related proteins
cell progress related proteins
signaling proteins
redox-related proteins
hormone metabolism proteins
development-related proteins
stress related proteins
DNA synthesis and chromatin structure proteins
glycosis proteins
cell wall protein
other proteins
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55
444
222
Supplemental Figure 4B!
Supplemental Figure 4. The amino acid alignment of DYT1 and several angiosperm DYT1 orthologs. The green arrows represent α-helixes, and the β-sheets are shown as purple rectangles. Boxes in blue are conserved or similar amino acid residues; the darker the color is, the higher the similarity is. Red and magenta boxes indicate the conserved sites, which we used to test for protein-protein interactions, and boxes in red highlight the critical amino acids for DYT1 in vivo function.
bHLH010
bHLH091
DYT1BIF YFP DAPI Merge YFP DAPI Merge
Supplemental Figure 5. The BIF domain is important for the dimerization of DYT1. BiFC results show the interactions between DYT1 BIF DYT1BIF and bHLH010, bHLH089, and bHLH091 in plants. The yellow fluorescence shows the interaction, the blue fluorescence from DAPI indicates the nucleus, and the merged signals are shown in white. Scale bar=20 µm.
Supplemental Figure 5!
DYT1 BIF
bHLH089
cYFP
DYT1
nYFPcYFP
Supplemental Figure 6. Y2H results showing interactions between various mutated DYT1 and DYT1, bHLH010, bHLH089, and bHLH091 proteins. Yeast containing both the AD and BD constructs grew on the SD/−Trp−Leu medium (the upper picture), and protein interactions were shown as the blue colonies on the lower SD/−Trp−Leu−His-Ade/AbA/X-α-gal plate.
Supplemental Figure 6!
DYT1
bHLH010
bHLH089
bHLH091
BD
DYT1
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DYT1
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DYT1
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Supplemental Figure 7!
anti-Myc antibody
anti-Tubulin antibody
Supplemental Figure 7. Immunoblot analysis of expression of various DYT1 proteins in yeast. BD fusion proteins contain the MYC tag, and tubulin serves as the internal control.
DYT1/mDYT1DYT1 BIF
DYT1BIF/DYT1bHLH
Myc
Supplemental Figure 8. Real time-PCR results showing the expression level of DYT1 in transgenic plants with various mutation and truncations. (A) Real-time PCR results showing the expression levels of EYFP tag which are fused with DYT1 and DYT1ΔBIF in the transgenic inflorescence; numbers indicate each independent lines. (B) Real-time PCR results showing the expression levels of the C-terminal DYT1 in transgenic flower buds; numbers indicate each independent line; the WT and dyt1-3 inflorescence were used as controls. Error bars indicate SD, n=3. �
Supplemental Figure 8!
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Supplemental Figure 9. Real time-PCR results showing the expression level of DYT1BIF in DYT1:DYT1BIF-EYFP/WT plants.Line-B4 to Line-B18 indicate each independent line. Line-B4 to Line-B9 showed reduced male fertility, and Line-B10 to Line-B18 exhibited normal fertility. WT inflorescence was used as a control. Error bars indicate SD, n=3. �
Supplemental Figure 9!
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RRKGRGKRKNKPFTTERERRCHLNERYEALKLLIPS
RCSWLQRKSKVTEVDVRIVDDEVTIKVVQKKKIN
BIF domainbHLH domain
327 score=5.3 360
204 score=5.2 239
bHLH089 bHLH domain
GRGSKKRKIF
RCSWLKRKSKFTDVDVRIIDDEVTIKIVQKK
BIF domain
312 score=6.3 343
209 score=11 218
bHLH010
RFRSKKRARVG
RCSWLKRKSKVTEVDVRIIDDEVTIKLVQKK
BIF domainbHLH domain
357 score=6.1 387
310 score=8.5 320
DYT1 BIF domainbHLH domain
RRKGRGKRKNKPFTTERERRCHLNERYEALKLLIPS 12 score=5.2 42
Supplemental Figure 10. Amino acid sequence comparison between DYT1, bHLH010, bHLH089, and bHLH091. (A) The BIF domain alignment of DYT1 and bHLH. Blue indicates the conserved amino acid. (B) Location of the NLSs are highlighted by red lines, and the amino acid sequences are shown. The score above or below the NLS sequences indicate the predicated NLS score by the website (http://nls-mapper.iab.keio.ac.jp). Grey boxes indicate basic regions, black boxes indicate the HLH domain, and green boxes represent the BIF domain.
Supplemental Figure 10!
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DYT1bHLH091bHLH089bHLH010
DYT1bHLH091bHLH089bHLH010
DYT1bHLH091bHLH089bHLH010
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M I KPEVE TSDL - - - - - - NEEMKK LG I EENVQLCK I GER - - - - K FWLK I I TEKRDG I F TK FMEVMREKKPESDV I DQC - - - SSNNS LRCSWLQRKSKV TEVDVR I VDDEV T I KVVQKKK I NCL L LVSKV LDNFKAQSEVVEQCL I NKKNNA LRCSWLKRKSK F TDVDVR I I DDEV T I K I VQKKK I NCL L FVSKVVDNYKPQSEVDQSCFNKNNNNS LRCSWLKRKSKV TEVDVR I I DDEV T I K LVQKKK I NCL L F T TKV LD
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F LGFE I I D I S - - L T TSNGA I L I SASVQTQE LCDVEQTKDF L LEVMR - - - - - - - - - - SNP - - - - - -QLQLDLHHVAGGQ I GEHYS F L FNTK I YEGS T I YASA I ANRV I EVVDKHYMAS LPN - SNY - - - - - -QLE LDLHHVAGAQ I GEHHS F L FNAK I SEGSSVYASA I ADRVMEV LKKQYMEA LSANNGYHCYSSDQLQLDLHHVAGGQ I GEHYS F L FNTK I CEGSCVYASG I ADT LMEVVEKQYMEAVPS - NGY - - - - - -
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bHLH010bHLH089bHLH091
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bHLH010bHLH089bHLH091
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Supplemental Figure 11!
Supplemental Figure 11. The expression levels of chimeric transgenic plants. The expression levels of chimeric genes were detected using the EYFP fusion-tag. DYT1-EYFP transgenic plants, in which the mutant phenotype was rescued, were used as the control. In DYT1N-bHLH010BIF-EYFP/dyt1-3 transgenic plants, Line#1-Line#3 show normal male fertility, Line#4-#6 show reduced fertility, and Line#7 exhibits the same male sterile phenotype as the mutant. In DYT1N-bHLH089BIF-EYFP/dyt1-3 transgenic plants, Line#1-Line#3 show normal male fertility, Line#4-Line#6 show reduced fertility, Line#7 and Line#8 exhibit more seriously reduced male fertility. Error bars indicate SD, n=3
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-EYFP/dyt1-3 �
35S NOS EYFP pGWB41 ccdB attR1 attR2
HindIII XbaI
pGWB41-DYT1pro DYT1 NOS EYFP ccdB attR1 attR2
HindIII XbaI
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SacI XhoI NotI
KpnI BamHI ECoRI
attB2
attP2
PCR products
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pGWB41-DYT1pro:DYT1
Supplemental Figure 12!
Supplemental Figure 12. Schematic maps of constructs to express various DYT1 proteins under the control of pGWB41-DYT1pro.
(A) Multiple restriction enzyme cloning sites and 5 Myc tag was obtained and inserted into the Gateway entry vector pDONR/zeo through a BP reaction. (B) The modified gateway entry vector pDONR/zeo-5 myc. (C) The Gateway binary vector pGWB41. (D) The 35S promoter of the Gateway binary vector pGWB41 was replaced by the DYT1 native promoter. (E) The modified binary vector harboring the DYT1 coding sequence driven by its native promoter.
5*myc attL1
SacI XhoI NotI
KpnI BamHI ECoRI
attL2
DYT1 NOS EYFP DYT1 attB1 attB2
HindIII XbaI