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�

kathleenfarquharson

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Supplemental Data. Cui et al. (2016). Plant Cell 10.1105/tpc.15.00986

0!

10!

20!

30!

40!

50!

60!

70!

80!

90!

100!

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. 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.

A

B

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

Am

ount

Per

cent

of D

YT1-

EYFP

(%)

I-Cyto/I-Total-Cell

I-Nu/I-Total-Cell

kathleenfarquharson

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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.


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

59

30

20

6

55

444

222

kathleenfarquharson

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Supplemental Figure 4A!

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.


DYT1 BIF

bHLH089

cYFP

DYT1

nYFPcYFP

kathleenfarquharson

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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.


DYT1

bHLH010

bHLH089

bHLH091

BD

DYT1

bHLH010

bHLH089

bHLH091

BD

A

DYT1

bHLH010

bHLH089

bHLH091

BD

B

DYT1

bHLH010

bHLH089

bHLH091

BD


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. �


B

0

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6

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xpre

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in dyt1-3�

Rel

ativ

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xpre

ssio

n Le

vel

A 4

in dyt1-3�

0

2

1

3

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. �


0

2

4

6

Rel

ativ

e E

xpre

ssio

n Le

vel 8

10

in WT

bHLH091

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.


B

A

DYT1bHLH091bHLH089bHLH010



1111

55626565

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

56636666

102120130123

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 - - - - - -

DYT1

bHLH010bHLH089bHLH091

DYT1

bHLH010bHLH089bHLH091

1111

56636666

102120130123

55626565


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

0

5

10

15R

elat

ive

Exp

ress

ion

Leve

l EYFP�

DYT1N-bHLH010BIF

-EYFP/dyt1-3 �DYT1N-bHLH089BIF

-EYFP/dyt1-3 �

35S NOS EYFP pGWB41 ccdB attR1 attR2

HindIII XbaI

pGWB41-DYT1pro DYT1 NOS EYFP ccdB attR1 attR2

HindIII XbaI

A

C

pDONR/zeo-5 myc

B

ccdB attP1

pDONR/zeo

5*myc attB1

SacI XhoI NotI

KpnI BamHI ECoRI

attB2

attP2

PCR products

D

E

pGWB41-DYT1pro:DYT1


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

supplemental figure 1 - plant cell filethe amount of dyt1-eyfp fluorescence in the tapetal cytoplasm...

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