!supplemental!data.!juranić!!etal.!(2012).!plantcell!10 ......dec 14, 2012 · os loc_os10g29050.1...

SUPPLEMENTAL FIGURES

Supplemental Data. Juranić et al. (2012). Plant Cell 10.1105/tpc.112.107169

Supplemental Figure S1: Sequence alignment of selected plant and animal MATH-BTB proteins. Entire protein sequences were aligned using MUSCLE (www.ebi.ac.uk/Tools/msa/muscle) and displayed with BOXSHADE 3.21 (www.ch.embnet.org/software/BOX_form.html) using a threshold of 55% sequence identity. Identical and similar amino acids are shaded in black and grey boxes, respectively. The position of the MATH domain is marked by a continuous grey line and the position of the BTB domain by a dashed black line. Domains are marked according to the position of domains from the well-characterized human MATH-BTB protein Hs- SPOP with the MATH domain streches from residues 28-166 and the BTB domain from residues 190-297 (Stogios et al., 2005). The residues important for binding to CUL3 are indicated by squares below sequences. Stars represent residues important for dimerization of BTB domains, while substrate-binding residues are presented with circles. All indicated residues are in accordance with experimental evidence from studies with Hs SPOP (shown in blue) and nematode protein MEL-26 (in red). MEL-26 residue Cys94 is crucial for interaction with its targets (AAA-ATPases MEI-1 and FIGL-1) and corresponds to Tyr87 in the SPOP MATH substrate-binding cleft. For sequence identifiers see Suppl. Table 1.


Supplemental Figure S2. Relative expression levels of Zm MAB1 in indicated reproductive tissues of five mab1 (RNAi) plants derived from two independent RNAi lines (#143 and #1149). Transcript levels were compared with corresponding wild-type (wt, inbred line A188) tissues whose levels were set as 1. (A) Anthers with microspores at the early uninucleate stage (UNI) and (B) at pollen mitosis stage I (PMI). (C) Ovules at the transition from meiosis (MEI) to stages FG1 and (D) FG2. Relative Zm MAB1 expression levels were determined after normalization with Zm GAPDH as described in Methods. Mean values are shown.

A B

C D

1,000 0,132 1,014 0,00

0,20

0,40

0,60

0,80

1,00

wt 143_3 1149_1

Rel

ativ

e ex

pres

sion

leve

l

1,000 0,095 0,304 0,00

0,20

0,40

0,60

0,80

1,00

wt 143_3 143_12

Rel

ativ

e ex

pres

sion

leve

l

1,000 0,332 0,829 0,00

0,20

0,40

0,60

0,80

1,00

wt 143_12 1149_8

Rel

ativ

e ex

pres

sion

leve

l

1,000 0,228 0,543 0,00

0,20

0,40

0,60

0,80

1,00

wt 143_3 1149_3

Rel

ativ

e ex

pres

sion

leve

l

Microspores: UNI Microspores: PMI

Ovules: MEI-FG1 Ovules: FG2


J K

MAB1-EGFP

GAPDH

MAB1-EGFP

GAPDH 597 bp

132 bp

772 bp

772 bp

A B C G

D E F

H I

* *

* total RNA

Supplemental Figure S3: In vivo instability of MAB1 in transgenic maize. Transgenic PZmMAB1:ZmMAB1-EGFP plants did not show expression of the EGFP fusion protein at any stage of male (A-C) or female gametophyte development (D-F), or after fertilization in the zygote (G). The construct that was used for stable maize transformation was tested in transiently transformed maize BMS suspension cells (H and I). To test the presence of Zm-MAB1-EGFP transcripts in transgenic maize plants, RNA was isolated from anthers containing uni- and binucleate microspores (J) and from ovules at the stage of meiosis (K). The numbers in (J and K) indicate the respective transgenic line. Asterisks in (B) indicate the germination pore and in (D) the position of the megaspore mother cell in the process of meiosis. Abbreviations: AP, antipodal cells; BIC, bicellular pollen stage; CC, central cell; CV, central vacuole; dSY, degenerated synergid cell; EC, egg cell; fCC, fertilized central cell; GC, generative cell; II, inner integuments; MN, microspore nucleus; MMC, megaspore mother cell; NC, nucellus cells; PMC, pollen mother cell; SY, synergid cell; UNC, unicellular pollen stage; Z; zygote. Scale bars are 20 µm.

MAB1-EGFP in BMS

MMC, meiosis FG4/5 FG7 (mature egg) zygote

PMC UNC BIC

*

* II

II

II

NC CV

NC

II

EC

CC

SY

dSY

Z

fCC

AP

GC

CV

MN


Supplemental Figure S4. The conservation among three identified maize CUL3 proteins and their putative orthologs in different organisms. Regions and domains of cullin 3 (CUL3) proteins indicated below the alignment correspond to the classification from the Pfam/SMART database. Cullin family members consist of an N-terminal helical region known as the cullin-repeat-like domain and a C-terminal region, which contains the cullin-homology region, RBX1-binding sites and a neddylation site. Neddylation results in the covalent conjugation of the small ubiquitin-like protein Nedd8/Rub1 onto a conserved lysine residue which is indicated by a red arrowhead at the C-terminus. Red dots at the N-terminal region above the alignment mark residues in helix II and V that were experimentally proven in C. elegans to be important for CUL3 binding to BTB. Violet squares mark predicted residues that interact with RBX1. A predicted nuclear localization signal (NLS, PxxxKKR) is indicated near the C-terminus. CUL3 protein sequences used for the alignement with MUSCLE are maize (Zm, Zea mays), rice (Os, Oryza sativa), Arabidopsis (At, Arabidopsis thaliana), nematode (Ce, Caenorhabdidis elegans), human (Hs, Homo sapiens) and yeast (Sc, Saccharomyces cerevisiae). Accession numbers are provided in Methods.

NLSPxxxKKR


Q R S T

EGFP-CUL3a mRFP-MAB1

Supplemental Figure S5.

A B C D

EGFP-MAB1 mRFP-MAB1 EGFP-MAB1 + mRFP-MAB1

E F G H

I J K L

+ MG132

M N O P

+ MG132





Supplemental Figure S5. Additional examples showing Zm MAB1 homodimerization, stability and subcellular localization of its interaction with Zm CUL3a in tobacco BY-2 cells. (A-D) and (E-H) Two examples showing that MAB1 forms homodimers in speckles in the cytoplasm and nucleus. (I-L) and (M-P) Two examples showing accumulation of MAB1 after treatment with proteasome inhibitor MG132 in large aggregates mainly in the cytoplasm. (Q and R) While EGFP-CUL3a alone shows an even distribution in the cytoplasm and is less abundant in the nucleus, (S and T) mRFP-MAB1 predominantly localizes in speckle-like structures in the nucleus, being less abundant in the cytoplasm of interphase nuclei. Scale bars: 20 µm.


Gene product name Sequence identifiers Number of splicing variants per gene

MAIZE MaizeSequence.org Transcript ID NCBI RefSeq Zm MAB1 AC195147.3_FGT001 EU344973 1

Zm MAB2 GRMZM2G404188_T01 / 1



Zm MAB5 GRMZM2G372171_T01 NM_001151836.1 1


Zm MAB7.1 GRMZM2G110531_T01 NM_001137076.1 2


Zm MAB9 GRMZM2G574887_T01 / 1 Zm MAB10.1 GRMZM2G154437_T01 NM_001138313.1 2

Zm MAB11.2 GRMZM2G077428_T02 NM_001155051.1 2

Zm MAB12.1 GRMZM2G181276_T01 / 2


Zm MAB14.1 GRMZM2G052985_T01 NM_001148597.1 3

Zm MAB15 GRMZM2G148213_T01 NM_001137047.1 1

Zm MAB16 GRMZM2G172210_T01 NM_001146883.1 1

Zm MAB17 GRMZM2G166049_T01 NM_001147165.1 1

Zm MAB18.1 GRMZM2G060765_T01 NM_001147635.1 5

Zm MAB19.1 GRMZM2G074323_T01 NM_001157458.1 3

Zm MAB20.1 GRMZM2G009724_T01 NM_001154292.1 2

Zm MAB21 GRMZM2G109738_T01 NM_001156453.1 1

Zm MAB22.1 GRMZM2G046238_T01 / 2

Zm MAB23 GRMZM2G143782_T01 NM_001156534.1 1

Zm MAB24 GRMZM2G103251_T01 NM_001148133.1 1

Zm MAB25 GRMZM2G088086_T01 NM_001158895.1 1

Zm MAB26 GRMZM2G161610_T01 NM_001146887.1 1




Zm MAB30 GRMZM2G319215_T01 NM_001138156.1 1

Zm MAB31 / EU951851 1

RICE TIGR Transcript ID Os MBTB3.1 LOC_Os03g57854.1 2

Os MBTB10.1 LOC_Os06g14060.1 2

Os MBTB11 LOC_Os06g45730.1 1














Os MBTB57 LOC_Os10g29790.1 1 ARABIDOPSIS TAIR ID

At BPM1.1 AT5G19000.1 2

At BPM2.1 AT3G06190.1 2

At BPM3.1 AT2G39760.1 2

At BPM4 AT3G03740.1 1

At BPM5 AT5G21010.1 1

At BPM6 AT3G43700.1 1

C. elegans Ensembl Transcript ID Ce MEL-26 ZK858.4 1

Ce BATH-4 F52C6.8 1

Ce BATH-13 F40B1.1 1

Ce BATH-43.1 T16H12.5a 3

Ce BATH-44.3 C07D10.2b.1 4

HUMAN Ensembl Transcript ID NCBI RefSeq Hs SPOP ENST00000347630 NM_003563 23

Hs SPOPL ENST00000280098 NM_001001664 4

Supplemental Table T1. Designated gene names and identifiers for MATH-BTB genes used in the phylogentic analysis and protein alignment. The included MATH-BTB genes are from maize, rice, Arabidopsis, C. elegans and human. Since some MATH-BTB genes generate more than one splicing variant, only the transcripts coding for proteins with both domains are included in this study. Genes without splicing variants are designated with a single number, whereas genes with more splicing variants additionally contain decimal numbers. The number after a decimal point presents a splicing variant used in the analysis.

SUPP

LEM

ENTA

L TA

BLE

S


Genotype

RNAi wt

1149_24 1149_19 x 143_113 x 143_14 x A188 A188xH99 x

Anther length (mm) 3.8 3.8 3.8 3.8 4 ND2 ND2 4 3.8 ND2 ND2

Two attached nuclei (Fig 3E-H)

18 (3.3%)

9 (2.3%) 3% 37

(4.5%) 59

(6.7%) 46

(6.6%) 6% 20 (3.5%)

3 (0.8%) 2% 0

(0%) 0

(0%) 0

(0%) 0

(0%) 0%

Cellularization defect (Fig 3K)

42 (7.7%)

11 (2.8%) 5% 7

(0.8%) 1

(0.1%) 30

(4.3%) 2% 76 (13.2%)

27 (7.5%) 10% 5

(1%) 0

(0%) 0

(0%) 0

(0%) 0%

Two separated nuclei (Fig 3I)

0 (0%)

0 (0%) 0% 3

(0.4%) 0

(0%) 12

(1.7%) 1% 1 (0.2%)

4 (1.1%) 1% 0

(0%) 0

(0%) 0

(0%) 0

(0%) 0%

Large nucleus with two nucleoli

(Fig 3J)

0 (0%)

0 (0%) 0% 3

(0.4%) 5

(0.6%) 5

(0.7%) 1% 0 (0%)

0 (0%) 0% 0

(0%) 0

(0%) 0

(0%) 0

(0%) 0%

Normal UNC 425 (77.4%)

269 (69.2%) 73% 3

(0.4%) 2

(0.2%) 0

(0%) 0% ND2 ND2 ND2 15 (3%)

3 (1.2%)

0 (0%)

0 (0%) 1%

Normal BIC 50 (9.1%)

83 (21.3%) 15% 15

(1.8%) 7

(0.8%) 157

(22.5%) 8% 128 (22.2%)

160 (44.3%) 33% 386

(77.7%) 219

(84.2%) 350

(90%) 440

(93.4%) 87%

Dead 14 (2.6%)

17 (4.4%) 4% 760

(91.7%) 803

(91.5%) 448

(64.2%) 82% 352 (61%)

167 (46.3%) 54% 91

(18.3%) 38

(14.6%) 39

(10%) 31

(6.6%) 12%

n1 549 389 828 877 698 577 361 497 260 389 471

n1: total number of microspores counted. ND2: not determined. 143_113: the line with the strongest expression of the Zm MAB1-RNAi-transgene.

Supplemental Table T2: Phenotypic classification of male gametophyte defects of heterozygous mab1 (RNAi) silencing lines. Inbred line A188 and wt hybrid line (A188xH99) with the identical genetic background as transgenic lines were used for phenotypic comparison. Microspores were freshly isolated from three larger anthers in the spikelet, viewed with DIC and counted within 1 h. At least two independent series of spore counting were performed for each line. Note that a developmental stage of spores correlates with the length of anthers. For confocal images of observed phenotypes, see Figure 3. ACD and germline identity phenotypes are highlighted in yellow.


Supplemental Table T3. Primers used for cDNA synthesis, RT-PCR and cloning.

ZmMAB1-fw 5’-GCAGGAGGACCCTCTCGACGTG-3’ ZmMAB1-rev 5’-GCCATTATTGGCTAGCTGCCGCTG-3’. ZmMAB1RNAi-Eco 5’-CGCTGAATTCGAAAAGGAAGTGCATT-3’ ZmMAB1RNAi-Bam 5’-CAGTGGATCCAGAGAACTCAAGCA-3’ ZmMAB1RNAi-Mlu 5’- CAGTACGCGTCAGAGAGAACTCAAGCA-3’ ZmMAB1RNAi-Bsr 5’- CGCGTGTACAGAAAAGGAAGTGCATT-3’ ZmMAB1F-Spe 5´-ATCGACTAGTATGGCCGGCCTCCTG-3’ ZmMAB1R-Bgl 5’-GGCCAGATCTTTGCTCCAACCTTC-3’ ZmMAB1-GwF 5’-CACCTGTATGGCCGGCCTCCTG-3’ ZmMAB1-GwR 5’-CGGATCCTATGCTCCAACCTTCTTC-3’ ZmMAB1-BstXF 5’-CGCCAGTGTGCTGGTATGGCCGGCCTCCTGC-3’ ZmMAB1-NotR 5’-CGAGCGGCCGCCTATGCTCCAACCTTCTTCC-3’ qMAB1-F 5’-CGAAGCGTCCTACAGTCGTT-3’ qMAB1-R 5’-CGAGATGGATCCCGAGGTTG-3’ ZmGAP-fw1 5’-AGGGTGGTGCCAAGAAGGTTG-3’ ZmGAP-rev1 5’-GTAGCCCCACTCGTTGTCGTA-3’ ZmGAP-fw2 5’-AGGGTCCACTCAAGGGTATCAT-3’ ZmGAP-rev2 5’-ACAAGCTTGACGAAGTGGTC-3’ ZmCul3-attB1F 5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCACCATGAGCAGCGGCGGCCCG-3’ ZmCul3-attB2R 5’- GGGGACCACTTTGTACAAGAAAGCTGGGTATGCAAGATAACGATATAA-3’ AtCUL3-BstXF 5’-CTGCAGAAGAGCTCATGAGTAATCAGAAGAAGAG-3’ AtCUL3-NotR 5’-AAGGAAAAAAGCGGCCGCTTAGGCTAGATAGCGGTAAA -3’ ZmCul3-BamF 5’-CCTAGGATCCATGAGCAGCGG-3’ ZmCul3-SphR 5’-CCTCTGCATGCTATGCAAGATAACGATATAATTTTC-3’ ZmCul3-BstXF 5’-CGCCAGTGTGCTGGCATGAGCAGCGGCGGCCCGC-3’ ZmCul3-truncR 5’-CGAGCGGCCGCCTACGAATTCAGAGCATTCTGG-3’ ZmTubG1-GwF 5’-CACCATGCCGCGCGAGATCATCAC-3’ ZmTubG1-GwR 5’-AGCGGATCMCACTACCAACTTAGAGTCAA-3’ AtKTN1-EcoF 5’-CCGGAATTCAGATGGTGGGAAGTAAT-3’ AtKTN1-NotR 5’-AAGGAAAAAAGCGGCCGCTTAAGCAGATCCAAACTCAGA-3’ GFP-attB1F 5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCACCATGGTGAGCAAGGGCGAG-3’ MBD-attB2R 5’-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAACCTCCTGCAGGAAAGTG-3’


SUPPLEMENTAL METHODS Generation of constructs for transient and stable transformation All primer sequences are presented in Supplemental Table T3. The following

constructs were generated:

MAB1-RNAi: a 249-bp fragment of the Zm MAB1 coding sequence (amplified from

nucleotide position 1922 in the Zm MAB1 genomic sequence (see Methods for

accession number) containing 135 bp of the ORF and 114 bp of the 3’ UTR was

cloned in the sense and anti-sense orientation, separated by the FAD2 omega-6

desaturase intron 1 from Arabidopsis (Genbank Acc. no. AJ271841) under control of

the maize Ubiquitin (Ubi1) promoter and the Agrobacterium tumefaciens OCS

terminator to generate the vector PUbi:ZmMAB1(AS):iF2:ZmMAB1(S):OCSt. The Zm

MAB1 fragment was amplified from a cDNA library of maize egg cells (Dresselhaus

et al., 1994) using the primers ZmMAB1RNAi-Eco and ZmMAB1RNAi-Bam and

cloned into the EcoRI/BamHI-digested vector PUbi-IF2 (DNA Cloning Service),

generating the intermediary vector PUbi:ZmMAB1(AS):iF2:OCSt. In a second step,

the identical Zm MAB1 fragment was amplified using the primer pair ZmMAB1RNAi-

Mlu and ZmMAB1RNAi-Bsr ligated into the MluI/BsrGI digested intermediary vector

PUbi:ZmMAB1(AS):iF2:OCSt, resulting in the MAB1-RNAi construct.

MAB1-EGFP: the open reading frame of Zm MAB1 (1041 bp without STOP codon)

was amplified from genomic DNA of maize cultivar B73 using the primer pair

ZmMAB1F-Spe and ZmMAB1R-Bgl containing SpeI and BglII restriction sites,

respectively. The PCR product was cloned into the ZeroBlunt TOPO vector

(Invitrogen) and subsequently into the SpeI and BamHI restriction sites of PLNU-

EGFP (DNA Cloning Service), encoding an improved EGFP protein for expression in

plants (Pang et al., 1996), thereby generating the vector PZmMAB1:ZmMAB1-

EGFP:NOSt.

mRFP-MAB1 and EGFP-MAB1: the ORF of Zm MAB1 DNA was amplified from the

plasmid PZmMAB1:ZmMAB1-EGFP (see above) with primers ZmMAB1-GwF and

ZmMAB1-GwR. The PCR product was cloned using the pENTRTM Directional TOPO®

Cloning Kit (Invitrogen) to generate the entry clone, which was used for Gateway LR

reaction (Gateway® LR ClonaseTM II Enzyme Mix, Invitrogen) with the destination

vectors pH7WGR2.0 and pB7WGF2.0 (Karimi et al., 2002) to generate the

expression vectors either with an N-terminal mRFP1 fusion to Zm MAB1


(P35S:mRFP-ZmMAB1:35St) or an N-terminal EGFP fusion to Zm MAB1

(P35S:EGFP-ZmMAB1:35St).

EGFP-CUL3a: the ORF of Zm CUL3a (Zea mays Cullin 3a; see Methods for

accession number) DNA (2211 bp with STOP codon) was amplified from leaf cDNA

using the primer pair ZmCul3-attB1F and ZmCul3-attB2R. The resulting PCR product

was cloned into a donor vector pDONR207 (Invitrogen) by Gateway BP reaction

(Gateway® BP ClonaseTM II enzyme mix, Invitrogen) and subsequently into the

destination vector pB7WGF2.0 (Karimi et al., 2002) by LR reaction, generating the

expression vector used for transient transformation experiments (P35S:EGFP-

ZmCUL3a:35St).

EGFP-TubG1: the ORF of Zm TubG1 (Zea mays gamma-tubulin 1; see Methods for

accession number) DNA (1410 bp with STOP codon) was amplified from ovule cDNA

using the primer pair ZmTubG1-GwF and ZmTubG1-GwR. The resulting PCR

product was cloned using the pENTRTM Directional TOPO® cloning kit. The resulting

entry clone was used for the Gateway LR reaction with the destination vector

pB7WGF2.0 to generate the expression vector with N-terminal EGFP fusion to

TubG1 (P35S:EGFP-ZmTubG1:35St).

2xEGFP-MBD: a 2032-bp chimeric gene carrying sEGFP (S65T green fluorescent

protein gene) and the microtubule binding domain (MBD) of MAP4 (the mouse

microtubule associated protein 4, GenBank acc. no. M72414) was amplified from

gDNA of transgenic Arabidopsis plants stably expressing EGFP-MBD (Marc et al.,

1998) with the primer pair EGFP-attB1F and MBD-attB2R. The resulting PCR

product was cloned into a donor vector pDONR207 by Gateway BP reaction and

subsequently into the destination vector pB7WGF2.0 by LR reaction, generating the

expression vector P35S:EGFP-sEGFP-MBD:35St.

MAB1-AD and MAB1-BD: the ORF of Zm MAB1 was amplified from the plasmid

PZmMAB1:ZmMAB1-EGFP (see above) using primers containing restriction sites

(ZmMAB1-BstXF and ZmMAB1-NotR) and cloned into a prey vector pYESTrp

(Invitrogen) in frame with the B42 activation domain, resulting in the ZmMAB1-AD

vector. Zm-MAB1 was also cloned into the bait vector pHybLex/Zeo (Invitrogen) in

frame with the LexA DNA binding domain (ZmMAB1-BD) by cutting out the Zm-

MAB1 cDNA from the ZmMAB1-AD vector with SacI/NotI restriction enzymes.

At-Cul3a-BD: the 2199-bp ORF of At CUL3a was amplified by RT-PCR from mRNA

extracted from Arabidopsis (Col-O) leaves using the primer pair AtCUL3-BstXF and


AtCUL3-NotR. The PCR product was cloned into the pCR®-BLUNT II-TOPO® vector,

retrieved by SacI and NotI digest and subsequently ligated into the bait vector

pHybLex/Zeo.

CUL3a-AD: the ORF of Zm CUL3a was amplified from the Zm CUL3a entry clone

(see above, EGFP-ZmCul3a) with the primer pair ZmCul3-BamF and ZmCul3-SphR

and inserted into the prey vector pYESTrp as a translational fusion to the B42

activation domain.

CUL3a∆-AD: a 1101-bp truncated version of Zm CUL3a was obtained by

amplification with the primer pair ZmCul3-BstXF and ZmCul3-truncR. Digested PCR

product was cloned into the pYESTrp vector.

At-KTN1-AD: a 1572-bp ORF of the p60 katanin gene At KTN1 (locus At1G80350)

was amplified by RT-PCR from Arabidopsis (Col-O) leaves using the primer pair

AtKTN1-EcoF and AtKTN1-NotR and the digested PCR product was cloned

thereafter into the pYESTrp vector.

SUPPLEMENTAL REFERENCES Dresselhaus, T., Lörz, H., and Kranz, E. (1994). Representative cDNA libraries

from few plant cells. Plant J. 5: 605-610.

Karimi, M., Inzé, D., and Depicker A. (2002). GATEWAY vectors for Agrobacterium-

mediated plant transformation. Trends Plant Sci. 7:193-195.

Marc, J., Granger, C.L., Brincat, J., Fisher, D.D., Kao, T.H., McCubbin, A.G., and Cyr, R.J. (1998). A EGFP-MAP4 reporter gene for visualizing cortical microtubule

rearrangements in living epidermal cells. Plant Cell 10: 1927–1939.

Pang, S.Z., DeBoer, D.L., Wan, Y., Ye, G., Layton, J.G., Neher, M.K., Armstrong, C.L., Fry, J.E., Hinchee, M.A., and Fromm, M.E. (1996). An improved green

fluorescent protein gene as a vital marker in plants. Plant Physiol 112: 893–900.

Stogios, P.J., Downs, G.S., Jauhal, J.J.S., Nandra, S.K., and Prive, G.G. (2005).

Sequence and structural analysis of BTB domain proteins. Genome Biol. 6: R82.


!supplemental!data.!juranić!!etal.!(2012).!plantcell!10 ......dec 14, 2012 · os loc_os10g29050.1...

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