supplemental data. luo et al. (2011). plant cell … tca ctt gaa ttc cat gat ca mips2 sense gggg aca...

Supplemental Figure 1. Phylogenetic tree of eukaryotic MIPS proteins.

Supplemental Data. Luo et al. (2011). Plant Cell 10.1105/tpc.111.083337

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Supplemental Figure 2. Expression patterns of the three MIPS genes in different

Arabidopsis tissues.

More than 20 transgenic lines were analyzed for each MIPS promoter. Bars = 1 mm.


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Supplemental Figure 3. Phenotypes of mips double and triple mutants and their

Trypan blue staining assays.

(A) to (E) Phenotype of seedlings grown in 16 hr light/8 hr dark growth condition.

Bars = 5 mm.

(F) to (H) Trypan blue staining of wild type (F), mips1 mips2+/- (G), and mips1 mips3

(H) rosette leaves. Dead cells were stained blue. Bars = 1 mm.


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Supplemental Figure 4. PI#1 localization in globular stage embryos of wild-type

and mips1 mips3 double mutant upon BFA treatment.

PIN1 localization in globular stage embryos for wild-type (B) and mips1 mips3

double mutant (D) after 10 min in 50 µM BFA (Invitrogen, Molecular Probes) treatment.

An equal amount of DMSO in 6% glucose solution was used as a negative control for

wild-type (A) and mips1 mips3 (C). Bars = 10 µm.


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Supplemental Figure 5. #umbers of trafficking vesicles in wild type, mips1 mips3

double mutant and PIS2-OX embryo cells.

Cells (n=15 for each line) with similar area of the cross section were used for

calculation. Error bars indicate standard error of mean.


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Supplemental Table 1. Primers used in this study.

T-D#A insertion

MIPS1 sense AGA ACA TTG TGC CAC TCC CTG GG

antisense CCT GAT CGC CAT ATC AAT TAG ACC

MIPS2 sense CGC TAT GGC TAG AGC TAG AGT TCT

antisense CGA TAT AGC CAA TTC GAT AAG ACC

MIPS3 sense GGA GGA TGG GAC ATA AGT GAC ATG

antisense TGA CAT GAG ATT CTC CGT CGT GTC

LBa1 TGG TTC ACG TAG TGG GCC ATC G

DL3 GCT TTC GCC TAT AAA TAC GAC GG

Real Time PCR

MIPS1 sense CGT TGG GAT GAA CGA TAC AAT

antisense ACA TTG TTC CTG ATC GCC ATA

MIPS2 sense GAT CAT GGA GTA CAA GTG AAG G

antisense TAA ACC GAC CAG ATC CAT ATC

MIPS3 sense ATG ATT CTC GAG TAC AAG TGA TC

antisense CGC GAG ATA CCAT TAC ATA TT

TUB2 sense GTT CTC GAT GTT GTT CGT AAG

antisense TGT AAG GCT CAA CCA CAG TAT

RT PCR

MIPS1 sense CGA CTT GCA GAA ACA GCT CAG G

antisense CGC AAC ATA CGG TAC ATA CTT

MIPS2 sense TCC TTG TTG GAG CTG GCA TT

antisense AAA CTT CCC CTC TCC TTC AGC

MIPS3 sense GCT GGT GTT ATC GCC AAT CGT

antisense CCT CAT CCT TGT CCA AAG ATG A

TUB2 sense GTT CTC GAT GTT GTT CGT AAG

antisense TGT AAG GCT CAA CCA CAG TAT

Promoter clone

MIPS1 sense CCA GCA CAT CAA CAT CAC CCT

antisense CTC AAT AAA CAT TTT TGC AGA G

MIPS2 sense TGT CGA TGC GGG ACG TTG AAC

antisense CTC GAT GAA CAT TTT CTC TGG

MIPS3 sense GGGG ACA AGT TTG TAC AAA AAA GCA GGC

TGA TGA AAT GTC TTT GTC CTG GT

antisense GGGG AC CAC TTT GTA CAA GAA AGC TGG

GTA GAA TTA ATT GAT GCT TGA GAG

CDS clone


TAT ATG TTT ATT GAG AGC TTC AA



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GTT TCA CTT GAA TTC CAT GAT CA


TAT ATG TTC ATC GAG AGC TTC AA


GTT TCA CTT GTA CTC CAT GAT CA


TAT ATG TTC ATC GAA AGC TTC AA


GTT TCA CTT GTA CTC GAG AAT CA

INO1 sense GGGG ACA AGT TTG TAC AAA AAA GCA GGC

TCA ATG ACA GAA GAT AAT AT


GTA TTA CAA CAA TCT CTC TTC

Over expression

PIS1 sense GGGG ACA AGT TTG TAC AAA AAA GCA GGC

TAA AGA GAT TCT CAC GAT CG


GTT TTG GTA ACG AGA TAA TGC TGA

PIS2 sense GGGG ACA AGT TTG TAC AAA AAA GCA GGC

TTT CCT GAC AGC GAG AAG AA


GTA ACA CGA TTC ACG GGC ACA

UGT84B1 sense GGGG ACA AGT TTG TAC AAA AAA GCA GGC

TAG CCT TGA AGA AGA AGA AA


GTC CTT GAT AAT GGT CAG CTA

OX lines T-D#A

insertion

PIS1 sense GTG TTT GGA ATC GGA GTA AGG A

PIS2 sense TTT TCT CCT GGC TTT GAC ATT G

UGT84B1 sense CTC ATA GCA ACG AAC CAA ACC

vector antisense GTT CAC CTT GAT GCC GTT CTT

Annotation:

MIPS1 sense/LBa1, MIPS2 antisense/LBa1, MIPS3 sense/DL3 primer pairs were used for mips

T-DNA insertion detection.

For the construction of gateway over expression vector, pH7FWG2 was digested by HindIII/SpeI

to release 35S promoter and filling in with the tetramer-35S-enhancer fragment.


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Supplemental Table 2. Accession numbers for the sequences used in the

alignment in Supplemental Figure 1 and Supplemental Dataset 1.

Species Abbreviation GI

Saccharomyces cerevisiae SceINO1 37362665

Ostreococcus tauri Ota 116061703

Arabidopsis thaliana (At5g10170) AthMIPS3 15236025

Brassica rapa subsp. Pekinensis Bra 72384416

Triticum aestivum Tae 14548098

Nicotiana tabacum Nta 14548096

Nicotiana paniculata Npa 14548099

Sesamum indicum Sin 14548095

Arabidopsis thaliana (At2g22240) AthMIPS2 15227169

Brassica napus MIPS1 BnaMIPS1 14548093






Glycine max (1) GmaMIPS1 119960405




Passiflora edulis f. flavicarpa Ped 95104695

Arabidopsis thaliana (AT4g39800) AthMIPS1 15236025

Suaeda maritima subsp. Salsa Sma 16755889

Medicago truncatula Mtr 92898152

Phaseolus vulgaris (1) Pvu_1 14548092

Phaseolus vulgaris (2) Pvu_2 60417544

Avicennia marina Ama 13447457

Spirodela polyrrhiza Spo 1170568

Zea mays (1) Zma_1 11762100

Zea mays (2) Zma_2 14548094


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Trifolium pratense Tpr 84468396

Avena sativa Asa 18181983

Xerophyta viscosa Xvi 32492882

Oryza sativa (japonica) (Os03g0192700) Osa_1 115451283

Oryza sativa (japonica) (Os10g0369900) Osa_2 115481722

Hordeum vulgare Hvu 14548089

Citrus x paradisi Cpa 1170567

Physcomitrella patens (1) Ppa_1 168055751

Physcomitrella patens (2) Ppa_2 168020111

Phaeodactylum tricornutum Ptric 219116747

Brassica juncea Bju 165929042

Picea sitchensis Psi 148907091

Vitis vinifera Vvi 157345093

Cicer arietinum Car_1 198401802

Cicer arietinum Car_2 198401804

Mesembryanthemum crystallinum Mcr 14548091

Arabidopsis arenosa Aare 217426800

Medicago sativa Msa 139478073

Vigna radiata Vra 189169790

Chlamydomonas reinhardtii Cre 159490574

Thalassiosira pseudonana Tps 220971094

Arthrocnemum glaucum Agl 145315805

Kluyveromyces lactis Kla 50307609

Candida glabrata Cgl 50290163

Debaryomyces hansenii Dha 50426419

Yarrowia lipolytica Yli 50545902

Aspergillus fumigatus Afu 70988765

Neosartorya fischeri Nfi 119497811

Laccaria bicolor Lbi_1 170100589

Laccaria bicolor Lbi_2 170100971

Pyrenophora tritici-repentis Ptrit 189198886

Cryptococcus neoformans Cne 58265488


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Penicillium marneffei Pma 212527356

Sclerotinia sclerotiorum Ssc 156066001

Gibberella zeae Gze 46136849

Drosophila melanogaster Dme 17137626

Aedes aegypti Aae 157132320

Anopheles gambiae Aga 158300873

Paramecium tetraurelia Pte 145499166

Trichomonas vaginalis Tva 123433216

Caenorhabditis briggsae Cbr 157750944

Ustilago maydis Uma 71022933

Malassezia globosa Mgl 164657384

Caenorhabditis elegans Cel 25147617

Tetrahymena thermophila Tth 118364145

Trypanosoma brucei Tbr 71747782

Strongylocentrotus purpuratus Spu 115640689

Coprinopsis cinerea Cci 169846152

Hydra magnipapillata Hma 221129143

Nematostella vectensis Nve 156381404

Mus musculus Mmus 12963757

Bos taurus Bta 114051253

Rattus norvegicus Rno 189163481

Canis familiaris Cfa 73986122

Salmo salar Ssa 213511546

Branchiostoma floridae Bfl 219472225

Macaca mulatta Mmul 109123938

Equus caballus Eca 194232542

Xenopus laevis Xla_1 148233209

Xenopus laevis Xla_2 147901823

Nasonia vitripennis Nvi 156555239

Xenopus (Silurana) tropicalis Xtr 187607836

Apis mellifera Ame 66546786

Dictyostelium discoideum Ddi 66808743


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Macaca fascicularis Mfa 75076346

Homo sapiens HsaMIPS1 7705558

Tribolium castaneum Tca 91088055

Ixodes scapularis Isc 215495900

Pediculus humanus corporis Phu 212515411

Entamoeba histolytica Ehi 67477740

Tetraodon nigroviridis Tni 47229914

Pan troglodytes Ptrog 114676130


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supplemental data. luo et al. (2011). plant cell … tca ctt gaa ttc cat gat ca mips2 sense gggg aca...

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