Lim et al, Supplemental Figure S1.

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Figure S1. Phenotypic effect of rice plants under As and Cd treatments. Rice (cv. Donganbyeo) seeds were germinated, and then grown in soil in the absence or presence of the indicated concentrations of As(V) (Na2HAsO4) or Cd (CdSO4) in a controlled environment with a 16 h light/8 h dark photoperiod at 22°C/18°C. Plant height was adjusted using the measuring rule at 4 weeks after transplanting. Error bars represent the standard error, and the same letters above each bar indicate no difference with treatment at a 0.05 significance level of the Duncan’s test. Data are from 3–4 in-dependent assays.

Lim et al, Supplemental Figure S2.

50 100 150 200 300 350 400250

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Figure S2. Hydropathy plot of the predicted OsHIR1 protein sequence. The hydropathy plot (mean values for a window of 17 amino acids) was elabo-rated using the Kyte–Doolittle (KD) hydrophobicity index (http://web.expasy.org/protscale/).

AtUBC21

OsHIR1

35S:EYFP # 1 # 11 # 12

35S:OsHIR1-EYFP

Lim et al, Supplemental Figure S3.

Figure S3. Semi-quantitative RT-PCR of OsHIR1-overexpress-ing Arabidopsis and control plants. Total RNAs from 2-week-old seedlings of 35S:OsHIR1-EYFP and 35S:EYFP (empty vec-tor) T3 transgenic plants were used for RT-PCR. AtUBC21 was used for the internal control.

Clone QDO/X/AX-α-Gal activ-

ityTIGR annotation (Rice Genome Annotation Resource)

Predicted subcellular local-ization

TIGR Locus

+ +++ sucrose-phosphate synthase Cytosol LOC_Os01g69030

+ +++ jacalin-like lectin domain containing protein Cytosol LOC_Os01g24710

+ +++ universal stress protein domain containing protein Chloroplast LOC_Os05g28740

+ + tRNA synthetases class II domain containing protein Nuclear LOC_Os02g04700

+ + elongation factor Tu Cytosol LOC_Os03g08020

+ + ADP-ribosylation factor Cytosol LOC_Os03g59740

+ +++ ferredoxin 1-like isoform 1 Mitochodria LOC_Os09g33950

+ +++ expressed protein Chloroplast LOC_Os09g13590

+ +++ aquaporin protein Cytosol LOC_Os05g14240

+ +++ DnaK family protein Cytosol LOC_Os11g47760

+ +++mitochondrial-processing peptidase subunit mitochondrial precursor Mitochondria LOC_Os03g11410

+ + Ubiquitin family domain containing protein Cytosol LOC_Os03g03920

+ +++ NADH dehydrogenase flavoprotein 2 mitochondrial precursor Chloroplast LOC_Os05g43360

+ +++ OTU-like cysteine protease family protein Nuclear LOC_Os02g07210

+ +++ VTC2 Chloroplast LOC_Os12g08810

+ + phosphosulfolactate synthase-related protein Cytosol LOC_Os06g46900

+ + ras-related protein Nuclear LOC_Os01g37800

+ + expressed protein Cytoskeleton LOC_Os03g04930

+ +++ transaldolase Chloroplast LOC_Os01g70170

+ ++ trehalose-6-phosphate synthase Nuclear LOC_Os09g20990

+ +++ expressed protein Nuclear LOC_Os06g36360

+ +++ WD domain G-beta repeat domain containing protein Mitochondria LOC_Os08g21660

+ +++ pollen-specific protein SF21 Chloroplast LOC_Os06g36740

+ +++ hypersensitive-induced response protein Cytosol LOC_Os08g30790

+ ++ CHIT3 - Chitinase family protein precursor Nuclear LOC_Os04g41680

+ + ubiquitin family protein Cytosol LOC_Os02g06640

+ + bZIP transcription factor domain containing protein Nuclear LOC_Os07g44950

+ ++ PRLI-interacting factor A Mitochodria LOC_Os02g58570

+ + calvin cycle protein CP12 Chloroplast LOC_Os03g19380

+ ++ receptor-like protein kinase HAIKU2 precursor Plasma membrane LOC_Os12g43640

+ ++ N-carbamoylputrescine amidase Cytosol LOC_Os02g33080

Lim et al, Supplemental Figure S4.

Figure S4. Positive interacting substrate proteins of the OsHIR1 RING E3 ligase. A yeast two-hybrid screening and their predicted subcellular distribution are presented. “+” indicates cell growth on QDO/X/A or density of α-GAL activity (“+,” weak; “++,” strong; “+++,” very strong). Subcellular local-izations of positive interacting proteins with OsHIR1 were predicted using the WoLF PSORT program (http://psort.hgc.jp/form.html).

Lim et al, Supplemental Figure S5.

35S::OsTIP4;1-EGFP

35S::OsHIR1-EGFP

/35S::OsTIP4;1-EGFP

35S::EGFP

OsHIR1

OsTIP4;1

Nt18S rRNA

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Figure S5. Coexpression of OsHIR1 and OsTIP4;1 in N. benthaminana leaves. A, Semi-quantitative RT-PCR was conducted to evaluate expression level of OsTIP4;1 in the present or present of OsHIR1 or. Each Agrobacterium (GV3101) containing the35S:OsHIR1-EGFP and 35S:OsTIP4:1-EGFP was transiently co-expressed in N. benthamiana leaves. The 35S:EGFP construct was used as negative control and the Nt18S rRNA gene was used as a internal control. B, The volume integration value of 35S:OsTIP4;1. Autoradiographs were digitized using a Bio-Rad Gel Doc XR+ system. The volume integration value for each band was determined after subtraction of local background by using Molecular Analyst Software (Bio-Rad) version 5.0. Bar indicates standard error of three replicate experiments.