Supplemental Figure 1. Mutation in NLA Causes Increased Pi Uptake Activity and
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1 Supplemental Figure 1. Mutation in NLA Causes Increased Pi Uptake Activity and PHT1 Protein Amounts. (A) Shoot morphology of 19-day-old nla mutants under Pi-sufficient conditions. (B) [ 33 P]Pi uptake activity of 19-day-old WT and nla mutants. N = 8; error bar = SEM; Student s t test, mutants versus WT, ***, P < FW, fresh weight; h, hour. (C) Protein expression of PHT1;1/2/3, PHT1;4 and PHF1 in the roots of WT and nla mutants under Pi-sufficient (+Pi) and -deficient conditions (-Pi).
2 Supplemental Figure 2. The Responses of WT and nla-2 Mutants after Recovery from Pi Deprivation. (A) Shoot morphology of the WT and nla-2 mutants under Pi-sufficient conditions (right) or after recovery from 4- day Pi deprivation (left). Severe necrosis at leaf edges appeared in the mutants after recovery for 3 days. (B) Expression of PHT1;1, PHT1;2, PHT1;3 and PHT1;4 in the roots of WT and nla-2 mutants after Pi replenishment. There was no significant difference in mrna level between the WT and nla mutants. N = 3; error bar = SEM. (C) Expression of primary mir827 (pri-mir827) in the WT after Pi replenishment. N = 3; error bar = SEM.
3 Supplemental Figure 3. Subcellular Localization of NLA. (A) Subcellular localization of GFP-NLA or NLA-GFP in Arabidopsis mesophyll protoplasts. Bar = 20 μm. (B) Colocalization of CFP-NLA with FM4-64 at the PM after staining for 15 minutes. Bar = 10 μm. (C) Subcellular localization of GFP-NLA in 3 independent nla-2 lines (12-day-old) overexpressing GFP-NLA. Bar = 20 μm. (D) Pi content of overexpressing GFP-NLA in 12-day-old nla-2 mutants grown in high Pi medium. N = 3; error bar = SEM; Student s t test, nla-2 mutants versus WT, ***, P < 0.005; overexpressing lines versus nla-2, +,P<0.05; +++, P < FW, fresh weight. (E) NLA proteins were detected only in the membrane protein fraction and not in the soluble protein fraction. PIP1, cfbpase and PHF1 were used as markers for PM proteins, cytosolic proteins and ER proteins, respectively.
4 Supplemental Figure 4. Expression Pattern of NLA and MIR827. GUS staining of roots of 12-day-old seedlings for examining the expression pattern of NLA (A) and MIR827 (B). The cross sections of roots were from the areas marked with arrowheads in the left panels. Expression pattern of NLA and MIR827 in shoots are shown in (C) and (D), respectively. Bar = 50 μm. Six independent transgenic lines of each construct were examined. Representative lines are shown.
5 Supplemental Figure 5. Analysis of Protein-Protein Interaction between NLA and PHT1;4. Coexpression of PHT1;4-cYFP with NLA C279A -nyfp in the tobacco leaves shown BiFC fluorescence in the PM (A) which was colocalized with FM4-64 after staining for 15 minutes (B). The merge panel is an overlaid image of YFP and bright field (A) or YFP, FM4-64 and bright field (B).
6 Supplemental Figure 6. The N-terminal Portion of PHT1;1 Is Sufficient for NLA-Mediated Degradation. (A) None of the mutated PHT1;1 proteins (PHT1;1 K524A,PHT1;1 K276/279A,PHT1;1 K276/279/524A ) were resistant to NLA-mediated degradation when coexpressed with NLA in tobacco leaves. (B) Coexpression of N- (PHT1;1 N1-294 ) or C- (PHT1;1 C ) terminal portion of PHT1;1 with NLA or NLA C279A in tobacco leaves. PHT1;1 N1-294 was degraded by NLA but not by NLA C279A. The signal of PHT1;1 C was too weak to be conclusive. Microsomal proteins were used for this experiment. PHT1;1 and its mutated and truncated forms were detected by anti-pht1;1/2/3 antibodies. Co-infiltration of 35S::GFP construct was used as an internal control. Note that "+++" and "++" denotes 5- and 2.5-fold increases in volume of infiltration relative to "+", respectively.
7 Supplemental Figure 7. NLA-Mediated Ubiquitination Triggers PHT1;1 Sorting into the Vacuole through the Endosomal Trafficking Pathway. The effect ectof E-64d treatment e t on PHT1;1-YFP trafficking ta in two additional a independent transgenic ta lines, #6 and #8 in the WT background (A) and #14 and #16 in nla-2 mutants (B). The merge panel is an overlaid image of YFP, FM4-64 and bright field. The aggregation of PHT1;1-YFP in the WT (A) is indicated by arrowheads. Bar = 20 μm.
8 Supplemental Figure 8. Subcellular localization of Os-NLA1. Subcellular localization of CFP-OsNLA1 (A) and OsNLA1-CFP (B) in tobacco leaves. The merge panel is an overlaid image of CFP and bright field. Bar = 20 μm.
9 Supplemental Figure 9. The Difference between NLA- and PHO2-Dependent Regulation of PHT1s. (A)The leaf phenotype of 19-day-old nla pho2 double and respective single mutants. The necrosis (arrowhead) was clearly observed at the leaf edge of double mutants. The Pi content (B) and protein expression of PHT1;1/2/3, PHT1;4 and PHF1 (C) in nla-1 pho2 double and the respective single mutants. N = 3; error bar = SEM. Student s t test, single mutants versus WT, *, P<0.05;**, P<0.01;***, P < 0.005; nla-1 pho2 versus pho2, +,P < FW, fresh weight. The detection of ARF1 was used as a loading control. (D) Immunoblot analysis of PHT1;1/2/3 and PHT1;4 in 10-day-old mutants and the corresponding WT. The detection of ARF1 was used as a loading control. (E) The mrna expression of NLA in 19-day-old WT and pho2 plants. There was no significant difference of NLA expression between WT and pho2 mutants. N = 3; error bar = SEM. (F) Expression of mature mir827 and mir399 in the roots of 19-day-old WT and pho2 plants. (G) The Pi content of nla-1 and pho2 mutants during plant growth. N = 3, error bar = SEM. Student s t test, single mutants versus WT, *, P < 0.05; ***, P < FW, fresh weight.
10 Supplemental Table 1. Sequence of Primers Used in This Study Gene Primer Sequence (5 3 ) AT1G02860 Forward CTTTTTTTTTGTTTGAGGGCTGAAT NLA Reverse TCATTTATTTTTTCTCTGAATTTCACAAC AT2G33770 Forward AGGTTTGAAGCTCCACCCTCA PHO2 Reverse CCCAAGATGTGATTGGAGTTCC AT2G38940 Forward GGTCCCAATAGTTTAGGTGAT PHT1;4 Reverse AGTTGCTAGAGACAAGGAGAA AT3G59884 Forward CCACGAAAGAGTTTGTTGATGGT MIR827 Reverse TCCTTGTGTTGATCGATTGGTT AT5G43350 Forward GCCATGACGAGAAATAATTATGT PHT1;1 Reverse TAACTTAAGGTCAACGAGCCAAT AT5G43360 Forward CGAGGCTGAGGTTGATAAATGAT PHT1;3 Reverse CACACATCGCAAAACCAATGAC AT5G43370 Forward AGCCATCATTGGAGCCTTC PHT1;2 Reverse ACCTTAGCCTTGTCTTGATT AT4G05320 Forward GGCCTTGTATAATCCCTGATGAATAAG UBQ10 Reverse AAAGAGATAACAGGAACGGAAACATAGT Construct Primer Sequence (5 3 ) 35S::NLA Forward AGCTCCATGAAGTTTTGTAAGAAGT 35S::GFP-NLA 35S::CFP-NLA Reverse ACCTCGAACTATATCATATTCCAGT 35S::NLA-GFP Forward AGCTCCATGAAGTTTTGTAAGAAGT 35S::NLA-CFP Reverse TATTCCAGTGAAGCTTCGGCA 35S::NLA C279A Forward GCCCGTGAGGATGGGGTTTATAAAGGTGC 35S::CFP-NLA C279A Reverse AAGCGGGCATTTTTCAGTTGCTTCTGCG Forward AGCTCCATGAAGTTTTGTAAGAAGT 35S::CFP-NLA SPX Reverse TCAAGAGAGCTCGCAGGAAAG 35S::NLA SPX -CFP Forward AGCTCCATGAAGTTTTGTAAGAAGT UBQ pro ::NLA SPX -nyfp Reverse AGAGAGCTCGCAGGAAAG 35S::CFP-NLA RING Forward ATGAAAATGCGAATAGAAATC Reverse ACCTCGAACTATATCATATTCCAGT Forward ATGAAAATGCGAATAGAAATC 35S:: NLA RING -CFP Reverse TATTCCAGTGAAGCTTCGGCA 35S::PHT1;1 Forward ATGGCCGAACAACAACTAGGAGT Reverse TTATTTCTCGTCATGGCTAACCTC UBQ pro ::PHT1;1-cYFP Forward ATGGCCGAACAACAACTAGGAGT Reverse TTTCTCGTCATGGCTAACCTCA Pro-for CCCGGGTCTAGGGACGTCCAAGTTCACTGAAT CACAGCTCCACCTCCACCTCCAGGCCGGCCAGAGAGTTCTTCAAGGGACTT P2 GCCT PHT1;1 pro ::PHT1;1-YFP FP-for GGCCGGCCTGGAGGTGGAGGTGGAGCTGTGAGCA FP-rev GGCCCCAGCGGCCGCAGCAGCACCAGCAGGATC P3 TGCTGGTGCTGCTGCGGCCGCTGGGGCCGGTGAGGCTGAGGTTAGCCATG P4 CGAAATATTAGACAAGATTTAAATGATATC
11 Supplemental Table 1. Sequence of Primers used in This Study (Continued) Construct Primer Sequence (5 3 ) P1 ATGGCCGAACAACAACTAGGAGT P2 CACAGCTCCACCTCCACCTCCAGGCCGGCCAGAGAGTTCTTCAAGGGACTT GCCT 35S::PHT1;1-YFP FP-for GGCCGGCCTGGAGGTGGAGGTGGAGCTGTGAGCA FP-rev GGCCCCAGCGGCCGCAGCAGCACCAGCAGGATC P3 TGCTGGTGCTGCTGCGGCCGCTGGGGCCGGTGAGGCTGAGGTTAGCCATG P4 CGAAATATTAGACAAGATTTAAATGATATC 35S::PHT1;4 Forward ATGGCAAGGGAACAATTACAAGTG Reverse CTAAACTATTGGGACCGTTCTACTATCA UBQ pro ::PHT1;4-cYFP Forward ATGGCAAGGGAACAATTACAAGTG Reverse AACTATTGGGACCGTTCTACTATCATT P1 ATGGCAAGGGAACAATTACAAGTG CACAGCTCCACCTCCACCTCCAGGCCGGCCGCTATTCTCATTGTCTTCATTTT P2 CACC 35S::PHT1;4-YFP FP-for GGCCGGCCTGGAGGTGGAGGTGGAGCTGTGAGCA FP-rev GGCCCCAGCGGCCGCAGCAGCACCAGCAGGATC P3 TGCTGGTGCTGCTGCGGCCGCTGGGGCCAACAATGATAGTAGAACGGTCCC P4 GGTCAATTACGAAGGAGAGCCTTCTACTT 35S::PHT1;1-HA Forward ATGGCCGAACAACAACTAGGAGT Reverse TTAAGCGTAATCTGGAACATCGTATGGGTATTTCTCGTCATGGCTAAC 35S::PHT1;1 K524A -HA 35S::PHT1;1 K276/279A -HA 35S::PHT1;1 K276/279/524A -HA 35S::OsNLA1 35S::CFP-OsNLA1 35S::OSNLA1-CFP UBQ pro ::IRT1-cYFP MIR827 pro ::GUS NLA pro ::GUS Forward GCATACCCATACGATGTTCCAGATTACGCT Reverse CTCGTCATGGCTAACCTCAGCCTCAC Forward GACCCCGCACAAAACTATGGCTTGTTCTCC Reverse TGCGACGTCATCCTCCACCCTTTCCTCAAG Forward ATGAAGTTTGCCAAGAAGTAC Reverse TCACATGCCCAAGAATGCC Forward ATGAAGTTTGCCAAGAAGTAC Reverse CATGCCCAAGAATGCCCTG Forward ATGGCTTCAAATTCAGCAC Reverse GCGGCCGCTTAAGCCCATTTGGCGATAA Forward CACTATGTTAAACTCAGCTT Reverse ACCTATAACGTTTCATGGAAGT Forward AGCCAAATAGGTATTAGTCT Reverse GAACATATCTTCTTCTTCGT Forward CGCTGGATCCATGAAGTTTTGTAAGAAGT pdl-nub-nla SPX Reverse TCAAGAGAGCTCGCAGGAAAG Forward ACATCTAGAATGGCAAGGGAACAATTAC ptmbv4-pht1;4-cub Reverse TATGTCTAGAAACTATTGGGACCGTTCTAC
12 Supplemental Table 2. Constructs Purpose promoter::reporter assay overexpression HA-tagged PHT1;1 and pointmutated PHT1;1 fluorescent protein-tagged fusion protein bimolecular fluorescence complementation (BiFC) split-ubiquitin yeast-two-hybrid Construct Plasmid backbone Insert or PCR product 2.2 kb upstream NLA pro ::GUS pmdc164 1 genomic sequence of NLA from start codon MIR827 pro ::GUS pmdc164 1 genomic sequence of 1.3 kb upstream MIR827 from stem loop Template used for cloning Arabidopsis genomic DNA Arabidopsis genomic DNA Cloning method 35S::PHT1;1 pmdc32 1 PHT1;1 CDS TOPO-PHT1;1 CDS 35S::PHT1;4 pmdc32 1 PHT1;4 CDS TOPO-PHT1;4 CDS 35S::NLA pmdc32 1 NLA CDS Arabidopsis cdna 35S::NLA C279A pmdc32 1 NLA C279A CDS TOPO-NLA CDS site-directed mutagenesis/ 35S::OsNLA1 pmdc32 1 OS-NLA1 CDS rice cdna 35S::PHT1;1-HA pmdc32 1 PHT1;1-HA TOPO-PHT1;1 CDS site-directed 35S::PHT1;1 K524A -HA pmdc32 1 PHT1;1 K524A -HA TOPO-PHT1;1-HA mutagenesis/ site-directed 35S::PHT1;1 K276/279A -HA pmdc32 1 PHT1;1 K276/279A -HA TOPO-PHT1;1-HA mutagenesis/ 35S::PHT1;1 K276/279/524A -HA pmdc32 1 PHT1;1 K276/279/524A -HA TOPO-PHT1;1K276/279A HA PHT1;1 pro ::PHT1;1-YFP pmdc99 1 PHT1;1 genomic DNA &YFP 35S::PHT1;1-YFP pmdc32 1 PHT1;1 CDS &YFP 35S::PHT1;4-YFP pmdc32 1 PHT1;4 CDS &YFP Arabidopsis genomic DNA and pk7wgy2 TOPO-PHT1;1 CDS and pk7wgy2 TOPO-PHT1;4 CDS and pk7wgy2 site-directed mutagenesis/ three piece PCR 4 / three piece PCR 4 / three piece PCR 4 / 35S::GFP-NLA pk7wgf2 2 NLA CDS TOPO-NLA CDS 35S::NLA-GFP pmdc83 1 NLA CDS TOPO-NLA CDS 35S::CFP-NLA pk7wgc2 2 NLA CDS TOPO-NLA CDS site-directed 35S::CFP-NLA C279A pk7wgc2 2 NLA C279A CDS TOPO-NLA CDS mutagenesis/ 35S::NLA-CFP pk7cwg2 2 NLA CDS TOPO-NLA CDS 35S::CFP-NLA SPX pk7wgc2 2 NLA CDS TOPO-NLA SPX 35S::CFP-NLA RING pk7wgc2 2 NLA CDS TOPO-NLA RING 35S::CFP-OsNLA1 pk7wgc2 2 Os-NLA1 CDS TOPO-OsNLA1 OsNLA1 CDS 35S::OsNLA1-CFP pk7cwg2 2 Os-NLA1 CDS TOPO-OsNLA1 CDS UBQ pro ::NLA-nYFP pubc-nyfp 3 NLA CDS TOPO-NLA CDS UBQ pro ::NLA SPX -nyfp pubc-nyfp 3 NLA CDS TOPO-NLA SPX UBQ pro ::PHT1;1-cYFP pubc-cyfp 3 PHT1;1 CDS TOPO-PHT1;1 CDS UBQ pro ::PHT1;4-cYFP pubc-cyfp 3 PHT1;4 CDS TOPO-PHT1;4 CDS UBQ pro ::CHL1-cYFP pubc-cyfp 3 CHL1 CDS pentrtm/d-topo- CHL1 UBQ pro ::IRT1-cYFP pubc-cyfp 3 IRT1 CDS pdl-nubg-irt1 (Shin et al., 2013) ptmbv4-pht1;4 ptmbv4 PHT1;4 CDS TOPO-PHT1;4 CDS XbaI pdl-nubg-nla SPX pdl-nx2 NLA CDS TOPO-NLA SPX BamHI/SmaI 1 Curtis, M.D., and Grossniklaus, U. (2003). A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol. 133: Karimi, M., Inzé, D., and Depicker, A. (2002). GATEWAY vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci. 7: Grefen, C., Donald, N., Hashimoto, K., Kudla, J., Schumacher, K., and Blatt, M.R. (2010). A ubiquitin-10 promoter-based vector set for fluorescent protein tagging facilitates temporal stability and native protein distribution in transient and stable expression studies. Plant J. 64: Tian, G.-W., Mohanty, A., Chary, S.N., Li, S., Paap, B., Drakakaki, G., Kopec, C.D., Li, J., Ehrhardt, D., Jackson, D., Rhee, S.Y., Raikhel, N.V., and Citovsky, V. (2004). High-Throughput Fluorescent Tagging of Full-Length Arabidopsis Gene Products in Planta. Plant Physiol. 135: Shin, L.J., Lo, J.C., Chen, G.H., Callis, J., Fu, H., and Yeh, K.C. (2013). IRT1 DEGRADATION FACTOR1, a RING E3 Ubiquitin Ligase, Regulates the Degradation of IRON-REGULATED TRANSPORTER1 in Arabidopsis. Plant Cell. PMID:
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