n. benthamiana - the plant cell · 21/04/2011 · fusion protein together with eres marker sar1....

YFP-

PHF

+ CFP

-PHT1

;2

YFP-

PHF

+ PH

T1;2-

CFP

!-GFP

YFP-

PHF1

CFP-P

HT1;2

PHT1

;2-CFP

Negati

ve co

ntrol

Supplemental Figure 1: PHT1;2 accumulation is PHF1dependent.Immunoblot analysis on total protein extract corresponding totransient expression in N. benthamiana leaf epidermis 48h post-infiltration presented in figure 2. Protein detection is done usinganti-GFP antibodies recognizing YFP-PHF1 (75kDa dark arrow)or CFP-PHT1;2 fusion proteins (90kDa white arrow).Coomassie blue acrylamide gel staining is shown as proteinloading control.

Supplemental Data. Bayle et al. (2011). Plant Cell 10.1105/tpc.110.081067

1

PHF1-GFP

PHF1-GFP ST-mRFP Merging

MergingST-mRFPD E F

BFA

50!M

120

min

Supplemental Figure 2: Strict ER localization of PHF1.(A-F) A. thaliana epidermal root cells expressing PHF1-GFP under control of its ownpromoter (A) and Golgi marker ST-mRFP (B). (C-D) same line with BFA treatment(50 !M, 2 hours). Scale bar:10 !m.


2

Supplemental Figure 3: PHT1;2-CFP export from ER is COPII-dependent(A-L) Transient protein expression in N. benthamiana epidermal cells analyzed byconfocal microscopy 48h post-infiltration. Transient expression of PHT1;2-CFPalone (B) showing weak plasma membrane fluorescence, together with Sec12-YFP(D-I) blue fluorescence is retained in ER structure with Sec12-YFP. Co-expression ofPHT1;2-CFP together with YFP-PHF1 (J-L) shows an increase of bluefluorescence, colocalization at the ER and correct targeting of PHT1;2-CFP to theplasma membrane. Scale bars are 10µm.

YFP-channel


3

Supplemental Figure 4: PHF1 is not associated with recruitment ofCOPII components to ERES.(A-L) Transient protein expression in N. benthamiana epidermal cells analyzed byconfocal microscopy 48h post-infiltration. Transient expression ERES markerYFP-Sec24 expressed alone (B), or together with ST-CFP (G-I), PHT1;2-CFP (J-L) or CFP-PHF1 (J-L). YFP-Sec24 mainly localizes to cytoplasm and is recruitedto ERES (white arrows). Scale bars are 10µm.


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Supplemental Figure 5: Co-expression of PHF1 and PHT1;2-CFPfusion protein together with ERES marker SAR1.(A-L) Transient protein expression in N. benthamiana epidermal cellsanalyzed by confocal microscopy 48h post-infiltrations. Transientexpression of SAR1-YFP alone (B) or together with CFP-PHF1 (D-F) orPHT1;2-CFP (J-L). Transient expression PHT1;2-CFP alone is indicatedas control (G-I) Scale bars = 10 !M.

G H I

J K LMerging

Merging

Sar1-YFP

Sar1-YFP

Sar1-YFP

CFP-PHF1

PHT1;2-CFP

PHT1;2-CFP

Merging

MergingCFP-channel

YFP-channel


5

Supplemental Figure 6: MS/MS fragmentation spectrum of the diphosphopeptideSLEEL(pS)GEAEV(pS)HDEK (PHT1.1).Attributed y and b ions are indicated on the spectrum. Double Neutral Loss (NL) waspresent. S*: identified phosphorylated serine after neutral loss.


6

Con

trol

treat

men

t

Supplemental Figure 7:Sorting endosomeslocalization of PHT1;1independently of Piexternal content.A. thaliana root tip cellsco-expressing PHT1;1-GFP and the sortingendosome marker SNX1-mRFP, 60 minutes after33 !M Wm treatment.Plants were cultivated onPi depleted medium (A-F)or Pi (500 !M) containingmedium (G-L) prior totreatment. (M)Quantitative analysis ofintracellular co-localization betweenPHT1;1-GFP and SNX-mRFP, cytofluorogramobtain for two images, (N)average Pearson’scoefficient (r) and afterCoste randomizationbased colocalization (rr)are given. Scale bars = 5!M. The Pearson’scoefficient was calculatedbased on the analysis of350 root tip cells from 20plants per condition.

Con

trol

treat

men

t

PH

T1;1

-GFP

SNX1-mRFP

PH

T1;1

-GFP

SNX1-mRFP

M 500!M Pi0!M Pi Nr =0,51±0,09rr =0,0±0,034

r =0,53±0,09rr =0,0±0,05

0!M

Pi

500!

M P

i

r = 0,55r = 0,53


7

T0 T7 T2

T0 T7 T2

Control treatmentCHX 50!M T7

T7

A B

E F

C D

G H

+P

-P

Supplemental Figure 8: Kinetic analysis of Pi-induced PHT1 degradation.A. thaliana root tip cells co-expressing PHT1;1-GFP cultivated on medium containing 0 !M(A-D) Pi or 500 !M (E-H). (A and E) prior to drug treatment; (B and F) 2 hours and (C and G)7 hours after CHX (50 !M) treatment. (H and L) control untreated after 7 hours. Scale barsare 10 !m


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Effect of pho2-1 mutation on PHT1;1:GFP degradation

0

10000

20000

30000

PHT1;1 pho2 N°1 pho2 N°2

rela

tive

fluor

esce

nce

T02h CHX

P500

Inhibition 26S proteasome test

0

10000

20000

30000

40000

50000

T0 2h CHX 6h MG132 2h CHX

rela

tive

fluor

esce

nce

(AU

)

P500

A

B

Supplemental Figure 9: Pi induced PHT1 degradation is not affected by MG-132 treatment or pho2-1 mutation. Relative fluorescence intensity of PHT1;1-GFP observed in root ofplant grown on +Pi (500 !M). (A) Effect of MG132 treatment (6h, 50!M), CHX was added during the two last hours (50 !M). (B) Effect ofpho2-1 mutation, PHT1;1-GFP marker was introgressed into pho2-1mutant and treated with CHX drugs as previously described.For each experiment, 12 lateral roots were observed. Thefluorescence from 20 (A) or 25 (B) regions of interests for each samplewas quantified. The mean and standard deviation from themeasurements are indicated here for one of the experimentsperformed.


9

500!

M P

i0!

M P

i

T0 T15min T60min

T0 T15min T60min

A B C

D E F

Supplemental Figure 10 : Phosphate starvationdoes not affect internalization of endocytic tracerFM4-64 in root cells. Seedlings cultivated eitherin 500!M Pi (A to C) or Pi deprived (D to F) mediumwere subjected to kinetic analysis of FM4-64internalization. Short time (A, B, D and E) showsendocytic tracer at the plasma membrane andendosomes, longer time shows tonoplast staining (Cand F). Scale bar=5 !M


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Table 1 : Primers used for amplification and site-directed mutagenesis of PHT1;1

forward primer reverse primer PHT1;1 cDNA 5'-CACCATGGCCGAACAACAACTAGG-3' 5'-TTTCTCGTCATGGCTAACCTCA-3' PHT1;1-S148A 5'-GTGACTACCCACTTGCTGCCACCATCATGTC-3' 5'-GACATGATGGTGGCAGCAAGTGGGTAGTCAC-3' PHT1;1-S148D 5'-GTGACTACCCACTTGATGCCACCATCATGTC-3' 5'-GACATGATGGTGGCATCAAGTGGGTAGTCAC-3' PHT1;1-S153A 5'-TGCCACCATCATGGCTGAATACGCAAACA-3' 5'-TGTTTGCGTATTCAGCCATGATGGTGGCA-3' PHT1;1-S153D 5'-TGCCACCATCATGGATGAATACGCAAACA-3' 5'-TGTTTGCGTATTCATCCATGATGGTGGCA-3' PHT1;1-T160A 5'-CGCAAACAAGAAGGCCCGTGGGGCTTTCA-3' 5'-TGAAAGCCCCACGGGCCTTCTTGTTTGCG-3' PHT1;1-T160D 5'-CGCAAACAAGAAGGACCGTGGGGCTTTCA-3' 5'-TGAAAGCCCCACGGTCCTTCTTGTTTGCG-3' PHT1;1-T239A 5'-GAAGATGCCTGAAGCTGCCCGTTACACCG-3' 5'-CGGTGTAACGGGCAGCTTCAGGCATCTTC-3' PHT1;1-T239D 5'-GAAGATGCCTGAAGATGCCCGTTACACCG-3' 5'-CGGTGTAACGGGCATCTTCAGGCATCTTC-3' PHT1;1-T368A 5'-GCGTTTATTGATGCCATTGGAAGGTTT-3' 5'-AAACCTTCCAATGGCATCAATAAACGC-3' PHT1;1-T368D 5'-GCGTTTATTGATGACATTGGAAGGTTT-3' 5'-AAACCTTCCAATGTCATCAATAAACGC-3' PHT1;1-S438A 5'-GGCCAGGCTAAGGGCTACATGTCATGGAA-3' 5'-TTCCATGACATGTAGCCCTTAGCCTGGCC-3' PHT1;1-S438D 5'-GGCCAGGCTAAGGGATACATGTCATGGAA-3' 5'-TTCCATGACATGTATCCCTTAGCCTGGCC-3' PHT1;1-S509A 5'-GCCCAAAGGCAAGGCCCTTGAAGAACTCT-3' 5'-AGAGTTCTTCAAGGGCCTTGCCTTTGGGC-3' PHT1;1-S509D 5'-GCCCAAAGGCAAGGACCTTGAAGAACTCT-3' 5'-AGAGTTCTTCAAGGTCCTTGCCTTTGGGC-3' PHT1;1-S514A 5'-CCTTGAAGAACTCGCTGGTGAGGCTGAGG-3' 5'-CCTCAGCCTCACCAGCGAGTTCTTCAAGG-3' PHT1;1-S514D 5'-CCTTGAAGAACTCGATGGTGAGGCTGAGG-3' 5'-CCTCAGCCTCACCATCGAGTTCTTCAAGG-3' PHT1;1-S520A 5'-TGAGGCTGAGGTTGCCCATGACGAGAAA-3' 5'-TTTCTCGTCATGGGCAACCTCAGCCTCA-3' PHT1;1-S520D 5'-TGAGGCTGAGGTTGACCATGACGAGAAA-3' 5'-TTTCTCGTCATGGTCAACCTCAGCCTCA-3'

The CACC sequence allowing a directional cloning of PHT1;1 cDNA into pENTR is shown in bold . For site-directed mutagenesis, the subsitution sites for Ser and Thr in the primers to generate the mutations are underlined.


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n. benthamiana - the plant cell · 21/04/2011 · fusion protein together with eres marker sar1....

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