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Page 1: Supporting Information - pnas. · PDF file1/2MS plates containing 5 μM β-estradiol (Sigma-Aldrich) for 24 h. DMSO instead of β-estradiol served as a control. ... (P5726; Sigma-Aldrich)

Supporting InformationOrtiz-Morea et al. 10.1073/pnas.1605588113SI Materials and MethodsPlant Material and Growth Conditions. All mutants and transgeniclines used were in the background of the Arabidopsis thaliana (L.)Heynh. accession Columbia 0 (Col-0). Seeds were sterilized,maintained for 2 d at 4 °C in the dark, and germinated on verticalhalf-strength Murashige and Skoog (1/2 MS) medium [1% (wt/vol)sucrose] agar plates, pH 5.8, at 22 °C in a 16-h/8-h light–darkcycle for 5 d with a light intensity of 120 μE m−2 s−1 (E, Einstein;1E = 1 mol of photons). The following mutant and transgenicArabidopsis lines have been described previously: pepr1pepr2 (2);chc2-1 and chc-2 (11); ap1m2-1 and hapless (hap13) (24); ap2m-2(21); ap2s (22); autophagy5-1 (atg5-1) (31) and atg7-2 (32); GFP-VAMP727 (33); VHA-a1-GFP (13); YFP-ARA7, YFP-RabA1eand YFP-MEMB12 (17); ARA6-GFP (18); and CLC1-GFP,CLC2-GFP, CHC1-GFP, and CHC2-GFP (16). The pUBQ10::ATG8-YFP/Col-0 line was a kind gift of M. Nowack andM. Fendrych, VIB–Ghent University, Ghent, Belgium. Thehap13 mutant was isolated in Wassilewskija (Ws) ecotype.

Generation of Constructs. The PEPR1 and PEPR2 promoters andcoding sequences were amplified by PCR with KaPaHIFI poly-merase (Sopachem) from genomic DNA (accession Col-0) withspecific primers (Table S1). The fragments thus obtained wereintroduced into pDONR entry vectors (pDONRP4-P1R pro-moter sequences and pDONR221 coding sequences) by meansof the Gateway system-compatible attB sites (Invitrogen). Tocreate transcriptional reporter vectors of PEPR1 and PEPR2,the entry clones pDONRP4-P1R-pPEPR1/PEPR2 were intro-duced into the destination vector pMK7S*NFm14GW with theGateway-based cloning (Invitrogen) to yield pMK7S*NFm14GW-pPEPR1/2::NLS-GFP. The entry vectors pDONRP4-P1R-pPEPR1/PEPR2 and pDONR221-PEPR1/PEPR2 were used in a triple LRreaction (MultiSite-Gateway; Invitrogen), combining pDONRP2-P3R-GFP and pB7m34GW to yield pB7m34GW-pPEPR1/PEPR2::PEPR1/PEPR2-GFP. To express PEPR1/PEPR2 underthe RPS5A promoter, the entry vector pDONRP4-P1R-pPEPR1/PEPR2 was replaced by pDONRP4-P1R-proRPS5A, producingthe pB7m34GW-pRPS5A::PEPR1/PEPR2-GFP construct. TheXVE>>AX2 line was generated using the coding sequence of theArabidopsis Auxilin2 (At4g12770) gene, which was introduced into theestradiol-inducible vector pMDC7B(UBQ10) via pENTR/D-TOPOGateway (Invitrogen), according to the manufacturer’s instructions.

Generation of Arabidopsis Transgenic Lines. Arabidopsis plants weretransformed with Agrobacterium tumefaciens by means of thefloral dip method. Plants expressing NLS-GFP under the en-dogenous promoters of PEPR1 and PEPR2 were transformedinto a Col-0 background. Primary transformants were selectedon 1/2MS medium containing kanamycin (50 mg/L). ThepRPS5A::PEPR1/PEPR2-GFP constructs were dipped into thepepr1pepr2 or XVE>>AX2 background, and transformants wereselected on 1/2MS medium containing 10 mg/L phosphino-thricin. For imaging line XVE>>AX2, seedlings were germi-nated as described previously for 4 d and then transferred to1/2MS plates containing 5 μM β-estradiol (Sigma-Aldrich) for24 h. DMSO instead of β-estradiol served as a control.

Peptides. The peptide pep1 (ATKVKAKQRGKEKVSSGRPG-QHN) with an HPLC purity of 95.16% and molecular weightof 2,491.78, and the peptide pep1 labeled with 5′-carboxyte-tramethylrhodamine at the N-terminal (TAMRA-pep1) with aHPLC purity of 97.07% and molecular weight of 2,905.75,

were purchased from Life Technologies. The peptide flg22(QRLSTGSRINSAKDDAAGLQIA), with an HPLC purity of95% and a molecular weight of 2,272.50, was acquired fromGenscript (catalog no. RP19986). The peptides were dissolved inwater to obtain peptide stocks of 100 μM. Further dilutions weredone with 1/2MS medium.

TAMRA-pep1 Analyses by LC-MS. Here 5-d-old pRPS5A:PEPR1-GFP–expressing pepr1pepr2 seedlings were dipped into 1 mL of50 μMTAMRA-pep1 dissolved in 1/2MS medium, pulsed for 10 s,washed three times with 1/2MS liquid medium, and transferred toagar plates. Shoots and roots were separated at the indicated timepoints, and roots were ground in liquid nitrogen to a fine powder.Then 100 μL of extraction buffer (20% CH3CN containing 20 mMTris·HCl, pH 6.8) was added to 100 mg of powder. After in-cubation for 30 min in a shaker, the mixture was centrifuged(13,000 × g for 30 min at 4 °C), and the supernatant was filteredthrough a 0.2-μm filter (Corning Costar Spin-X cellulose acetatecentrifuge tube filter) and analyzed by LC-MS. LC-MS data werecollected on an Agilent 1100 Series instrument with a Phenom-enex Kinetex C18 100Å column (150 × 4.6 mm, 5 μm at 35 °C)connected to an ES-MSD type VL mass detector (quadrupole iontrap mass spectrometer) with a flow rate of 1.5 mL/min with thefollowing solvent systems: (A) 0.1% HCOOH in H2O and (B)CH3CN. The column was flushed with 100% A for 2 min, then agradient from 0 to 100% B over 6 min was used, followed by 2 minof flushing with 100% B. MS spectra were collected in positivemode. A deconvolution procedure was applied to deliver the totalmolecular weight of the TAMRA-pep1 as calculated from thedifferent signals for the multiply charged species.

Imaging. Arabidopsis seedlings were imaged on an OlympusFluoView 1000 inverted confocal microscope equipped with awater-corrected 60× objective (NA 1.2) at digital zoom 3 or on aZeiss LSM 880 equipped with a water-corrected 63× objective(C-Apochromat). For the evaluation of the TAMRA-pep1 PM la-beling on different cell layers of the root meristem and of the rootexpression patterns of the AtPep1 receptors, digital zoom 1 wasused. The excitation wavelength was 488 nm for GFP and YFP,458 nm for CFP, and 559 nm for TAMRA and FM4-64. Emissionwas detected at 500–530 nm for GFP and YFP, 460–500 nm forCFP, and 570–670 nm for TAMRA and FM4-64. For TAMRA-pep1, the intensity was manipulated with the Olympus software.

TAMRA-pep1 Application and Competition Assays. For the PM-labeling assay, 5-d-old seedlings were dipped into 500 μL of100 nM TAMRA-pep1 dissolved in 1/2MS medium, pulsed fordifferent times as indicated, washed three times with 1/2MSliquid medium, and transferred to coverslips for visualization ofmeristem epidermal cells under the confocal microscope. For thecompetition assay, seedlings were pretreated with 0, 0.1, 1, 10,100, 1,000, and 10,000 nM of pep1 or flg22 for 5 min and thenwashed three times with 1/2MS medium before being dippedinto TAMRA-pep1 for 10 s.

Quantification of PM Fluorescent Intensity. For quantification, im-ages were converted to 8-bit images, and fixed regions of interestwere selected to measure the fluorescent intensity of the PM andbackground into the intracellular space with ImageJ. Then therelative PM fluorescence was calculated by dividing the PM in-tensity by the background intensity. Six epidermal cells from eightplants were quantified.

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TAMRA-pep1 Internalization Assay. The 5-d-old seedlings weredipped into 500 μL of 100 nM TAMRA-pep1 dissolved in 1/2MSmedium for 10 s, washed three times with 1/2MS liquid medium,kept in 500 μL of 1/2MS medium over a piece of parafilm placedin a Petri dish, and then imaged for the indicated time points. Tocompare TAMRA-pep1 uptake between the clathrin mutantsand the Col-0 WT, images were quantified with ImageJ. To thisend, images were first converted to 8-bit images, and then theentire PM and intracellular space were selected with a brush toolsize of 5 pixels and the polygon selection tool, respectively. Thenthe average intensity of the top 50 highest pixels for both the PMand the intracellular space was used to obtain a ratio betweenintracellular and PM fluorescence. Six epidermal cells from eightplants were quantified.

Colocalization Analysis. For the colocalization analysis of confocalimages acquired by confocal laser microscopy, ImageJ was usedwith the plugin PSC colocalization to obtain the Pearson cor-relation coefficients as colocalization readouts, as well as a pixeldistribution scatterplot in which the pixels in the green imageserved as the y-coordinates and the intensity of the corre-sponding pixels in the red image served as the x-coordinates. Forcolocalization between TAMRA-pep1 and PEPR1-GFP andPEPR2-GFP, the regions of interest were selected, and coloc-alization analysis was carried out with a threshold of 10. Todetermine the percentage of TAMRA-pep1 vesicles colocalizedwith different endomembrane markers, each individual TAM-RA-pep1 spot was selected as an individual region of interestand colocalization was analyzed with a threshold of 10, consid-ering the spots for which the Person correlation value was >0.5as positively labeled endosomes. A threshold of 10 was used toavoid noise. Calculations were done by measuring the back-ground gray values present in the analyzed images.

Chemical Treatments. The inhibitors BFA (50 mM DMSO stock),ConcA (2 mM DMSO stock), and CHX (50 mM DMSO stock)were purchased from Sigma-Aldrich. FM4-64 was acquired fromMolecular Probes (2 mM water stock). Five-day-old seedlingswere incubated for the indicated times into 1 mL of liquid 1/2MSmedium containing 2 μM ConcA, 50 μM BFA, 50 μM CHX,or a combination of BFA and CHX. For seedlings expressingPEPR1-GFP, inhibitor treatments were performed together withthe endosomal marker FM4-64 at room temperature. For FM4-64 staining, seedlings were incubated for 5 min in 1 mL of 1/2MSliquid medium containing 4 μM of the dye plus the respectiveinhibitor treatment. Control treatments were done with equalamounts of DMSO. All washing steps and imaging were carriedout in the presence of the respective inhibitors.

MAPK Activation Assay. chc2-1, chc2-2, ap2s, and ap2m2 seedlingswere germinated and grown on 1/2MS medium containing agarfor 5 d. ap1m-2 and hap13 seedlings were grown for 10 d toidentify homozygous mutants. Seedlings were transferred tomultiwell plates (∼10 seeds per well) containing 1 mL per well of1/2MS medium supplemented with 0.5% (wt/vol) sucrose andtreated with 20 nM pep1 for the indicated times. At each timepoint, 20 seedlings were snap-frozen in liquid nitrogen. Proteinswere extracted with a buffer containing 50 mM Tris, pH 7.5,200 mM NaCl, 1 mM EDTA, 10% (vol/vol) glycerol, 0.1% (vol/vol)Tween 20, 1 mM phenylmethylsulfonyl fluoride, 1 mM DTT, 1×phosphatase inhibitor mixture 2 (P5726; Sigma-Aldrich) and 1×protease inhibitor mixture (P9599; Sigma-Aldrich). Equal amountsof proteins (30 μg) were resolved on 7.5% (wt/vol) polyacrylamidegels and transferred onto a PVDF membrane (Bio-Rad). Primaryantibodies against MPK3 (1:2,500; Sigma-Aldrich) and MPK6

(1:8,000; Sigma-Aldrich), against phospho-p44/42 MAP kinase(1:2,500; Cell Signaling Technology), and against tubulin (1:15,000;Sigma-Aldrich) were used with horseradish peroxidase-conjugatedanti-rabbit as a secondary antibody (1:8,000; GE Healthcare). Sig-nal detection was performed using Western Lightning Plus ECL(PerkinElmer). For induction of the β-estradiol–inducible lineXVE>>AX2, seedlings were germinated and grown as describedabove for 5 d. β-estradiol was added to the medium at a finalconcentration of 5 μM for 24 h before the pep1 treatments. DMSOserved as a control.

Alkalinization Assay. For this assay, 1 mL of cell suspension (5 dafter subculture) was transferred into each well of a 24-well plate(Corning) and allowed to equilibrate on an orbital shaker at 120rpm for 1 h. The pH of the medium was measured at the timepoints of the indicated peptide concentrations with a EA940pH meter (Orion) with a semimicro pH electrode (Orion).

RT-PCR. All gene expression experiments were conducted using6-d-old seedlings. Total RNA was extracted with TriZol reagent(Life Technologies) according to the manufacturer’s instructions,followed by DNase I treatment (Life Technologies) to removeany residue of genomic DNA, and quantified with a Nanodropspectrophotometer (Thermo Fisher Scientific). cDNA was syn-thesized from 1 μg of total RNA with Improm-II ReverseTranscriptase (Promega).The AtPep1-induced gene PEPR1 was analyzed. The GAPDH

gene served as a control. The primers used are listed in Table S1.For the PCR reactions, 1 μL of cDNA was used. The reactionswere placed in the thermocycler under the following conditions:94 °C for 2 min and appropriate numbers of cycles of 94 °C for20 s, 60 °C for 30 s, and 72 °C for 45 s. These experiments wererepeated at least three times.

Ca2+Mobilization Assay.The induced Ca2+ signaling was evaluatedby monitoring the level of cytosolic Ca2+ in Arabidopsis seed-lings. Seedlings of the Arabidopsis line homozygous for a singleinsertion of a transgene encoding a cytoplasmically expressedaquorin-driven by the cauliflower mosaic virus 35S promoterwere studied. Seeds were sterilized as described previously andplated on 1/2MS medium without vitamins. The plates were keptunder constant light at 24 °C (150 μE m−2 s−1) for 4 d. A singleseedling was transferred into each well of a 96-well white mi-croplate (Thermo Labsystems) containing 200 mL of 1/2MSmedium supplemented with 2.5 mM coelenterazine cp (Biotium)and incubated in the dark at 24 °C for 16 h. In each plate wellcontaining a single seedling, TAMRA-pep1 or pep1 was addedat a final concentration of 10 nM. The resulting luminescenceemission was monitored with a microplate reader (Biotec ELx800) for 24 time points over ∼360 s after addition of the re-spective peptide. Water served as a control.

Root Growth Assay. Seeds were sown on 1/2MS solid medium,stratified for 2 d at 4 °C in the dark, and placed vertically in thelight. At 10 d (Fig. S2) or 3 d (Fig. S8) after germination,seedlings were transferred to square transparent Petri disheswith solid 1/2MS medium supplemented with or without theindicated amounts of peptides and incubated for another 4 d,after which the plates were scanned and root growth was mea-sured. For measurements, scanned images were processed andevaluated with ImageJ.

Statistical Analysis. P values were calculated with the two-tailedStudent’s t test using Microsoft Excel.

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Fig. S1. Identification of TAMRA-pep1 by LC-MS in plant tissues. (A) TAMRA-pep1 was first identified in the medium at a final concentration of 50 μM. (Left)Chromatogram at 254 nm showing the tagged peptide eluting at 3.25 min. (Right) LC-MS peak chromatogram of the fraction at 3.26 min. (B) The correctexpected molecular weight of the TAMRA-pep1 (2,904) was assessed by a deconvolution procedure. (C–E) Chromatogram at 254 nm (Left) and LC-MS peakchromatogram at 3.26 min (Right) of pRPS5A::PEPR1-GFP–expressing root extracts not incubated with TAMRA-pep1 (mock control) (C) or incubated with thepeptide (50 μM) for 1 h (D) or 2 h (E). Note that TAMRA-pep1 was not detected after 2 h. Arrows indicate TAMRA-pep1–specific peaks.

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0

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Fig. S2. Biologically active TAMRA-pep1. (A) Measurement of the pH of Arabidopsis cell suspensions after 20 min of exposure to different concentrations ofpep1 or TAMRA-pep1 (n = 9). (B) Total luminescence of aquorin-expressing Arabidopsis plants subjected to the aquaporin Ca2+ assay and treated with 10 nMpep1 or TAMRA-pep1 (n = 9). (C) Root growth inhibition of Col-0 in the presence of pep1 or TAMRA-pep1 (n = 10). (D) RT-PCR analysis of PEPR1 gene in 6-d-oldArabidopsis seedlings treated for 60 min with 100 nM pep1 or TAMRA-pep1. GAPDH (At1g13440) and water served as loading control and control, respectively.These experiments were repeated at least twice, with similar results. Error bars indicate SD. n, number of wells in A and number of seedlings in B and C.

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pep1

flg22

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Fig. S3. PM labeling of TAMRA-pep1. (A) PM of Arabidopsis WT (Col-0) and pepr1perp2 double-mutant root meristem epidermal cells labeled within secondsafter application by TAMRA-pep1. The 5-d-old seedlings were treated with TAMRA-pep1 (100 nM) for different time points, washed three times, and thenimaged. (Right) Quantification of fluorescence intensity in the PM (n = 48). Error bars indicate SD. (B) Quantification of the FM4-64 signal intensity in Col-0 andpepr1perp2 double mutant. Values represent the intracellular/PM signal intensity ratio in root meristem epidermal cells of 5-d-old seedlings treated with FM4-64 (4 μM, 5 min, three washouts) and kept in the presence of 100 nM pep1 for another 90 min (n >32). Error bars indicate SEM. No statistically significantdifference (*P ≤ 0.01, Student’s t test) was found. (C) pep1, but not flg22, is able to compete with TAMRA-pep1. Five-day-old Arabidopsis seedlings wereincubated with various concentrations of pep1 or flg22 for 5 min, then treated with TAMRA-pep1 for 10 s, washed, and imaged. The fluorescence intensity ofthe PM is quantified in Fig. 1D. (D) Temperature-dependent internalization of TAMRA-pep1. Five-day-old Arabidopsis seedlings were incubated at 4 °C for 2 hbefore treatment with TAMRA-pep1 (100 nM, 10 s), washed, and imaged after incubation for 40 min at 4 °C. The same TAMRA-pep1 treatment was done withseedlings at room temperature. n, number of cells analyzed. (Scale bars: 10 μM.)

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Relat

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pPEPR2::NLS-GFP

Fig. S4. Expression patterns and subcellular localization of PEPR1 and PEPR2 in the Arabidopsis roots. (A) pPEPR1::NLS-GFP and pPEPR2::NLS-GFP expression inthe differentiation zone (Top) and meristem (Bottom) of the roots of 5-d-old Arabidopsis seedlings. (B) PEPR1 and PEPR2 fused to GFP and expressed undertheir endogenous promoters or under the RPS5A promoter complemented with the pepr1pepr2 double mutant. Root growth inhibition analysis of ArabidopsisCol-0, pepr1pepr2, pPEPR1::PEPR1-GFP/pepr1pepr2, pPEPR2::PEPR2-GFP/pepr1pepr2, pRPS5A::PEPR1-GFP/pepr1pepr2, and pRPS5A::PEPR2-GFP/pepr1pepr2seedlings (n = 10). Seedlings were germinated on 1/2MS medium and then transferred for 4 d to 1/2MS medium supplemented with pep1 (100 nM). Rootgrowth is presented relative to the untreated control. Four independent transgenic lines were analyzed for each construct. n, number of seedlings analyzed.Error bars indicate SD. *P < 0.001 (Student’s t test) relative to the untreated control. (C) Localization of PEPR1-GFP and PEPR2-GFP expressed under theirendogenous promoter in the pepr1pepr2 mutant in the differentiation zone of the roots of 5-d-old Arabidopsis seedlings. (Scale bars: 30 μm.)

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Fig. S5. AtPep1 and PEPR1 internalize with similar dynamics. (A) Internalization of TAMRA-pep1 in root epidermis of 5-d-old Arabidopsis seedlings afterincubation with TAMRA-pep1 (100 nM, 10 s), three washouts, and imaging at the indicated time points. Root meristem epidermal cells are shown. (B) PEPR1-GFP internalization was triggered by pep1 in a dose-dependent manner. Seedlings were treated with different concentrations of pep1 for 10 s, washed threetimes, and imaged after chases of 5 min and 40 min. (C) Internalization of PEPR1-GFP after treatment with pep1 (100 nM, 10 s) as in A. Application of flg22 andmock (water) did not change the PEPR1-GFP localization. (D) PEPR1-GFP internalization on constant elicitation with pep1. The 5-d-old pepr1pepr2 seedlingscomplemented with PEPR1-GFP expressed from the RPS5A promoter were used in B, C, and D. (Scale bars: 10 μm.)

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r(P) = 0.80 r(P) = 0.74 r(P) = 0.83 r(P) = 0.67 r(P) = 0.49

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[min]A

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Fig. S6. (A) Cointernalization of PEPR1-GFP and FM4-64 after elicitation with pep1. Here 5-d-old pepr1pepr2 seedlings complemented with pRPS5A::PEPR1-GFP construct were stained with FM4-64 (4 μM, 5 min, three washouts), maintained in the presence of pep1 (100 nM), and imaged at the indicated times.(B) Cointernalization of AtPep1 and its receptor PEPR2. pRPS5A::PEPR2-GFP/pepr1pepr2 seedlings as in A were treated with TAMRA-pep1 (100 nM, 10 s),washed three times, and imaged at the indicated times. As a colocalization indicator, the Pearson correlation, r(P), was calculated for merged images. Arrowspoint to colocalized endosomes. (Scale bar: 10 μm.)

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CLC

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FP-A

RA

7M

VB

AR

A6-

GFP

MV

BG

FP-V

AM

P727

Vacu

ole

D

ARA7

TA

MR

A-p

ep1

endo

som

es c

olab

eled

(%)

60

40

20

0

100

80

TA

MR

A-e

p1

endo

som

es c

olab

eled

(%)

60

40

20

0 ARA6

40’

ATG8

100

80

TA

MR

A-p

ep1

endo

som

es c

olab

eled

(%)

60

40

20

0

ATG

8-Y

FPA

utop

hagy

TAMRA-pep1Merge

Merge

A C

Col-0 atg5-1 atg7-20

0.4

Col-0 atg5-1 atg7-2

TAM

RA

-pep

1 flu

ores

cenc

ein

tens

ity in

trace

llula

r/PM

0.8

TAMRA-pep1 Merge

MergeF

B

YFP

-MEM

B12

Gol

gi

[100nM]

Merge100

80

TA

MR

A-A

tPep

1 en

doso

mes

col

abel

ed (%

)

60

40

20

0MEMB12

Marker

MarkerTAMRA-pep1

[100nM]

TAMRA-pep1[100nM]

TAMRA-pep1[100nM]

TAM

RA

-pep

1[1

00nM

]E

Fig. S7. Endocytic route of TAMRA-pep1 in Arabidopsis root epidermal meristem cells. (A) Only partial colocalization of TAMRA-pep1–labeled vesicles withthe TGN markers VHA-a1-GFP, SYP61-CFP, GFP-SYP42, CLC1-GFP, CLC2-GFP, CHC1-GFP, CHC2-GFP, and YFP-RabA1e. (B) Lack of colocalization of TAMRA-pep1–labeled vesicles with the Golgi compartments marked by YFP-MEMB12 and with the autophagy compartments marked by ATG8-YFP (C). (D) TAMRA-pep1internalization was not impaired in the autophagy mutants atg5-1 and atg7-2. (Top) Representative root epidermis images of seedlings treated with TAMRA-pep1 (100 nM, 10 s, three washouts, 40-min chase). (Bottom) Quantification of TAMRA-pep1 uptake. The graph illustrates the intracellular/PM fluorescenceintensity ratio (n >45). n, number of analyzed cells. Error bars indicate SEM. (E) Labeling of most TAMRA-pep1 vesicles with the MVB markers YFP-ARA7 andARA6-GFP. (F) Accumulation of TAMRA-pep1 fluorescence in the vacuoles, delimited by the tonoplast marker GFP-VAMP727. The 5-d-old seedlings weretreated with TAMRA-pep1 (100 nM, 10 s) and washed three times with medium. Root tip epidermal cells were imaged after chases of 40 min (A–E) and 90 min(F). The graphs in A, B, C, and E show the percentage of TAMRA-pep1–positive vesicles labeled by the markers (n >300). n, TAMRA-pep1–positive vesicles. Onlythe TAMRA-pep1–positive vesicles with a colocalization value >0.5 as calculated with the Pearson’s correlation coefficient were considered colocalized. Arrowsand arrowheads point colocalized and uncolocalized endosomes, respectively. (Scale bars: 10 μm.)

Ortiz-Morea et al. www.pnas.org/cgi/content/short/1605588113 9 of 12

Page 10: Supporting Information - pnas. · PDF file1/2MS plates containing 5 μM β-estradiol (Sigma-Aldrich) for 24 h. DMSO instead of β-estradiol served as a control. ... (P5726; Sigma-Aldrich)

A

TAM

RA-p

ep1

[100

nM

]

* * *

Col-0 chc2-1 chc2-2 ap2m2-2 ap2s

D

10 25 50 100pep1 [nM]

Rela

tive

mai

n ro

ot le

nght

to

unr

eate

d co

ntro

l (%

)

0

20

40

60

80 Col-0chc2-1chc2-2

C

Col-0 chc2-1 chc2-2 ap2m2-2 ap2s0

0.3

0.6

0.9

TAM

RA-p

ep1

fluor

esce

nce

inte

nsity

intra

cellu

lar/P

M

**

Col

-0

ConcA (2 μM)

0

1

2

3

ConcA (2 μM)

TAM

RA

-pep

1 en

doso

mes

per

cel

l [60

min

] **

DMSO

TAMRA-pep1 [100nM, 90-min chase]

B

DMSO

Fig. S8. Clathrin-dependent TAMRA-pep1 internalization and AtPep1-mediated responses. (A) Impaired activity of vacuolar H+-ATPase by the presence ofConcA slightly delaying the pass of TAMRA-pep1 to the vacuole. Quantification of the number of TAMRA-pep1 endosomes per cell (n = 36). n, number of cellsanalyzed. Error bars indicate SEM. **P ≤ 0.001 (Student’s t test) relative to Col-0. The 5-d-old seedlings were pretreated with ConcA (2 μM, 30 min), then treatedwith TAMRA-pep1 (100 nM, 10 s), washed three times, and kept in the presence of ConcA (2 μM) until imaging (60-min chase). DMSO served as a control. (Scalebars: 10 μm.) (B) TAMRA-pep1 uptake in WT Arabidopsis (Col-0; control) and chc2-1, chc2-2, ap2m-2, and ap2s homozygous mutants. The 5-d-old seedlings weretreated with TAMRA-pep1 (100 nM, 10 s), washed three times, and imaged after a 40-min chase. (C) Quantification of the TAMRA-pep1 uptake in B. Graphsillustrate the intracellular/PM fluorescence intensity ratio (n >45). n, number of analyzed cells. Error bars indicate SEM. *P ≤ 0.01 (Student’s t test) relative toCol-0. (Scale bar: 10 μm.) (D) Root growth analysis of Col-0, chc2-1, and chc2-2 seedlings grown in the presence of various concentrations of pep1. Data are thesum of two biological repeats, and the main root length is presented as relative to the mock control. Error bars indicate SEM.

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Page 11: Supporting Information - pnas. · PDF file1/2MS plates containing 5 μM β-estradiol (Sigma-Aldrich) for 24 h. DMSO instead of β-estradiol served as a control. ... (P5726; Sigma-Aldrich)

Col-0+ pep1 [min]

WB: anti-tubulin

WB: anti-p42/44

pMPK6

Tubulin

0 5 10 15 30 0 5 10 15 30 0 5 10 15 30

chc2-1 chc2-2

+ pep1 [min]

WB: anti-tubulin

WB: anti-p42/44

Col-00 5 10 15 30 0 5 10 15 30

ap2s

Tubulin

WB: anti-tubulin

WB: anti-p42/44

Col-00 5 10 15 30 0 5 10 15 30

ap2m2-2

Tubulin

+ pep1 [min]

pMPK3

pMPK6pMPK3

pMPK6pMPK3

Col-0 ap1m2-1

Ws hap13

+ pep1 [min] 0 15 30 0 15 30

WB: anti-p42/44

WB: anti-MPK6anti MPK3

0 15 30 0 15 30+ pep1 [min]

WB: anti-p42/44

WB: anti-MPK6anti MPK3

pMPK6pMPK3

pMPK6pMPK3

MPK6MPK3

MPK6MPK3

TAM

RA-p

ep1

PM fl

uore

scen

t int

ensit

y

0

0.5

1.0

1.5

hap13 Ws

TAMRA-pep1 [100 nM, 10 s]

Ws hap13

A

B

Fig. S9. MAPK activation on pep1 application. (A) WT (Col-0 or Ws) Arabidopsis plants and chc1-1, chc2-1, chc2-2, ap2m-2, ap2s, ap1m2-1, and hap13 ho-mozygous mutants treated with pep1 (20 nM) for the indicated times. Phosphorylation of MPK6 and MPK3 was detected with anti–phospho-p44/p42-MPKantibody. The immunoblot was reprobed with anti-MPK6, anti-MPK3, and anti-tubulin antibodies for protein quantification. Individual MPKs were identifiedby molecular mass and are indicated by arrows. (B) PM labeling of TAMRA-pep1 in WT (Ws) and hap13 mutant root meristem epidermal cells. Seedlings weretreated with TAMRA-pep1 (100 nM) for 10 s, washed three times, and imaged. (Right) Quantification of PM fluorescence intensity (n = 32). n, number ofanalyzed cells. Error bars indicate SEM. No statistically significant difference was found (*P ≤ 0.01, Student’s t test). WB, Western blot.

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Page 12: Supporting Information - pnas. · PDF file1/2MS plates containing 5 μM β-estradiol (Sigma-Aldrich) for 24 h. DMSO instead of β-estradiol served as a control. ... (P5726; Sigma-Aldrich)

Table S1. Primers used for cloning and RT-PCR analysis

DNA region Primer Sequence 5′–3′

CloningGenomic PEPR1 Forward GGGGACAAGTTTGTACAAAAAAGCAGGCTCAATGAAGAATCTTGGGGGGTTGTTC

Reverse GGGGACCACTTTGTACAAGAAAGCTGGGTACCGAACTGAATCAGAGGAGCAGenomic PEPR2 Forward GGGGACAAGTTTGTACAAAAAAGCAGGCTCAATGAGGAATCTTGGGTTACTCG

Reverse GGGGACCACTTTGTACAAGAAAGCTGGGTAGTGAACTGAACCCGAAGTGCTTCTPromoter PEPR1 Forward GGGGACAACTTTGTATAGAAAAGTTGCTTCACTGATCTGTTTGTTGCAAAC

Reverse GGGGACTGCTTTTTTGTACAAACTTGCCTGAGTTTAAAGATCGAGAAACATGPromoter PEPR2 Forward GGGGACAACTTTGTATAGAAAAGTTGCTATTAGGGTGGTCTATCGGTCAG

Reverse GGGGACTGCTTTTTTGTACAAACTTGCATTAGAGCTCAAGAGACTGAAATATGCDS Auxilin2 Forward CACCATGGATGATTTCACAGGATTGTT

Reverse TCAAAAGAGTTCCTCTGAGTTGAATRT-PCR

GAPDH Forward TTGGTGACAACAGGTCAAGCAReverse AAACTTGTCGCTCAATGCAA

PEPR1 Forward GTTTTGGCTGAGGAAAGACGReverse ACATTGTACCGTGCAGACCA

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