Supporting Information - pnas.?1/2MS plates containing 5 ¼M ²-estradiol (Sigma-Aldrich)...
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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 lightdarkcycle for 5 d with a light intensity of 120 E m2 s1 (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, VIBGhent 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 manufacturers 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 M -estradiol (Sigma-Aldrich) for24 h. DMSO instead of -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 M. Further dilutions weredone with 1/2MS medium.
TAMRA-pep1 Analyses by LC-MS. Here 5-d-old pRPS5A:PEPR1-GFPexpressing pepr1pepr2 seedlings were dipped into 1 mL of50 MTAMRA-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 L of extraction buffer (20% CH3CN containing 20 mMTrisHCl, 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-m 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 100 column (150 4.6 mm, 5 m 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 500530 nm for GFP and YFP, 460500 nm forCFP, and 570670 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 L 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 L of 100 nM TAMRA-pep1 dissolved in 1/2MSmedium for 10 s, washed three times with 1/2MS liquid medium,kept in 500 L 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 M ConcA, 50 M BFA, 50 M 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 M 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 transf