www.sciencemag.org/cgi/content/full/science.aah5692/DC1

Supplementary Materials for

Regulation of sugar transporter activity for antibacterial defense in Arabidopsis

Kohji Yamada,* Yusuke Saijo, Hirofumi Nakagami, Yoshitaka Takano*

*Corresponding author. Email: kohjiyamada226@gmail.com (K.Y.); ytakano@kais.kyoto-u.ac.jp (Y.T.)

Published 24 November 2016 on Science First Release

DOI: 10.1126/science.aah5692

This PDF file includes:

Materials and Methods Figs. S1 to S13 Table S1 References

Materials and Methods Plant materials and growth conditions

The Columbia-0 (Col-0) ecotype of Arabidopsis plants was used as wild type. stp1 (SALK_048848), stp13 (SALK_045494) (9), bak1-4 (SALK_116202) (24) and fls2 (SAIL_691C4) (25) were used. BAK1-FLAG/bak1-4 bkk1 (26) was used. Plants were grown on soil or 0.5 x Murashige and Skoog (MS) agar medium (0.5 x MS salts, 0.5 x MS vitamins, 25 mM sucrose, 0.5 g/l MES (pH 5.7), 0.8% agar) at 22 °C under 10 h light/14 h dark conditions or 16 h light/8 h dark conditions, respectively.

Plasmid construction

GFP, 3 x HA, 3 x FLAG DNA fragments were inserted into the EcoRI/SacI sites of pRI 101-AN (Takara). These plasmids were named pRI 35S-GFP, pRI 35S-HA, pRI 35S-FLAG, respectively. To establish stable transgenic plants, genomic fragments containing the STP13 promoter and coding regions were inserted into the HindIII/NdeI sites of pRI 35S-GFP. For transient assays in N. benthamiana, an STP13 cDNA fragment was inserted into the NdeI/SalI sites of pRI 35S-FLAG. cDNA fragments of FLS2, EFR and PEPR1 were inserted into the NdeI/SalI sites of pRI 35S-GFP. An LTI6b cDNA fragment was inserted into the BsrGI/SacI sites of pRI 35S-GFP. For transient assays in Arabidopsis protoplasts, fragments containing the 35S promoter, 3 x HA or 3 x FLAG and the NOS terminator were amplified by PCR from pRI 35S-HA or pRI 35S-FLAG and inserted into the KpnI/SacI sites of pBluescript SK. These were named pSK 35S-HA and pSK 35S-FLAG, respectively. cDNA fragments of FLS2, BAK1, BIK1 and GFP were inserted into the NdeI/SalI sites of pSK 35S-HA. The STP13 cDNA fragment was inserted into the NdeI/SalI sites of pSK 35S-FLAG. For the in vitro kinase assay, fragments encoding the middle loop and C-terminal tail of STP13 were inserted into the BamHI/EcoRI sites of pGEX-2TK (GE Healthcare). Fragments encoding the cytoplasmic kinase domains of FLS2, BAK1 and PEPR1, and full-length BIK1, were inserted into the BamHI/EcoRI sites of pMAL-c2 (New England Biolabs). For yeast assays, a GFP fragment was inserted into the SalI/XhoI sites of pDR196 (27); this plasmid was named pDR196 GFP. The STP13 cDNA fragment was inserted into the EcoRI/SalI sites of pDR196 GFP. Site-directed mutagenesis of STP13 was carried out by PCR. For bacterial effector delivery assay, a genomic fragment containing the avrPto promoter and the coding region were inserted into the EcoRI/KpnI sites of pHMCya (28). All cloning reactions were performed using an In-Fusion HD Cloning Kit (Takara) following the manufacturer’s instructions. Primers used for plasmid construction are listed in Table S1.

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Pathogen inoculation Pseudomonas syringae pv. tomato (Pst) DC3000 and ΔhrcC strain (29) were

cultured in NYGA medium (5 g/l bactopeptone, 3 g/l yeast extract, 20 ml/l glycerol) with 50 μg/ml rifampicin at 28 °C overnight. They were washed twice and resuspended in 10 mM MgCl2. Pst DC3000 (OD600 = 0.2) was sprayed with 0.01% Silwet L-77 onto, and ΔhrcC (OD600 = 0.02) was syringe-infiltrated into, 4-week-old Arabidopsis. Plants were placed under plastic covers for 3 days and 4 days after Pst DC3000 and Pst DC3000ΔhrcC, respectively, was inoculated. Four leaf discs obtained from four independent leaves were pooled. Serially diluted suspensions in 10 mM MgCl2 were plated on NYGA agar medium and incubated at 28 °C for 2 days to enumerate colonies. Bacterial effector delivery assay

pHM avrPto-Cya was introduced into Pst DC3000 by electroporation. The bacterium was cultured in NYGA medium with 50 μg/ml rifampicin and 100 μg/ml spectinomycin at 28 °C overnight. The cells were washed twice and resuspended in 10 mM MgCl2. The bacterium (OD600 = 0.2) was sprayed with 0.005% Silwet L-77 onto 4-week-old Arabidopsis. Six leaves were pooled 10 h after inoculation and ground into a powder under liquid nitrogen. cAMP was extracted with 0.1 N HCl at 4 °C and centrifuged at 21,500 x g for 10 min at 4 °C twice to remove debris. The amounts of cAMP in the supernatants were measured by cAMP Enzyme Immuno Assay (EIA) System (GE Healthcare, RPN2251) following the manufacturer’s instructions. cAMP levels were normalized to total protein amounts in each extract. Sugar absorption in plants and yeast cells

For plant assays, whole seedlings of ten-day-old Arabidopsis plants grown on 0.5 x MS agar medium were used. Seedlings were transferred into 10 ml of transport buffer (0.5 x MS salts, 25 mM MES-KOH (pH 5.5), without sucrose) with or without 4 μM flg22 in 6-well plates. After 24 h, the buffer was replaced with 9 ml of fresh buffer. Seedlings were equilibrated for 1 h, and then 1 ml of 100 mM glucose or fructose containing 74 kBq of 14C-labeled glucose or fructose was added. After 2 h, the seedlings were washed twice with water, and pooled into groups of five seedlings. After measuring their fresh weights, whole seedlings were placed in vials with Clear-sol scintillation cocktail (Nacalai Tesque). For yeast assays, plasmids were introduced into the hexose-deficient yeast mutant EBY.S7 strain (30). Yeast cells were cultured in medium containing 2% maltose overnight at 30 °C. The OD600 was adjusted to 1.0 with 20 mM MES-KOH (pH 5.5) buffer and the cells were equilibrated for 30 min. Twenty microliters of solution with 7.4 kBq of 14C-labeled sugars were added to 180 μl of the yeast cells. Sugar concentration was adjusted with unlabeled sugars. After incubation for 10 min at 30 °C, the reactions were stopped by adding 5 ml of sterile water and then filtered

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through a nitrocellulose membrane (pore size 0.8 μm, Sigma, N8772). The filters were washed twice with 5 ml of sterile water and placed in vials with scintillation cocktail. Apoplastic sugar measurement

Water (mock) or 1 μM flg22 was infiltrated into mature leaves of 5-week-old Arabidopsis. After 24 h, extraction buffer (20 mM Tris-HCl, pH 7.5, 50 mM NaCl) was vacuum-infiltrated into 2 g of leaves. After being blotted dry with a paper towel, leaves were carefully packed into a 20 ml needleless syringe, which was then placed in a 50 ml tube and centrifuged at 560 x g for 5 min in a swing rotor. Apoplastic fluid was collected at the bottom of the tube, and 100 µl was used for sugar measurement. Glucose concentration was measured enzymatically by an NADPH-based assay (10716260035, Roche) following the manufacturer’s instructions. Invertase assay

Cell wall-bound invertase activity was measured using a previously described method (9). Water (mock) or 1 μM flg22 was infiltrated into mature leaves of 5-week-old Arabidopsis. After 24 h, the leaves were harvested and homogenized in extraction buffer (20 mM sodium phosphate, pH 6.5, 1 mM EDTA, 1 mM EGTA, 1 mM MgCl2, 1 mM MnCl2, 5 mM DTT) and centrifuged at 21,500 x g for 10 min at 4 °C. The pellets were washed twice with extraction buffer, and cell wall-bound invertase was solubilized from the pellets with extraction buffer containing 1 M NaCl by rotation for 5 h at 4 °C. After centrifugation, the supernatant was used as an invertase fraction for this assay. To measure invertase activity, we mixed 40 μl of 0.4 M sodium acetate (pH 5.0), 10 μl of 1 M sucrose and 50 μl of invertase fraction containing 5 μg of protein. The reaction mixtures were incubated at 37 °C for 30 min, heat-inactivated at 95 °C for 5 min, and then neutralized with 10 μl of 1 M KOH. Glucose concentration was measured by an NADPH-based assay (10716260035, Roche) as invertase activity.

In vitro kinase assay

The in vitro kinase assay followed a previously described method (31) with some modifications. GST-fused and MBP-fused recombinant proteins were purified from Escherichia coli BL21 (DE3) strain with Glutathione Sepharose 4B (GE Healthcare) and amylose resin (New England Biolabs), respectively. GST-fused substrates (500 ng) and 100 ng of MBP-fused kinases were incubated in kinase reaction buffer (25 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 1.5 mM MnCl2, 0.5 mM CaCl2, 5 mM DTT) in the presence of 10 μM non-radiolabeled ATP containing 370 kBq of [γ-32P]ATP for 1 h at 30 °C. The reactions were stopped by the addition of SDS sample buffer. Proteins were separated by 12.5% SDS-PAGE. Radioactivity in the dried gel was detected by BAS2000 (Fujifilm). As controls, proteins were separated in independent gels and visualized with Coomassie Brilliant Blue (CBB).

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qRT-PCR analysis Ten-day-old seedlings were submerged in 0.5 μM flg22, and then harvested after

the indicated times. Total RNA was isolated from the plant samples using RNAiso reagent (Takara) following the manufacturer’s instructions, and reverse-transcribed using a PrimeScript RT reagent kit (Perfect Real Time) (Takara) with oligo(dT) primer and random primer. Quantitative PCR was performed with SYBR premix ExTaq II (Takara) on a Thermal Cycler Dice Real Time system TP800 (Takara). The expression levels of genes of interest were normalized relative to those of a reference gene, ACT2. Relative expression (log2) was calculated by subtracting Ct values of genes of interest from those of ACT2. Fold change was based on values of relative expression (log2), which was calculated by two to the power of relative expression (log2).

Immunoblot analysis

Proteins of interest were detected with the following antibodies: anti-GFP (B-2, Santa Cruz Biotechnology), anti-HA (3F10, Roche), anti-FLAG (M2, Sigma), anti-BAK1 and anti-FLS2 (Agrisera). Quantification of immunoblots was performed with CSAnalyzer4 (ATTO). Band intensities were normalized with the values of Ponceau S-stained bands, and relative band intensities were then calculated based on those of the corresponding controls (1.0).

Co-immunoprecipitation analysis

Arabidopsis protoplasts were transfected with plasmids by a previously described protocol (32). Agrobacterium GV3101 strain was infiltrated into N. benthamiana using a previously described protocol (16). Proteins were extracted from protoplasts incubated for 6 h after transfection, from 10-day-old Arabidopsis seedlings exposed to 1 μM flg22 for 10 h, or from leaves of N. benthamiana at 3 dpi with Agrobacterium, using extraction buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 2 mM DTT, 0.5% IGEPAL CA-630) at 4 °C for 30 min. For co-IP analysis, samples were centrifuged at 21,500 x g for 15 min at 4 °C. Supernatants were incubated with anti-HA affinity matrix (Roche) for 1 h or with GFP-trap (Chromotek) for 3 h at 4 °C. Stomatal aperture measurement

Stomatal aperture measurement followed previously described methods (33). Epidermal peels were obtained from 4-week-old Arabidopsis leaves exposed to at least 3 h light conditions. Peels were incubated in buffer containing 25 mM MES-KOH (pH 6.15) and 10 mM KCl with or without 5 μM flg22 for 1 h. Pictures of stomata were taken under a microscope, and stomatal aperture (width/length) was measured by Photoshop (Adobe).

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Statistical analysis All data displayed in this study were analyzed using Excel (Microsoft) or R. Two-

tailed unpaired t-tests were performed to measure differences from at least three independent biological replicates.

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Figure S1. flg22-induced stomatal closure remains unaffected in the absence of STP1 and STP13. Stomatal aperture was determined in epidermal peels derived from rosette leaves of 4-week-old Arabidopsis after exposure to 5 µM flg22 for 1 h. Results are averages ± SE of at least 25 stomata (*, p < 0.05 compared to the corresponding values of each mock treatment using a two-tailed t-test).

Figure S2. Apoplastic glucose levels are elevated in response to flg22 in the absence of STP1 and STP13. (A) Relative amounts of apoplastic glucose in rosette leaves of 5-week-old Arabidopsis were determined after water (mock) or flg22 application for 24 h. Results are averages ± SE of three biological replicates (*, p < 0.05 compared to the corresponding values of each mock treatment using a two-tailed t-test). (B) Cell wall-bound invertase activity in rosette leaves of 5-week-old Arabidopsis after water (mock) or flg22 application for 24 h. Results are averages ± SE of three biological replicates (*, p < 0.05 compared to the value of mock treatment using a two-tailed t-test). (C) Relative amounts of apoplastic glucose in rosette leaves of 5-week-old Arabidopsis were determined after flg22 application for 24 h. Results are averages ± SE of three biological replicates (*, p < 0.05 compared to the corresponding value in WT plants using a two-tailed t-test).

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Figure S3. STP13 is induced in response to flg22. qRT-PCR analysis of STP1 and STP13 in 10-day-old Arabidopsis seedlings exposed to 0.5 µM flg22. Results are averages ± SE of three biological replicates. Values at 0 h are set to 1.0 for fold change.

Figure S4. STP13-GFP induction is detectable in response to flg22 in mature leaves. Anti-GFP immunoblot analysis for STP13-GFP in 4-week-old Arabidopsis mature leaves infiltrated with water (mock) or 1 µM flg22. Ponceau S-stained loading controls are shown (bottom).

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Figure S5. STP13 associates with membrane-localized PRRs when co-introduced into N. benthamiana leaves. Co-immunoprecipitation analysis was conducted in N. benthamiana leaves for the indicated membrane proteins at 3 dpi with Agrobacterium. IP and IB denote immunoprecipitation and immunoblotting with the indicated antibodies, respectively.

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Figure S6. STP13 interacts with FLS2 in plants. Co-immunoprecipitation analysis between STP13-GFP and FLS2 in 10-day-old Arabidopsis seedlings. + and - indicate 0.5 µM flg22 and mock treatment for 10 h, respectively.

Figure S7. FLS2-BAK1 association is detectable 10 h after flg22 application. Co-immunoprecipitation analysis in 10-day-old Arabidopsis seedlings exposed to 0.5 µM flg22 for the indicated times.

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Figure S8. The C terminus of STP13 is phosphorylated by BAK1, but not by PEPR1 or BIK1, in vitro. (A) Schematic view of the structure of STP13. (B) Amino acid sequences of STP13-ML and STP13-CT. (C) Autoradiographs of in vitro kinase assays performed by incubating GST-STP13 ML and GST-STP13 CT with MBP-BAK1 CD, MBP-PEPR1 CD or MBP-BIK1 for 1 h with [γ-32P]ATP (upper images); CBB-stained controls are shown below.

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Figure S9. Residue T485 is conserved among STP13 orthologs in various plant species, but not among STP homologs in Arabidopsis. T485 of Arabidopsis STP13 and the corresponding residues of STP13 orthologs (A) or Arabidopsis STP homologs (B) are marked with a red rectangle. At, Os, Ta, Vv and Gm indicate Arabidopsis thaliana, Oryza sativa, Triticum aestivum, Vitis vinifera and Glycine max, respectively.

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Figure S10. T485D substitution leads to altered STP13 activity in yeast cells. (A) Complementation assay for glucose uptake in yeast cells. Four independent transformed yeast cell populations were grown on medium containing 2% maltose (control) or 10 mM glucose for 2 days. (B) Transformed yeast cells were grown on 10 mM, 1 mM and 0.1 mM glucose medium. (C) 14C-labeled glucose uptake assay in yeast cells expressing non-tagged STP13 variants. Yeast cells were incubated with 1 mM glucose containing 14C-labeled glucose. Results are averages ± SE of three biological replicates (*, p < 0.05 in two-tailed t-tests compared to the corresponding values of STP13-GFP cells). (D) Anti-GFP immunoblot analysis for STP13-GFP in yeast cells. (E) Microscopic imaging of GFP fluorescence in yeast cells. (F) Uptake rates for glucose in yeast cells (left). Hanes-Woolf plots of the same data are shown to the right. Results are averages ± SE of three biological replicates.

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Figure S11. STP13-GFP and STP13 (T485A)-GFP are comparably induced after flg22 application. Anti-GFP immunoblot analysis in 10-day-old Arabidopsis seedlings exposed to water (mock) or 4 µM flg22 for 24 h. Ponceau S-stained loading controls are shown (bottom). The numbers below the immunoblot represent relative intensities of STP13-GFP bands, normalized to the backgrounds in the Ponceau S-stained loading controls and with the value upon mock treatment (STP13-GFP) set as 1.0.

Figure S12. Increased growth of Pst DC3000 is not detectable in stp1 stp13 plants at 10 hpi. Growth of spray-inoculated Pst DC3000 (avtPto-Cya) (OD600 = 0.2) on rosette leaves of 4-week-old plants. Bacterial titers were determined at 10 hpi. Results are averages ± SE of at least three independent leaf disc pools. n.s. indicates non-significance (p > 0.05) between indicated values using a two-tailed t-test.

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Figure S13. A model for sugar competition at the interface between plants and pathogens. Apoplastic sucrose is cleaved by cwINV to glucose and fructose. Upon ligand perception, FLS2 complexes induce STP13 expression and enhance the sugar uptake activity of STP13, via T485 phosphorylation. This results in limitation of apoplastic sugar availability to the pathogen, thereby reducing both a metabolic energy source and sugar-induced effector delivery.

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Table. S1 Primers used in this study

name sequence description

STP1_Fw 5'-ATGCCGTCTTTCCTCAAGCG-3'

For qPCR

STP1_Rv 5'-TAGCGTCGGGCTATCGTACT-3'

STP13_Fw 5'-TGTTTCTGTTTAACTCCTTGAGAGA-3'

STP13_Rv 5'-TTCGCCGAAGTCGCAAATCCTCCTC-3'

ACT2_Fw 5'-CTTGCACCAAGCAGCATGAA-3'

ACT2_Rv 5'-CCGATCCAGACACTGTACTTCCTT-3'

sGFP_Fw_EcoRI 5'-TACCGGATCCGAATTCATGGTGAG CAAGGGCGAGGAGCTGT-3'

For pRI 35S GFP sGFP_Rv_SacI 5'-GATCGGGGAAATTCGAGCTCTTAC

TTGTACAGCTCGTCCATGCCG-3'

3xFLAG_sense

5'-AATTCGACTACAAGGATCACGATG GAGACTACAAGGATCACGATATTG ACTACAAGGATGACGATGACAAAT AAGAGCT-3' For pRI 35S FLAG

3xFLAG_antisense 5'-CTTATTTGTCATCGTCATCCTTGTA GTCAATATCGTGATCCTTGTAGTCT CCATCGTGATCCTTGTAGTCG-3'

3xHA_sense

5'-AATTCTACCCATACGATGTTCCTG ACTATGCGTATCCCTATGACGTCC CGGACTATGCAGGATATCCATACG ACGTTCCAGATTACGCTTAAGAGCT-3'

For pRI 35S HA

3xHA_antisense

5'-CTTAAGCGTAATCTGGAACGTCGTA TGGATATCCTGCATAGTCCGGGACG TCATAGGGATACGCATAGTCAGGAA CATCGTATGGGTAG-3'

nYFP_Fw_EcoRI 5'-TACCGGATCCGAATTCATGGAGCAG AAGCTGATCAGCGAGG-3'

For pRI 35S nY nYFP_Rv_SacI 5'-GATCGGGGAAATTCGAGCTCTTAGG

CCATGATATAGACGTTGTG-3'

cYFP_Fw_EcoRI 5'-TACCGGATCCGAATTCATGTACCCAT ACGATGTTCCAGATT-3'

For pRI 35S cY cYFP_Rv_SacI 5'-GATCGGGGAAATTCGAGCTCTTACTT

GTACAGCTCGTCCATGCC-3'

STP13pro_Fw_HindIII 5'-GGCCAGTGCCAAGCTTCGTGACTTA GCAGACTCGTTGAAAT-3'

For pRI genomic STP13 GFP STP13_SalI_Rv 5'-TACCCCCGGGGTCGACAAGCCGTGT

TGAAGGATCAAAGCCA-3'

EFR_Fw_NdeI 5'-CACTGTTGATACATATGATGAAGC TGTCCTTTTCACTTGTTT-3'

For pRI 35S EFR-GFP EFR_Rv_Sal 5'-TACCCCCGGGGTCGACCATAGTATG

CATGTCCGTATTTAAC-3'

PEPR1_Fw_Nde 5'-CACTGTTGATACATATGATGAAGA ATCTTGGGGGGTTGTTCA-3'

For pRI 35S PEPR1-GFP PEPR1_Rv_Sal 5'-TACCCCCGGGGTCGACCCGAACTGA

ATCAGAGGAGCAGCTT-3'

pRI_35S upstream_Fw 5'-TATAGGGCGAATTGGGTACCTACAGT CTCAGAAGACCAAAGGGC-3'

For pSK 35S HA and pSK 35S FLAG pRI_NosT_downstream_Rv 5'-GGGAACAAAAGCTGGAGCTCCCATG

ATTACGAATTAATTCCCGA-3'

STP13_Fw_NdeI 5'-CACTGTTGATACATATGATGACCGGA GGAGGATTTGCGACTT-3'

For pSK 35S STP13-FLAG STP13_Rv_SalI 5'-TACCCCCGGGGTCGACAAGCCGTGT

TGAAGGATCAAAGCCA-3'

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Table. S1 continued

FLS2_Fw_NdeI 5'-CACTGTTGATACATATGATGAAGTTA CTCTCAAAGACCTTTT-3'

For pSK 35S FLS2-HA and pRI 35S FLS2-GFP FLS2_Rv_SalI 5'-TACCCCCGGGGTCGACAACTTCTCG

ATCCTCGTTACGATCT-3'

BAK1_Fw_NdeI 5'-CACTGTTGATACATATGATGGAACGA AGATTAATGATCCCTT-3'

For pSK 35S BAK1-HA BAK1_Rv_SalI 5'-TACCCCCGGGGTCGACTCTTGGACCC

GAGGGGTATTCGTTT-3'

BIK1_Fw_NdeI 5'-CACTGTTGATACATATGATGGGTTCTT GCTTCAGTTCTCGAG-3'

For pSK 35S BIK1-HA BIK1_Rv__SalI 5'-TACCCCCGGGGTCGACCACAAGGTGC

CTGCCAAAAGGTTTT-3'

GFP_Fw_NdeI 5'-CACTGTTGATACATATGATGGTGAGCA AGGGCGAGGAGCTGT-3'

For pSK 35S GFP-HA GFP_Rv_SalI 5'-TACCCCCGGGGTCGACTTACTTGTACA

GCTCGTCCATGCCG-3'

STP13_ML_Fw_BamHI 5'-TGCATCTGTTGGATCCGTGACAGAGAC ACCAAACAGT-3'

For pGEX GST-STP13 ML STP13_ML_Rv_EcoRI 5'-CAGTCACGATGAATTCGACGAGCTGAG

GACGGTTTCT-3'

STP13_CT_Fw_BamHI 5'-TGCATCTGTTGGATCCCCGGAGACTAA GAATATTCCT-3'

For pGEX GST-STP13 CT STP13_Rv_EcoRI 5'-CAGTCACGATGAATTCTTAAAGCCGTG

TTGAAGGATCAAA-3'

BAK1_CD_Fw_EcoRI 5'-AAGGATTTCAGAATTCAAAAAGCCGCA GGACCACTTCTTTG-3'

For pMAL MBP-BAK1 CD BAK1_CD_Rv_SalI 5'-TTGCCTGCAGGTCGACTTATCTTGGACC

CGAGGGGTATT-3'

FLS2_CD_Fw_EcoRI 5'-AAGGATTTCAGAATTCGAAAATTCATCA GAGTCCTCATTACCG-3'

For pMAL MBP-FLS2 CD FLS2_CD_Rv_SalI 5'-TTGCCTGCAGGTCGACCTAAACTTCTCG

ATCCTCGTTAC-3'

PEPR1_CD_Fw_EcoRI 5'-AAGGATTTCAGAATTCATTTGCCTACG TCGTCGCAAAGGA-3'

For pMAL MBP-PEPR1 CD PEPR1_CD_Rv_SalI 5'-TTGCCTGCAGGTCGACTTACCGAACTGA

ATCAGAGGAGCAG-3'

BIK1_Fw_EcoRI 5'-AAGGATTTCAGAATTCATGGGTTCTTGC TTCAGTTCTCGA-3'

For pMAL MBP-BIK1 BIK1_Rv_SalI 5'-TTGCCTGCAGGTCGACCTACACAAGGTG

CCTGCCAAAAGGT-3'

GFP_Fw_SalI 5'-TATCGATACCGTCGACATGGTGAGCAA GGGCGAGGA-3'

For pDR 196 GFP GFP_Rv_XhoI 5'-CGGGCCCCCCCTCGAGTTACTTGTACA

GCTCGTCCA-3'

STP13_Fw_EcoRI 5'-CGGGCTGCAGGAATTCATGACCGGAGG AGGATTTGC-3'

For pDR STP13-GFP STP13_Rv_SalI 5'-TGCTCACCATGTCGACAAGCCGTGTTGA

AGGAT-3'

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Table. S1 continued STP13_S513A_Fw 5'-GTGAATGGTGAGAAGGCTAATGGT-3'

For site-directed mutagenesis

STP13_S513A_Rv 5'-ACCATTAGCCTTCTCACCATTCAC-3'

STP13_S517A_S523A_S524A_Fw 5'-AAGGCTAATGGTAAAGCTAATGGCTTTG ATCCTGCAGCACGG-3'

STP13_S517A_S523A_S524A_Rv 5'-TTTACCATTAGCCTTCTCACCATTCACGA ACTC-3'

STP13_T485A_Fw 5'-AATATTCCTATCGAGGAGATGGCTGAGAGA-3'

STP13_T485D_Fw 5'-AATATTCCTATCGAGGAGATGGATGAGAGA-3'

STP13_T485_Rv 5'-CTCGATAGGAATATTCTTAGTCTCCGGAA GTAG-3'

avrPto_Fw_EcoRI 5'-GACGGCCAGTGAATTCGGTCCAGGAAATG AATACCCAGCTT-3'

For pHM avrPto-Cya avrPto_Rv_KpnI 5'-TGCTGAGATCCCCGGGTACCTTGCCAGTTA

CGGTACGGGCTAGG-3'

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References and Notes 1. R. T. Voegele, K. W. Mendgen, Nutrient uptake in rust fungi: How sweet is parasitic life?

Euphytica 179, 41–55 (2011). doi:10.1007/s10681-011-0358-5

2. J. Doidy, E. Grace, C. Kühn, F. Simon-Plas, L. Casieri, D. Wipf, Sugar transporters in plants and in their interactions with fungi. Trends Plant Sci. 17, 413–422 (2012). doi:10.1016/j.tplants.2012.03.009 Medline

3. K. Wang, M. Senthil-Kumar, C. M. Ryu, L. Kang, K. S. Mysore, Phytosterols play a key role in plant innate immunity against bacterial pathogens by regulating nutrient efflux into the apoplast. Plant Physiol. 158, 1789–1802 (2012). doi:10.1104/pp.111.189217 Medline

4. L. Q. Chen, B.-H. Hou, S. Lalonde, H. Takanaga, M. L. Hartung, X.-Q. Qu, W.-J. Guo, J.-G. Kim, W. Underwood, B. Chaudhuri, D. Chermak, G. Antony, F. F. White, S. C. Somerville, M. B. Mudgett, W. B. Frommer, Sugar transporters for intercellular exchange and nutrition of pathogens. Nature 468, 527–532 (2010). doi:10.1038/nature09606 Medline

5. M. Cohn, R. S. Bart, M. Shybut, D. Dahlbeck, M. Gomez, R. Morbitzer, B.-H. Hou, W. B. Frommer, T. Lahaye, B. J. Staskawicz, Xanthomonas axonopodis virulence is promoted by a transcription activator-like effector-mediated induction of a SWEET sugar transporter in cassava. Mol. Plant Microbe Interact. 27, 1186–1198 (2014). doi:10.1094/MPMI-06-14-0161-R Medline

6. R. Wahl, K. Wippel, S. Goos, J. Kämper, N. Sauer, A novel high-affinity sucrose transporter is required for virulence of the plant pathogen Ustilago maydis. PLOS Biol. 8, e1000303 (2010). doi:10.1371/journal.pbio.1000303 Medline

7. T. Li, B. Liu, M. H. Spalding, D. P. Weeks, B. Yang, High-efficiency TALEN-based gene editing produces disease-resistant rice. Nat. Biotechnol. 30, 390–392 (2012). doi:10.1038/nbt.2199 Medline

8. J. S. Eom, L.-Q. Chen, D. Sosso, B. T. Julius, I. W. Lin, X.-Q. Qu, D. M. Braun, W. B. Frommer, SWEETs, transporters for intracellular and intercellular sugar translocation. Curr. Opin. Plant Biol. 25, 53–62 (2015). doi:10.1016/j.pbi.2015.04.005 Medline

9. K. Yamada, M. Kanai, Y. Osakabe, H. Ohiraki, K. Shinozaki, K. Yamaguchi-Shinozaki, Monosaccharide absorption activity of Arabidopsis roots depends on expression profiles of transporter genes under high salinity conditions. J. Biol. Chem. 286, 43577–43586 (2011). doi:10.1074/jbc.M111.269712 Medline

10. P. Lemonnier, C. Gaillard, F. Veillet, J. Verbeke, R. Lemoine, P. Coutos-Thévenot, S. La Camera, Expression of Arabidopsis sugar transport protein STP13 differentially affects glucose transport activity and basal resistance to Botrytis cinerea. Plant Mol. Biol. 85, 473–484 (2014). doi:10.1007/s11103-014-0198-5 Medline

11. J. W. Moore, S. Herrera-Foessel, C. Lan, W. Schnippenkoetter, M. Ayliffe, J. Huerta-Espino, M. Lillemo, L. Viccars, R. Milne, S. Periyannan, X. Kong, W. Spielmeyer, M. Talbot, H. Bariana, J. W. Patrick, P. Dodds, R. Singh, E. Lagudah, A recently evolved hexose transporter variant confers resistance to multiple pathogens in wheat. Nat. Genet. 47, 1494–1498 (2015). doi:10.1038/ng.3439 Medline

19

12. X. F. Xin, S. Y. He, Pseudomonas syringae pv. tomato DC3000: A model pathogen for probing disease susceptibility and hormone signaling in plants. Annu. Rev. Phytopathol. 51, 473–498 (2013). doi:10.1146/annurev-phyto-082712-102321 Medline

13. W. Zeng, S. Y. He, A prominent role of the flagellin receptor FLAGELLIN-SENSING2 in mediating stomatal response to Pseudomonas syringae pv tomato DC3000 in Arabidopsis. Plant Physiol. 153, 1188–1198 (2010). doi:10.1104/pp.110.157016 Medline

14. K. H. Liu, Y. F. Tsay, Switching between the two action modes of the dual-affinity nitrate transporter CHL1 by phosphorylation. EMBO J. 22, 1005–1013 (2003). doi:10.1093/emboj/cdg118 Medline

15. D. Couto, C. Zipfel, Regulation of pattern recognition receptor signalling in plants. Nat. Rev. Immunol. 16, 537–552 (2016). doi:10.1038/nri.2016.77 Medline

16. K. Yamada, M. Yamashita-Yamada, T. Hirase, T. Fujiwara, K. Tsuda, K. Hiruma, Y. Saijo, Danger peptide receptor signaling in plants ensures basal immunity upon pathogen-induced depletion of BAK1. EMBO J. 35, 46–61 (2016). doi:10.15252/embj.201591807 Medline

17. T. S. Nühse, A. Stensballe, O. N. Jensen, S. C. Peck, Phosphoproteomics of the Arabidopsis plasma membrane and a new phosphorylation site database. Plant Cell 16, 2394–2405 (2004). doi:10.1105/tpc.104.023150 Medline

18. B. Schwessinger, M. Roux, Y. Kadota, V. Ntoukakis, J. Sklenar, A. Jones, C. Zipfel, Phosphorylation-dependent differential regulation of plant growth, cell death, and innate immunity by the regulatory receptor-like kinase BAK1. PLOS Genet. 7, e1002046 (2011). doi:10.1371/journal.pgen.1002046 Medline

19. T. V. Huynh, D. Dahlbeck, B. J. Staskawicz, Bacterial blight of soybean: Regulation of a pathogen gene determining host cultivar specificity. Science 245, 1374–1377 (1989). doi:10.1126/science.2781284 Medline

20. J. L. Stauber, E. Loginicheva, L. M. Schechter, Carbon source and cell density-dependent regulation of type III secretion system gene expression in Pseudomonas syringae pathovar tomato DC3000. Res. Microbiol. 163, 531–539 (2012). doi:10.1016/j.resmic.2012.08.005 Medline

21. L. M. Schechter, K. A. Roberts, Y. Jamir, J. R. Alfano, A. Collmer, Pseudomonas syringae type III secretion system targeting signals and novel effectors studied with a Cya translocation reporter. J. Bacteriol. 186, 543–555 (2004). doi:10.1128/JB.186.2.543-555.2004 Medline

22. A. Rico, G. M. Preston, Pseudomonas syringae pv. tomato DC3000 uses constitutive and apoplast-induced nutrient assimilation pathways to catabolize nutrients that are abundant in the tomato apoplast. Mol. Plant Microbe Interact. 21, 269–282 (2008). doi:10.1094/MPMI-21-2-0269 Medline

23. J. C. Anderson, Y. Wan, Y.-M. Kim, L. Pasa-Tolic, T. O. Metz, S. C. Peck, Decreased abundance of type III secretion system-inducing signals in Arabidopsis mkp1 enhances resistance against Pseudomonas syringae. Proc. Natl. Acad. Sci. U.S.A. 111, 6846–6851 (2014). doi:10.1073/pnas.1403248111 Medline

20

24. D. Chinchilla, C. Zipfel, S. Robatzek, B. Kemmerling, T. Nürnberger, J. D. G. Jones, G. Felix, T. Boller, A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence. Nature 448, 497–500 (2007). doi:10.1038/nature05999 Medline

25. C. Zipfel, S. Robatzek, L. Navarro, E. J. Oakeley, J. D. G. Jones, G. Felix, T. Boller, Bacterial disease resistance in Arabidopsis through flagellin perception. Nature 428, 764–767 (2004). doi:10.1038/nature02485 Medline

26. V. Ntoukakis, B. Schwessinger, C. Segonzac, C. Zipfel, Cautionary notes on the use of C-terminal BAK1 fusion proteins for functional studies. Plant Cell 23, 3871–3878 (2011). doi:10.1105/tpc.111.090779 Medline

27. A. Weise, L. Barker, C. Kühn, S. Lalonde, H. Buschmann, W. B. Frommer, J. M. Ward, A new subfamily of sucrose transporters, SUT4, with low affinity/high capacity localized in enucleate sieve elements of plants. Plant Cell 12, 1345–1355 (2000). doi:10.1105/tpc.12.8.1345 Medline

28. A. Furutani, M. Takaoka, H. Sanada, Y. Noguchi, T. Oku, K. Tsuno, H. Ochiai, S. Tsuge, Identification of novel type III secretion effectors in Xanthomonas oryzae pv. oryzae. Mol. Plant Microbe Interact. 22, 96–106 (2009). doi:10.1094/MPMI-22-1-0096 Medline

29. J. Yuan, S. Y. He, The Pseudomonas syringae Hrp regulation and secretion system controls the production and secretion of multiple extracellular proteins. J. Bacteriol. 178, 6399–6402 (1996). doi:10.1128/jb.178.21.6399-6402.1996 Medline

30. R. Wieczorke, S. Dlugai, S. Krampe, E. Boles, Characterisation of mammalian GLUT glucose transporters in a heterologous yeast expression system. Cell. Physiol. Biochem. 13, 123–134 (2003). doi:10.1159/000071863 Medline

31. Y. Kadota, J. Sklenar, P. Derbyshire, L. Stransfeld, S. Asai, V. Ntoukakis, J. D. G. Jones, K. Shirasu, F. Menke, A. Jones, C. Zipfel, Direct regulation of the NADPH oxidase RBOHD by the PRR-associated kinase BIK1 during plant immunity. Mol. Cell 54, 43–55 (2014). doi:10.1016/j.molcel.2014.02.021 Medline

32. S. D. Yoo, Y. H. Cho, J. Sheen, Arabidopsis mesophyll protoplasts: A versatile cell system for transient gene expression analysis. Nat. Protoc. 2, 1565–1572 (2007). doi:10.1038/nprot.2007.199 Medline

33. M. Melotto, W. Underwood, J. Koczan, K. Nomura, S. Y. He, Plant stomata function in innate immunity against bacterial invasion. Cell 126, 969–980 (2006). doi:10.1016/j.cell.2006.06.054 Medline

21