Supplemental Figure 1. Conserved regions of the kinase domain of PEPR1, PEPR2, CLV1 and BRI1.

Transcription

1 Supplemental Figure 1. Conserved regions of the kinase domain of PEPR1, PEPR2, CLV1 and BRI1. The asterisks and colons indicate the important residues for ATP-binding pocket and substrate binding pocket, respectively. ******************* *********************************** ***************************: PEPR1 827 LNEKYTIGRGAHGIVYRASLGSGKVYAVKRLVFASHIRANQSMMREIDTIGKVRHRNLIKLEGFWLRKDDGLMLYRYMPKGSLYDVLHGVSPKENVLDWSARYNVALGVAHGLAYLHYDC 946 PEPR2 794 LDDKYIIGRGAHGVVYRASLGSGEEYAVKKLIFAEHIRANQNMKREIETIGLVRHRNLIRLERFWMRKEDGLMLYQYMPNGSLHDVLHRGNQGEAVLDWSARFNIALGISHGLAYLHHDC 913 CLV1 692 LKEENIIGKGGAGIVYRGS MPNNVDVAIKRLVGRGTGRSDHGFTAEIQTLGRIRHRHIVRLLGYVANKDTNLLLYEYMPNGSLGELLHG--SKGGHLQWETRHRVAVEAAKGLCYLHHDC 809 BRI1 883 FHNDSLIGSGGFGDVYKAI LKDGSAVAIKKLIHVSG-QGDREFMAEMETIGKIKHRNLVPLLGYCKVGDERLLVYEFMKYGSLEDVLHDPKKAGVKLNWSTRRKIAIGSARGLAFLHHNC 1001 *********:********* ************************::#:#:: PEPR1 947 HPPIVHRDIKPENILMDSDLEPHIGDFGLARLLDDSTVST--ATVTGTTGYIAP ENAFKTVR-GRESDVYSYGVVLLELVTRKRAVD KSFPESTDIVSWVRSALSSSNNN-VEDMVTTIVD 1063 PEPR2 914 HPPIIHRDIKPENILMDSDMEPHIGDFGLARILDDSTVST--ATVTGTTGYIAP ENAYKTVR-SKESDVYSYGVVLLELVTGKRALD RSFPEDINIVSWVRSVLSSYED--EDDTAGPIVD 1029 CLV1 810 SPLILHRDVKSNNILLDSDFEAHVADFGLAKFLVDGAASECMSSIAGSYGYIAP EYAYTL-KVDEKSDVYSFGVVLLELIAGKKPVG -EFGEGVDIVRWVRNTEEEITQPSDAAIVVAIVD 927 BRI SPHIIHRDMKSSNVLLDEN LEARVSDFGMARLMSAMDTHLSVSTLAGTPGYVPPEYYQSF-RCSTKGDVYSYGVVLLELLTGKRPTD SPDFGDNNLVGWVKQHAK LRISDVFD 1112 Catalytic loop Activation loop (P+1 loop) Guanilyl cyclase catalytic domain PEPR PILVDELLDSSLREQVMQV TELALSCTQQDPAMRPTMRDAVKLL 1107 PEPR PKLVDELLDTKLREQAIQV TDLALRCTDKRPENRPSMRDVVKDL 1073 CLV1 928 PRLTGYPLTS-----VIHV FKIAMMCVEEEAAARPTMREVVHML 968 BRI PELMKE--DPALEIELLQHLKVAVACLDDRAWRRPTMVQVMAMF

2 Supplemental Figure 2. The effect of MeSA and ACC on the expression patterns of PEPR1 and PEPR2 in four-week-old Arabidopsis. Relative expression of PEPR1 (A) and PEPR2 (B) after spraying with H 2 O (0.1 % Triton X-100), methyl salicylate (MeSA, 2 mm in 0.01 % Triton X-100) and 1-aminocyclopropan-1-carboxylic acid (ACC, 0.1 mm in 0.01 % Triton X-100). Relative expressions of PR-1 (C) and PDF1.2 (D) after treatment with MeSA and ACC, respectively, are shown as positive control for the treatments. Similar results were obtained in different experiments. 2

3 Supplemental Figure 3. The effect of Pep1 on the expression patterns of WRKY22, WRKY29, WRKY53, WRKY55, and PR-1 genes for the T-DNA insertional mutants. Two-week-old Arabidopsis seedlings grown in liquid medium were incubated with 10 nm Pep1 for 30 min, and expression was analyzed by quantitative RT-PCR. Error bars indicate standard error from three different experiments. The letters indicate groupings by the one-way analysis of variance (ANOVA) with Tukey multiple comparison test (P<0.05). 3

4 Supplemental Figure 4. P. syringae pv. tomato DC3000 (Pst DC3000) infection assay of T- DNA insertional mutants pretreated with either H 2 O or Pep1 (1 µm). (A) Symptoms of WT, pepr1-2, pepr2-2 and pepr1-2/pepr2-2 four days after infection with Pst DC3000. (B) Pst DC3000 proliferation in WT, pepr1-2, pepr2-2 and pepr1-2/pepr2-2 with or without Pep1. Peptides or H 2 O was infiltrated one day prior to infection. Leaf discs from infected leaves were homogenized and the number of colony-forming units (cfu/cm 2 ) was estimated by their growth on nutrient broth agar plates. Values presented are the average ± SE from nine plants from 3 independent experiments. The asterisks indicate samples that are significantly different from corresponding samples supplied with H 2 O (t-test, P<0.0006). 4

5 Supplemental Figure 5. The effect of Pep peptides on the expression pattern of WRKY33 gene in the T-DNA mutants. Two-week-old Arabidopsis seedlings grown in liquid medium were incubated with 10 nm peptide for 30 min, and expression was analyzed by quantitative RT-PCR. Expression levels are indicated relative to the expression in wild type seedlings supplied with H 2 O. Error bars indicate standard error for five different experiments. The number of asterisks indicates samples that are significantly different from corresponding samples supplied with H 2 O (t-test: one asterisk, P<0.05; two asterisks, P<0.01). 5

6 Supplemental Table 1. Primers used in this study. Name Sequence (5-3 ) PEPR2-4 1 PEPR2-7 1 LBb1 1 PEPR2-FS 2 PEPR2-RS 2 PEPR1-KpnI 3 PEPR1-3 3 PEPR2-2 3 PEPR2-7 3 GUS-f 3 GUS-r 3 TUB2-f 3 TUB2-r 3 EF1β-f 3 EF1β-r 3 PROPEP1-a 4 PROPEP1-b 4 PEPR1-a 4 PEPR1-b 4 PEPR2-a 4 PEPR2-b 4 MPK3-a 4 MPK3-b 4 WRKY22-a 4 WRKY22-b 4 WRKY29-a 4 WRKY29-b 4 WRKY33-a 4 WRKY33-b 4 WRKY53-a 4 WRKY53-b 4 WRKY55-a 4 WRKY55-b 4 PDF1.2-a 4 PDF1.2-b 4 PR1-a 4 PR1-b 4 UBQ5-a 4 UBQ5-b 4 TTGGATCCAACTCATTGAACG ACGCCAGTCCATGTGAAATC GCGTGGACCGCTTGCTGCAACT (at left border of T-DNA) CCCGGGTTTCAGTCTCTTGAGCTCTAATCT CCCGGGCAAATCTTCACGGTATTCAATGG CGGGGTACCTCGATCTTTAAACTCAGATGAAGAA TCCCAAGTTGTGGAGGTA TCCCATCATCAATGGGTATGT ACGCCAGTCCATGTGAAATC CAACGAACTGAACTGGCAGA GGCACAGCACATCAAAGAGA CAACGCTACTCTGTCTGTCC TCTGTGAATTCCATCTCGTC ACCAGATCAATGAGCCCAAG GAAAACCAAGGCACCTCAAA ATCAGATAGACGAAGCGAAG CTAATTATGTTGGCCAGGAC CAACAACAATGTGGAGGATA AACGAGATTACCGAACTGAA AAGAAGATGGCTTAATGCTG CAGTTGTGCCAGTAACAGTG ATGCGCTTATTGACAGAGTT TCAGAGCTTGTTCAACAGTG CCGTCGGACGACAAAGTAAT CTGCTGCTACATGGCACACT ACCCTTTCTCCACACAAACG TGGGTTTCTGCCCGTATTTA GAAACAAATGGTGGGAATGG TGTCGTGTGATGCTCTCTCC GCGACAAGACACCAGAGTCA ACCGTTGGATTGAACCAGTC ACTGCGTTAAACAAAACCAA ACCGTTTGTTCTTCTCCTTC CTTATCTTCGCTGATCTTGT CGTAACAGATACACTTGTGTGC CTAAGAGGCAACTGCAGACT GTATGGCTTCTCGTTCACAT AGAAGATCAAGCACAAGCAT CAGATCAAGCTTCAACTCCT 1 For screening of T-DNA insertional lines. 2 For making binary vector expressing AtPEPR2 in tobacco cells. 3 For RT-PCR in T-DNA insertional lines and transgenic tobacco cells. 4 For qrt-pcr analysis 6