Supplemental Figure S1. Nucleotide and deduced amino acid sequences of pepper CaHSP70a (Capsicum annuum heat shock protein 70a) cdna.

Transcription

1 Supplemental Figure S1. Nucleotide and deduced amino acid sequences of pepper (Capsicum annuum heat shock protein 70a) cdna. Translation initiation codon is shown in bold typeface; termination codon is annotated with an asterisk. Yellow and green shades indicate the two actin-like ATPase domains. The peptide-binding domain is shaded with gray. The sequences of the three heat shock protein signature domains are boxed..

2 A ******** Actin-like ATPase domain MAGKGEGPAIGIDLGTTYSCVGVWQHDRVEIIANDQGNRTTPSYVGFTDSERLIGDAAKN 60 NtHSP70.3 MAGKGEGPAIGIDLGTTYSCVGVWQHDRVEIIANDQGNRTTPSYVGFTDSERLIGDAAKN 60 SlHSP70 MAGKGEGPAIGIDLGTTYSCVGVWQHDRVEIIANDQGNRTTPSYVGFTDTERLIGDAAKN 60 OsHSP70 MAGKGEGPAIGIDLGTTYSCVGVWQHDRVEIIANDQGNRTTPSYVGFTDSERLIGDAAKN 60 AtHSP70 MAGKGEGPAIGIDLGTTYSCVGVWQHDRVEIIANDQGNRTTPSYVAFTDSERLIGDAAKN 60 QVAMNPINTVFDAKRLIGRRFSDASVQSDIKHWPFKVISGPGDKPMIVVNYKGEEKQFAA 120 NtHSP70.3 QVAMNPINTVFDAKRLIGRRFSDASVQSDIKLWPFKVISGPGDKPMIVVNYKGEEKQFAA 120 SlHSP70 QVALNPINTVFDAKRLIGRRFSDASVQEDMKLWPFKVIPGPGDKPMIVVTYKGEEKEFAA 120 OsHSP70 QVAMNPINTVFDAKRLIGRRFSDASVQSDIKLWPFKVIAGPGDKPMIVVQYKGEEKQFAA 120 AtHSP70 QVAMNPTNTVFDAKRLIGRRYSDPSVQADKSHWPFKVVSGPGEKPMIVVNHKGEEKQFSA 120 EEISSMVFIKMREIAEAFLGSTVKNAVVTVPAYFNDSQRQATKDAGVIAGLNVMRIINEP 180 NtHSP70.3 EEISSMVLIKMKEIAEAFLGSTVKNAVVTVPAYFNDSQRQATKDAGVISGLNVMRIINEP 180 SlHSP70 EEISSMVLTKMKEIAEAFLGSTVKNAVVTVPAYFNDSQRQATKDAGVISGLNVMRIINEP 180 OsHSP70 EEISSMVLIKMREIAEAYLGTTIKNAVVTVPAYFNDSQRQATKDAGVIAGLNVMRIINEP 180 AtHSP70 EEISSMVLIKMREIAEAFLGSPVKNAVVTVPAYFNDSQRQATKDAGVISGLNVMRIINEP 180 ************** TAAAIAYGLDKKATSVGEKNVLIFDLGGGTFDVSLLTIEEGIFEVKATAGDTHLGGGGLC 240 NtHSP70.3 TAAAIAYGLDKKATSVGEKNVLIFDLGGGTFDVSLLTIEEGIFEVKATAGDTHLGGEDFD 240 SlHSP70 TAAAIAYGLDKKATSAGEKNVLIFDLGGGTFDVSLLTIEEGIFEVKATAGDTHLGGEDFD 240 OsHSP70 TAAAIAYGLDKKATSVGEKNVLIFDLGGGTFDVSLLTIEEGIFEVKATAGDTHLGGEDFD 240 AtHSP70 TAAAIAYGLDKKASSVGEKNVLIFDLGGGTFDVSLLTIEEGIFEVKATAGDTHLGGEDFD 240 HRMVNHFVQEFKRKNKKDISGNPRALRRLRTACERAKRTLSSTAQTTIEIDSLYEGIDFY 300 NtHSP70.3 NRMVNHFVQEFKRKHKKDITGNPRALRRLRTACERAKRTLSSTAQTTIEIDSLYEGVDFY 300 SlHSP70 NRMVNHFVHEFKRKHKKDITGNPRALRRLRTACERAKRTLSSTAQTTIEIDSLYEGVDFY 300 OsHSP70 NRMVNHFVQEFKRKNKKDITGNPRALRRLRTACERAKRTLSSTAQTTIEIDSLYEGIDFY 300 AtHSP70 NRMVNHFVQEFKRKNKKDITGNPRALRRLRTACERAKRTLSSTAQTTIEIDSLFEGIDFY 300 Actin-like ATPase domain *************** STITRARFEELNMDLFRKCMEPVEKCLRDAKMDKSTIHDVVLVGGSTRIPKVQQLLQD-F 359 NtHSP70.3 STITRARFEELNMDLFRKCMEPVEKCLRDAKMDKSTVHDVVLVGGSTRIPKVQQLLQD-F 359 SlHSP70 STITRARFEELNMDLFRKCMEPVEKCLRDAKMDKSTVHDVVLVGGSTRIPKVQQVAMTNF 360 OsHSP70 STITRARFEELNMDLFRKCMEPVEKCLRDAKMDKSSVHDVVLVGGSTRIPRVQQLLQD-F 359 AtHSP70 TTITRARFEELNMDLFRKCMEPVEKCLRDAKMDKSSVHDVVLVGGSTRIPKVQQLLQD-F 359 FNGKELCKSINPDEAVAYGAAVQAAILSGEGNEKVQDLLLLDVTPLSLGLETAGGVMTVL 419 NtHSP70.3 FNGKELCKSINPDEAVAYGAAVQAAILSGEGNEKVQDLLLLDVTPLSLGLETAGGVMTVL 419 SlHSP70 FNGKELCKSINPDEAVAYGAAVQAAILSGEGNEKVQDLLLLDVTPLSLGLETAGGVMTVL 420 OsHSP70 FNGKELCKNINPDEAVAYGAAVQAAILSGEGNEKVQDLLLLDVTPLSLGLETAGGVMTVL 419 AtHSP70 FNGKELCKSINPDEAVAYGAAVQAAILSGEGNEKVQDLLLLDVTPLSLGLETAGGVMTVL 419 IPRNTTIPTKKEQVFSTYSDNQPGVLIQVFEGER-ARTRDNNLLGKFELSGIPPAPRGVP 478 NtHSP70.3 IPRNTTIPTKKEQVFSTYSDNQPGVLIQVYEGER-ARTRDNNLLGKFELSGIPPAPRGVP 478 SlHSP70 IPRNTTIPTKKEQVFSTYSDNQPGVLIQVFEGERRARTRDNNLLGKFELSVIPPAPRVVP 480 OsHSP70 IPRNTTIPTKKEQVFSTYSDNQPGVLIQVYEGER-TRTRDNNLLGKFELSGIPPAPRGVP 478 AtHSP70 IPRNTTIPTKKEQIFSTYSDNQPGVLIQVYEGER-ARTKDNNLLGKFELSGIPPAPRGVP 478 Peptide binding domain QITVCFDVDANGILNVSAEDKTTGQKNKITITNDKGRLSKEEIEKMVQDAEKYKAEDEEL 538 NtHSP70.3 QITVCFDIDANGILNVSAEDKTTGQKNKITITNDKGRLSKEEIEKMVQEAEKYKAEDEEH 538 SlHSP70 QITVCFDIDANGILNVSAEDKTTGQKNKITITNDKGRLSKEEIEKMVQEAEKYKAEDEEL 540 OsHSP70 QITVCFDIDANGILNVSAEDKTTGQKNKITITNDKGRLSKEEIEKMVQEAEKYKSEDEEH 538 AtHSP70 QITVCFDIDANGILNVSAEDKTTGQKNKITITNDKGRLSKEEIEKMVQEAEKYKAEDEEH 538 KKKVEAKNSLENYAYNMRNTVKDEKIGSKLSPDDKKKIEDAIDQAISWLDSNQLAEVDEF 598 NtHSP70.3 KKKVEAKNALENYAYNMRNTIKDEKIGSKLSSDDKKKIEDAIDQAISWLDSNQLAEADEF 598 SlHSP70 KKKVEAKNSLENYAYNMRNTVKDEKIGSKLSSDDKKKIEDAVDQAISWLESNQLAEVDEF 600 OsHSP70 KKKVESKNALENYAYNMRNTIKDEKIASKLPAADKKKIEDAIDQAIQWLDGNQLAEADEF 598 AtHSP70 KKKVDAKNALENYAYNMRNTIKDEKIASKLDAADKKKIEDAIDQAIEWLDGNQLAEADEF 598 DKMKELEG I CNPIIAK MYQGAG DDAPP AGGS S-AGPKIEEVD EGEAGVPMD NtHSP70.3 DKMKELES I CNPIIAK MYQGAG DDAPP AGGS S-AGPKIEEVD EGEAGAPMD-- 648(96%) SlHSP70 DKMKELEG I CNPIIAK MYQGAG DDAPP SGGS S-AGPKIEEVD EGDAGVPMD-- 650(94%) OsHSP70 DKMKELEG I CNPIIAK MYQGAG DDAPP AGGS G-AGPKIEEVD DADMAGGMDE- 649(93%) AtHSP70 DKMKELES L CNPIIAR MYQGAG DDDTP AGGS GGAGPKIEEVD EPDMGGAGGM- 650(91%) Actin-like ATPase Actin-like ATPase peptide binding domain Supplemental Figure S2. Alignment of with other HSP70 proteins from tobacco (NtHSP70-3, accession no.:aar17080), tomato (SlHSP70, accession no.:caa37970), rice (OsHSP70, accession no.

3 AAX95352), and Arabidopsis (AtHSP70, accession no. AAG51030). A, Positions of amino acid residues in the corresponding proteins are numbered. Gaps marked with dashes were introduced to maximize sequence alignment. Identical residues are shaded in black. Dark- and light-gray shades indicate conserved and semiconserved residues, respectively. The three HSP70 signature domains are marked with asterisks above the sequence., Schematic diagram of primary structure. A Xanthomonas campestris pv. vesicatoria Mock Ds1 Ds1( avrst ) H h rrna kda H Mock Xanthomonas campestris pv. vesicatoria h C Ds1 Ds1(avrsT) C Nuclear fraction Cytoplasmic fraction 70 I: anti - CaHSP70 20 I: anti - H3 15 I: anti - Hsc70 70 kda Xcv Ds1 Xcv Ds1(avrsT ) Xcv Ds1 Xcv Ds1( avrst ) h Supplemental Figure S3. RNA gel blot and immunoblot analyses of expression in pepper plants. RNA extracted from pepper plants was blotted to nylon membrane and hybridized with 32 P-labeled probes. rrna is shown as a loading control. H: healthy leaves, Mock: treated with 10 mm MgCl2. A, expression in leaves infected with virulent Ds1 (compatible) or avirulent Ds1 (avrst) (incompatible) strains of Xanthomonas campestris pv. vesicatoria (Xcv)., Immunoblot analysis of protein levels in leaves infected with Xcv Ds1 or Xcv Ds1 (avrst) using anti- antibodies. Mock: treated with 10 mm MgCl2.. Equal protein loading was visualized by Coomassie brilliant blue (C) staining. C, protein levels in nuclear and cytoplasmic fractions of the protein extracts from leaves infected with Xcv Ds1 or Xcv Ds1 (avrst). Anti-H3 and Anti-Hsc70 antibodies were used to detect nuclear histone histone 3 (H3) and cytoplasmic heat shock complex 70 (Hsc70) proteins as positive controls, respectively.

4 35S:EV 35S: h CaPR1 CaPIK1 CaRP1 18 S rrna Supplemental Figure S4. RT-PCR analyses of defense- and cell- deathrelated gene expression in pepper leaves during transient expression of.

5 A EV avrst avrst:nls avrst:nes OD 600 : c c 2 1 a b 0 EV :NLS :NES OD 600 : a a a a Supplemental Figure S5. Cell death phenotypes and quantification in leaves 2 days after infiltration with Agrobacterium carrying (A) avrst, avrst:nls, and avrst:nes, and (), :NLS, and :NES. Cell-death levels were rated based on a 0 3 scale: 0, no cell death (<10%); 1, weak cell death (10 30%); 2, partial cell death (30 80%); and 3, full cell death (80 100%). Data represent the means ± SD from three independent experiments. Different letters indicate statistically significant differences (LSD, P < 0.05).

6 Supplemental Figure S6. and CaSGT1 expression contributes independently to the resistance to Xanthomonas campestris pv. vesicatoria (Xcv) infection. A, Xcv growth in, CaSGT1 or /CaSGT1-silenced pepper leaves infected with Xcv Ds1 (EV) and Ds1 (avrst) ( cfu ml -1 )., H2O2 quantification in Xcv-inoculated leaves ( cfu ml -1 ). C, Electrolyte leakage measurement in Xcv-inoculated ( cfu ml -1 ) leaves. hai: hours after inoculation. Data represent the means ± SD of three independent experiments. Different letters indicate statistically significant differences (LSD, P < 0.05). A 35S: WT #2 #3 #4 UQ 5 WT #2 #3 #4 Days after inoculation

7 Supplemental Figure S7. overexpression (OX) in Arabidopsis plants does not alter the susceptibility to Pseudomonas syringae pv. tomato (Pst) infection. A, Ectopic expression in -OX transgenic lines., acterial growth in leaves of wild-type and transgenic plants at 0, 1, and 3 days after inoculation with P. syringae pv. tomato DC3000 and DC3000 (avrrpm1). A WT #2 #3 # Sporangiophores/cotyledon WT #2 #3 #4 14.8(±1.8) a 13.1(±0.1) a 14.3(±1.1) a 14.1(±2.1) a Supplemental Figure S8. overexpression (OX) in Arabidopsis plants does not alter susceptibility to Hyaloperospora arabidopsidis infection. A, Disease symptoms in cotyledons of wild-type and transgenic seedlings 7 days after inoculation with Hp. arabidopsidis Noco2., Numbers of sporangiophores per cotyledon in wild-type and transgenic plants 5 days after inoculation. Data were rated using four scales: no sporulation (0 sporangiophore/cotyledon), light sporulation (1 to 9 sporangiophores/cotyledon), medium sporulation (10 to 19 sporangiophores/cotyledon), or heavy sporulation (>20 sporangiophores/cotyledon). More than 50 cotyledons of wild-type and transgenic plant lines were counted. Average numbers of sporangiophores are shown below. Data represent the means ± SD from three independent experiments. Different letters indicate statistically significant differences (LSD, P < 0.05). A ifc Visible Merged ifc DAPI Merged

8 / AvrsT/ AvrsT / CaSGT1/ CaSGT1 / CaPIK1/ CaPIK1 Input IP: HA Input IP: HA Input IP: HA AvrsT:cMyc CaSGT1:cMyc CaPIK1:cMyc :HA :HA :HA α-cmycα-cmycα-cmyc α-haα-haα-ha CCC Supplemental Figure S9. interacts with AvrsT, but not with CaSGT1 or CaPIK1 in planta. A, imolecular fluorescence complementation images of interactions of with AvrsT, CaSGT1 or CaPIK1 in Nicotiana benthamiana leaves. YFP signals were visualized by confocal microscopy. Cell nuclei were counter-stained with DAPI. ars=50 µm., Immunoblot and coimmunoprecipitation analyses of transient :HA co-expression with AvrsT:cMyc, CaSGT1:cMyc or CaPIK1:cMyc in N. benthamiana leaves. Extracted proteins were immunoprecipitated with α-ha beads, and immunoblotted with α-cmyc or α-ha antibodies. Protein loading was visualized by Coomassie brilliant blue (C) staining.

9 AvrsT CaSGT 1 Heat stress or Xcv (avrst) challenge AvrsT bound to AvrsT T3E Defense genes Cell death Nucleus SA JA ROS Cell deathtriggering protein? bound to cell death-triggering clients Supplemental Figure S10. Proposed model for the and AvrsT complex, together with the CaPIK1-CaSGT1-AvrsT complex, in plant cell death and defense signaling. T3E, type III effector; SA, salicylic acid; JA, jasmonic acid; ROS, reactive oxygen species.