Supplement Table 1: Constructs used in BiFC interactions and as effectors for promoter activation

Gene / Insert domain / Res. sites / Primer / Locus in genbank / Amino acids
(aa) / Vector / Name of constructs
1# / HsfA1a / protein coding cDNA / XbaI-AscI / HSF1XbaI HSF1AscI-rev. / AT4G17750 / 1-495 / pUC-SPYNE-152 / HsfA1a-YFPN152
2 / HsfA1a / protein coding cDNA / XbaI-AscI / HSF1XbaI HSF1AscI-rev. / AT4G17750 / 1-495 / pUC-SPYCE / HsfA1a-YFPC
3 / HsfA1a / OD / XbaI-AscI / HSF1-olim-XbaI HSF1-olim-AscI-rev. / AT4G17750 / 161-262 / pUC-SPYNE-152 / ODHsfA1a-YFPN152
4 / HsfA1a / OD / XbaI-AscI / HSF1-olim-XbaI HSF1-olim-AscI-rev. / AT4G17750 / 161-262 / pUC-SPYCE / ODHsfA1a-YFPC
5 / HsfA1a / ΔOD / XbaI-AscI / hsf1-mu-forw. hsf1-mu-rev. / AT4G17750 / 1-160+263-495 / pUC-SPYNE-152 / HsfA1aΔOD-YFPN152
6 / HsfA1a / ΔOD / XbaI-AscI / hsf1-mu-forw. hsf1-mu-rev. / AT4G17750 / 1-160+263-495 / pUC-SPYCE / HsfA1aΔOD-YFPC
7# / HsfA1b / protein coding cDNA / XbaI-AscI / HSF3XbaI HSF3AscI-rev. / AT5G16820 / 1-481 / pUC-SPYNE-152 / HsfA1b-YFPN152
8 / HsfA1b / protein coding cDNA / XbaI-AscI / HSF3XbaI HSF3AscI-rev. / AT5G16820 / 1-481 / pUC-SPYCE / HsfA1b-YFPC
9 / HsfA1b / OD / XbaI-AscI / HSF3-olim-XbaI HSF3-olim-AscI-rev. / AT5G16820 / 141-229 / pUC-SPYNE-152 / ODHsfA1b-YFPN152
10 / HsfA1b / OD / XbaI-AscI / HSF3-olim-XbaI HSF3-olim-AscI-rev. / AT5G16820 / 141-229 / pUC-SPYCE / ODHsfA1b-YFPC
11# / HsfA2 / protein coding cDNA / XbaI-SmaI / atHSFA2 forw. XbaI atHSFA2 rev. SmaI / AT2G26150 / 1-345 / pUC-SPYNE-152 / HsfA2-YFPN152
12 / HsfA2 / protein coding cDNA / XbaI-SmaI / atHSFA2 forw. XbaI atHSFA2 rev.SmaI / AT2G26150 / 1-345 / pUC-SPYCE / HsfA2-YFPC
13 / HsfA2 / OD / XbaI-SmaI / HSFA2ODForw.XbaI HSFA2ODRev. SmaI / AT2G26150 / 150-229 / pUC-SPYNE-152 / ODHsfA2-YFPN152
14 / HsfA2 / OD / XbaI-SmaI / HSFA2ODForw.XbaI HSFA2ODRev. SmaI / AT2G26150 / 150-2 / pUC-SPYCE / ODHsfA2-YFPC
15# / HsfB2b / protein coding cDNA / XbaI-SmaI / Hsf7 Forw. XbaI Hsf7 Rev. Sma / AT4G11660 / 1-377 / pUC-SPYNE-152 / HsfB2b-YFPN152

# also used as effector in promoter GFP reporter assay

Supplement Table 2: HSF constructs used in Yeast 2-hybrid interactions

Gene / Insert / Res. site / Primer / Locus in genbank / Amino acids
(aa) / Vector / Name of constructs
1 / HsfA1a / protein coding cDNA / SmaI / Hsf1 SmaI Forw.2 Hsf1 SmaI Rev.2 / AT4G17750 / 2-496 / pGADT7 / AD-HsfA1a
2 / HsfA1a / protein coding cDNA / SmaI / Hsf1 SmaI Forw.2 Hsf1 SmaI Rev.2 / AT4G17750 / 2-496 / pGBKT7 / BD-HsfA1a
3 / HsfA1b / protein coding cDNA / EcoRI / Hsf3 EcoRI Forw. Hsf3 EcoRI Rev. / AT5G16820 / 2-482 / pGADT7 / AD-HsfA1b
4 / HsfA1b / protein coding cDNA / EcoRI / Hsf3 EcoRI Forw. Hsf3 EcoRI Rev. / AT5G16820 / 2-482 / pGBKT7 / BD-HsfA1b
5 / HsfA2 / protein coding cDNA / SmaI / hsfA2 SmaI Forw. hsfA2 SmaI Rev. / AT2G26150 / 2-346 / pGADT7 / AD-HsfA2
6 / HsfA2 / protein coding cDNA / SmaI / hsfA2 SmaI Forw. hsfA2 SmaI Rev. / AT2G26150 / 2-346 / pGBKT7 / BD-HsfA2

Supplement Table 3: Promoter GFP reporter constructs used in activation assays

Gene / Insert / Insert enzyme / Primer / Locus in genbank / Length of
upstream sequence
(bp)* / Vector / Name of constructs
1 / pHSP26.5 / 5´upstream / BamHI / Hsp26.5-P Forw Hsp26.5-P Rev / AT1g52560 / 1680 / pGTkan / pHsp26.5-GFP
2 / pHSP18.1-CI / 5´upstream / SacI / AT5G59720 / 895 / pGTkan / pHsp18.1-CI GFP
3 / pHSP21 / 5´upstream / BamHI / Hsp21 Forw Hsp21 Rev / AT4G27670 / 1367 / pGTkan / pHsp25.3-GFP
4 / pHSP17.6C-CI / 5´upstream / BamHI / Hsp17.6C-CI Forw Hsp17.6C-CI Rev / AT1G53540 / 731 / pGTkan / pHsp17.6C-CI-GFP
5 / pHSP17.6CII / 5´upstream / BamHI / Hsp17.6-CII Forw Hsp17.6CII Rev / AT5G12020 / 1337 / pGTkan / pHsp17.6CII-GFP
6 / pHsfA2 / 5´upstream / BamHI / HSFA2 Forw HSFA2 Rev / AT2G26150 / 729 / pGTkan / pHsfA2-GFP

*)Sequences starting immediately upstream of the ATG of the protein coding region

Supplement Table 4: Primers used in Hsf and reporter gene constructions

Name / Forward (F) and reverse (R) primer sequences / Length / Restriction enzyme
HSF1XbaI / F 5'-CTGCTCTAGAGCATGTTTGTAAATTTCAAATACTTCTCTTTC-3' / 42 / XbaI
HSF1AscI-rev. / R 5'-TTGGCGCGCCTGTGTTCTGTTTCTGATGTGAGAAG-3' / 35 / AscI
HSF1-olim-XbaI / F 5'-CTGCTCTAGAGCATGTCTCAGGGTCAAGGTTC-3' / 32 / XbaI
HSF1-olim-AscI-rev. / R 5'-TTGGCGCGCCTATTGGCCTCGGTTACATG-3' / 29 / AscI
hsf1-mu-forw. / F 5'-CATGACAAGAAGCGGAGACTCAGAGAG-3' / 27 / -
hsf1-mu-rev. / R 5'-GTACTGTAATTGCTGAGATTGTGGATTACTAC-3' / 31 / -
Hsf1 SmaI Forw.2 / F 5'-TCCCCCGGGGGGATTTGTAAATTTCAAATACTTCTCTTTC-3' / 40 / SmaI
Hsf1 SmaI Rev.2 / R 5'-TCCCCCGGGGGGACTAGTGTTCTGTTTCTGATGTGAG-3' / 37 / SmaI
HSF3XbaI / F 5'-CTGCTCTAGAGCATGGAATCGGTTCCCG-3' / 28 / XbaI
HSF3AscI-rev. / R 5'-TTGGCGCGCCTTTTCCTCTGTGCTTCTGAG-3' / 30 / AscI
HSF3-olim-XbaI / F 5'-CTGCTCTAGAGCATGGAGGTGGGGAAGTTTG-3' / 31 / XbaI
HSF3-olim-AscI-rev. / R 5'-TTGGCGCGCCTGTTGCTTCCTGGAATTTGTCTG-3' / 33 / AscI
Hsf3 EcoRI Forw. / F 5'-CCGGAATTCCGGGAATCGGTTCCCGAATC-3' / 29 / EcoRI
Hsf3 EcoRI Rev. / R 5'-CCGGAATTCCGGTTATTTCCTCTGTGCTTCTGAG-3' / 34 / EcoRI
atHSFA2 forw.XbaI / F 5'-GCTCTAGAGCATGGAAGAACTGAAAGTGGAAA-3' / 32 / XbaI
atHSFA2 rev. SmaI / R 5'-TCCCCCGGGGGAAGGTTCCGAACCAAGAAAAC-3' / 32 / SmaI
HSFA2 OD Forw.XbaI / F 5'-GCTCTAGAGCATGTCATGTGTTGAGGTTGGG-3' / 31 / XbaI
HSFA2 OD Rev. SmaI / R 5'-TCCCCCGGGGGAACTCATAACCGCAAACTGCT-3' / 32 / SmaI
hsfA2 SmaI Forw. / F 5'-TCCCCCGGGGGGAGAAGAACTGAAAGTGGAAATGG-3' / 35 / SmaI
hsfA2 SmaI Rev. / R 5'-TCCCCCGGGGGATTAAGGTTCCGAACCAAGAA-3' / 32 / SmaI
Hsf7 Forw. XbaI / F 5'-GCTCTAGAGCATGCCGGGGGAACAA-3' / 25 / XbaI
Hsf7 Rev. SmaI / R 5'-TCCCCCGGGGGATTTTCCGAGTTCAAGCCAC-3' / 31 / SmaI
Hsp26.5-P Forw / F 5'-CGCGGATCCGCGCATCATCATGCTGTGGTTTATAT-3' / 35 / BamHI
Hsp26.5-P Rev / R 5'-CGCGGATCCGCGTGTTTTCAAATCGGTAAATTTCTTA-3' / 37 / BamHI
Hsp21 Forw / F 5'-CGCGGATCCGCGTCTCTTCTAAAAATTCAACCTTTC-3' / 36 / BamHI
Hsp21 Rev / R 5'-CGCGGATCCGCGTTGTTTCGAGTATGAGCCA-3' / 31 / BamHI
Hsp17.6C-CI Forw / F 5'-CGCGGATCCGCGATTATATACACATACACATTCAGG-3' / 36 / BamHI
Hsp17.6C-CI Rev / R 5'-CGCGGATCCGCGTTTCACTTCCTCTTGTG-3' / 29 / BamHI
Hsp17.6-CII Forw / F 5'-CGCGGATCCGCGTTTGTTTGTGATCGTGTTTT-3' / 32 / BamHI
Hsp17.6CII Rev / R 5'-CGCGGATCCGCGTGTTAGTTGTGTTGTGTTTGC-3' / 33 / BamHI
HSFA2 Forw / F 5'-CGCGGATCCGCGCAATCAAATCTCTCCTTCACG-3' / 33 / BamHI
HSFA2 Rev / R 5'-CGCGGATCCGCGTTTCGTTGTTTATCTCAAATCCA-3' / 35 / BamHI

Supplement Figures S1-S3

Figure S1: Western blot immunodetection of expression levels of recombinant HsfA1a and HsfA1b in protoplasts in the promoter activation assay (corresponding with the data depicted in Figure 3).

The protein samples were isolated from the same transformed protoplasts as used in one of the promoter activation assays (promoter-GFP constructs tested: pHsp17.6C-CI, pHsp17.6CII, pHsfA2, pHsp26.5-P(r),pHsp 18.1-CI, pHsp-25.3-P) contributing to the results shown in Fig. 3 of the manuscript. Hsf expression was detected using Myc-antibody.

Transformed effectors: 1) bZIP63; 2) HsfA1a; 3) HsfA1b; 4) HsfA1a/HsfA1b.

Figure S2: Western blot immunodetection of expression levels of HsfA1, HsfA1b and HsfA2 in transformed protoplasts in the promoter activation assay (corresponding with the data depicted in Figure 4).

The protein samples were isolated from the same transformed protoplasts as used in one of the promoter activation assays (Promoter: pHsp 18.1-CI) by early and (HsfA1a, Hsf A1b) late (HsfA2) class A Hsfs. Hsf expression was detected using Myc-antibody.

Transformed effectors 1) bZIP63; 2) HsfA1a; 3) HsfA2; 4) HsfA1a/A2; 5) HsfA1b/A2.

Figure S3: System test of promoter activation assay - activation of the pHSP25.6-P-GFP reporter by different effectors.

Arabidopsis protoplasts were transformed with the promoter pHSP25.6-P-GFP reporter plasmids in combinations different DNAs tested as effectors. Plasmids (25 µg) expressing the effectors YFPN152 (expressing only the N-terminal part of YFP), HsfA1a-YFPN152 (HSF-effector), bZIP63-YFPN152 (b-ZIP) or only carrier DNAs (DNA 1: bovine, DNA 2: herring sperm)

GFP fluorescence was quantified by flow cytometry. Data were normalized for transformation efficiency using co-transformation of luciferase (LUC) expression plasmid. HS: heat stress treatment (3 hrs, 37°C); RT: room temperature (3Hrs 25°C).

The bZIP63-YFPN152 dummy effector was used as a negative control for determining endogenous HSF-activities of protoplasts, which results in similar background levels of the GFP reporter activities as the samples treated only with carrier DNAs, with the dummy effector YFPN152, or without any effector DNA. Heat stress causes a minor increase in GFP reporter activities of theses controls, probably reflecting the promoter activation by the endogenous Hsfs. The HsfA1a-YFPN152 effector shows strongly increased promoter activation with and without heat stress.

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