Supplemental Table 1. Primers used for cloning and PCR amplification in this study
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1 Supplemental Table 1. Primers used for cloning and PCR amplification in this study Target Gene Primer sequence NATA1 (At2g393) forward GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT CAT GGC GCC TCC AAC CGC AGC ACC A GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC CTA GAT GTT TAG CTT GTC AAT AGC TTG AAG NATA2 (At2g392) forward GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT CAT GGC AGC CGC CGC ACC G GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC CTA GAT GTT GAC CTG ATC AAA AGC TTC GFP (from pmdc83) forward GGGG ACA AGT TTG TAC AAA AAA GCA GGC TAA ATG AGT AAA GGA GAA GAA CTT TT GGG GAC CAC TTT GTA CAA GAA AGC TGG GTT TAG TGG TGG TGG TGG TG NATA1 promoter region forward TAA GCT AAA GAA TTA AAG TAG TAA (for ecotype analysis) GAA GAT TAG GGT TTC GAG AAT CC
2 Supplemental Table 2. Primers used for quantitative RT-PCR Target Gene Primer sequence EF1-α (At5g639) forward TGAGCACGCTCTTCTTGCTTTCA GGTGGTGGCATCCATCTTGTTACA PR1 (At2g1461) forward TGATCCTCGTGGGAATTATGT TGCATGATCACATCATTACTTCAT PR2 (At3g5726) forward AGCTTCCTTCTTCAACCACACAGC TGGCAAGGTATCGCCTAGCATC PR3 (At3G5442) forward TCTAGCTTGAACGGTGGCTGTG CGTGCCATTAACGGTGCTTTGG PR4 (At3G472) forward TCCAAATCCAAGCCTCCGTTGC GCGGCAAGTGTTTAAGGGTGAAG PR5 (At1G754) forward AGCAATGCCGCTTGTGATGAAC ATCACCCACAGCACAGAGACAC PR6 (At2G389) forward ATACATGTCTAGCCGGCGGTTG TTGCTACCGCGTTAGGGTTTGG PAO1 (At5g137) forward GCTCTAACATTCTGGTGGTGACG CCCAAACATGTCCCTGAGAACAC PAO2 (At2g432) forward AGATTGTAGGATGCGAGTC TTAGACGATATAAGAAGAGG PAO3 (At3g595) forward ACAAACCTCACGACCTCTATG TCAAGCACACGCATCCTG PAO4 (At1g6584) forward GGGAACAGTGACATTCTCGAAAC AATTGGAACCCTGCTTCTGTCTG PAO5 (At4g2972) forward GATGACCTAGACGCAATG ATGAGTTGTGGAGTAATGG NATA1 (At2g393) forward AGCAGATGGGTGCGCAGGTT TCGCTCGATGGGTCTCATGCA SPDS1 (AT1G2382) forward GAAGAGGATAACGGCGGC CAGAGAACCACCCAGGAATAACAG SPDS2 (AT1G731) forward TGATTTGCCCGTGAAGAGACC GAGAACCATCCAGGAATAATAGAGG SPMS (AT5G5312) forward GTTGTTGGTGGAGGTGATGGTG GGATTTACGGAGGAACTCAGCAG
3 A B Supplemental Figure S1. Spatial and temporal expression of (A) NATA1 and (B) NATA2 from Botany Array Resource (
4 Arabidopsis thaliana NATA1 (gi: ) Arabidopsis lyrata subsp. lyrata (gi: ) Capsella rubella (gi: ) Arabidopsis thaliana NATA2 (gi: ) Capsella rubella (gi: ) Arabidopsis lyrata subsp. lyrata (gi: ) Brassica napus (gi:155877) Brassica rapa subsp. Pekinensis (gi: ) Solanum lycopersicum (gi: ) Solanum tuberosum (gi: ) Glycine max (gi: ) Populus trichocarpa (gi: ) Populus trichocarpa (gi: ) Zea mays (gi: ) Sorghum bicolor (gi: ) Oryza sativa Indica Group (gi: ) Oryza sativa Japonica Group (gi: ) Nicotiana benthamiana (gi:nbs1545g1.1) Solanum tuberosum (gi: ) Nicotiana tabacum (gi: ) Homo sapiens (gi:633) Supplemental Figure S2. Phylogenetic tree of plant proteins with similarity to polyamine acetyltransferases. Phylogenetic tree of NATA1 protein homologs. Human (Homo sapiens) spermidine/spermine N 1 -acetyl-transferase (SSAT) was used as an outgroup. A consensus phylogenetic tree was produced with 1 replicates. Values at the branch points indicate bootstrap percentages.
5 Mouse SSAT MAKFVIRPATAADCSDILRLIKELAKYEYMEE At2g MAPPTAAPEPNTV-PETSPTGHRMFSRIRLATPTDVPFIHKLIHQMAVFERLTH At2g392 MAAAAPPPPPTAAPEPNMVAPLISPIGHPMFSRIRLATPSDVPFIHKLIHQMAVFERLTH ** ** :* * :**:::* :* :. Mouse SSAT QVILTEKDLLEDGFGEHPFYHC LVAEVP-- At2g393 LFVATESGLASTLFNSRPFQAVTVFLLEISPSPFPTTHD-ASSPDFTPFLETHKVDLPIE At2g392 LFSATESGLASTLFTSRPFQSFTVFLLEVSRSPFPATITSSPSPDFTPFFKTHNLDLPID. **. *. *.:** ::* Mouse SSAT ---KEHWTPE---GHSIVGFAMYYFTYDPWIGK-LLYLEDFFVMSDYRGFGIGSEILKNL At2g393 DPDREKFLPDKLNDVVVAGFVLFFPNYPSFLAKQGFYIEDIFMREPYRRKGFGKLLLTAV At2g392 DPESYNFSPDMLNDVVVAGFVLFFPNYSSFLSKPGFYIEDIFVREPYRRKGFGSMLLTAV.: *: :.**.:::.* ::.* :*:**:*:. ** *:*. :*. : Mouse SSAT SQVAMRCRCSSMHFLVAEWNEPSINFYKRRGASDLSSEEGWRLFKIDKEYLLKMATEE- At2g393 AKQAVKLGVGRVEWIVIDWNVNAINFYEQMGAQVF---KEWRLCRLTGDALQAIDKLNI At2g392 AKQAVKMGYGRVEWVVLDWNVNAIKFYEQMGAQIL---QEWRVCRLTGDALEAFDQVNI :: *::. :.::* :** :*:**:: **. : : **: :: : * : : Supplemental Figure S3. Active site amino acid sequence alignment of Arabidopsis NATA1 and NATA2 with mouse spermidine/spermine acetyltransferase (SSAT; GI: ). Orange: CoA binding region, blue: spermine binding residues, and green: proposed catalytic site. Sequences were aligned with Clustal Omega (
6 A B C 1.1 kb Supplemental Figure S4. Arabidopsis ecotypes with natural nata1 knockout mutations were identified through the 11 Genomes Project ( (A) NATA1 (AT2G393) promoter regions from the Amel-1, Bur-, Cal-, Ped-, Zal-1, TDr-1 and Col- ecotypes were amplified with the indicated primers. (B) Agarose gel showing bands corresponding to the NATA1 promoter regions of the indicated Arabidopsis ecotypes. (C) Sequence alignment of Chr2: from Bur- and Col-. The NATA1 transcript is marked in grey and the ATG translation start codon is indicated with an arrow. Sequences were aligned with Clustal Omega (
7 5 kda 37 kda 25 kda 6His-NATA1, c.a. 27 kda Supplemental Figure S5. Heterologously expressed NATA1 protein was purified with Ni-NTA and visualizedd using a silver-stained protein gel. Shown is a representative gel.
8 A ornithine Ornithine (μm) Acetyl-CoA (μm) B putrescine Putrescine (μm) Acetyl-CoA (μm) C 1,3-diaminopropane ,3-Diaminopropane (μm) Acetyl-CoA (μm) Supplemental Figure S6. Representative Michaelis Menten curves. The kinetics of heterologously expressed NATA1 were determined by Michaelis-Menten curves for (A) ornithine, (B) putrescine and (C) 1,3-diaminopropane by changing concentration of the potential substrate and acetyl-coa, respectively. Shown are the means of three technical replicates.
9 OD6 ± SE Guazatine concentration umol 1 umol 1 umol 1 umol. h 1h 3h 6h 9h 12h 24h 48h time (hours) Supplemental Figure S7. Growth of P. syringae in LB medium supplemented with guazatine. Mean ± SE of N =4.
10 Total salicylic acid ng gram FW -1 ± SE WT nata1 WT nata1 WT nata1 DAY DAY 2 DAY 4 Supplementary Figure S8. Time course of total salicylic acid accumulation in response to P. syringae infection. Mean ± SE of n = 12.
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