SUPPLEMENTAL DATA. Supplementary data

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1 SUPPLEMENTAL DATA Supplementary data Figure S1: Verification of pla-iα1 knockdown lines a: Semiquantitative RT-PCR analysis of PLA-Iα1 transcript expression in different transgenic lines and wild type control (WT) using total RNA isolated from leaves. b: Semiquantitative RT-PCR analysis of PLA-Iα1 (Iα1), PLA-Iα2 (Iα2), PLA-Iβ2 (Iβ2), PLA-Iγ1 (Iγ1), PLA-Iγ2 (Iγ2) and PLA-Iγ3 (Iγ3) in line pla-iα1 8-1 (8-1) and wild type (+) using total RNA isolated from leaves. Expression of the house-keeping gene RPL2 (ribosomal protein large subunit 2) was used as quantitative control. Figure S2: Gene silencing phenotype of pla-ia1-lines after treatment with Pseudomonas DC3000(avrRPM1) Infiltration symptoms on wild type leaves and those from transgenic lines pla-ia1 7-1 and 8-1 up to 3 days after infiltration with Pseudomonas DC3000(avrRPM1) strain. Leaves were infiltrated with a 1x10 6 cfu/ml bacterial suspension. Figure S3: Bacterial growth of Pseudomonas DC3000(avrRPM1) Comparison of bacterial growth of Pseudomonas DC3000(avrRPM1 on wild type plants, pla-iα1 7-1 and pla-iα1 8-1 plants three days post inoculation (dpi). Plants were inoculated with 1x10 6 cfu/ml suspension of bacteria. Figure S4: Verification of quadruple mutant pla-iβ2xpla-iγ1xpla-iγ2xpla-iγ3 line c1x11 a: Verification of homozygous T-DNA insertion using PCR with gene specific and with T-DNA specific primers. b: Transcription levels of the genes PLA-Iβ2, PLA-Iγ1, PLA-Iγ2 and PLA-Iγ3 in quadruple mutant line c1x11 and wild type. Expression of the house-keeping gene RPL2 (ribosomal protein large subunit 2) was used as quantitative control. The quadruple mutant was gained out of serial crosses from the single mutants. Therefore a and b are also a proof for the single mutants. Figure S5: Colocalization studies of egfp fusion proteins in Arabidopsis protoplast. The first column on the left shows the GFP channel and full length PLA-Iα1:eGFP fusion protein (indicated in green) followed by Nile Red-stained lipid bodies (indicated

2 in red) and the protoplast in bright field. The column on the right side shows an overlay of all previously described channels. Supplementary experimental methods Gene expression studies using semiquantitative RT-PCR Total RNA was extracted from leaves using the Trizol reagent (Invitrogen) And RT- PCR was performed with 3 μg of RNA using a cdna synthesis kit of Fermentas..Expression levels of RPL2 (At2g07715) were monitored to serve as a quantifying control. After PCR cycles of amplification, aliquots of each PCR product were resolved by agarose gel electrophoresis, and were visualized with ethidium bromide under UV light. See Table S2 in the supplementary part for full information about the primers. Bacteria growth assay The leaves of six-week-old plants were inoculated with 1x10 6 cfu/ml bacteria suspension. Bacteria growth assay was conducted as described in Katagiri et al., 2002.

3 Tab. S1: Primers used for verification of T-DNA insertion mutants and pla-iα1 knockdown line Primer Sequence Primers used for verification of the RNAi construct ( Hilson et al., 2004) Agri51 5'-CAACCACGTCTTCAAAGCAA-3' Agri56 5'-CTGGGGTACCGAATTCCTC-3' Agri64 5'-CTTGCGCTGCAGTTATCATC-3' Agri69 5'-AGGCGTCTCGCATATCTCAT-3' Primers used for verification of the T-DNA insertion Spm32sense 5'-TACGAATAAGAGCGTCCATTTTAGAGTGA-3' LB1b prok2anti 5'-GCGTGGACCGCTTGCTGCAACT-3' PLA-Iβ2anti 5'-TGATTTCGCCGACCAAAGACTCG-3' LIP3sense 5'-CTT TTGAACTTCTCAACAATG-3' LIP4anti 5 -CAAACGTGATCCACACATTAATTACGAAA-3 PLA-Iγ1sense 5'-AGGCTTCAATATCTACAAGGCT-3' PLA-Iγ1anti 5'-CTCAATCCTCTCCTTGAATCG-3' SAIL_646_E09sense 5'-GAGGGTGGAGAGAATTTGAGG-3' SAIL_646_E09anti 5'-CGGTATAGATCCAGTCGATGG-3' SAIL_897_D11sense 5'-CTCAATGTCAATCTCATGGGG-3' SAIL_897_D11anti 5'-ACATGAATTACAAGAACAGGACAC-3' SALK_014323sense 5'-CTACTCTACCCTCCTCCACCG-3' SALK_014323anti 5'-TACGTGCTGAGGTTAGCTTCC-3' SALK_139011Csense 5'-TTTAGCAGGATCTTCTGCTGG-3' SALK_139011Canti 5'-TTGCTCTGTTTAAAGGGTTGC-3' SALK_139280sense 5'-ATGCATTGTTCGACCTCAGAC-3' SALK_139280anti 5'-TACTGAATGGAATATTCCGCG-3' SAIL_716_F08sense 5'-AATAAATACACCATCATATGAAGATTTG-3' SAIL_716_F08anti 5'-CGGAGAATCCTAATTGTGTGC-3' SALK_079693sense 5'-CTTTGGTACCACCATGAATGG-3' SALK_079693anti 5'-TTGTCACACAAGAAGCATTGC-3' SALK csense 5'-ATATGGGGTTACCAAAATCCG-3' SALK canti 5'-ATTCGATTCGCGTAATTTGTG-3' SALK csense 5'-CTCTTGGTCGTGAATAGCGAG-3' SALK canti 5'-GATGTTTGAGCTCAATTTCGC-3' SALK_076354csense 5'-GCGACTAGTGAATTAACTGCG-3' SALK_076354canti 5'-TTTCCCATTCACTTCATTTGC-3' SALK_147687csense 5'-TTATTACCGGAGCGACAACAC-3' SALK_147687canti 5'-TCCAATAACGGTTAAGCAACG-3' SALK_088821csense 5'-CGGATAATCTCCTTCCTCCAC-3' SALK_088821canti 5'-ACAAAGCCCATTCCTTGTACC-3'

4 Tab. S2: Primers used for gene expression analyses Primer Sequence Primers used in semiquantitative gene expression studies PLA-Iα1RTsense 5'-CCAAAGTCTTCACTCAGAAC-3' PLA-Iα1RTanti 5'-CGCTATGTCGTAAGCTAGAA-3' PLA-Iα2RTsense 5'-CCGGAGGATTGGCAAGGGACA-3' PLA-Iα2RTanti 5'-CACTGTCCTCATCTTCATCGG-3' PLA-Iβ2RTsense 5'-TTACGCCTAACGCCGACATATTCC-3' PLA-Iβ2RTanti 5'-CATTTAGGTAAACGAACGGATGACG-3' PLA-Iγ1RTsense 5'-AGGCTTCAATATCTACAAGGCT-3' PLA-Iγ1RTanti 5'-CTCAATCCTCTCCTTGAATCG-3' PLA-Iγ2 RTsense 5'-CTTTTGAACTTCTCAACAATG-3' PLA-Iγ2 RTanti 5'-CTGAGAATCTCGCGAATTTGC-3' PLA-Iγ3 RTsense 5'-GAGGTTCAAGGACGATGTGACGAAC-3' PLA-Iγ3 RTanti 5'-CAAACGTGATCCACACATTAATTACGAAA-3' PR1aRTsense 5'-CACAAGATTATCTAAGGGTTC-3' PR1aRTanti 5'-CTTCATTAGTATGGCTTCTCG-3' RPL2sense 5'-GTGGTGCTCCTCTTGCTCGT-3 RPL2anti 5'-GGAGGTGCATCACGCCTAAC-3' Primers used in quantitative Real Time analyses Actin2/8sense 5'-GGTGATGGTGTGTCT-3' Actin2/8anti 5'-ACTGAGCACAATGTTAC-3' PLA-Iα1Qsense 5'-TTACGACATAGCGGAAC-3' PLA-Iα1Qanti 5'-GTAGATATGCCGCATT-3'

5 Tab. S3: Primers used for cloning and verification egfp fusion constructs Primer Sequence Restriction site PLAIα1:eGFPsense 5'-GGATCCATGGCGGCCAAAGTCTTCACTC-3' BamH1 PLAIα1:eGFPanti 5'-GAATTCAGCTTGGATCAATCTAGACCTA-3' EcoR1 PLAIα1Δ373anti 5'-ATGGTCGACTCCTAGCTCCACACACCG-3' Sal1 PLAIα1Δ206anti 5'-ATGGTCGACAGTGCCACGAAACGTCAC-3' Sal1 TPΔPLAIα1anti 5'-ATGGTCGACCCCTTGTATCTCTCTCCAA-3' Sal1 rbcssense 5'-CTAAGGTACCATGGCTTCTATGATATCC-3' Kpn1 rbcsanti 5'-AGAGTAAAGTCCATGGCCGGATCCAATC-3' BamH1 cpsrp43:gfpsense 5'-CGGGGATCCATGGCTTCTATGATATCC-3' cpsrp43:gfpanti 5'-CAACAACCAATGAATGAACTGCAGGTC-3' Pst1 PLA-Iβ2:GFPsense 5'-GGTACCATGCAGACACTTACGCC-3' Kpn1 BamH1 PLA-Iβ2:GFPanti 5'-CTGCAGTGAAGACGACGGACTAG-3' Pst1 PFF19anti 5'-GCCCTCGAACTTCACCTC-3'

6 Figure S1 a PLA-Iα1 RPL2 b WT WT RPL2 Iα1 Iα2 Iβ2 Iγ1 I γ2 Iγ Figure S1: Verification of pla-iα1 knockdown lines a: Semiquantitative RT-PCR analysis of PLA-Iα1 transcript expression in different transgenic lines and wild type control (WT) using total RNA isolated from leaves. b: Semiquantitative RT-PCR analysis of PLA-Iα1 (Iα1), PLA-Iα2 (Iα2), PLA-Iβ2 (Iβ2), PLA-Iγ1 (Iγ1), PLA-Iγ2 (Iγ2) and PLA-Iγ3 (Iγ3) in line pla-iα1 8-1 (8-1) and wild type (+) using total RNA isolated from leaves. Expression of the house-keeping gene RPL2 (ribosomal protein large subunit 2) was used as quantitative control.

7 Figure S2 0 dpi 1 dpi pla-la1 7-1 pla-la dpi pla-la1 7-1 pla-la dpi pla-la1 7-1 pla-la1 8-1 Figure S2: Gene silencing phenotype of pla-ia1-lines after treatment with Pseudomonas DC3000(avrRPM1) Infiltration symptoms on wild type leaves and those from transgenic lines pla-ia1 7-1 and 8-1 up to 3 days after infiltration with Pseudomonas DC3000(avrRPM1) strain. Leaves were infiltrated with a 1x10 6 cfu/ml bacterial suspension.

8 Figure S3 Figure S3: Bacterial growth of Pseudomonas DC3000(avrRPM1) Comparison of bacterial growth of Pseudomonas DC3000(avrRPM1 on wild type plants, pla-iα1 7-1 and pla-iα1 8-1 plants three days post inoculation (dpi). Plants were inoculated with 1x10 6 cfu/ml suspension of bacteria.

9 Figure S4 a T-DNA WT gene T-DNA WT gene T-DNA WT gene PLA-Iβ2 PLA-Iγ1 PLA-Iγ2 PLA-Iγ3 C1X11 heterozygous line b RPL2 PLA-Iβ2 PLA-Iγ2 PLA-Iγ3 PLA-Iγ1 c1x11 c1x11 c1x11 c1x11 c1x11 Figure S4: Verification of quadruple mutant pla-iβ2xpla-iγ1xpla-iγ2xpla-iγ3 line c1x11 a: Verification of homozygous T-DNA insertion using PCR with gene specific and with T-DNA specific primers. b: Transcription levels of the genes PLA-Iβ2, PLA-Iγ1, PLA-Iγ2 and PLA-Iγ3 in quadruple mutant line c1x11 and wild type. Expression of the house-keeping gene RPL2 (ribosomal protein large subunit 2) was used as quantitative control. The quadruple mutant was gained out of serial crosses from the single mutants. Therefore a and b are also a proof for the single mutants.

10 Figure S5 egfp Nile Red bright field overlay i Figure S5: Colocalization studies of egfp fusion proteins in Arabidopsis protoplast. The first column on the left shows the GFP channel and full length PLA-Ia1:eGFP fusion protein (indicated in green) followed by Nile Red-stained lipid bodies (indicated in red) and the protoplast in bright field. The column on the right side shows an overlay of all previously described channels.

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