Arabidopsis actin depolymerizing factor AtADF4 mediates defense signal transduction triggered by the Pseudomonas syringae effector AvrPphB

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1 Arabidopsis actin depolymerizing factor mediates defense signal transduction triggered by the Pseudomonas syringae effector AvrPphB Files in this Data Supplement: Supplemental Table S1 Supplemental Table S2 Supplemental Figure S1 Supplemental Figure S2 Supplemental Figure S3

2 Supplemental Table S1. Characterization of null mutants from collected T- DNA insertion lines Gene name Locus T-DNA insertion lines Null mutant? Primers and size of PCR product At3g46010 Salk_ Yes F: 5 -gctctgatattagtgtttctcta-3 R: 5 -gatctttcaaggggatgatgata-3 492bp At3g46000 Salk_ No F: 5 -tcggtttgatttagccatggca-3 Salk_ No R: 5 -cttattcaaatgcccttacagg-3 Salk_ No 466bp At5g59880 Salk_ Yes F: 5 -gactccattattctcctgcttc-3 R: 5 -gcatcactgagtaatagtacga-3 507bp At5g59890 Salk_ No F: 5 -tgacaccgttttgattcagcc-3 Salk_ No R: 5 -cgaatataatcggaagcttattc-3 Torrey Mesa insertion Yes 478bp line#garlic_823_a11.b.1 b.lb3fa AtADF5 At2g16700 Salk_ No F: 5 -tcaaactttgtaaccctttagc-3 Salk_ No R: 5 -gacaaagtgagtcaaatcatattctc-3 Salk_ No 499bp Salk_ No Salk_ No AtADF9 At4g34970 Salk_ Yes F: 5 -atgacggatgattgcaagaaa-3 R: 5 -tcatttggcccggtcctggat-3 395bp Supplemental Table S2. Primer sequences of AtADFs, PR1 and PDF1.2 used for quantitative RT-PCR PR1 PDF1.2 S: 5 -tgagtgatggtactggtacttga-3 ; A: 5 -caagaccgaaacaccgatagaatg-3 S: 5 -tctggagtgaatatgtttcctctg-3 ; A: 5 -acatacaataataccaagtagaatg-3 S: 5 -tcggttgaatcaaactttttcgt-3 ; A: 5 -ggtaccgtcacagcaaactttagg-3 S: 5 -gatacacttgacacccttcattctatct-3 ; A: 5 -aggaagcaaacacagcacaaaccttgtg-3 S: 5 -gatgtgatcatgcatacacacgtaca-3 ; A: 5 -atcctgcatatgatgctccttattga-3 S: 5 -ccagcacacaagtgtatctgttacgt-3 ; A: 5 -gttactcatagagtgacagagacttat-3

3 Supplemental Figure S1. RT-PCR analysis of gene expression of AtADF genes in wild type Col-0 and Atadf mutant plants. Amplication of Arabidopsis β-tubublin gene was used as a control to determine the relative expression of AtADF genes. Supplemental Figure S2. Molecular characterization of transgenic lines of Atadf4 complemented with genomic DNA with T7 tag sequence at the C-terminus. Genomic DNA was extracted from Col-0, Atadf4, and two complementing lines Atadf4(g) #1 and #2. The upper panel shows the PCR product with primers F and R (Table S1). As expected, the product can only be amplified from wild type Col-0, but not from Atadf4 mutant and two transgenic lines due to T-DNA insertion in the endogenous genomic DNA. The lower panel shows the PCR product with primers F (Table S1) and StopT7-R (5 - ttaacccatttgttgaccacctg), which were designed to amplify the transgene. As desired, the band was successfully amplified from two complementing lines, but not from Col-0 and Atadf4 plants.

4 Supplemental Figure S3. Oligonucleotides and the procedure to make a RNAi construct targeting through. Inverted-repeat PCR (IR-PCR) was used to construct a single stem-loop transgene. Synthetic oligos each encoding 90 nucleotides of, 2, 3, and 4 unique 3 UTR sequence and 10 nucleotides of overlapping sequence of the neighboring AtADF oligo were added to two steps of 8 cycle PCR reactions to allow a minimal overlapping product to form containing all four ADF target sequences (steps 1 and 2). This product was then used as the template in two separate PCR reactions using primer pairs (S1 and A1, and S2 and A2) that created the sense and antisense oriented products (step 3). These two products, termed 1234 and were purified and added to a new PCR reaction also containing a linker sequence from the β-glucoronidase coding sequence. Together these three products were allowed to form a minimally overlapping product in an 8 cycle PCR reaction (step 4) and then used as the template in a PCR reaction containing terminal primers (Clamp-S and Clamp-A) to get a clean product (step 5). This final product was cloned into pbluescript and then sub-cloned into the pbin19 binary vector for transformation into Agrobacterium and eventually Col-0 plants.

5 A1 primer Tga tag tga tag tga tag tga gga tcc tct aac agt cac aag aga ctg taa A2 primer Agc gtt agc gtt agc gtt agc gag ctc tct aac agt cac aag aga ctg taa S1 primer Ttt ctt gcc gtt ttc gtc gga gag taa aaa cca aag ttt att at S2 primer Act gga aaa aga act tct gga gag taa aaa cca aag ttt att at Clamp-S Agc gtt agc gtt agc gtt agc gag ctc Clamp-A Tga tag tga tag tga tag tga gga tcc oligo Aga gta aaa acc aaa gtt tat tat cat cat ccc ctt gaa aga tct att ctc tta ttc tgt tat ttt tga gtg atg gta ctg gta ctt gat oligo Ggt gtt aaa aag ttt tca taa cag tca gag gaa aca tat tca ctc cag aaa cca aac tta ttc aaa tgc cct tac agg gtt ttg ttt tac atc aag tac c oligo Ttt taa cac ctg aaa gaa atc ccc tga aaa ttt cgg ttg aat caa act ttt tcg gac tat tac ctc agt gat gct ttt tcc taa agt ttc gtg acg gta c oligo Tct aac agt cac aag aga ctg taa gag aat aga aaa cac cat ttc ata ata gtc gaa tat aat cgg aag ctt att caa aga gag ttt ctt gta ccg tca c S1 3. A1 S2 A2 4. GUS sequence 5. CLAMP-S GUS sequence CLAMP-A 5 3