Figure S1. Amino acid alignment of PsAvh163, HaRxL96 and PITG_10341.1 in which functional domains are highlighted. Signal peptide (SP), RXLR and EER host targeting motifs (HTS), and W, Y, L motifs.

Figure S2. Amino acid alignment of PsAvh163 alleles in which functional domains are highlighted. Signal peptide (SP), RXLR and EER host targeting motifs (HTS), and W, Y, L motifs.

Figure S3. Schematic of the strategy to test the function of predicted host targeting sequences from Ha96 and Ps163. (a) Chimeric genes were produced by fusing the host targeting sequence (HTS, Yellow) from the target effector between the soybean PR-1a signal peptide (SP, Black) and the Avr1B C-terminus (Purple). (b) The chimeric protein is produced in soybean cells and directed for secretion by the SP which is cleaved in the endoplasmic reticulum. If the HTS is functional, then the protein will translocate from the apoplast back to the cytosol where it is recognized by RPS1b. This recognition will trigger the HR and block GUS expression. (c) Schematic depicting the double barrel gene gun. Tungsten particles with the chimeric gene (Chi) and GUS were co-bombarded along with a control shot of empty vector (EV) and GUS. A functional HTS will deliver the secreted, chimeric protein back into the cell triggering HR in Rps1b soybean, reducing the amount of cells expressing GUS relative to the control bombardment. Percent living cells were calculated relative to co-bombarded controls.

Figure S4. HaRxL96 suppresses cell death in N.  benthamiana. (a) A.  tumefaciens GV3101 containing HaRxL96 was infiltrated in N.  benthamiana. Two days later the same sites were challenged with INF1 or PsAvh163 and cell death was monitored visually over a period of 7 days. (b) Quantification of suppression of INF1-induced cell death by HaRxL96. Bars depict the percentage of sites with macroscopic cell death. Error bars represent SE from three independent biological replicates. *P<0.05, t-test compared to INF1 control.

Figure S5. Quantification of transgene transcript levels in independently transformed lines containing 35S:HaRxL96 and 35S:PsAvh163, using quantitative PCR. Transcript accumulation is expressed as a fold change relative to AtActin. Error bars represent SE from technical replicates.

Figure S6. Multiple independent Col lines expressing HaRxL96 and PsAvh163 display enhanced susceptibility to virulent Hpa Emco5. Hpa growth was quantified in seedlings infected with virulent Hpa Emco5 at 10 days after germination. Genomic DNA was extracted from seedlings collected at 6 days post inoculation. qPCR was used to measure the relative abundance of HpaActin relative to AtActin2, as a proxy for pathogen biomass. Error bars represent SE. *< 0.05; t-test comparisons with Col-0. This experiment is representative of three independent biological replicates.

Figure S7. HaRxL96 and PsAvh163 suppress defense gene induction in response to avirulent Hpa Emoy2. Two additional replicates (a,b) of the experiment described in Figure 8 are shown. *ddCt values are statistically significant (< 0.05) relative to Col-0 samples.

Figure S8. HaRxL96 and PsAvh163 do not affect accumulation of salicylic acid. Ten to twelve day-old Col:HaRxL96 or Col:PsAvh163 were challenged with 5 × 104 spores mL−1 Emoy2, tissue was collected at 0, 12, 24, 48, and 96 h post inoculation, and SA was quantified. Fold change is in reference to 0 HPI. Error bars represent SE from three independent biological replicates, each with leaves from six plants. There are no statistically significant changes (< 0.05).

Table S1. Table summarizing screens with HaRxL96 and PsAvh163 for a visible hypersensitive response in Arabidopsis ecotypes, when delivered from P.  syringae via the effector detector vector.

Table S2. Primers used in this study.

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