Figure 5

Deletion of the mimp in the SIX1 promoter does not impair SIX1 expression, but a small region with a conserved motif is required for SIX1 expression during plant infection. (A) Schematic representation of the SIX1 locus. Black lines: deleted promoter fragments (deletion length in bp); pink box: mimp; yellow arrow: SIX gene; orange circles: sequence matching AAGTCGGCAGTT[AG] motif enriched in SIX1-7 promoters. (B) In vitro expression of SIX1 in the promoter deletion strains. Mycelium of the indicated Fol strains was collected after growth in minimal medium, From the collected mycelium RNA was extracted and RT-PCR was performed to detect transcripts of SIX1 and, as control, the constitutively expressed FEM gene. (C) In planta expression of SIX1 in the promoter deletion strains. Ten days old susceptible (without resistance genes) or resistant (encoding the I-3 resistance protein that recognizes Six1) tomato seedlings were inoculated with the indicated Fol strains or with water (mock). Roots were harvested 9 dpi (days post inoculation, RNA was extracted and RT-PCR was performed as described above. (D) Disease assay of tomato plants. Ten days old seedlings of susceptible or resistant tomato seedlings (as above) were inoculated with the indicated strains or with water. Three weeks after inoculation disease was scored by determining the plant weight above the cotyledons and by phenotypic scoring according to a disease index ranging from zero (no disease) to four (heavily diseased or dead plants). (1) mock, (2) WT, (3) SIX1p1189#1, (4) SIX1p1189#2, (5) SIX1p1230#1, (6) SIX1p1230#2. Please note that, SIX1p1189#1 was not included in the bioassay with the resistant cultivar, because it is not pathogenic (see D). A black box marks interactions where recognition of Six1 by I-3 is broken or where no disease is caused. Error bars indicate the 95% confidence interval of the mean.