Fig. 4.

ALD1 accumulation at the site of infection fully restores defense responses in local leaves. (A) Treatment schemes in local leaves in (B–E). Local leaves (the third to fifth leaves) were painted with DEX (30 μM) or mock treated for 1 d, and then inoculated with PmaDG6. The primary (1°) local leaves were then collected at the indicated times for further analysis. (B) Titer of PmaDG6 in local leaves of the WT, ald1-T2 (ald1), and DEX- or mock-treated transgenic pDEX::ALD1 lines #6 and #10. Colony-forming unit (CFU) number was measured in local leaves on day 3 after infection with PmaDG6 (OD₆₀₀=0.0001). Error bars indicate the SEM of eight biological replicates. The experiment was repeated three times with similar results. Another experiment that employed DEX spraying also showed similar results. (C) PR1 gene expression level in DEX- (30 μM) painted local leaves at 0 h (no treatment, NT) and 9 h after PmaDG6 (DG6, OD₆₀₀=0.01) infection in the indicated genotypes: wild type (WT), ald1-T2 (ald1), and pDEX::ALD1#6 (#6). Error bars indicate the SEM from at least two biological replicates and three technical replicates. Each biological replicate consists of 6–9 leaves from at least three plants. The experiment was repeated twice with similar results. (D) Endogenous salicylic acid (SA) levels in local leaves were measured by HPLC in the indicated genotypes. DEX- (30 μM) or mock-painted local leaves were collected at 0 h (no treatment, NT) or 9 h after PmaDG6 (DG6, OD₆₀₀=0.01) infection. Free SA is shown in the left panel, and total SA is shown in the right panel. Error bars indicate the SEM of at least three biological replicates. Each biological replicate consists of 6–9 leaves from at least three plants. (E) Defense-related metabolite levels measured by GC-MS in local leaves of the indicated genotypes after 48 h infection. Error bars indicate the SEM from four biological replicates. (F) Pip and NHP levels in petiole exudates are not rescued in pDEX::ALD1 plants. Plants at ~4 weeks old of the WT, ald1, and pDEX::ALD1#6 (#6) were sprayed with 30 μM DEX for 1 d before infection. Petiole exudates were collected during 12–72 h post-local inoculation of the SAR-inducing PmaDG6 strain (OD₆₀₀=0.01). Metabolite levels measured by GC-MS. Results are the average with the SE from six biological replicates. Each biological replicate contains 12 leaves in 1.4 ml of 1 mM Na₂-EDTA (pH 8.0) solution. Different letters indicate statistically significant differences (P<0.05, ANOVA, Fisher’s LSD test). ND, not detected; hpi, hours post-infection.