Figure 3. Transgenic N. attenuata plants showed in planta antimicrobial activity against Bacillus pumilus DSM 1794, but not against Pseudomonas syringae pv tomato DC3000. .

(A) Bacteria were pressure infiltrated into fully expanded rosette-stage leaves and re-isolated at 0, 2, 4 and 6 d post infiltration (dpi). The pictures show two technical replicates per genotype from a dilution series. (B) The mean colony forming units (CFU) were plotted as log CFU cm⁻² leaf area (±SD, n = 4 plants). Asterisks indicate statistically significant differences between WT and the transgenic plants (students t-test; **p≤0.01; ***p≤0.001). (C) Summary of the CFU fold reduction (non-log scale) compared to WT averaged among genotypes.

Figure 3—figure supplement 1. Leaf infiltration method for the determination of in planta antimicrobial activity against Bacillus pumilus DSM 1794.

(A) Illustration of the pressure infiltration procedure. About 300–400 µL bacterial solution were injected into fully expanded leaves on both sides of the midrib. The infiltrated area was marked with a pen and bacteria re-isolated using a cork borer. Leaf disks were squeezed with a pestle and serial dilutions of 10⁻¹ to 10⁻³ were spotted on LB media for CFU counting. (B) The lines ICE 1.1.1, ICE 6.4.2 and ICE 8.4.1 showed antimicrobial activity from 3 to 9 d post infiltration (dpi). ICE 1.5.2 plants showed a lack of antibacterial activity (red arrow). The mean colony forming units (CFU) were plotted as log CFU cm⁻² leaf area (±SD, n = 4; ICE 1 and ICE 8 n = 2). Asterisks indicate statistically significant differences between WT and transgenic plants (t-test;*, p≤0.05; **p≤0.01; ***p≤0.001). (C) Serial dilutions of re-isolated B. pumilus infiltrated into the first homozygous generation (T₃) of the single insertion ICE lines. (D) A diagnostic PCR as described in Gase et al. (2011) revealed an incomplete expression cassette for line ICE 1.5.2 (red arrow) which explains the lack in activity.