Figure 6.

HopZ5 variants are compromised in their ability to trigger defence when overexpressed in Arabidopsis thaliana Col-0. (a) hopZ5-transgene expression was determined in hopZ5 stable expression lines in the Col-0 background. 15–20 T2 seedlings from two or three independent lines per hopZ5 variant (hopZ5-wild-type: Z-1, Z-4, Z-5; C218A: C-1, C-4; K278R: K-2, K-6; G2A: G-2, G-3) were harvested after selection for three weeks on solid media containing kanamycin. RNA was extracted and cDNA synthesized and used for quantitative polymerase chain reaction (qPCR) for the hopZ5-YFP transgene. Expression is relative to internal EF1α expression only. Error bars indicate standard error from three technical replicates. Asterisks indicate results of Student’s t-test between selected sample and Col-0 wild-type also grown on plates but without kanamycin; ***(P < 0.0001). This experiment was conducted three times with similar results. (b) Stable transgenic lines expressing wild-type HopZ5 show altered morphology. 2-week-old T2 plants as in (a) were transferred to soil after selection on kanamycin. Photographs are of representative 5-week-old plants that showed a differential phenotype only. Typically between 1-in-3 (Z-1) to 1-in-4 plants (Z-4 and Z-5) showed a stunted/early flowering phenotype, putatively representative of Mendelian segregation and individuals homozygous for the transgene. Yellow arrows indicate the inflorescence in the early-flowering plants. (c) PR1 marker gene expression was determined in hopZ5 stable expression lines as in (a). Expression is relative to internal EF1α expression and PR1 expression for wildtype. Error bars indicate standard error from three technical replicates. Asterisks indicate results of Student’s t-test between selected sample and Col-0 wild-type also grown on plates but without kanamycin; ***(P < 0.0001). This experiment was conducted three times with similar results. (d) Pto DC3000 growth in hopZ5 stable expression lines in Col-0. Pto DC3000 was blunt syringe-infiltrated at 5 × 10⁵ CFU/mL into leaves of each stable expression line from (a) and bacterial growth was determined 4 days post-infection (4 dpi). Error bars represent standard error from six technical replicates. Asterisks indicate results of Student’s t-test between selected sample and Col-0 wild-type; ns (P > 0.05), *(P < 0.05), **(P < 0.01). The experiment was conducted three times with similar results.