Phenotypes of avrRpt2-transgenic seedlings. (A) Seven-day-old light-grown Col-0 rps2 seedlings (Left) and Col-0 rps2-transgenic seedlings expressing pRPS2-avrRpt2 (Right). (B) Number of lateral roots per seedling of 7-day-old Col-0 rps2 seedlings (empty bar) and Col-0 rps2-transgenic seedlings expressing pRPS2-avrRpt2 (filled bar). Each bar represents the mean number of lateral roots of a minimum of 10 seedlings ± SEM. (C and D) Primary root length (C) and fresh weight (D) of 7-day-old seedling populations of indicated genotypes. 1, Col-0 rps2 parent; 2, Col-0 rps2 pRPS2-avrRpt2 parent; 3, F2 seedlings that do not carry the pRPS2-avrRpt2 transgene; 4, F2 seedlings carrying the pRPS2-avrRpt2 transgene. Each bar represents the mean primary root length of a minimum of 10 seedlings ± SEM. For data shown in A and B, similar results were obtained in experiments using seedlings from a second independent Col-0 rps2 pRPS2-avrRpt2-transgenic line, as well as in experiments using seedlings of two independent No-0 rps2 p35S-avrRpt2-transgenic lines, nontransgenic No-0 rps2 seedlings, or No-0 rps2 seedlings transformed with the pMON10098 empty vector (data not shown). For data shown in C and D, similar results were obtained in an experiment using No-0 rps2 p35S-avrRpt2-transgenic seedlings, No-0 rps2-nontransgenic seedlings, and an F2 population of a No-0 rps2 population segregating for the p35S-avrRpt2 transgene (see Materials and Methods) (data not shown). The asterisks indicate a significant difference with P < 0.05, when compared with data from control seedlings, based on the results of an unpaired one-tailed Student's t test.

AvrRpt2 promotes auxin-induced gene expression in Arabidopsis protoplasts. Col-0 or Col-0 rps2-101c protoplasts were cotransfected with constructs encoding the indicated type III effector and a GH3-LUC reporter (29) and incubated for 3 h before treatment with 1 μM NAA. Expression of GH3-LUC was assayed 3 h after NAA treatment. The activities of GH3-LUC were detected in the absence (A) and presence (B) of NAA.

The presence of AvrRpt2 is associated with enhanced increases in free IAA levels in PstDC3000-infected plants. Free IAA levels in rosette leaves of 3- to 4-week-old No-0 rps2 p35S-avrRpt2 plants (filled bars) or No-0 rps2 plants transformed with the pMON10098 empty vector (empty bars) 2 days after mock inoculation with 10 mM MgCl₂ containing 0.02% L-77 (M, bars 1 and 4), inoculation with PstDC3000 (DC, bars 2 and 5), or PstDC3000(avrRpt2) [DC(a), bar 3] at a concentration of ≈5 × 10⁸ cfu/ml in 10 mM MgCl₂ containing 0.02% Silwet L-77. Each bar represents the mean of three replicates ± SEM. a, Significant difference when compared with the values obtained for uninoculated or mock-inoculated samples. b, Significant difference from the value obtained for No-0 rps2 pMON10098 plants inoculated with DC3000 (based on the results of an unpaired one-tailed Student's t test; P < 0.05). Similar results were obtained in a second independent experiment.

The avrRpt2-transgenic seedlings exhibit increased sensitivity to the auxin analog 2,4-D. Seeds of Col-0 rps2 plants (open squares) and Col-0 rps2-transgenic plants expressing pRPS2-avrRpt2 (filled squares) were germinated and grown on 1/2 × MS plates containing the indicated concentrations of the auxin analog 2,4-D for 7 days and then measured for root length. (A) Length of primary roots of seedlings on plates containing the indicated concentration of 2,4-D. Data points represent the average root length of a minimum of 10 seedlings ± SEM. (B) Data from A expressed as relative root length, normalizing the average length of seedlings grown on 2,4-D to average length of seedlings grown in the absence of 2,4-D. Similar results were obtained in a second independent experiment when testing a second, independent Col-0 rps2 pRPS2-avrRpt2-transgenic line, as well as in experiments using No-0 rps2 p35S-avrRpt2 and control seedlings (data not shown).

The avrRpt2-transgenic seedlings contain higher levels of free IAA. Free IAA levels were measured in shoot tissue of 7-day-old transgenic seedlings expressing avrRpt2 (filled bars) and nontransgenic (empty bars) seedlings. Results from experiments with Col-0 rps2 and Col-0 rps2-transgenic seedlings expressing pRPS2-avrRpt2 and No-0 rps2 and No-0 rps2-transgenic seedlings expressing p35S-avrRpt2 are shown. (A) Free IAA levels were normalized by fresh weight of the seedlings. (B) Data shown in A, with free IAA levels normalized for the number of seedlings assayed. Each bar represents the mean of three independent measurements ± SEM. The asterisks indicate a significant difference with P < 0.05, when compared with control tissue, based on the results of an unpaired one-tailed Student's t test. Similar results were obtained in two independent experiments, as well as in free IAA measurements of the root tissue of these seedlings (data not shown).

Plants treated with auxin analogs exhibit altered susceptibility to PstDC3000 infection. Four- to five-week-old wild-type Col-0 plants were dip-inoculated in PstDC3000 at a concentration of ≈5 × 10⁸ cfu/ml in 10 mM MgCl₂ containing 0.02% Silwet L-77. The plant on the Right was simultaneously treated with 5 × 10⁻⁵ M NAA at the time of infection. The photo was taken 4 days after inoculation. Similar results were observed in more than three independent experiments when plants were treated with 5 × 10⁻⁶ M 2,4-D (data not shown) and in experiments using No-0 plants (data not shown).