Figure 4.

Previous infection with avirulent Pst DC3000 avrRpt2 induces priming for enhanced PAL gene activation by virulent Pst DC3000, wounding, or infiltration of water. Three lower leaves of Arabidopsis plants were infiltrated with a cell suspension of SAR-inducing Pst DC3000 avrRpt2 (16 × 10⁴ cfu mL⁻¹) in MgCl₂ (+). Mock inoculations were performed by infiltrating three lower leaves of control plants with MgCl₂ in the absence of Pst DC3000 avrRpt2 (−). Three days later, two upper leaves of the mock-inoculated and the Pst DC3000 avrRpt2-infected plants were left untreated (−), dipped into a cell suspension of virulent Pst DC3000 (35 × 10⁶ cfu mL⁻¹) in MgCl₂/Silwet (+), slightly squeezed with forceps (+), or infiltrated with water (+). Mock challenge treatments were performed by leaving the upper leaves untreated (−; forceps and infiltration) or by dipping two of them into a solution of MgCl₂/Silwet in the absence of bacteria (−; Pst DC3000). Total RNA was extracted from the upper leaves 2 and 3 h after wounding/infiltration and bacterial inoculation, respectively, and was assayed for accumulation of PAL mRNA by RNA gel-blot analysis. Another aliquot of leaves was harvested at the 24-h time point postinoculation and was analyzed for the accumulation of PR-1 transcripts. The accumulation of PAL mRNA (2-h time point) and PR-1 transcripts (24-h time point) in plants that had been pretreated for 3 d with BTH (+) and then infiltrated on two leaves with water (+) served as a positive control. To document equal sample loading and transfer of RNA, the membrane of the PAL blot was stripped and reprobed with a ³²P-labeled Arabidopsis β-tubulin cDNA. The establishment of SAR in Pst DC3000 avrRpt2-inoculated plants was confirmed in a parallel assay in which two upper leaves of three mock-preinoculated or Pst DC3000 avrRpt2-preinfected plants were infiltrated, 3 d post-treatment, with a suspension of virulent Pst DC3000. Three days later, the mock-preinoculated plants were diseased and exhibited wet chlorotic lesions, whereas the Pst DC3000 avrRpt2-preimmunized plants remained free of visible symptoms (data not shown).