Figure 2.

Coinfiltration of the CaM antagonist W7 impairs NO generation in wild-type leaves inoculated with avirulent pathogen Pst avrRpt2⁺. A, NO generation was detected as fluorescence (green signal) within cells of wild-type leaves inoculated with mock inoculum, Pst avrRpt2⁺, or Pst avrRpt2⁺ and W7. Three leaves were evaluated per treatment; representative images are shown. B, Quantification of NO signal intensity in response to the treatments shown in A. Fluorescence of the entire area of each leaf captured in the camera field of view was quantified by measuring relative brightness within the defined range of 256 shades of gray per pixel (unit area). Results are presented as mean fluorescence intensity per pixel ± se (n =3). It should be noted that the NO binding fluorophore of DAF-2DA is generated only within cells upon cleavage of the ester linkage; hence, the dye responds only to intracellular NO. As the signal intensity of the entire leaf area within the field of view is quantified with the ImageJ software, the quantified signal per unit leaf area is an underestimation of the actual signal intensity within cells. Nonetheless, this analysis allowed for the quantification of treatment effects on NO generation. This experiment was repeated twice; results similar to those shown here were obtained in both experiments.