Figure 1.
Effect of exogenous and endogenous abscisic acid (ABA) on bacterial leaf blight (BLB) development in rice.
(A). Susceptible IRBB3 and resistant IRBB13 plants were pretreated with ABA (100 µM) and/or the ABA inhibitor fluridone (flu; 0.4 µM) for 3 and 6 days, respectively. Fifth and sixth stage leaves were inoculated with Xanthomonas oryzae pv. oryzae (Xoo) strain PXO99 using the standard leaf-clipping method. Fourteen days post inoculation (dpi), disease was evaluated by measuring the length of the water-soaked BLB lesions. Data are means ± SE of at least 10 plants. Different letters indicate statistically significant differences (Mann-Whitney: n ≥20; α = 0.05). (B). Effect of ABA (100 µM and fluridone (0.4 µM) on PXO99 titers in susceptible IRBB3 and resistant IRBB13. Data are means ± SE of three biological replicates. Asterisks indicate statistically significant differences compared to control treatments (LSD; n = 3; α = 0.05). (C). Symptom development on Ctrl, ABA or fluridone-pretreated IRBB3 leaves at 14 dpi. (D). Effect of fluridone (0.4 µM) on BLB development in OsMPK5 RNAi and WT Nipponbare plants. Data are means ± SE of at least 10 plants. Different letters indicate statistically significant differences (Mann-Whitney: n ≥20; α = 0.05). All experiments were repeated at least twice with similar results.
Figure 2.
Dynamics of ABA pathway in response to virulent Xanthomonas oryzae pv. oryzae (Xoo) infection.
(A) through (D). Effect of ABA pretreatment on ABA-biosynthesis (OsNCED3, OsNCED4) and ABA-responsive genes (OsLip9 and OsRab16) in IRBB3 leaves inoculated with Xoo strain PXO99. For details on ABA pretreatment and Xoo inoculation, see legend to Figure 1. Transcript levels were normalized using eukaryotic elongation factor eEF1α as an internal reference and, for each treatment, expressed relative to the normalized expression levels in mock-inoculated control plants at the appropriate time point. Data are means ± SD of two technical and two biological replicates from a representative experiment, each biological replicate representing a pooled sample from 3 individual plants. Two sets of independent experiments were carried out with similar results. Asterisks indicate statistically significant differences per treatment compared to either control (0 dpi) or mock-treated samples (1, 2, 4 and 8 dpi).
Figure 3.
Cross-talk experiments demonstrating mutual antagonism between ABA and SA.
IRBB3 leaf segments were incubated for 8 h in aequous solutions containing 50 µM ABA and/or 500 µM SA and subsequently tested for expression of the ABA responsive gene OsLip9 and SA marker genes OsNPR1 and OsWRKY45. Transcript levels were normalized using eukaryotic elongation factor eEF1α as an internal reference and for each treatment expressed relative to the normalized expression levels in non-treated control plants. Data are means ± SD of two technical and two biological replicates from a representative experiment, each biological replicate representing a pooled sample from 13 individual plants. The experiment was repeated once with similar results. Asterisks indicate statistically significant differences compared to control, non-treated samples.
Figure 4.
ABA counteracts SA-mediated defenses to Xoo.
(A) through (C). Expression of SA marker genes OsWRKY45, OsNPR1 and OsWRKY13 in control (Ctrl) and ABA pretreated IRBB3 leaves inoculated with PXO99. Transcript levels were normalized using eukaryotic elongation factor eEF1α as an internal reference and for each treatment expressed relative to the normalized expression levels in mock-inoculated control plants at the appropriate time point. Data are means ± SD of two technical and two biological replicates from a representative experiment, each biological replicate representing a pooled sample from 3 individual plants. Asterisks indicate statistically significant differences per treatment compared to either control (0 dpi) or mock-treated samples (1, 2, 4 and 8 dpi). (D). Effect of single and combined pretreatment with ABA (100 µM) and/or SA (500 µM) on BLB development in susceptible IRBB3 plants. Lesions were measured 14 days after inoculation with PXO99. Data are means ± SE of at least 10 plants. Different letters indicate statistically significant differences (Mann-Whitney; n ≥20; α = 0.05) (E) and (F). Effect of exogenous ABA treatment (100 µM) on BLB development in OsNPR1-OX and OsWRKY13-OX lines and their respective WT Taipei and Mudanjiang. Data are means ± SE of at least 10 plants. Different letters indicate statistically significant differences (Mann-Whitney; n ≥20; α = 0.05). Repetition of experiments led to results similar to those shown.
Figure 5.
Fluridone suppresses pathogen-induced transcription of ABA biosynthesis and response genes.
(A) through (C). Relative expression of ABA biosynthesis and responsive genes, OsNCED3, OsLip9 and OsRab16, in control (Ctrl) and fluridone-pretreated (0.4 µM) IRBB3 leaves inoculated with PXO99. Transcript levels were normalized using eukaryotic elongation factor eEF1α as an internal reference and expressed relative to the normalized expression levels in mock-inoculated control plants at the appropriate time point. Data are means ± SD of two technical and two biological replicates from a representative experiment, each biological replicate representing a pooled sample from 3 individual plants. Two sets of independent experiments were carried out with similar results. Asterisks indicate statistically significant differences per treatment compared to either control (0 dpi) or mock-treated samples (1, 2, 4 and 8 dpi).
Figure 6.
Fluridone induced Xoo resistance is independent of SA.
(A) through (C). Transcript levels of the SA regulatory genes OsWRKY45, OsNPR1 and OsWRKY13 in control and fluridone-treated (0.4 µM) IRBB3 leaves inoculated with PXO99. Data are means ± SD of two technical and two biological replicates, each biological replicate representing a pooled sample from 3 individual plants. Asterisks indicate statistically significant differences per treatment compared to either control (0 dpi) or mock-treated samples (1, 2, 4 and 8 dpi). (D) and (E). Effect of fluridone (0.4 µM) on BLB development in OsNPR1 RNAi and NahG-expressing lines and their respective WT Taipei and Nipponbare. Data are means ± SE of at least 10 plants. Different letters indicate statistically significant differences (Mann-Whitney; n ≥20; α = 0.05). Repetition of experiments led to results similar to those shown.
Figure 7.
Model illustrating how dynamic interplay between ABA and SA molds innate immunity of rice against the BLB pathogen Xoo.
Sharp arrows represent stimulatory effects, blunt arrows depict antagonistic interactions.