Figure 8. Dual regulatory role of WRKY33 in modulating host defenses to B. cinerea 2100.
WRKY33 positively regulates target genes involved in camalexin biosynthesis thereby contributing to host resistance towards B. cinerea 2100. Target genes involved in ET/JA biosynthesis and signaling can either be positively or negatively regulated by WRKY33. On the other hand, WRKY33 negatively regulates ABA levels by directly targeting and repressing NCED3 and NCED5 expression, or inducing expression of CYP707A3, a gene involved in ABA metabolism. Thus, WRKY33 has both activator and repressor functions that may depend on promoter context. Red arrows indicate positive regulation, whereas black bars indicate negative regulation. The curved red arrow highlights positive feedback regulation of WRKY33 on its own gene promoter.
Figure 8—figure supplement 1. Schematic representation showing WRKY33-dependent host immunity towards the necrotroph B. cinerea 2100 through repressing the ABA network.
In WT Arabidopsis Col-0 plants (upper panel), B. cinerea inoculation induces WRKY33 expression and protein accumulation that controls ABA levels by repressing the ABA biosynthetic target genes NCED3 and NCED5, and by inducing the ABA metabolic gene CYP707A3. WRKY33 also directly or indirectly represses expression of other genes in different hormone signaling pathways as well as genes encoding WRKY, and NAC TFs. WRKY33 also positively affects resistance towards B. cinerea 2100 by directly targeting and inducing the expression of several genes involved in camalexin biosynthesis. In the wrky33 mutant (lower panel), B. cinerea infection fails to activate host camalexin biosynthesis genes and increases ABA levels by up-regulation of NCED3 and NCED5, which activates downstream ABA-dependent genes and ABA signaling. Among the ABA-dependent genes are genes involved in SA signaling, WRKY TFs, NAC TFs, and genes associated with auxin or JA conjugation indicating that ABA may act as a key sub-node in WRKY33-dependent host immunity to B. cinerea. Whereas WT-like resistance is restored in the wrky33 nced3 nced5 triple mutant, all currently tested mutants of WRKY33-regulated genes acting downstream of ABA failed to restore resistance in the wrky33 mutant background (marked by an asterisk). Thus, additional crucial genes causal for WRKY33-dependent resistance to B. cinerea 2100 remain to be discovered, as do ABA signaling components affecting WRKY33 expression (indicated by the question marks). The solid lines with arrows indicate induction or positive modulation, the bar heads indicate suppression. Direct WRKY33 targets are indicated by the green color.
Figure 8—figure supplement 2. AMT1 and CYP71A12 expression is positively regulated by WRKY33.
(A–B) IGV images of ChIP-seq data revealing high infection-dependent WRKY33 binding at the promoters of AMT1 (A) and CYP71A12 (B). (C–D) qRT-PCR analyses of AMT1 (C) and CYP71A12 (D) in WT and wrky33 at the indicated time points after B. cinerea 2100 spore inoculation. qRT-PCR data were normalized to the expression of At4g26410. Error bars represent SD of three biological replicates (n = 3). Asterisks indicate significant differences between WT and wrky33 (*, p < 0.05; **, p < 0.001; two-tailed t-test).
Figure 8—figure supplement 3. The role of GH3 genes in WRKY33-mediated host resistance to B. cinerea 2100.
(A) Expression levels of GH3.2 and GH3.3 in WT and wrky33 plants determined by qRT-PCR at the indicated time points after B. cinerea 2100 spore inoculation. Data were normalized to the expression of At4g26410. Error bars represent SD of three biological replicates (n = 3). Asterisks indicate significant differences between WT and wrky33 (*, p < 0.05; **, p < 0.001; two-tailed t-test). (B) B. cinerea 2100 infection phenotypes of WT, wrky33, gh3.2, gh3.3, gh3.2 gh3.3, and wrky33 gh3.2 gh3.3 plants are shown at 3 days post infection. Two 2 μl droplets containing 2.5 × 105 spores were applied to each leaf.