Mutation of the Glucosinolate Biosynthesis Enzyme Cytochrome P450 83A1 Monooxygenase Increases Camalexin Accumulation and Powdery Mildew Resistance

Abstract

Small secondary metabolites, including glucosinolates and the major phytoalexin camalexin, play important roles in immunity in Arabidopsis thaliana. We isolated an Arabidopsis mutant with increased resistance to the powdery mildew fungus Golovinomyces cichoracearum and identified a mutation in the gene encoding cytochrome P450 83A1 monooxygenase (CYP83A1), which functions in glucosinolate biosynthesis. The cyp83a1-3 mutant exhibited enhanced defense responses to G. cichoracearum and double mutant analysis showed that this enhanced resistance requires NPR1, EDS1, and PAD4, but not SID2 or EDS5. In cyp83a1-3 mutants, the expression of genes related to camalexin synthesis increased upon G. cichoracearum infection. Significantly, the cyp83a1-3 mutant also accumulated higher levels of camalexin. Decreasing camalexin levels by mutation of the camalexin synthetase gene PAD3 or the camalexin synthesis regulator AtWRKY33 compromised the powdery mildew resistance in these mutants. Consistent with these observations, overexpression of PAD3 increased camalexin levels and enhanced resistance to G. cichoracearum. Taken together, our data indicate that accumulation of higher levels of camalexin contributes to increased resistance to powdery mildew.

Keywords: Arabidopsis thaliana; CYP83A1; camalexin; plant immunity; powdery mildew.

FIGURE 1

The cyp83a1-3 mutant displays enhanced resistance to Golovinomyces cichoracearum. (A) Four-weeks-old Arabidopsis wild-type and cyp83a1-3 mutant plants were infected with G. cichoracearum, and representative leaves were removed and photographed at 8 dpi. (B) Leaves were stained with trypan blue at 8 dpi, bar = 200 μm. (C) Leaves were stained with DAB and trypan blue at 2 dpi; arrows indicate H₂O₂ accumulation, bar = 200 μm. (D) Quantification of fungal growth in plants at 5 dpi by counting the number of conidiophores per colony. Results represent the mean and standard deviation in three independents experiments (n = 30). Asterisk represents statistically significant differences from wild-type (P < 0.01, nested ANOVA). (E) Schematic representation of the CYP83A1 protein, arrows indicate mutation sites of three cyp83a1 mutant alleles. (F) Four-weeks-old wild-type, cyp83a1-3, cyp83a1-1, cyp83a1-2, and transgenic cyp83a1-3 mutant plants complemented with the wild-type CYP83A1 gene (gCYP83A1) were infected with G. cichoracearum and representative leaves were stained with trypan blue at 8 dpi, bar = 200 μm. (G) Quantification of fungal growth in plants at 5 dpi by counting the number of conidiophores per colony. Results represent the mean and standard deviation in three independent experiments (n = 30; P < 0.01, nested ANOVA).

FIGURE 2

The resistance in cyp83a1-3 is SA-independent. (A) Four-weeks-old Arabidopsis wild-type, cyp83a1-3 mutant, and double mutant plants were infected with G. cichoracearum, and representative leaves were removed and stained with trypan blue at 8 dpi, bar = 200 μm. (B) Quantification of fungal growth of the plants in (a) at 5 dpi by counting the number of conidiophores per colony. Results represent the mean and standard deviation in three independent experiments (n = 30). Different letters represent statistically significant differences (P < 0.01, nested ANOVA). (C) Free SA levels were measured in the uninfected and infected (3 dpi) leaves after inoculation with G. cichoracearum. FW, fresh weight. (D) Accumulation of PR1, PR2, and FRK1 transcripts in 4-weeks-old plants infected by G. cichoracearum examined by quantitative real-time PCR. Results represent the mean and standard deviation in three independent experiments (n = 4).

FIGURE 3

Transcript accumulation of genes related to glucosinolate or camalexin biosynthesis upon G. cichoracearum infection. (A) Four-weeks-old wild-type plants were infected with G. cichoracearum, and the accumulation of CYP83A1, SUR2, CYP71A13, and PAD3 transcripts was examined by quantitative real-time PCR. Results represent the mean and standard deviation in three independent experiments (n = 4). (B) Four-weeks-old CYP83A1-GFP transgenic plants were infected with G. cichoracearum and immunoblot analysis was performed using an anti-GFP antibody. The large subunit of Rubisco was used as a protein loading control.

FIGURE 4

The cyp83a1-3 mutant accumulates high levels of camalexin upon G. cichoracearum infection, which is suppressed by mutation of PAD3. (A) Four-weeks-old wild-type, cyp83a1-3, pad3 mutant, and double-mutant plants were infected with G. cichoracearum. Representative leaves were removed and stained with trypan blue at 8 dpi, bar = 200 μm. (B) Quantification of fungal growth of the plants in (a) at 5 dpi by counting the number of conidiophores per colony. Results represent the mean and standard deviation in three independent experiments (n = 30; P < 0.01, nested ANOVA). (C) Four-weeks-old plants were infected with G. cichoracearum. Camalexin accumulation was determined at 0 and 5 dpi. Results represent the mean and standard deviation in three experiments (n = 3). Asterisk represents statistically significant difference from wild-type (P < 0.01, nested ANOVA). (D) The transcript accumulation of PAD3 was examined by quantitative real-time PCR on samples from 4-weeks-old wild-type, cyp83a1-3 and pad3 mutant plants. (E) The transcript accumulation of CYP71A13 examined by quantitative real-time PCR. Results represent the mean and standard deviation in three independent experiments (n = 4).

FIGURE 5

The resistance phenotype and high levels of camalexin in cyp83a1-3 is suppressed by mutation of WRKY33. (A) Four-weeks-old wild-type, cyp83a1-3, wrky33, and wrky33 cyp83a1-3 double-mutant plants were infected with G. cichoracearum. Representative leaves were removed and stained with trypan blue at 8 dpi, bar = 200 μm. (B) Quantification of fungal growth of the plants in (a) at 5 dpi by counting the number of conidiophores per colony. Results represent the mean and standard deviation in three independent experiments (n = 30; P < 0.01, nested ANOVA). (C) Camalexin accumulation of the plants in (A) was determined at 0 and 5 dpi. Results represent the mean and standard deviation in three independent experiments (n = 3). Asterisk represents statistically significant difference from wild-type (P < 0.01, nested ANOVA).

FIGURE 6

PAD3-overexpressing plants accumulate higher levels of camalexin and display a cyp83a1-3-like resistance phenotype. (A) The accumulation of PAD3 transcript in 4-weeks-old plants was examined by quantitative real-time PCR. Results represent the mean and standard deviation in three independent experiments (n = 4). (B) Four-weeks-old plants were infected with G. cichoracearum and camalexin accumulation was determined at 0 and 5 dpi. Three PAD3-OX independent lines were tested, and similar results were obtained in these lines. One representative PAD3-OX line (PAD3-OX-1) is shown. Results represent the mean and standard deviation in three experiments (n = 3). (C) Quantification of fungal growth of wild-type, cyp83a1-3, and PAD3-OX plants at 5 dpi by counting the number of conidiophores per colony. Results represent the mean and standard deviation in three independent experiments (n = 30). Different letters represent statistically significant differences (P < 0.01, nested ANOVA). (D) Four-weeks-old wild-type, cyp83a1-3, and PAD3-OX-1 plants were infected with G. cichoracearum. Three PAD3-OX independent lines were examined, and similar results were obtained in these three lines. Representative leaves were removed and stained with trypan blue at 8 dpi, bar = 200 μm.