Arabidopsis cytochrome P450 monooxygenase 71A13 catalyzes the conversion of indole-3-acetaldoxime in camalexin synthesis

Abstract

Camalexin (3-thiazol-2-yl-indole) is an indole alkaloid phytoalexin produced by Arabidopsis thaliana that is thought to be important for resistance to necrotrophic fungal pathogens, such as Alternaria brassicicola and Botrytis cinerea. It is produced from Trp, which is converted to indole acetaldoxime (IAOx) by the action of cytochrome P450 monooxygenases CYP79B2 and CYP79B3. The remaining biosynthetic steps are unknown except for the last step, which is conversion of dihydrocamalexic acid to camalexin by CYP71B15 (PAD3). This article reports characterization of CYP71A13. Plants carrying cyp71A13 mutations produce greatly reduced amounts of camalexin after infection by Pseudomonas syringae or A. brassicicola and are susceptible to A. brassicicola, as are pad3 and cyp79B2 cyp79B3 mutants. Expression levels of CYP71A13 and PAD3 are coregulated. CYP71A13 expressed in Escherichia coli converted IAOx to indole-3-acetonitrile (IAN). Expression of CYP79B2 and CYP71A13 in Nicotiana benthamiana resulted in conversion of Trp to IAN. Exogenously supplied IAN restored camalexin production in cyp71A13 mutant plants. Together, these results lead to the conclusion that CYP71A13 catalyzes the conversion of IAOx to IAN in camalexin synthesis and provide further support for the role of camalexin in resistance to A. brassicicola.

Figure 1.

Positions of T-DNA Insertions in At2g30770. The gene model from The Arabidopsis Information Resource (www.arabidopsis.org) is shown. Exons are represented by rectangles and introns and untranscribed regions by lines. Black fill indicates translated sequences. The protein sequence is represented by the bottom rectangle. The filled region labeled “H” represents the heme-iron ligand signature. Positions of the T-DNA insertions are indicated by arrows. aa, amino acids.

Figure 2.

Susceptibility of Various Arabidopsis Mutants to A. brassicicola. Leaves 3 d after inoculation. Plants were 24 d old when they were inoculated with A. brassicicola. The fifth true leaves from two plants of each genotype are shown.

Figure 3.

Disease Index Scoring of A. brassicicola–Infected Plants. Plants (21 d old) were inoculated as above. After 3 d, 20 to 24 leaves per genotype were assigned a disease index score. 1, necrosis confined to the area of the inoculation droplet; 2, some chlorosis around the necrotic spot; 3, spreading chlorosis and/or some necrosis beyond the inoculation droplet; 4, spreading necrosis.

Figure 4.

Camalexin Levels in Various Arabidopsis Mutants after Infection by Pathogens. (A) Infection by A. brassicicola. Plants were inoculated with A. brassicicola spores as described in Methods. After 3 d, entire leaves were excised and camalexin was determined. Each sample consisted of two leaves, and each bar represents the mean and sd of eight replicate samples. Col, Columbia. (B) Infection by P. syringae. Plants were inoculated with bacteria at a starting density of 4 × 10⁴ colony-forming units/cm² (OD₆₀₀ = 0.004). After 48 h, samples consisting of 1 cm² of leaf were excised using a cork borer, and camalexin was determined. Bars represent the means and sd of eight replicates. No camalexin was observed in uninfected leaves of any genotype (data not shown).

Figure 5.

Expression Patterns of Selected Genes during P. syringae Infection as Measured by Microarray Experiments. (A) Expression changes in response to infection by Psm ES4326. Expression values were determined using robust multichip average and divided by 1000 for convenience. Each bar represents the mean and sd of three independent replicates. (B) Comparison of PAD3 and CYP71A13 expression patterns. Data points represent log₂-transformed values from the Genevestigator database. The underlying data are provided as Supplemental Table 1 online.

Figure 6.

Carbon Monoxide Difference Spectrum of CYP71A13. CYP71A13 was functionally expressed in E. coli as observed by the CO-difference spectrum of purified membranes. The dotted and solid lines represent the spectrum before and 15 min after CO exposure. The spectra were recorded at room temperature.

Figure 7.

The Catalytic Properties of CYP71A13 toward IAOx as Analyzed by LC-MS. LC-MS chromatograms from E. coli spheroplasts reconstituted with recombinant NADPH:cytochrome P450 reductase from Arabidopsis (ATR1) and incubated with IAOx. The reaction mixtures were extracted with ethyl acetate, and the organic phase was analyzed by LC-MS. IAOx is indicated by arrows. The two geometric isomers appear as separate peaks but with an elevated baseline in between due to partial on-column equilibration.

Figure 8.

Analysis of CYP71A13 by Optical Difference Spectroscopy. A saturated type II spectrum was obtained with 100 μM tryptamine in the sample cuvette (thick solid line). The addition of 100 μM tryptamine to the reference cuvette gave a baseline (dotted line). The spectra were recorded at room temperature and did not increase over time. (A) Increasing concentrations of IAOx in the sample cuvette (2.5, 10, and 100 μM) gradually displaced tryptamine, giving the reverse type II spectrum. (B) Increasing concentrations of phenylacetaldoxime in the sample cuvette (20, 40, and 100 μM) gradually displaced tryptamine, giving the reverse type II spectrum.

Figure 9.

Transient Expression of CYP79B2 and CYP71A13 in N. benthamiana Results in Conversion of Trp to IAN via IAOx. Microsomes from leaves infiltrated with p19 (thin solid line), CYP79B2 (dotted line), or CYP79B2 and CYP71A13 (thick solid line) in combination with p19 were incubated with Trp. After incubation, the reaction mixtures were extracted with ethyl acetate, and the organic phases were analyzed by LC-MS. m/z, mass-to-charge ratio. (A) Reconstructed ion chromatogram of m/z 175 (IAOx) from the different microsomal assays. (B) Reconstructed ion chromatogram of m/z 155 (IAN) from the different microsomal assays.

Figure 10.

In Vivo Feeding of IAN to AgNO₃-Treated Arabidopsis Mutants. After spraying with silver nitrate, rosette leaves of Col-0, cyp71A13-1, cyp79B2 cyp79B3, and pad3 plants were incubated with 250 μM IAN (black bars) or water (gray bars). Bars represent means and sd of six replicates. Significance of differences between water- and IAN-treated plants was tested using an unpaired, two-tailed t test. The difference for Col-0 was not significant (P = 0.16), while the differences for cyp71A13-1 and cyp79B2 cyp79B3 were highly significant (P = 0.00028 and 0.0000049, respectively).