Tryptophan-derived metabolites are required for antifungal defense in the Arabidopsis mlo2 mutant

Abstract

Arabidopsis (Arabidopsis thaliana) genes MILDEW RESISTANCE LOCUS O2 (MLO2), MLO6, and MLO12 exhibit unequal genetic redundancy with respect to the modulation of defense responses against powdery mildew fungi and the control of developmental phenotypes such as premature leaf decay. We show that early chlorosis and necrosis of rosette leaves in mlo2 mlo6 mlo12 mutants reflects an authentic but untimely leaf senescence program. Comparative transcriptional profiling revealed that transcripts of several genes encoding tryptophan biosynthetic and metabolic enzymes hyperaccumulate during vegetative development in the mlo2 mlo6 mlo12 mutant. Elevated expression levels of these genes correlate with altered steady-state levels of several indolic metabolites, including the phytoalexin camalexin and indolic glucosinolates, during development in the mlo2 single mutant and the mlo2 mlo6 mlo12 triple mutant. Results of genetic epistasis analysis suggest a decisive role for indolic metabolites in mlo2-conditioned antifungal defense against both biotrophic powdery mildews and a camalexin-sensitive strain of the necrotrophic fungus Botrytis cinerea. The wound- and pathogen-responsive callose synthase POWDERY MILDEW RESISTANCE4/GLUCAN SYNTHASE-LIKE5 was found to be responsible for the spontaneous callose deposits in mlo2 mutant plants but dispensable for mlo2-conditioned penetration resistance. Our data strengthen the notion that powdery mildew resistance of mlo2 genotypes is based on the same defense execution machinery as innate antifungal immune responses that restrict the invasion of nonadapted fungal pathogens.

Figure 1.

Developmentally controlled mlo2-associated leaf chlorosis and necrosis resembles an authentic leaf senescence program. A, Time-course analysis of photosynthetic performance (F_v/F_m) of Col-0 wild-type (black circles) and mlo2 mlo6 mlo12 (white circles) mutant plants. Data represent means ± sd of four independent rosette leaves (leaf 7) measured at the time points indicated (days after sowing). Plants were grown in long-day conditions. The experiment was repeated twice with similar results (Supplemental Fig. S2; data not shown). B, Time-course analysis of chlorophyll content in leaves of Col-0 wild-type (black circles) and mlo2 mlo6 mlo12 (white circles) mutant plants. Data represent means ± sd of two independent rosette leaves (leaf 7) collected at the time points indicated (days after sowing) and measured with three technical replicates. Plants were grown in long-day conditions. Note that the very same leaves were taken for measuring chlorophyll content and for determining photosynthetic performance. The experiment was repeated once with similar results (Supplemental Fig. S2). FW, Fresh weight. Asterisks denote statistically significant differences (P < 0.05; Student's t test) from the Col-0 wild type. C, Habitus of representative unchallenged (pathogen-free) plants at 7 weeks after sowing (top row) and macroscopic phenotypes of detached leaves (leaf 5) from 4-week-old plants dark treated for 4 d (bottom row). Plants were grown in long-day conditions. The experiment was repeated four times with similar results.

Figure 2.

Transcript accumulation of MLO2 peaks around the onset of leaf senescence. A, Time-course analysis of MLO2 promoter-driven GUS expression in a transgenic Col-0 wild-type plant during vegetative development. Plants were grown in short-day conditions (10 h of light), and entire plants of the indicated ages were stained for GUS activity. The photographs depict exemplary plants from one experiment; similar results were obtained in two independent replicate experiments. B, Schematic representation of AtMLO2 expression data on publicly accessible microarray databases. The cartoon represents leaves at various developmental stages, color coded with the respective AtMLO2 expression level (according to the reference color bar shown at the bottom). Microarray source data are from the AtGenExpress project (Schmid et al., 2005). The pictograph is a screenshot from the Arabidopsis eFP browser (http://bbc.botany.utoronto.ca/efp/cgi-bin/efpWeb.cgi; Winter et al., 2007). c, Cauline leaf; s, senescent leaf.

Figure 3.

Altered gene expression levels and secondary metabolite accumulation in the Arabidopsis mlo2 mlo6 mlo12 mutant. A, Expression patterns of genes involved in secondary metabolite biosynthesis and of senescence-associated genes are altered in the mlo2 mlo6 mlo12 mutant compared with the Col-0 wild type (wt). Gene expression levels determined by quantitative RT-PCR are each presented relative to the expression level in the wild type at the early time point. Values shown represent means ± sd of a representative experiment comprising at least three technical replicates per genotype and time point. Leaf samples of plants grown in short-day conditions were collected at 5 weeks (early time point) and 7 weeks (late time point) after sowing; leaf samples of plants grown in long-day conditions were collected at 4 weeks (early time point) and 5 weeks (late time point) after sowing. Light gray bars show early time point; dark gray bars show late time point. Similar results were obtained in an independent biological experiment (Supplemental Fig. S3). B, Secondary metabolite profile of leaves of the mlo2 mlo6 mlo12 triple mutant compared with the Col-0 wild type. Rosette leaf 7 of independent plants grown in short-day conditions was collected at 5, 6, 7, and 8 weeks after sowing and used for metabolite analysis. The graphs show the average ± sd of a representative experiment comprising three leaf samples. The experiment was repeated once with similar results. The left y axis refers to camalexin and I3A, whereas the right y axis applies to I3G and 4MI3G. A statistically significant difference from Col-0 is indicated either by an asterisk (P < 0.01; Student's t test) or the number sign (P < 0.05; Student's t test). FW, Fresh weight.

Figure 4.

Altered levels of indolic metabolite in leaves of mlo mutant plants depend on the conventional biosynthetic route and are not the primary cause of the early leaf senescence phenotype. A, Leaf 7 from nine independent plants grown in short-day conditions was collected at 7 weeks after sowing, and HPLC analysis was performed on metabolite extracts. The graphs show averages ± sd of a representative experiment comprising three technical replicates. One typical experiment out of three is shown. Asterisks denote statistically significant differences (P < 0.05; Student's t test) from the Col-0 wild type, and number signs denote statistically significant differences (P < 0.05; Student's t test) from the mlo2 mutant. FW, Fresh weight. B, Habitus of representative unchallenged (pathogen-free) plants at 7 weeks after sowing (top row) and macroscopic phenotypes of detached leaf 5 from 4-week-old plants dark treated for 4 d (bottom row). Plants were grown in long-day conditions. The experiment was repeated once (except for Col-0, mlo2, and mlo2 mlo6 mlo12, which had five replicates) with similar results.

Figure 5.

Indolic secondary metabolites are essential for mlo2-mediated powdery mildew resistance. A, Representative photographs depicting macroscopic infection phenotypes of wild-type and mutant plants upon challenge with G. orontii. Images were taken at 10 d postinoculation. B, Quantitative analysis of G. orontii host cell entry (determined at 48 h postinoculation; gray bars) and conidiophore formation (determined at 7 d postinoculation; black bars). Results represent means ± sd of four to eight independent experiments per genotype. Statistically significant differences from the Col-0 wild type are indicated by asterisks (*** P < 0.01, * P < 0.05; Student's t test), and statistically significant differences from mlo2 are indicated by number signs (^### P < 0.01, ^# P < 0.05; Student's t test). C, Quantitative analysis of E. pisi entry into Arabidopsis epidermal cells determined at 7 d postinoculation. Results represent means ± sd of three to six samples per genotype derived from at least three independent experiments. Statistically significant differences from the Col-0 wild type are indicated by asterisks (*** P < 0.01, * P < 0.05; Student's t test), and statistically significant differences from mlo2 are indicated by number signs (^### P < 0.01, ^# P < 0.05; Student's t test).

Figure 6.

Camalexin is essential for mlo2-mediated resistance against B. cinerea. A, Representative photographs depicting macroscopic infection phenotypes on leaves of wild-type and mutant plants upon challenge with a camalexin-sensitive strain of B. cinerea. Detached leaves of 4-week-old plants grown in 16-h-light/8-h-dark cycles (low light intensity and humidity) were inoculated with two 5- μ L droplets of a suspension containing 5 × 10⁵ spores mL⁻¹. Images were taken at 2 d postinoculation. The experiment was repeated twice with similar results. B, Quantitative assessment of lesion diameter determined at 2 d postinoculation with B. cinerea. Results originate from one experiment and represent means ± sd of at least nine lesions per genotype. Statistically significant differences from the Col-0 wild type are indicated by asterisks (*** P < 0.01, * P < 0.05; Student's t test), and statistically significant differences from mlo2 are indicated by number signs (P < 0.01; Student's t test). The experiment was repeated twice with similar results.

Figure 7.

The GSL5/PMR4 callose synthase is required for spontaneous callose deposition but dispensable for penetration resistance of the mlo2 mutant. A, Representative micrographs of spontaneous callose deposition in unchallenged (pathogen-free) plants grown in long-day conditions. Leaves were collected at the time points indicated. The experiment was repeated once and additionally performed once with plants grown in short-day conditions, yielding similar results. Bar = 50 μ m. B, Quantitative analysis of G. orontii host cell entry (determined at 48 h postinoculation; gray bars) and conidiophore formation (determined at 7 d postinoculation; black bars). Results represent means ± sd of three to eight independent experiments per genotype. Statistically significant differences from the Col-0 wild type are indicated by asterisks (*** P < 0.01, * P < 0.05; Student's t test), and statistically significant differences from mlo2 are indicated by number signs (^### P < 0.01, ^# P < 0.05; Student's t test). C, Habitus of representative unchallenged (pathogen-free) plants at 7 weeks after sowing (top row) and macroscopic phenotypes of detached leaf 5 from 4-week-old plants dark-treated for 4 d (bottom row). In both experiments, plants were grown in long-day conditions. The experiment was repeated twice with similar results.

Figure 8.

Proposed model for MLO-mediated control of defense to powdery mildew fungi and early leaf senescence/callose deposition. The model integrates previous data (Consonni et al., 2006) and findings of this work. The font size of the components symbolizes the weight of their functional contributions. Components marked by red crosses are not required for a given pathway. [See online article for color version of this figure.]