Benzoxazinoid metabolites regulate innate immunity against aphids and fungi in maize

Abstract

Benzoxazinoids (BXs), such as 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA), are secondary metabolites in grasses. The first step in BX biosynthesis converts indole-3-glycerol phosphate into indole. In maize (Zea mays), this reaction is catalyzed by either BENZOXAZINELESS1 (BX1) or INDOLE GLYCEROL PHOSPHATE LYASE (IGL). The Bx1 gene is under developmental control and is mainly responsible for BX production, whereas the Igl gene is inducible by stress signals, such as wounding, herbivory, or jasmonates. To determine the role of BXs in defense against aphids and fungi, we compared basal resistance between Bx1 wild-type and bx1 mutant lines in the igl mutant background, thereby preventing BX production from IGL. Compared to Bx1 wild-type plants, BX-deficient bx1 mutant plants allowed better development of the cereal aphid Rhopalosiphum padi, and were affected in penetration resistance against the fungus Setosphaeria turtica. At stages preceding major tissue disruption, R. padi and S. turtica elicited increased accumulation of DIMBOA-glucoside, DIMBOA, and 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one-glucoside (HDMBOA-glc), which was most pronounced in apoplastic leaf extracts. Treatment with the defense elicitor chitosan similarly enhanced apoplastic accumulation of DIMBOA and HDMBOA-glc, but repressed transcription of genes controlling BX biosynthesis downstream of BX1. This repression was also obtained after treatment with the BX precursor indole and DIMBOA, but not with HDMBOA-glc. Furthermore, BX-deficient bx1 mutant lines deposited less chitosan-induced callose than Bx1 wild-type lines, whereas apoplast infiltration with DIMBOA, but not HDMBOA-glc, mimicked chitosan-induced callose. Hence, DIMBOA functions as a defense regulatory signal in maize innate immunity, which acts in addition to its well-characterized activity as a biocidal defense metabolite.

Figure 1.

Contribution of Bx1 and Igl to basal resistance against the cereal aphid R. padi. Batches of neonate nymphs in clip cages were allowed to feed for 7 d from the first leaf of igl mutant lines, bx1 mutant lines, and bx1 igl double mutant lines, which had been selected from two independent crosses between the bx1 mutant and igl mutant of maize. A, Average weights (±sem; n = 15) of neonate nymphs after 7 d. B, Average percentages of batch survival (±sem) after 7 d. Different letters indicate statistically significant differences (ANOVA, followed by Fisher’s lsd test; α = 0.05). Wild-type alleles are indicated in black and mutant alleles in gray. The comparison between igl and bx1 igl mutant lines was repeated in two additional experiments with similar results.

Figure 2.

HPLC-DAD quantification of DIMBOA-glc, DIMBOA, and HDMBOA-glc in whole-tissue extracts (A) and apoplastic extracts (B) from mock- and R. padi-infested maize leaves. Material was collected at 48 h after aphid feeding in clip cages. Mock treatments consisted of clip cages without aphids. Data represent mean values in μg g⁻¹ fresh weight (±sem) from four biologically replicated leaf samples. Asterisks indicate statistically significant differences compared to mock-treated leaves (Student’s t test; α = 0.05). The experiment was repeated with similar results.

Figure 3.

Contribution of Bx1 and Igl to penetration resistance against the necrotrophic fungus S. turcica. Leaves of igl mutant lines, bx1 mutant lines, and bx1 igl double mutant lines were inoculated with 5 × 10⁴ spores mL⁻¹ and 3 d later collected for lactophenol trypan blue staining and microscopy analysis. A, Average hyphal lengths (μm) emerging from fungal spores (±sem) in the epidermal cell layer. Different letters indicate statistically significant differences (ANOVA, followed by Fisher’s lsd test; α = 0.05; n = 16). B, Frequency distributions between developing germination hyphae (class I; white) and arrested germination hyphae (class II; gray). Asterisks indicate statistically significant differences compared to BX-producing igl mutant lines from each cross (χ² test; α = 0.05; n = 16). The comparison between igl and bx1 igl mutant lines was repeated in two additional experiments with similar results.

Figure 4.

HPLC-DAD quantification of DIMBOA-glc, DIMBOA, and HDMBOA-glc in whole-tissue extracts (A) and apoplastic extracts (B) from mock- and S. turcica-inoculated maize leaves. Material was collected at 2 and 5 dpi. Data represent means in μg g⁻¹ fresh weight (±sem) from three biologically replicated samples. Asterisks indicate statistically significant differences compared to mock-treated leaves (Student’s t test; α = 0.05). P values indicate levels of statistical significance.

Figure 5.

HPLC-DAD quantification of DIMBOA-glc, DIMBOA, and HDMBOA-glc in whole-tissue extracts (A) and apoplastic extracts (B) at 24 h after infiltration of leaf segments with 0.2% chitosan or mock buffer. Data represent means in μg g⁻¹ fresh weight (±sem) from three biologically replicated samples. Asterisks indicate statistically significant differences compared to mock-treated leaves (Student’s t test; α = 0.05). The experiment was repeated twice with similar results.

Figure 6.

Transcriptional feedback regulation of the BX pathway by DIMBOA. Apoplastic infiltration with BX-inducing concentrations of chitosan (0.2%; A) and exposure to the volatile BX precursor indole (B) repressed Bx gene expression. BX-deficient bx igl double mutant lines displayed enhanced Bx gene transcription compared to BX-producing igl single mutant lines (C and D). Apoplastic leaf infiltration with DIMBOA (E and F) repressed Bx gene expression, whereas HDMBOA-glc had no effect (G), suggesting transcriptional feedback regulation by apoplastic DIMBOA. Shown are average fold-change values (±sem) of genes with a statistically significant level of induction (red) or repression (green) compared to mock treatments (A, B, E, and G) or BX-producing igl single mutant lines (C and D). Differences in expression between three biologically replicated samples from independent experiments were tested for statistical significance, using Student’s t tests or a nonparametric Wilcoxon-Mann-Whitney test when values did not follow normal distributions (α = 0.05). n.d., Not determined.

Figure 7.

Bx1 regulates chitosan-induced callose deposition. Leaf segments from igl single mutant lines and bx1 igl double mutant lines were infiltrated with chitosan (0.05%) or mock solution. At 24 h after infiltration, leaf segments were collected for aniline blue staining, UV-epifluoresence microscopy, and digital quantification of callose intensity. Shown are fold-induction values of callose (±sem; n = 15), relative to the average callose intensity in mock-treated Bx1 igl lines from each cross. Photographs show representative differences in fluorescent callose signals under UV-epifluoresence microscopy.

Figure 8.

DIMBOA-induced callose deposition. Infiltration with 20 μg mL⁻¹ DIMBOA elicits similar levels of callose deposition as infiltration with chitosan (0.1%), whereas infiltration with 20 μg mL⁻¹ HDMBOA-glc had no effect in comparison to the corresponding mock treatment. Shown are fold-induction values of callose deposition (±sem; n = 15), relative to average callose intensities in mock treatments at 24 h after infiltration treatment. Different letters indicate statistically significant differences (ANOVA, followed by Fisher’s lsd test; α = 0.05). Photographs show representative differences in fluorescent callose signals by UV-epifluorescence microscopy.

Figure 9.

Model of BX-dependent innate immunity against aphids and fungi. Activation of maize innate immunity leads to apoplastic deposition of HDMBOA-glc and DIMBOA-glc. Subsequent hydrolysis into biocidal aglycones can provide chemical defense against pests and diseases (Cambier et al., 2001; Rostás, 2007). Both HDMBOA and DIMBOA are degraded into MBOA and BOA (Maresh et al., 2006), indicating that DIMBOA (red), and not HDMBOA, has an additional function in the regulation of Bx gene expression and callose deposition. I, Intracellular space; A, apoplast.