Identity, regulation, and activity of inducible diterpenoid phytoalexins in maize

Abstract

Phytoalexins constitute a broad category of pathogen- and insect-inducible biochemicals that locally protect plant tissues. Because of their agronomic significance, maize and rice have been extensively investigated for their terpenoid-based defenses, which include insect-inducible monoterpene and sesquiterpene volatiles. Rice also produces a complex array of pathogen-inducible diterpenoid phytoalexins. Despite the demonstration of fungal-induced ent-kaur-15-ene production in maize over 30 y ago, the identity of functionally analogous maize diterpenoid phytoalexins has remained elusive. In response to stem attack by the European corn borer (Ostrinia nubilalis) and fungi, we observed the induced accumulation of six ent-kaurane-related diterpenoids, collectively termed kauralexins. Isolation and identification of the predominant Rhizopus microsporus-induced metabolites revealed ent-kaur-19-al-17-oic acid and the unique analog ent-kaur-15-en-19-al-17-oic acid, assigned as kauralexins A3 and B3, respectively. Encoding an ent-copalyl diphosphate synthase, fungal-induced An2 transcript accumulation precedes highly localized kauralexin production, which can eventually exceed 100 μg · g(-1) fresh weight. Pharmacological applications of jasmonic acid and ethylene also synergize the induced accumulation of kauralexins. Occurring at elevated levels in the scutella of all inbred lines examined, kauralexins appear ubiquitous in maize. At concentrations as low as 10 μg · mL(-1), kauralexin B3 significantly inhibited the growth of the opportunistic necrotroph R. microsporus and the causal agent of anthracnose stalk rot, Colletotrichum graminicola. Kauralexins also exhibited significant O. nubilalis antifeedant activity. Our work establishes the presence of diterpenoid defenses in maize and enables a more detailed analysis of their biosynthetic pathways, regulation, and crop defense function.

Fig. 1.

Identity and GC/MS spectra of kauralexin A1–A3 and B1–B3 methyl esters. (A) GC retention times and predominant positive-CI MS [M + H]⁺ ions of kauralexins A1–A3 and B1–B3 (e.g., K-A1, K-B1) methyl esters. Structures and EI spectra of kauralexins A1 (B), A2 (C), A3 (D), B1 (E), B2 (F), and B3 (G), respectively, are shown as methyl ester derivatives. In each panel, the y axis denotes relative abundance of ions.

Fig. 2.

Insect and fungal attack rapidly induces kauralexin concentrations. Average (n = 3, ±SEM) induced kauralexin A1 (A), A2 (B), A3 (C), B1 (D), B2 (E), and B3 (F) concentrations in maize stems experiencing no treatment (○), mechanical damage (●), and O. nubilalis herbivory (▼). Average (n = 4, ±SEM) induced kauralexin A1 (G), A2 (H), A3 (I), B1 (J), B2 (K), and B3 (L) concentrations in control maize stems (○) or those treated with a combination of damage plus H₂O (●), pectinase elicitor (■), C. graminicola spores (△), or R. microsporus spores (▼). Because of the similarity between control/damage + H₂O and pectinase/C. graminicola treatments, plot symbols are partly obscured. Within plots, different letters (a–c) represent significant differences at final time points (P < 0.01 for all ANOVAs; P < 0.05 for Tukey test corrections for multiple comparisons).

Fig. 3.

Insights into hormonal, transcriptional, spatial, and developmental regulation of kauralexins. Average (n = 4, ±SEM) trans-JA (A), OPDA (B), and OPEA (C) concentrations in control stems (○) or those treated with a combination of damage plus either H₂O (●) or R. microsporus spores (▼). (D) Average (n = 4, ±SEM) total kauralexins in stems 24 h after treatment with damage plus H₂O, EP, JA, OPDA, OPEA, or a combination of JA + EP, OPDA + EP, and OPEA + EP. (E) Average (n = 3, ±SEM) qRT-PCR transcript levels of An2 in control stems (○) or those treated with a combination of damage plus either H₂O (●) or R. microsporus spores (▼) and subsequent kauralexin (◆) accumulation. (F) Average (n = 4, ±SEM) total kauralexins in successively removed layers of R. microsporus-inoculated stems. (G) Average (n = 4, ±SEM) total kauralexin accumulation in the roots (○), shoots (●), and scutella (▲) of healthy untreated seedlings. Within plots, different letters (a–f) represent significant differences at indicated time points (P < 0.01 for all ANOVAs; P < 0.05 for Tukey test corrections for multiple comparisons).

Fig. 4.

Kauralexins display antifungal and insect antifeedant activities at physiologically relevant concentrations. Average (n = 8, ±SEM) R. microsporus growth time course in nutrient broth containing kauralexin A3 (A) and kauralexin B3 (B) at concentrations of 0 (○), 10 (●), and 100 (▼) μg·mL⁻¹ and, similarly, C. graminicola in the presence of kauralexin A3 (C) and kauralexin B3 (D). Average (n = 8, ±SEM) mass (grams) of maize stem consumed by O. nubilalis larvae in a 24-h choice assay containing 0 (Control) or 50 μg·g⁻¹ FW of kauralexin A3 (E) or, similarly, kauralexin B3 (F). K-A3, kauralexin A3; K-B3, kauralexin B3. Average (n = 6, ±SEM) 24-h growth (% mass gain) of O. nubilalis larvae on a diet containing a range of kauralexin A3 (G) or kauralexin B3 (H) concentrations. Within plots (A–F), different letters (a–c) represent significant differences (P < 0.01 for all ANOVAs; P < 0.05 for Tukey test corrections for multiple comparisons). Not statistically different (n.s.d.) indicates ANOVA P values >0.05.