Novel acidic sesquiterpenoids constitute a dominant class of pathogen-induced phytoalexins in maize - PubMed (original) (raw)
Novel acidic sesquiterpenoids constitute a dominant class of pathogen-induced phytoalexins in maize
Alisa Huffaker et al. Plant Physiol. 2011 Aug.
Abstract
Nonvolatile terpenoid phytoalexins occur throughout the plant kingdom, but until recently were not known constituents of chemical defense in maize (Zea mays). We describe a novel family of ubiquitous maize sesquiterpenoid phytoalexins, termed zealexins, which were discovered through characterization of Fusarium graminearum-induced responses. Zealexins accumulate to levels greater than 800 μg g⁻¹ fresh weight in F. graminearum-infected tissue. Their production is also elicited by a wide variety of fungi, Ostrinia nubilalis herbivory, and the synergistic action of jasmonic acid and ethylene. Zealexins exhibit antifungal activity against numerous phytopathogenic fungi at physiologically relevant concentrations. Structural elucidation of four members of this complex family revealed that all are acidic sesquiterpenoids containing a hydrocarbon skeleton that resembles β-macrocarpene. Induced zealexin accumulation is preceded by increased expression of the genes encoding TERPENE SYNTHASE6 (TPS6) and TPS11, which catalyze β-macrocarpene production. Furthermore, zealexin accumulation displays direct positive relationships with the transcript levels of both genes. Microarray analysis of F. graminearum-infected tissue revealed that Tps6/Tps11 were among the most highly up-regulated genes, as was An2, an ent-copalyl diphosphate synthase associated with production of kauralexins. Transcript profiling suggests that zealexins cooccur with a number of antimicrobial proteins, including chitinases and pathogenesis-related proteins. In addition to zealexins, kauralexins and the benzoxazinoid 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one-glucose (HDMBOA-glucose) were produced in fungal-infected tissue. HDMBOA-glucose accumulation occurred in both wild-type and benzoxazine-deficient1 (bx1) mutant lines, indicating that Bx1 gene activity is not required for HDMBOA biosynthesis. Together these results indicate an important cooperative role of terpenoid phytoalexins in maize biochemical defense.
Figures
Figure 1.
Identity and GC/MS spectra of zealexin A1-A3 and B1 as methyl esters. A, Damage. B, Damage plus _F. graminearum_-inoculated (106 spores mL−1) maize stem samples after 48 h, analyzed as GC/(+)CI-MS total ion chromatograms (TIC). Predominant fatty acid methyl esters are labeled as palmitic and steric acid. C, Expanded GC/(+)CI-MS selected ion trace of predominant fungal-induced sesquiterpenoids as methyl esters. Analytes 1 to 5, denoted by [M+H]+ 249 ions, are unmodified sesquiterpene acids. For analyte 6, a partially unsaturated sesquiterpene acid, 247 is the parent [M+H]+ ion. As oxygenated acidic sesquiterpenoids, analytes 7 and 8 to 11 are denoted by 249 [M+H-H20]+ ions and 247 [M+H-H20]+ ions, respectively. Analytes 12 to 14 are denoted by 261 and 263 [M+H]+ ions, consistent with partially unsaturated, oxygenated acidic sesquiterpenoids. D, Zealexin structures deduced by NMR. EI spectra (_mass_-_to_-charge ratio) of: Zealexin A1 (E); analyte 5, Zealexin B1 (F); analyte 6, Zealexin A2 (G); analyte 10, and Zealexin A3 (H); analyte 11. In each section (A–C and E–H), the y axis denotes relative abundance of ions.
Figure 2.
With the exception of a maize specialist (C. graminicola), zealexins accumulate in response to an array of fungal pathogens. Average (n = 4, ±
sem
) of zealexin A1 (A), zealexin A2 (B), zealexin A3 (C), and zealexin B1 (D) levels in stems 24 h after plants were untreated (control, C), or damaged and treated with either water (D), or a water suspension of the following phytopathogenic fungi; C. graminicola (C.g.), C. sublineolum (C.s.), C. heterostrophus (C.h.), A. flavus (A.f.), and R. microsporus (R.m.) at a concentrated inoculum (1 × 107 spores mL−1). Within plots different letters (a–d) represent significant differences (all ANOVAs, P < 0.0001; Tukey test corrections for multiple comparisions, P < 0.05).
Figure 3.
Relationship of pathogen-induced zealexin concentrations with Tps6 and Tps11 transcript levels. Average (n = 4; ±
sem
). A, Zealexin A1. B, Zealexin A3 levels in maize stems experiencing no treatment (○), damage plus water (•), or damage plus water suspensions of R. microsporus (▾, 107 spores mL−1), A. flavus (△, 107 spores mL−1), or F. graminearum (106 spores mL−1) at time 0 or 4, 12- and 24-h postinoculation. C, Tps6. D, Tps11 corresponding average (n = 4; ±
sem
) qRT-PCR fold change of transcripts as compared to levels of Tps6 in control stems. E, Tps6. F, Tps11 relationship between fungal-induced fold change in gene expression and total zealexins at 24 h. Within plots, different letters (a–d) represent significant differences at 24 h (all ANOVAs, P < 0.0001; Tukey test corrections for multiple comparisons, P < 0.05).
Figure 4.
Dose-response relationships between fungal-induced terpenoid phytoalexins and fold change in candidate terpene synthase transcripts. Average (n = 4; ±
sem
). A, Zealexin A1. B, Zealexin A3. C, Total kauralexin levels in damaged stems treated with either water (D) or with water suspensions of increasing concentrations of F. graminearum (▪) or A. flavus (△) spores after 48 h. D, Tps11. E, Tps6. F, An2 corresponding average (n = 4; ±
sem
) qRT-PCR fold change in transcript abundance 48 h after fungal inoculation. G, β-Macrocarpene. H, Total zealexin relationship to fungal-induced fold change in Tps6 expression. I, Relationship between qRT-PCR fold change in transcript abundance of Tps6 and An2. J, Relationship between fungal-induced total zealexins and total kauralexins. Plots G to J represent all treatment groups from the corresponding 48-h F. graminearum and A. flavus dose-response experiment, and thus general trends. Within plots different letters (a–e) represent significant differences (all ANOVAs, P < 0.0001; Tukey test corrections for multiple comparisons, P < 0.05).
Figure 5.
Transcripts encoding Tps6/11 and antimicrobial proteins are coregulated following F. graminearum infection. A, Scatter plot of average probe intensity (Log2 scale) for Affymetrix maize microarray chips hybridized with cDNA from mock-inoculated stems versus _F. graminearum_-inoculated stems. Probes corresponding to genes up-regulated in _F. graminearum_-inoculated stems are plotted in red whereas the down-regulated are plotted in blue. Linear regression (_r_2 = 0.901) is indicated in magenta; green lines indicate threshold of fold change >2. Student’s t test with Benjamini-Hochberg FDR analysis was applied. The 26 genes up-regulated >100-fold in response to F. graminearum (Ps < 0.015; _t_ > 12.4) are numbered in descending order. Tps6/11 was the second-most up-regulated probe set, whereas An2 was 12th (green numbers). Among the other 24 genes, the majority were pathogenesis-protein related (PR, blue numbers); including chitinases (underlined;1, 3, 4, 8, 9, 15), Pr10 (5), Pr5 (11), proteinase inhibitors (6, 16, 23), Ser endopeptidase (14), peroxidase (17), β-1,3 glucanase (19), zeamatin-like protein (22), and polygalacturonase inhibitor (26). Non-PR genes (black numbers) included Bak1 (6), an _O_-methyl transferase (10) and GST 2 (18). Genes of unknown function are marked with gray numbers (13, 20, 21, 24, 25). B to
D
, Average (n = 4; ±
sem
) qRT-PCR fold change in transcript levels of genes encoding antimicrobial proteins at 48 h in damaged stems treated with either water (D) or water containing increasing inoculum concentrations of F. graminearum (▪) or A. flavus (△) spores after 48 h. B, Bowman-Birk proteinase inhibitor Wip1. C, Endochitinase A (Eca). D, Pathogenesis-response protein 10 (Pr10). Within each plot different letters (a–e) represent significant differences (all ANOVAs, P < 0.001; Tukey test corrections for multiple comparisons, P < 0.05).
Figure 6.
Zealexin and benzoxazinoid defenses are concurrently induced by fungal infection. Average levels (n = 4; ±
sem
) of defense metabolites in H88 and the H88-derived bx1 mutant line. Stems were either untreated (C), or damaged and treated with water (D) or water containing 1 × 106 spores mL−1 F. graminearum (F.g.). A, Total zealexins. B, Total measured benzoxazinoid hydroxamic acids. C, Cinnamic acid. D, JA. Within each plot different letters (a–d) represent significant differences (all ANOVAs, P < 0.0001; Tukey test corrections for multiple comparisions, P < 0.05).
Figure 7.
Fungal-induced JA and ethylene production promotes zealexin accumulation as demonstrated by the synergistic activity of pharmacological applications. Average (n = 4; ±
sem
). A, JA. B, Ethylene levels in stems that were either untreated (○), or damaged and treated with water (•) or water containing F. graminearum (1 × 106 spores mL−1, ▪). Within each plot and time point, different letters (A–C) represent significant differences (all ANOVAs, P < 0.003; Tukey test corrections for multiple comparisons, P < 0.05). C, Average (n = 4, ±
sem
) total zealexins in maize stems 24 h following damage plus either water, the ethylene releasing chemical ethephon (EP), ethylphosphonic acid (EPA), phosphonic acid (PA), JA, or a combination of JA + EP, JA + EPE, or JA + P. JA was applied at 100 nmol plant−1 as a Na+ salt while EP, EPA, and PA were applied at 33 nmol plant−1 in 10 μL water. Within this plot different letters (a and b) represent significant differences (all ANOVAs, P < 0.0001; Tukey test corrections for multiple comparisons, P < 0.05).
Figure 8.
Zealexins are ubiquitous in maize and inducible in stems by insect herbivory, mycotoxigenic fungi, and nontoxigenic fungi. A, Average (n = 4, ±
sem
) total zealexins, arranged from low to high, in the scutella of 10-d-old maize seedlings from 23 diverse inbred maize lines. B, Time course of average total zealexin (n = 3, ±
sem
) accumulation in maize stems following no treatment (○), damage (•) or damage + O. nubilalis herbivory (▾). C, Average (n = 4, ±
sem
) total zealexins in maize stems 48 h after the plants were damaged and treated with either water (D) or water containing mycotoxigenic A. flavus (△) and nonmycotoxigenic A. sojae (▲) at a range of inoculum levels. D, Average (n = 4, ±
sem
) total zealexins in maize stems 48 h after either no treatment (C) or damage plus either water (D), YEP, or U. maydis: a1b1 a2b2, or mated, each at final concentration of 1 × 106 cells mL−1 in YEP. Within plots, different letters (a–d) represent significant differences (ANOVA P value < 0.001; Tukey test corrections for multiple comparisons: P < 0.05).
Figure 9.
Zealexins inhibit fungal growth at physiologically relevant concentrations. Average (n = 8, ±
sem
). A, Zealexin A1. D, Zealexin A2. G, Zealexin A3 influence on the time course of R. microsporus growth in nutrient broth at 0 (▿), 25 (gray triangle), and 100 (▾) μg mL−1. B, Zealexin A1. E, Zealexin A2. H, Zealexin A3 influence on the time course of A. flavus growth in nutrient broth at 0 (△), 25 (gray triangle), and 100 (▲) μg mL−1. C, Zealexin A1. F, Zealexin A2. I, Zealexin A3 influence on the time course of F. graminearum growth in nutrient broth at 0 (□), 25 (gray square), and 100 (▪) μg mL−1. Within plots, different letters (a–c) represent significant differences (all ANOVAs P < 0.006; Tukey test corrections for multiple comparisons: _P_ < 0.05). Not statistically different (NSD) indicates ANOVA _P_ > 0.05.
References
- Bhatnagar D, Yu J, Ehrlich KC. (2002) Toxins of filamentous fungi. Chem Immunol 81: 167–206 -PubMed
- Bily AC, Reid LM, Taylor JH, Johnston D, Malouin C, Burt AJ, Bakan B, Regnault-Roger C, Pauls KP, Arnason JT, et al. (2003) Dehydrodimers of ferulic acid in maize grain pericarp and aleurone: resistance factors to Fusarium graminearum. Phytopathology 93: 712–719 -PubMed
- Boué SM, Carter CH, Ehrlich KC, Cleveland TE. (2000) Induction of the soybean phytoalexins coumestrol and glyceollin by Aspergillus. J Agric Food Chem 48: 2167–2172 -PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Medical
Research Materials
Miscellaneous