Oxylipins from both pathogen and host antagonize jasmonic acid-mediated defence via the 9-lipoxygenase pathway in Fusarium verticillioides infection of maize

Abstract

Oxylipins are a newly emerging group of signals that serve defence roles or promote virulence. To identify specific host and fungal genes and oxylipins governing the interactions between maize and Fusarium verticillioides, maize wild-type and lipoxygenase3 (lox3) mutant were inoculated with either F. verticillioides wild-type or linoleate-diol-synthase 1-deleted mutant (ΔFvlds1D). The results showed that lox3 mutants were more resistant to F. verticillioides. The reduced colonization on lox3 was associated with reduced fumonisin production and with a stronger and earlier induction of ZmLOX4, ZmLOX5 and ZmLOX12. In addition to the reported defence function of ZmLOX12, we showed that lox4 and lox5 mutants were more susceptible to F. verticillioides and possessed decreased jasmonate levels during infection, suggesting that these genes are essential for jasmonic acid (JA)-mediated defence. Oxylipin profiling revealed a dramatic reduction in fungal linoleate diol synthase 1 (LDS1)-derived oxylipins, especially 8-HpODE (8-hydroperoxyoctadecenoic acid), in infected lox3 kernels, indicating the importance of this molecule in virulence. Collectively, we make the following conclusions: (1) LOX3 is a major susceptibility factor induced by fungal LDS1-derived oxylipins to suppress JA-stimulating 9-LOXs; (2) LOX3-mediated signalling promotes the biosynthesis of virulence-promoting oxylipins in the fungus; and (3) both fungal LDS1- and host LOX3-produced oxylipins are essential for the normal infection and colonization processes of maize seed by F. verticillioides.

Keywords: Fusarium; jasmonic acid; linoleate diol synthase; maize; oxylipin cross-talk; susceptibility genes.

Figure 1

Dynamics of disease progression of Fusarium verticillioides (FvWT)‐infected B73 maize kernels. (A) Colonization. (B) Conidiation. (C) Fumonisin B1 accumulation. (D) Fungal biomass‐dependent conidiation. (E) Fungal biomass‐dependent fumonisin B1 accumulation. (F) Overview of disease progression. These factors were determined for B73 seed infected by the FvWT strain over a time course of 15 days post‐inoculation (dpi). Error bars indicate ± standard error (SE).

Figure 2

Colonization (based on ergosterol) (A), fumonisin accumulation (B) and conidiation as a function of fungal biomass as measured through ergosterol (C) on wild‐type (B73) and lox3 mutant maize kernels inoculated with wild‐type (FvWT) and oxylipin‐deficient (ΔFvlds1D) Fusarium verticillioides strains at 6 and 9 days post‐inoculation (dpi). Error bars indicate ± standard error (SE). The same letters above the histograms denote insignificant differences between means of the treatments within the same time point, resulting from Tukey's honestly significant difference test (P < 0.05).

Figure 3

Relative expression of ZmLOX1 (A), ZmLOX2 (B), ZmLOX3 (C), ZmLOX4 (D), ZmLOX5 (E) and ZmLOX12 (F) in wild‐type (B73) and lox3 mutant maize kernels inoculated with wild‐type (FvWT) and oxylipin‐deficient (ΔFvlds1D) Fusarium verticillioides strains at 6 and 9 days post‐inoculation (dpi). Error bars indicate ± standard error (SE). The same letters above the histograms denote insignificant differences between means of the treatments within the same time point, resulting from Tukey's honestly significant difference test (P < 0.05).

Figure 4

Production of conidia of Fusarium verticillioides (FvWT) was increased when grown on kernels of the lox4 and lox5 mutants compared to the wild type (B73). (A, C) Visual observation of fungal mycelial growth on lox4 and lox5 mutants, and near‐isogenic wild type kernels (NIL WT) 13 days post inoculation with F. verticillioides. The fungus easily colonized lox4 and lox5 mutant kernels more aggressively compared with wild type kernels. (B, D) Number of conidia produced on infected seed of maize lox4 and lox5 mutants, and NIL WTs, at 13 days post inoculation. (E, F) 12‐OPDA and JA content of lox4, lox5, and lox12 mutant kernels compared to WT. Vertical bars indicate ± se. Unconnected letters represent statistical differences within day post infection (DPI) by Tukey's HSD on log‐transformed data.

Figure 5

Relative expression of FvLOX1 (A), FvLDS1 (B), FvLDS2 (C) and FvFAO (D) genes in wild‐type (B73) and lox3 mutant maize kernels inoculated with wild‐type (FvWT) and oxylipin‐deficient (ΔFvlds1D) Fusarium verticillioides strains at 6 and 9 days post‐inoculation (dpi). Error bars indicate ± standard error (SE). The same letters above the histograms denote insignificant differences between means of the treatments within the same time point, resulting from Tukey's honestly significant difference test (P < 0.05).

Figure 6

Multiple reaction monitoring (MRM) quantification of linoleate diol synthase (LDS)‐derived oxylipins [8‐hydroperoxyoctadecenoic acid, 8‐HpODE (A); 8,13‐dihydroxyoctadecenoic acid, 8,13‐diHODE (B); 9,10‐dihydroxymonoenoic acid, 9,10‐diHOME (C); 12,13‐dihydroxymonoenoic acid, 12,13‐diHOME (D); 9(10)‐epoxymonoenoic acid, 9(10)‐epoOME (E); 12(13)‐epoxymonoenoic acid, 12(13)‐epoOME (F)] in wild‐type (B73) and lox3 mutant maize kernels inoculated with wild‐type (FvWT) and oxylipin‐deficient (ΔFvlds1D) Fusarium verticillioides strains at 6 and 9 days post‐inoculation (dpi). Error bars indicate ± standard error (SE).

Figure 7

Multiple reaction monitoring (MRM) quantification of lipoxygenase (LOX)‐derived oxylipins [9‐oxo‐octadecenoic acid, 9‐oxoODE (A); 13‐oxo‐octadecenoic acid, 13‐oxoODE (B); 9‐hydroxyoctadecenoic acid, 9‐HODE (C); 13‐hydroxyoctadecenoic acid, 13‐HODE (D); 9‐hydroxyoctatrienoic acid, 9‐HOTrE (E); 13‐hydroxyoctatrienoic acid, 13‐HOTrE (F)] in wild‐type (B73) and lox3 mutant maize kernels inoculated with wild‐type (FvWT) and oxylipin‐deficient (ΔFvlds1D) Fusarium verticillioides strains at 6 and 9 days post‐inoculation (dpi). Error bars indicate ± standard error (SE).

Figure 8

Model depicting the oxylipin‐mediated cross‐talk between maize and Fusarium verticillioides uncovered in this study. Polyunsaturated fatty acids (PUFAs) are substrates for maize or F. verticillioides oxygenases. In the host, the pathogenicity‐promoting ZmLOX3 is the primary isoform responsible for the biosynthesis of 9‐KOT and 9‐HOT in seed. In the fungus, the virulence factor, FvLDS1, appears to be responsible for the production of 9‐oxylipins that induce the expression of ZmLOX3 in infected seed. The increased production of ZmLOX3‐derived 9‐KOT, 9‐HOT or their downstream products suppresses the transcription of the defence‐related LOX isoforms, ZmLOX4, ZmLOX5 and ZmLOX12, which would normally catalyse the production of additional 9‐oxylipins to positively promote JA‐mediated defences to suppress F. verticillioides seed colonization. Symbols in red and blue represent metabolites and enzymes, respectively. The enzymes functionally characterized by this study are shown in bold. AOS, allene oxide synthase; DOX, dioxygenase; FAO, fatty acid oxygenase; 9‐HOT, 9‐hydroxyoctadecatrienoic acid; JA, jasmonic acid; 9‐KOT, 9‐ketooctadecatrienoic acid; LDS, linoleate diol synthase; LOX, lipoxygenase.