Aspergillus flavus Exploits Maize Kernels Using an "Orphan" Secondary Metabolite Cluster

Abstract

Aspergillus flavus is a saprophytic cosmopolitan fungus, capable of infecting crops both pre- and post-harvest and exploiting different secondary metabolites, including aflatoxins. Aflatoxins are known carcinogens to animals and humans, but display no clear effect in host plants such as maize. In a previous study, we mined the genome of A. flavus to identify secondary metabolite clusters putatively involving the pathogenesis process in maize. We now focus on cluster 32, encoding for fungal effectors such as salicylate hydroxylase (SalOH), and necrosis- and ethylene-inducing proteins (npp1 domain protein) whose expression is triggered upon kernel contact. In order to understand the role of this genetic cluster in maize kernel infection, mutants of A. flavus, impaired or enhanced in specific functions (e.g., cluster 32 overexpression), were studied for their ability to cause disease. Within this frame, we conducted histological and histochemical experiments to verify the expression of specific genes within the cluster (e.g., SalOH, npp1), the production of salicylate, and the presence of its dehydroxylated form. Results suggest that the initial phase of fungal infection (2 days) of the living tissues of maize kernels (e.g., aleuron) coincides with a significant increase of fungal effectors such as SalOH and Npp1 that appear to be instrumental in eluding host defences and colonising the starch-enriched tissues, and therefore suggest a role of cluster 32 to the onset of infection.

Keywords: Aspergillus flavus; effectors; histology; maize kernel; npp1; quercetin; salicylate hydroxylase.

Figure 1

Histological assays showing the progression of the infection by different strains at 2, 3 and 4 dai. MockB73 (a–c) tissues were not infected at any time point. Wild-type AF3357 (d–f) spores are present just outside the kernel at 2 dai (d), and at 4 dai the fungus had directly invaded the aleuronic cells as shown by the arrow (f). Starting at 2 dai, the AFC-1 mutant (g–i) experienced visible vacuolisation of aleuronic cells (arrows in g) as symptoms of PCD onset, progressing to 4 dai with ongoing infection (i). The transcription factor overexpressing mutant, Zn₂Cys₆-OE-GFP (j–l), as a consequence of fungal invasion caused detachment of the periderm from the aleuronic layer (arrow in j), then proceeds just outside the aleuronic layer (arrow in k) until the kernel is collapsed (l). Magnification 400×.

Figure 2

TUNEL assay at 2 dai in all the studied strains: MockB73, AF3357 (wild type); AFC-1 (−pyrG, −argD mutant); Zn₂Cys₆-OE-GFP (overexpressing the transcription factor). In all three cases, the cells belong to the aleuronic layer. This assay shows apoptotic (A) nuclei in blue indicated by the arrows. Magnification 1000×.

Figure 3

Relative expression of transcriptional levels for cluster 32 genes SalOH (a), npp1 (b) and AFLA_096260 (c) in fungal strains AF3357, Zn₂Cys₆-OE-GFP and AFC-1 from 14 hrs to 5 dai. Mean values and standard deviations were used to perform t-tests comparing Zn₂Cys₆-OE-GFP or AFC-1 to AF3357 (* p < 0.05; ** p < 0.01).

Figure 3

Relative expression of transcriptional levels for cluster 32 genes SalOH (a), npp1 (b) and AFLA_096260 (c) in fungal strains AF3357, Zn₂Cys₆-OE-GFP and AFC-1 from 14 hrs to 5 dai. Mean values and standard deviations were used to perform t-tests comparing Zn₂Cys₆-OE-GFP or AFC-1 to AF3357 (* p < 0.05; ** p < 0.01).

Figure 4

Relative abundances of salicylic acid (a) and catechol (b) detected in maize when not infected (MockB73) or infected with one of three A. flavus strains. The colours in each bar represent different infection time points.

Figure 5

Histochemical assay for nepA∆-GUS B9-5 and for nepA-OE-GUS B5-12 strains. Timeline follows infection process starting from the pericarp and the endosperm to the embryo region.