A novel gene, ROA, is required for normal morphogenesis and discharge of ascospores in Gibberella zeae

Abstract

Head blight, caused by Gibberella zeae, is a significant disease among cereal crops, including wheat, barley, and rice, due to contamination of grain with mycotoxins. G. zeae is spread by ascospores forcibly discharged from sexual fruiting bodies forming on crop residues. In this study, we characterized a novel gene, ROA, which is required for normal sexual development. Deletion of ROA (Δroa) resulted in an abnormal size and shape of asci and ascospores but did not affect vegetative growth. The Δroa mutation triggered round ascospores and insufficient cell division after spore delimitation. The asci of the Δroa strain discharged fewer ascospores from the perithecia but achieved a greater dispersal distance than those of the wild-type strain. Turgor pressure within the asci was calculated through the analysis of osmolytes in the epiplasmic fluid. Deletion of the ROA gene appeared to increase turgor pressure in the mutant asci. The higher turgor pressure of the Δroa mutant asci and the mutant spore shape contributed to the longer distance dispersal. When the Δroa mutant was outcrossed with a Δmat1-2 mutant, a strain that contains a green fluorescence protein (GFP) marker in place of the MAT1-2 gene, unusual phenotypic segregation occurred. The ratio of GFP to non-GFP segregation was 1:1; however, all eight spores had the same shape. Taken together, the results of this study suggest that ROA plays multiple roles in maintaining the proper morphology and discharge of ascospores in G. zeae.

Fig. 1.

Targeted deletion and complementation of ROA. For each panel, the left and right sides show the strategy and Southern analysis, respectively. (A) Targeted deletion of ROA from the genome of the wild-type strain. E, EcoRV; gen, Geneticin resistance gene; lane 1, wild-type strain; lane 2, Δroa mutant. The 5′ flanking region of ROA (black bar) was used as a probe. (B) Complementation of ROA in the Δroa strain. H, HindIII; hygB, hygromycin resistance gene; lane 1, wild-type strain; lane 2, Δroa; lanes 3, Δroa::ROA. The partial ROA ORF (black bar) and hygB ORF (gray bar) were used as probes for the left and right blots, respectively. The sizes of the standards (kb) are indicated on the left of each blot.

Fig. 2.

Morphology of asci rosettes and ascospores. (A and D) Microscopic observation of 8-DAI asci rosettes of the wild-type (WT) (A) or Δroa (D) strain. (B and E) Discharged ascospores of the wild-type (B) or Δroa (E) strain. (C and F) Nuclei of the discharged ascospores were stained with acriflavin. Scale bar = 20 μm.

Fig. 3.

Light microscopy of developing asci and ascospores. Perithecia of wild-type (A, B, and C) or Δroa (D, E, and F) strains were stained with toluidine blue. Perithecia were collected from carrot agar 5, 7, and 9 days after sexual induction (DAI). Scale bar = 20 μm.

Fig. 4.

Forcible ascospore discharge of the wild-type (WT), Δroa, and Δroa::ROA strains. Photographs were taken 48 h after the assay was initiated. A semicircular agar block (arrowhead) covered with perithecia was placed on a coverslip in the chamber. This placement allowed for ascospores (arrow) to be discharged horizontally down the length of the chamber onto the coverslip.

Fig. 5.

Number of ascospores discharged at indicated distances in still air. Wild-type (black bar), Δroa (dashed bar), and complement Δroa::ROA strains (white bar) are shown. One thousand spores from each strain were assessed.

Fig. 6.

Mass of epiplasmic fluid components per ascus. Wild-type (black bar), Δroa (dashed bar), and complement Δroa::ROA (white bar) strains are shown.

Fig. 7.

High-performance liquid chromatography (HPLC) chromatogram of polyol standards (A), polyols in the epiplasmic fluid of the wild-type strain (B), or polyols in the epiplasmic fluid of the Δroa strain (C). Peaks 1 to 5 are glycerol, erythritol, arabitol, mannitol, and glucose, respectively.

Fig. 8.

Outcrosses between Δmat1 female and Δroa male. In the heterothallic Δmat1 strain, the histone H1 gene was fused with GFP. DIC, differential interference contrast image; GFP, GFP fluorescence image; DIC + GFP, DIC images merged with GFP fluorescence image. Scale bar = 20 μm.

Fig. 9.

Localization of ROA in mycelia (A and B) or ascospores (C and D). Young asci before spore delimitation and ascospores within asci are indicated by arrowheads and arrows, respectively. DIC, DIC image; GFP, GFP fluorescence image. Scale bar = 20 μm.