The L-type calcium ion channel cch1 affects ascospore discharge and mycelial growth in the filamentous fungus Gibberella zeae (anamorph Fusarium graminearum)

Abstract

Cch1, a putative voltage-gated calcium ion channel, was investigated for its role in ascus development in Gibberella zeae. Gene replacement mutants of CCH1 were generated and found to have asci which did not forcibly discharge spores, although morphologically ascus and ascospore development in the majority of asci appeared normal. Additionally, mycelial growth was significantly slower, and sexual development was slightly delayed in the mutant; mutant mycelia showed a distinctive fluffy morphology, and no cirrhi were produced. Wheat infected with Deltacch1 mutants developed symptoms comparable to wheat infected with the wild type; however, the mutants showed a reduced ability to protect the infected stalk from colonization by saprobic fungi. Transcriptional analysis of gene expression in mutants using the Affymetrix Fusarium microarray showed 2,449 genes with significant, twofold or greater, changes in transcript abundance across a developmental series. This work extends the role of CCH1 to forcible spore discharge in G. zeae and suggests that this channel has subtle effects on growth and development.

FIG. 1.

Southern analysis of Δcch1 mutants and complements compared to the wild type using a probe internal to CCH1 (A) and a probe specific to hph1 (B). Lanes from left to right: 1-kb ladder, wild type (WT), transformant Δcch1-T10, Δcch1-T11, Δcch1-T12, Δcch1-T13, Δcch1-T14, and complements cch1comp6 and cch1comp5. There is an empty lane between the two complements. Transformant Δcch1-T11 is an ectopic insertion of the replacement construct, including hph. Ten micrograms was loaded per lane. Genomic DNA was digested with BamHI. The CCH1 internal probe hybridizes to a BamHI fragment of 2,891 bp in the wild type. The hph1 probe hybridizes to a BamHI fragment of 3,465 bp in Δcch1 replacement mutants. Numbers on the left indicate size in kb.

FIG. 2.

Vegetative growth on carrot agar 96 h after inoculation. (A) Wild type. (B) Ectopic transformant Δcch1-T11. (C and D) Mutants Δcch1-T12 and Δcch1-T14. Note the dense, fluffy growth. (E) Δcch1comp6. Complementation of the Δcch1 deletion mutant restores the wild-type phenotype.

FIG. 3.

Forcible ascospore discharge in the wild type (A), ectopic transformant Δcch1-T11 (B), mutant Δcch1-T12 (C), complement Δcch1comp6 (D), and mutant Δcch1-T12 supplemented with 38.46 mM CaCl₂ (E). Photographs were taken 24 h after the assay was initiated. Other mutants shared a phenotype similar that of to Δcch1-T12.

FIG. 4.

Cirrhus formation and ascospore morphology. Cirrhi were present in the wild type (A; circled) and absent in the mutant Δcch1 (B). Occasional clusters of conidia (arrowheads) were seen on the surface of the mutant perithecia. A moderate number of cch1 mutant asci harbored abnormal spores (C; arrowhead). For panels A and B an Axioskop 2 Plus microscope (Carl Zeiss, Inc., Hallbergmoos, Germany) equipped with differential interference contrast optics was used. An AxioCam HRC (Hallbergmoos) photomicrographic system attached to the microscope was used to capture images. The photograph in panel C was taken on a Nikon Labophot 2 microscope using phase optics and a Kodak DCS Pro camera (Rochester, NY). Scale bars, 180 μm (A and B) and 5 μm (C).

FIG. 5.

The effect of supplemental calcium (CaCl₂) on vegetative growth. (A) Wild-type PH-1. (B) Mutant Δcch1-T12 (other Δcch1 mutants showed the same effect). From left to right, the CaCl₂ concentrations are 0.77, 7.69, 38.46, and 76.92 mM.

FIG. 6.

The effects of the calcium chelator BAPTA on growth of the wild type (A), ectopic transformant Δcch1-T11 (B), and mutant Δcch1-T12 (C) at 9 days postinoculation. Mutant Δcch1-T14 (not shown) gave a similar phenotype to Δcch1-T12.