The FgNot3 Subunit of the Ccr4-Not Complex Regulates Vegetative Growth, Sporulation, and Virulence in Fusarium graminearum

Abstract

The Ccr4-Not complex is evolutionarily conserved and important for multiple cellular functions in eukaryotic cells. In this study, the biological roles of the FgNot3 subunit of this complex were investigated in the plant pathogenic fungus Fusarium graminearum. Deletion of FgNOT3 resulted in retarded vegetative growth, retarded spore germination, swollen hyphae, and hyper-branching. The ΔFgnot3 mutants also showed impaired sexual and asexual sporulation, decreased virulence, and reduced expression of genes related to conidiogenesis. Fgnot3 deletion mutants were sensitive to thermal stress, whereas NOT3 orthologs in other model eukaryotes are known to be required for cell wall integrity. We found that FgNot3 functions as a negative regulator of the production of secondary metabolites, including trichothecenes and zearalenone. Further functional characterization of other components of the Not module of the Ccr4-Not complex demonstrated that the module is conserved. Each subunit primarily functions within the context of a complex and might have distinct roles outside of the complex in F. graminearum. This is the first study to functionally characterize the Not module in filamentous fungi and provides novel insights into signal transduction pathways in fungal development.

Fig 1. Molecular characterization of FgNot3.

(A) Occurrences of subunit genes of the Ccr4-Not complex homologs in representative species. The image was constructed using the STRING database [45]. Sc, Saccharomyces cerevisiae; Cg, Candida glabrata; Sp, Schizosaccharomyces pombe; Af, Aspergillus fumigatus; Mo, Magnaporthe oryzae; Fg, Fusarium graminearum; Nc, Neurospora crassa; Cn, Cryptococcus neoformans; Um, Ustilago maydis; Osi, Oryza sativa Indica; Zm, Zea mays; At, Arabidopsis thaliana; Pi, Phytophthora infestans; Pu, Pythium ultimum; Mm, Mus musculus; Hs, Homo sapiens; Dm, Drosophila melanogaster; Ce, Caenorhabditis elegans; Dd, Dictyostelium discoideum. (B) Phylogenetic tree of homologs of the Not3 subunit from the Ccr4-Not complex from representative species constructed using amino acid sequence comparison. (C) Schematic presentation of the conserved regions of Not3 subunit homologs between S. cerevisiae and F. graminearum. The percentage of identity between two proteins was calculated using the ALIGN algorithm (global alignment with no short-cuts). Different shadings denote the domain entries in the InterPro database (http://www.ebi.ac.uk/interpro/) and the HMMPham database (http://pfam.sanger.ac.uk/).

Fig 2. The vegetative growth of ΔFgnot3 mutants.

(A) Mycelial growth on complete medium (CM) and minimal medium (MM). The pictures were taken 5 days after inoculation. The pictures were taken from the upper (top) and the side (middle) of the plates. (B) Microscopic observation of hyphae. The differential interference contrast (DIC) images were taken 2 days after inoculation. Scale bar = 50 μm. (C) Swollen hyphae of ΔFgnot3 mutants on CM agar. Scale bar = 50 μm. WT, F. graminearum wild-type strain Z-3639; ΔFgnot3, FgNOT3 deletion mutant; FgNot3c, ΔFgnot3-derived strain complemented with FgNOT3.

Fig 3. Conidiation and germination of ΔFgnot3 mutants.

(A) Conidial production. The number of conidia was counted after 5 days of incubation in CMC. The values were generated based on three biological replicates. Significant differences (P < 0.05) are indicated with an asterisk. (B) Conidial morphology. Conidia were induced on YMA and subsequently observed by DIC. Scale bar = 20 μm. (C) Morphology of conidiophores in F. graminearum strains. Pictures were taken 1 to 3 days after conidium induction. Scale bar = 10 μm. (D) Relative transcript levels of genes related to conidiation. Total RNA of the wild-type and ΔFgnot3 strains was extracted 18 h after inoculation in CMC. The relative transcript levels of each subunit gene in the wild-type were arbitrarily set to 1. Significant differences (P < 0.05) are indicated with an asterisk. (E) Germination rate. The percentage of conidium germination in CM and MM. WT, F. graminearum wild-type strain Z-3639; ΔFgnot3, FgNOT3 deletion mutant; FgNot3c, ΔFgnot3-derived strain complemented with FgNOT3.

Fig 4. The sexual development and virulence of ΔFgnot3 mutants.

(A) Fertility tests. Each strain was inoculated on carrot agar and mock-fertilized (self-cross) or outcrossed with the corresponding male strains (WT, ΔFgnot3 and Δmat1). The photographs were taken 7 days after sexual induction. Scale bar = 500 μm. (B) Asci of an outcross Δmat1 × ΔFgnot3. Eight ascospores of an ascus from the Δmat1 × ΔFgnot3 outcross showing 1:1 segregation with and without Gfp-tagged histone H1. The photographs were taken 9 days after sexual induction. Scale bar = 10 μm. (C) Virulence on wheat heads. The center spikelet of each wheat head was injected with 10 μl of a conidial suspension. Pictures were taken 21 days after inoculation. Mock, negative control mock-inoculated with 0.01% of Tween 20; WT, F. graminearum wild-type strain Z-3639; ΔFgnot3, FgNOT3 deletion mutant; FgNot3c, ΔFgnot3-derived strain complemented with FgNOT3.

Fig 5. Sensitivity to thermal stress.

(A) Mycelial growth at different temperatures. (B) Mycelial growth inhibition rate. Each strain was inoculated on CM and incubated at 25°C, 29°C, and 31°C for 5 days. The percentage of the mycelial radial growth inhibition was calculated using the following equation: [(C−N)/C] × 100, where C is colony diameter of the control (at 25°C), and N is that of treatments (at 29°C and 31°C) as previously described [48]. The values were generated based on three biological replicates. The significance of differences between the wild-type and each strain was calculated using Student’s t-test. Significant differences (P < 0.05) are indicated with an asterisk for the 29°C condition or a double asterisk for the 31°C condition. WT, F. graminearum wild-type strain Z-3639; ΔFgnot2, FgNOT2 deletion mutant; ΔFgnot3, FgNOT3 deletion mutant; ΔFgnot4, FgNOT4 deletion mutant; FgNot2c, ΔFgnot2-derived strain complemented with FgNOT2; FgNot3c, ΔFgnot3-derived strain complemented with FgNOT3; FgNot4c, ΔFgnot4-derived strain complemented with FgNOT4.

Fig 6. Relative transcript levels of subunits of the Ccr4-Not complex during the conidium induction stage.

Total RNA of the wild-type and ΔFgnot3 strains was extracted 18 h after inoculation in CMC. The relative transcript levels of each subunit in the Ccr4-Not complex in wild type were arbitrarily set to 1. Significant differences (P < 0.05) are indicated with an asterisk.

Fig 7. The vegetative growth of ΔFgnot2 and ΔFgnot4 mutants.

Pictures were taken 4 days after inoculation on CM and MM. WT, wild-type strain Z-3639; ΔFgnot2, FgNOT2 deletion mutant; FgNot2c, ΔFgnot2-derived strain complemented with FgNOT2; ΔFgnot4, FgNOT4 deletion mutant; FgNot4c, ΔFgnot4-derived strain complemented with FgNOT4.

Fig 8. Phenotypes of ΔFgnot2 and ΔFgnot4 mutants.

(A) Conidial morphology. Conidia were induced on YMA and subsequently observed by DIC. Scale bar = 20 μm. (B) Morphologies of conidiophores. Scale bar = 20 μm. (C-D) Fertility tests of ΔFgnot2 (C) and ΔFgnot4 (D). Each strain was inoculated on carrot agar and mock fertilized (self-cross) or outcrossed with a respective male strain (WT, ΔFgnot2, ΔFgnot4, and Δmat1). Pictures were taken 7 days after sexual induction. Scale bar = 500 μm. (E) Virulence on wheat heads. The center spikelet of each wheat head was injected with 10 μl of a conidial suspension. Pictures were taken 21 days after inoculation. WT, wild-type strain Z-3639; ΔFgnot2, FgNOT2 deletion mutant; FgNot2c, ΔFgnot2-derived strain complemented with FgNOT2; ΔFgnot4, FgNOT4 deletion mutant; FgNot4c, ΔFgnot4-derived strain complemented with FgNOT4.

Fig 9. Mycotoxin production of ΔFgnot2, ΔFgnot3, and ΔFgnot4 mutants.

Total trichothecene production or zearalenone (ZEA) production was normalized to fungal ergosterol levels with the following equation as previously described [41]: [trichothecene or ZEA production (μg/g) / ergosterol contents (μg/g)] × 100. The values were generated based on three biological replicates. The significance of differences between the wild-type and each strain was calculated using Student’s t-test. Significant differences (P < 0.05) are indicated with an asterisk. WT, wild-type strain Z-3639; ΔFgnot2, FgNOT2 deletion mutant; FgNot2c, ΔFgnot2-derived strain complemented with FgNOT2; ΔFgnot3, FgNOT3 deletion mutant; FgNot3c, ΔFgnot3-derived strain complemented with FgNOT3; ΔFgnot4, FgNOT4 deletion mutant; FgNot4c, ΔFgnot4-derived strain complemented with FgNOT4.