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Functional roles of FgLaeA in controlling secondary metabolism, sexual development, and virulence in Fusarium graminearum.

Kim HK, Lee S, Jo SM, McCormick SP, Butchko RA, Proctor RH, Yun SH - PLoS ONE (2013)

Bottom Line: In addition, ΔFgLaeA strains exhibited an earlier induction of sexual fruiting body (perithecia) formation and drastically reduced disease symptoms in wheat, indicating that FgLaeA seems to negatively control perithecial induction, but positively control virulence toward the host plant.A split luciferase assay demonstrated that FgLaeA was able to interact with FgVeA, a homolog of A. nidulans veA.Taken together, these results demonstrate that FgLaeA, a member of putative FgVeA complex, controls secondary metabolism, sexual development, and virulence in F. graminearum, although the specific regulation pattern differs from that of LaeA in A. nidulans.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biotechnology, Soonchunhyang University, Asan, Republic of Korea.

ABSTRACT
Fusarium graminearum, the causal agent of Fusarium head blight in cereal crops, produces mycotoxins such as trichothecenes and zearalenone in infected plants. Here, we focused on the function of FgLaeA in F. graminearum, a homolog of Aspergillus nidulans LaeA encoding the global regulator for both secondary metabolism and sexual development. Prior to gene analysis, we constructed a novel luciferase reporter system consisting of a transgenic F. graminearum strain expressing a firefly luciferase gene under control of the promoter for either TRI6 or ZEB2 controlling the biosynthesis of these mycotoxins. Targeted deletion of FgLaeA led to a dramatic reduction of luminescence in reporter strains, indicating that FgLaeA controls the expression of these transcription factors in F. graminearum; reduced toxin accumulation was further confirmed by GC-MS analysis. Overexpression of FgLaeA caused the increased production of trichothecenes and additional metabolites. RNA seq-analysis revealed that gene member(s) belonging to ~70% of total tentative gene clusters, which were previously proposed, were differentially expressed in the ΔFgLaeA strain. In addition, ΔFgLaeA strains exhibited an earlier induction of sexual fruiting body (perithecia) formation and drastically reduced disease symptoms in wheat, indicating that FgLaeA seems to negatively control perithecial induction, but positively control virulence toward the host plant. FgLaeA was constitutively expressed under both mycotoxin production and sexual development conditions. Overexpression of a GFP-FgLaeA fusion construct in the ΔFgLaeA strain restored all phenotypic changes to wild-type levels and led to constitutive expression of GFP in both nuclei and cytoplasm at different developmental stages. A split luciferase assay demonstrated that FgLaeA was able to interact with FgVeA, a homolog of A. nidulans veA. Taken together, these results demonstrate that FgLaeA, a member of putative FgVeA complex, controls secondary metabolism, sexual development, and virulence in F. graminearum, although the specific regulation pattern differs from that of LaeA in A. nidulans.

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Hyphal growth, pigmentation, and conidiation.(A) Colony morphology of the WT (Z3643), ΔFgLaeA, OE (FgLaeA-overexpression), add-back, ΔFgVeA[32], and ΔFgVelB[33] strains on CM plates. Upper and lower panels show the morphology in surface and undersurface of plates, respectively. (B) The number of conidia formed in CMC liquid medium under dark and light conditions. The y-axis represents the number of conidia (×105 or ×106/mL). Data shown are the mean values obtained from three independent samples. Statistical analysis was performed with ANOVA and Duncan’s multiple range test. The same letter above bars represents no significant difference.
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pone-0068441-g002: Hyphal growth, pigmentation, and conidiation.(A) Colony morphology of the WT (Z3643), ΔFgLaeA, OE (FgLaeA-overexpression), add-back, ΔFgVeA[32], and ΔFgVelB[33] strains on CM plates. Upper and lower panels show the morphology in surface and undersurface of plates, respectively. (B) The number of conidia formed in CMC liquid medium under dark and light conditions. The y-axis represents the number of conidia (×105 or ×106/mL). Data shown are the mean values obtained from three independent samples. Statistical analysis was performed with ANOVA and Duncan’s multiple range test. The same letter above bars represents no significant difference.

Mentions: Compared to wild-type (WT) progenitors Z3643, FLTRI6, and FLZEB2, all ΔFgLaeA strains examined exhibited a slight reduction in radial growth, but a significant reduction in red hyphal pigmentation on potato dextrose agar (PDA; Difco Laboratories, Detroit, MI, USA), CM [3], and carrot agar (Fig. 2). However, this reduction was relatively modest compared with the phenotypes caused by the deletion of FgVeA[33] or FgVelB[34], which showed fewer aerial hyphae with reduced or abolished pigmentation (Fig. 2). The reduced pigmentation phenotype caused by ΔFgLaeA was less robust than a typical albino mutant of F. graminearum (e.g., that generated by a deletion of PKS12, the gene responsible for biosynthesis of the red pigment aurofusarin [37]). Aurofusarin production was still observed in young marginal hyphae of mutant colonies, but older regions of the colony were albino, resulting in ring-shaped pigmentation (Fig. 2). This pigmentation pattern was also seen in the marginal regions between different growing colonies (Fig. S1). Fewer aerial mycelia, with lower hydrophobicity were observed only on the albino region in the ΔFgLaeA strains; however, the formation of thick aerial hyphae was retained in the pigmented margin areas, similar to those from WT strains. This reduced pigmentation phenotype was restored to WT levels in both the FgLaeA-add-back and OE strains (Fig. 2). However, this phenotype is dependent upon growth conditions, as the same ΔFgLaeA strain was completely albino when grown in complete liquid medium (Fig. S1).


Functional roles of FgLaeA in controlling secondary metabolism, sexual development, and virulence in Fusarium graminearum.

Kim HK, Lee S, Jo SM, McCormick SP, Butchko RA, Proctor RH, Yun SH - PLoS ONE (2013)

Hyphal growth, pigmentation, and conidiation.(A) Colony morphology of the WT (Z3643), ΔFgLaeA, OE (FgLaeA-overexpression), add-back, ΔFgVeA[32], and ΔFgVelB[33] strains on CM plates. Upper and lower panels show the morphology in surface and undersurface of plates, respectively. (B) The number of conidia formed in CMC liquid medium under dark and light conditions. The y-axis represents the number of conidia (×105 or ×106/mL). Data shown are the mean values obtained from three independent samples. Statistical analysis was performed with ANOVA and Duncan’s multiple range test. The same letter above bars represents no significant difference.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3713025&req=5

pone-0068441-g002: Hyphal growth, pigmentation, and conidiation.(A) Colony morphology of the WT (Z3643), ΔFgLaeA, OE (FgLaeA-overexpression), add-back, ΔFgVeA[32], and ΔFgVelB[33] strains on CM plates. Upper and lower panels show the morphology in surface and undersurface of plates, respectively. (B) The number of conidia formed in CMC liquid medium under dark and light conditions. The y-axis represents the number of conidia (×105 or ×106/mL). Data shown are the mean values obtained from three independent samples. Statistical analysis was performed with ANOVA and Duncan’s multiple range test. The same letter above bars represents no significant difference.
Mentions: Compared to wild-type (WT) progenitors Z3643, FLTRI6, and FLZEB2, all ΔFgLaeA strains examined exhibited a slight reduction in radial growth, but a significant reduction in red hyphal pigmentation on potato dextrose agar (PDA; Difco Laboratories, Detroit, MI, USA), CM [3], and carrot agar (Fig. 2). However, this reduction was relatively modest compared with the phenotypes caused by the deletion of FgVeA[33] or FgVelB[34], which showed fewer aerial hyphae with reduced or abolished pigmentation (Fig. 2). The reduced pigmentation phenotype caused by ΔFgLaeA was less robust than a typical albino mutant of F. graminearum (e.g., that generated by a deletion of PKS12, the gene responsible for biosynthesis of the red pigment aurofusarin [37]). Aurofusarin production was still observed in young marginal hyphae of mutant colonies, but older regions of the colony were albino, resulting in ring-shaped pigmentation (Fig. 2). This pigmentation pattern was also seen in the marginal regions between different growing colonies (Fig. S1). Fewer aerial mycelia, with lower hydrophobicity were observed only on the albino region in the ΔFgLaeA strains; however, the formation of thick aerial hyphae was retained in the pigmented margin areas, similar to those from WT strains. This reduced pigmentation phenotype was restored to WT levels in both the FgLaeA-add-back and OE strains (Fig. 2). However, this phenotype is dependent upon growth conditions, as the same ΔFgLaeA strain was completely albino when grown in complete liquid medium (Fig. S1).

Bottom Line: In addition, ΔFgLaeA strains exhibited an earlier induction of sexual fruiting body (perithecia) formation and drastically reduced disease symptoms in wheat, indicating that FgLaeA seems to negatively control perithecial induction, but positively control virulence toward the host plant.A split luciferase assay demonstrated that FgLaeA was able to interact with FgVeA, a homolog of A. nidulans veA.Taken together, these results demonstrate that FgLaeA, a member of putative FgVeA complex, controls secondary metabolism, sexual development, and virulence in F. graminearum, although the specific regulation pattern differs from that of LaeA in A. nidulans.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biotechnology, Soonchunhyang University, Asan, Republic of Korea.

ABSTRACT
Fusarium graminearum, the causal agent of Fusarium head blight in cereal crops, produces mycotoxins such as trichothecenes and zearalenone in infected plants. Here, we focused on the function of FgLaeA in F. graminearum, a homolog of Aspergillus nidulans LaeA encoding the global regulator for both secondary metabolism and sexual development. Prior to gene analysis, we constructed a novel luciferase reporter system consisting of a transgenic F. graminearum strain expressing a firefly luciferase gene under control of the promoter for either TRI6 or ZEB2 controlling the biosynthesis of these mycotoxins. Targeted deletion of FgLaeA led to a dramatic reduction of luminescence in reporter strains, indicating that FgLaeA controls the expression of these transcription factors in F. graminearum; reduced toxin accumulation was further confirmed by GC-MS analysis. Overexpression of FgLaeA caused the increased production of trichothecenes and additional metabolites. RNA seq-analysis revealed that gene member(s) belonging to ~70% of total tentative gene clusters, which were previously proposed, were differentially expressed in the ΔFgLaeA strain. In addition, ΔFgLaeA strains exhibited an earlier induction of sexual fruiting body (perithecia) formation and drastically reduced disease symptoms in wheat, indicating that FgLaeA seems to negatively control perithecial induction, but positively control virulence toward the host plant. FgLaeA was constitutively expressed under both mycotoxin production and sexual development conditions. Overexpression of a GFP-FgLaeA fusion construct in the ΔFgLaeA strain restored all phenotypic changes to wild-type levels and led to constitutive expression of GFP in both nuclei and cytoplasm at different developmental stages. A split luciferase assay demonstrated that FgLaeA was able to interact with FgVeA, a homolog of A. nidulans veA. Taken together, these results demonstrate that FgLaeA, a member of putative FgVeA complex, controls secondary metabolism, sexual development, and virulence in F. graminearum, although the specific regulation pattern differs from that of LaeA in A. nidulans.

Show MeSH
Related in: MedlinePlus