A Large-Scale Functional Analysis of Putative Target Genes of Mating-Type Loci Provides Insight into the Regulation of Sexual Development of the Cereal Pathogen Fusarium graminearum.

Fusarium graminearum, the causal agent of Fusarium head blight in cereal crops, produces sexual progeny (ascospore) as an important overwintering and dissemination strategy for completing the disease cycle. This homothallic ascomycetous species does not require a partner for sexual mating; instead, it carries two opposite mating-type (MAT) loci in a single nucleus to control sexual development. To gain a comprehensive understanding of the regulation of sexual development in F. graminearum, we used in-depth and high-throughput analyses to examine the target genes controlled transcriptionally by two-linked MAT loci (MAT1-1, MAT1-2). We hybridized a genome-wide microarray with total RNAs from F. graminearum mutants that lacked each MAT locus individually or together, and overexpressed MAT1-2-1, as well as their wild-type progenitor, at an early stage of sexual development. A comparison of the gene expression levels revealed a total of 1,245 differentially expressed genes (DEGs) among all of the mutants examined. Among these, genes involved in metabolism, cell wall organization, cellular response to stimuli, cell adhesion, fertilization, development, chromatin silencing, and signal transduction, were significantly enriched. Protein binding microarray analysis revealed the presence of putative core DNA binding sequences (ATTAAT or ATTGTT) for the HMG (high mobility group)-box motif in the MAT1-2-1 protein. Targeted deletion of 106 DEGs revealed 25 genes that were specifically required for sexual development, most of which were regulated transcriptionally by both the MAT1-1 and MAT1-2 loci. Taken together with the expression patterns of key target genes, we propose a regulatory pathway for MAT-mediated sexual development, in which both MAT loci may be activated by several environmental cues via chromatin remodeling and/or signaling pathways, and then control the expression of at least 1,245 target genes during sexual development via regulatory cascades and/or networks involving several downstream transcription factors and a putative RNA interference pathway.

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

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.

A split luciferase complementation assay for studying in vivo protein-protein interactions in filamentous ascomycetes.

Protein-protein interactions play important roles in controlling many cellular events. To date, several techniques have been developed for detection of protein-protein interactions in living cells, among which split luciferase complementation has been applied in animal and plant cells. Here, we examined whether the split luciferase assay could be used in filamentous ascomycetes, such as Gibberella zeae and Cochliobolus heterostrophus. The coding sequences of two strongly interacting proteins (the F-box protein, FBP1, and its partner SKP1) in G. zeae, under the control of the cryparin promoter from Cryphonectria parasitica, were translationally fused to the C- and N-terminal fragments of firefly luciferase (luc), respectively. Each fusion product inserted into a fungal transforming vector carrying the gene for resistance to either geneticin or hygromycin B, was transformed into both fungi. We detected complementation of split luciferase proteins driven by interaction of the two fungal proteins with a high luminescence intensity-to-background ratio only in the fungal transformants expressing both N-luc and C-luc fusion constructs. Using this system, we also confirmed a novel protein interaction between transcription factors, GzMCM1 and FST12 in G. zeae, which could hardly be proven by the yeast two-hybrid method. This is the first study demonstrating that monitoring of split luciferase complementation is a sensitive and efficient method of studying in vivo protein-protein interactions in filamentous ascomycetes.