The Magnaporthe oryzae genes FLB3 and FLB4, orthologues of the Aspergillus nidulans regulators of conidiation FlbC and FlbD, were inactivated. These genes encode C2H2 zinc finger and Myb-like transcription factors, respectively, in A. nidulans. Analysis of the resultant mutants demonstrated that FLB4 is essential for spore formation and that strains lacking this gene are fluffy in their colony morphology due to an inability to complete conidiophore formation. Meanwhile, FLB3 is required for normal levels of aerial mycelium formation. We identified genes dependent on both transcription factors using microarray analysis. This analysis revealed that the transcription of several genes encoding proteins implicated in sporulation in Magnaporthe oryzae and other filamentous fungi are affected by FLB3 or FLB4 inactivation. Furthermore, the microarray analysis indicates that Flb3p may effectively reprogramme the cell metabolically by repressing transcription of genes encoding biosynthetic enzymes and inducing transcription of genes encoding catabolic enzymes. Additionally, qRT-PCR was employed and showed that FLB3 and FLB4 transcripts are enriched in synchronously sporulating cultures, as were the transcripts of other genes that are necessary for normal conidiation, consistent with a role for their gene products in this process.

A forward genetics approach was applied in order to investigate the molecular basis of morphological transition in the wheat pathogenic fungus Zymoseptoria tritici. Z. tritici is a dimorphic plant pathogen displaying environmentally regulated morphogenetic transition between yeast-like and hyphal growth. Considering the infection mode of Z. tritici, the switching to hyphal growth is essential for pathogenicity allowing the fungus the host invasion through natural openings like stomata. We exploited a previously developed Agrobacterium tumefaciens-mediated transformation (ATMT) to generate a mutant library by insertional mutagenesis including more than 10,000 random mutants. To identify genes involved in dimorphic switch, a plate-based screening system was established. With this approach eleven dimorphic switch deficient random mutants were recovered, ten of which exhibited a yeast-like mode of growth and one mutant predominantly growing filamentously, producing high amount of mycelium under different incubation conditions. Using genome walking approach previously established, the T-DNA integration sites were recovered and the disrupted genomic loci of corresponding mutants were identified and validated within reverse genetics approach. As prove of concept, two of the random mutants obtained were selected for further investigation using targeted gene inactivation. Both genes deduced were found to encode known factors, previously characterized in other fungi: Ssk1p being constituent of HOG pathway and Ade5,7p involved in de novo purine biosynthesis. The targeted mutant strains defective in these genes exhibit a drastically impaired virulence within infection assays on whole wheat plants. Moreover exploiting further physiological assays the predicted function for both gene products could be confirmed in concordance with conserved biological role of homologous proteins previously described in other fungal organisms.

Diseases caused by dimorphic phytopathogenic and systemic dimorphic fungi have markedly increased in prevalence in the last decades, and understanding the morphogenic transition to the virulent state might yield novel means of controlling dimorphic fungi. The dimorphic fungus Z. tritici causes significant economic impact on wheat production, and yet the regulation of the dimorphic switch, a key first step in successful plant colonization, is still largely unexplored in this fungus. The fungus is amenable to suppression by fungicides at this switch point, and the identification of the factors controlling the dimorphic switch provides a potential source of novel targets to control Septoria tritici blotch (STB). Inhibition of the dimorphic switch can potentially prevent penetration and avoid any damage to the host plant. The aim of the current work was to unveil genetic determinants of the dimorphic transition in Z. tritici by using a forward genetics strategy. Using this approach, we unveiled two novel factors involved in the switch to the pathogenic state and used reverse genetics and complementation to confirm the role of the novel virulence factors and further gained insight into the role of these genes, using transcriptome analysis via RNA-Seq. The transcriptomes generated potentially contain key determinants of the dimorphic transition.

Different external stimuli are perceived by multiple sensor histidine kinases and transmitted by phosphorylation via the phosphotransfer protein Ypd1p in the multistep phosphorelay system of the high osmolarity glycerol signaling pathway of filamentous fungi. How the signal propagation takes place is still not known in detail since multiple sensor histidine kinase genes in most filamentous fungi are coded in the genome, whereas only one gene for Ypd1p exists. That raises the hypothesis that various Ypd1p isoforms are produced from a single gene sequence, perhaps by alternative splicing, facilitating a higher variability in signal transduction. We found that the mRNA of MoYPD1 in the rice blast fungus Magnaporthe oryzae is subjected to an increased structural variation and amplified putative isoforms on a cDNA level. We then generated mutant strains overexpressing these isoforms, purified the products, and present here one previously unknown MoYpd1p isoform on a proteome level. Alternative splicing was found to be a valid molecular mechanism to increase the signal diversity in eukaryotic multistep phosphorelay systems.