Fusarium fujikuroi causes bakanae (“foolish seedling”) disease of rice which is characterized by hyper-elongation of seedlings resulting from production of gibberellic acids (GAs) by the fungus. This plant pathogen is also known for production of harmful mycotoxins, such as fusarins, fusaric acid, apicidin F and beauvericin. Recently, we generated the first de novo genome sequence of F. fujikuroi strain IMI 58289 combined with extensive transcriptional, epigenetic, proteomic and chemical product analyses. GA production was shown to provide a selective advantage during infection of the preferred host plant rice. Here, we provide genome sequences of eight additional F. fujikuroi isolates from distant geographic regions. The isolates differ in the size of chromosomes, most likely due to variability of subtelomeric regions, the type of asexual spores (microconidia and/or macroconidia), and the number and expression of secondary metabolite gene clusters. Whilst most of the isolates caused the typical bakanae symptoms, one isolate, B14, caused stunting and early withering of infected seedlings. In contrast to the other isolates, B14 produced no GAs but high amounts of fumonisins during infection on rice. Furthermore, it differed from the other isolates by the presence of three additional polyketide synthase (PKS) genes (PKS40, PKS43, PKS51) and the absence of the F. fujikuroi-specific apicidin F (NRPS31) gene cluster. Analysis of additional field isolates confirmed the strong correlation between the pathotype (bakanae or stunting/withering), and the ability to produce either GAs or fumonisins. Deletion of the fumonisin and fusaric acid-specific PKS genes in B14 reduced the stunting/withering symptoms, whereas deletion of the PKS51 gene resulted in elevated symptom development. Phylogenetic analyses revealed two subclades of F. fujikuroi strains according to their pathotype and secondary metabolite profiles. Fusarium fujikuroi causes bakanae disease of rice. Infected seedlings appear to be taller and more slender when compared to healthy seedlings due to its ability to produce gibberellic acids (GAs). The disease is responsible for high yield losses, and its incidence varies with regions, rice cultivars grown and the aggressiveness of the fungal isolates. However, not all infected seedlings show bakanae symptoms: one of the isolates, B14, causes stunting and early withering of infected seedlings. The reason for the two pathotypes is not well understood. Researchers thought that the stunting phenotype was mostly caused by fungal-derived secondary metabolites such as fusaric acid, but there is no experimental evidence yet. B14 differs from the other strains by the presence of more PKS gene clusters, low expression of GA genes, lack of detectable levels of GAs and the production of high amounts of fumonisins in rice. Analysis of additional field isolates revealed a strong correlation between the pathotype (bakanae or stunting) and either GA or fumonisin production. Based on phylogenetic analyses, F. fujikuroi strains can be divided into two phylogenetically distinct subclades according to their pathotype and secondary metabolite profiles. This study provides new insights into the genomic variations and the population structure inside the species F. fujikuroi which will help to develop disease control strategies for this rice pathogen.

In this work, we present a comprehensive analysis of the H3K36 histone methyltransferases Set2 and Ash1 in the filamentous ascomycete Fusarium fujikuroi. In Saccharomyces cerevisiae, one single methyltransferase, Set2, confers all H3K36 methylation, while there are two members of the Set2 family in filamentous fungi, and even more H3K36 methyltransferases in higher eukaryotes. Whereas the yeast Set2 homolog has been analyzed in fungi previously, the second member of the Set2 family, designated Ash1, has not been described for any filamentous fungus. Western blot and ChIP-Seq analyses confirmed that F. fujikuroi Set2 and Ash1 are H3K36-specific histone methyltransferases that deposit H3K36me3 at specific loci: Set2 is most likely responsible for H3K36 methylation of euchromatic regions of the genome, while Ash1 methylates H3K36 at the subtelomeric regions (facultative heterochromatin) of all chromosomes including the accessory chromosome XII. Our data indicate that H3K36me3 cannot be considered a hallmark of euchromatin in F. fujikuroi, and likely also other filamentous fungi, making them different to what is known about nuclear characteristics in yeast and higher eukaryotes. We suggest that the H3K36 methylation mark exerts specific functions when deposited at euchromatic or subtelomeric regions by Set2 or Ash1, respectively. We found an enhanced level of H3K27me3, an increased instability of subtelomeric regions and losses of the accessory chromosome XII over time in Δash1 mutants, indicating an involvement of Ash1 in DNA repair processes. Further phenotypic analyses revealed a role of H3K36 methylation in vegetative growth, sporulation, secondary metabolite biosynthesis and virulence in F. fujikuroi.

Summary: Here we present the identification and characterization of the H3K4‐specific histone methyltransferase Set1 and its counterpart, the Jumonji C demethylase Kdm5, in the rice pathogen Fusarium fujikuroi . While Set1 is responsible for all detectable H3K4me2/me3 in this fungus, Kdm5 antagonizes the H3K4me3 mark. Notably, deletion of both SET1 and KDM5 mainly resulted in the upregulation of genome‐wide transcription, also affecting a large set of secondary metabolite (SM) key genes. Although H3K4 methylation is a hallmark of actively transcribed euchromatin, several SM gene clusters located in subtelomeric regions were affected by Set1 and Kdm5. While the regulation of many of them is likely indirect, H3K4me2 levels at gibberellic acid (GA) genes correlated with GA biosynthesis in the wild type, Δ kdm5 and OE:: KDM5 under inducing conditions. Whereas Δ set1 showed an abolished GA ₃ production in axenic culture, phytohormone biosynthesis was induced in planta , so that residual amounts of GA ₃ were detected during rice infection. Accordingly, Δ set1 exhibited a strongly attenuated, though not abolished, virulence on rice. Apart from regulating secondary metabolism, Set1 and Kdm5 function as activator and repressor of conidiation respectively. They antagonistically regulate H3K4me3 levels and expression of the major conidiation‐specific transcription factor gene ABA1 in F. fujikuroi .

The fungus F. fujikuroi is well known for its production of gibberellins causing the ‘bakanae’ disease of rice. Besides these plant hormones, it is able to produce other secondary metabolites (SMs), such as pigments and mycotoxins. Genome sequencing revealed altogether 45 potential SM gene clusters, most of which are cryptic and silent. In this study we characterize a new non-ribosomal peptide synthetase (NRPS) gene cluster that is responsible for the production of the cyclic tetrapeptide apicidin F (APF). This new SM has structural similarities to the known histone deacetylase inhibitor apicidin. To gain insight into the biosynthetic pathway, most of the 11 cluster genes were deleted, and the mutants were analyzed by HPLC-DAD and HPLC-HRMS for their ability to produce APF or new derivatives. Structure elucidation was carried out be HPLC-HRMS and NMR analysis. We identified two new derivatives of APF named apicidin J and K. Furthermore, we studied the regulation of APF biosynthesis and showed that the cluster genes are expressed under conditions of high nitrogen and acidic pH in a manner dependent on the nitrogen regulator AreB, and the pH regulator PacC. In addition, over-expression of the atypical pathway-specific transcription factor (TF)-encoding gene APF2 led to elevated expression of the cluster genes under inducing and even repressing conditions and to significantly increased product yields. Bioinformatic analyses allowed the identification of a putative Apf2 DNA-binding (“Api-box”) motif in the promoters of the APF genes. Point mutations in this sequence motif caused a drastic decrease of APF production indicating that this motif is essential for activating the cluster genes. Finally, we provide a model of the APF biosynthetic pathway based on chemical identification of derivatives in the cultures of deletion mutants.

The pathogenic fungus Aspergillus fumigatus is able to adapt to extremely variable environmental conditions. The A. fumigatus genome contains four genes coding for mitogen-activated protein kinases (MAPKs), which are important regulatory knots involved in diverse cellular responses. From a clinical perspective, MAPK activity has been connected to salvage pathways, which can determine the failure of effective treatment of invasive mycoses using antifungal drugs. Here, we report the characterization of the Saccharomyces cerevisiae Fus3 ortholog in A. fumigatus, designated MpkB. We demonstrate that MpkB is important for conidiation and that its deletion induces a copious increase of dihydroxynaphthalene (DHN)-melanin production. Simultaneous deletion of mpkB and mpkA, the latter related to maintenance of the cell wall integrity, normalized DHN-melanin production. Localization studies revealed that MpkB translocates into the nuclei when A. fumigatus germlings are exposed to caspofungin stress, and this is dependent on the cross-talk interaction with MpkA. Additionally, DHN-melanin formation was also increased after deletion of genes coding for the Gα protein GpaA and for the G protein-coupled receptor GprM. Yeast two-hybrid and coimmunoprecipitation assays confirmed that GpaA and GprM interact, suggesting their role in the MpkB signaling cascade. IMPORTANCEAspergillus fumigatus is the most important airborne human pathogenic fungus, causing thousands of deaths per year. Its lethality is due to late and often inaccurate diagnosis and the lack of efficient therapeutics. The failure of efficient prophylaxis and therapy is based on the ability of this pathogen to activate numerous salvage pathways that are capable of overcoming the different drug-derived stresses. A major role in the protection of A. fumigatus is played by melanins. Melanins are cell wall-associated macromolecules classified as virulence determinants. The understanding of the various signaling pathways acting in this organism can be used to elucidate the mechanism beyond melanin production and help to identify ideal drug targets.