Comparative genomics of geographically distant Fusarium fujikuroi isolates revealed two distinct pathotypes correlating with secondary metabolite profiles.

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.

Set1 and Kdm5 are antagonists for H3K4 methylation and regulators of the major conidiation-specific transcription factor gene ABA1 in Fusarium fujikuroi.

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, Δkdm5 and OE::KDM5 under inducing conditions. Whereas Δset1 showed an abolished GA3 production in axenic culture, phytohormone biosynthesis was induced in planta, so that residual amounts of GA3 were detected during rice infection. Accordingly, Δset1 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.

Secondary metabolism in Fusarium fujikuroi: strategies to unravel the function of biosynthetic pathways.

The fungus Fusarium fujikuroi causes bakanae disease of rice due to its ability to produce the plant hormones, the gibberellins. The fungus is also known for producing harmful mycotoxins (e.g., fusaric acid and fusarins) and pigments (e.g., bikaverin and fusarubins). However, for a long time, most of these well-known products could not be linked to biosynthetic gene clusters. Recent genome sequencing has revealed altogether 47 putative gene clusters. Most of them were orphan clusters for which the encoded natural product(s) were unknown. In this review, we describe the current status of our research on identification and functional characterizations of novel secondary metabolite gene clusters. We present several examples where linking known metabolites to the respective biosynthetic genes has been achieved and describe recent strategies and methods to access new natural products, e.g., by genetic manipulation of pathway-specific or global transcritption factors. In addition, we demonstrate that deletion and over-expression of histone-modifying genes is a powerful tool to activate silent gene clusters and to discover their products.

Analysis of the global regulator Lae1 uncovers a connection between Lae1 and the histone acetyltransferase HAT1 in Fusarium fujikuroi.

The fungus Fusarium fujikuroi causes "bakanae" disease of rice due to its ability to produce gibberellins (GAs), a family of plant hormones. Recent genome sequencing revealed the genetic capacity for the biosynthesis of 46 additional secondary metabolites besides the industrially produced GAs. Among them are the pigments bikaverin and fusarubins, as well as mycotoxins, such as fumonisins, fusarin C, beauvericin, and fusaric acid. However, half of the potential secondary metabolite gene clusters are silent. In recent years, it has been shown that the fungal specific velvet complex is involved in global regulation of secondary metabolism in several filamentous fungi. We have previously shown that deletion of the three components of the F. fujikuroi velvet complex, vel1, vel2, and lae1, almost totally abolished biosynthesis of GAs, fumonisins and fusarin C. Here, we present a deeper insight into the genome-wide regulatory impact of Lae1 on secondary metabolism. Over-expression of lae1 resulted in de-repression of GA biosynthetic genes under otherwise repressing high nitrogen conditions demonstrating that the nitrogen repression is overcome. In addition, over-expression of one of five tested histone acetyltransferase genes, HAT1, was capable of returning GA gene expression and GA production to the GA-deficient Δlae1 mutant. Deletion and over-expression of HAT1 in the wild type resulted in downregulation and upregulation of GA gene expression, respectively, indicating that HAT1 together with Lae1 plays an essential role in the regulation of GA biosynthesis.

Gibepyrone Biosynthesis in the Rice Pathogen Fusarium fujikuroi Is Facilitated by a Small Polyketide Synthase Gene Cluster.

The 2H-pyran-2-one gibepyrone A and its oxidized derivatives gibepyrones B-F have been isolated from the rice pathogenic fungus Fusarium fujikuroi already more than 20 years ago. However, these products have not been linked to the respective biosynthetic genes, and therefore, their biosynthesis has not yet been analyzed on a molecular level. Feeding experiments with isotopically labeled precursors clearly supported a polyketide origin for the formal monoterpenoid gibepyrone A, whereas the terpenoid pathway could be excluded. Targeted gene deletion verified that the F. fujikuroi polyketide synthase PKS13, designated Gpy1, is responsible for gibepyrone A biosynthesis. Next to Gpy1, the ATP-binding cassette transporter Gpy2 is encoded by the gibepyrone gene cluster. Gpy2 was shown to have only a minor impact on the actual efflux of gibepyrone A out of the cell. Instead, we obtained evidence that Gpy2 is involved in gene regulation as it represses GPY1 gene expression. Thus, GPY1 was up-regulated and gibepyrone A production was enhanced both extra- and intracellularly in Δgpy2 mutants. Furthermore, expression of GPY genes is strictly repressed by members of the fungus-specific velvet complex, Vel1, Vel2, and Lae1, whereas Sge1, a major regulator of secondary metabolism in F. fujikuroi, affects gibepyrone biosynthesis in a positive manner. The gibepyrone A derivatives gibepyrones B and D were shown to be produced by cluster-independent P450 monooxygenases, probably to protect the fungus from the toxic product. In contrast, the formation of gibepyrones E and F from gibepyrone A is a spontaneous process and independent of enzymatic activity.

The SAGA complex in the rice pathogen Fusarium fujikuroi: structure and functional characterization.

Post-translational modification of histones is a crucial mode of transcriptional regulation in eukaryotes. A well-described acetylation modifier of certain lysine residues is the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex assembled around the histone acetyltransferase Gcn5 in Saccharomyces cerevisiae. We identified and characterized the SAGA complex in the rice pathogen Fusarium fujikuroi, well-known for producing a large variety of secondary metabolites (SMs). By using a co-immunoprecipitation approach, almost all of the S. cerevisiae SAGA complex components have been identified, except for the ubiquitinating DUBm module and the chromodomain containing Chd1. Deletion of GCN5 led to impaired growth, loss of conidiation and alteration of SM biosynthesis. Furthermore, we show that Gcn5 is essential for the acetylation of several histone 3 lysines in F. fujikuroi, that is, H3K4, H3K9, H3K18 and H3K27. A genome-wide microarray analysis revealed differential expression of about 30% of the genome with an enrichment of genes involved in primary and secondary metabolism, transport and histone modification. HPLC-based analysis of known SMs revealed significant alterations in the Δgcn5 mutant. While most SM genes were activated by Gcn5 activity, the biosynthesis of the pigment bikaverin was strongly increased upon GCN5 deletion underlining the diverse roles of the SAGA complex in F. fujikuroi.

A Fungal N-Dimethylallyltryptophan Metabolite from Fusarium fujikuroi.

The range of secondary metabolites (SMs) produced by the rice pathogen Fusarium fujikuroi is quite broad. Several polyketides, nonribosomal peptides and terpenes have been identified. However, no products of dimethylallyltryptophan synthases (DMATSs) have been elucidated, although two putative DMATS genes are present in the F. fujikuroi genome. In this study, the in vivo product derived from one of the DMATSs (DMATS1, FFUJ_09179) was identified with the help of the software MZmine 2. Detailed structure elucidation showed that this metabolite is a reversely N-prenylated tryptophan with a rare form of prenylation. Further identified products probably resulted from side reactions of DMATS1. The genes adjacent to DMATS1 were analyzed; this showed no influence on the biosynthesis of the product.

Knock-down of the methyltransferase Kmt6 relieves H3K27me3 and results in induction of cryptic and otherwise silent secondary metabolite gene clusters in Fusarium fujikuroi.

Filamentous fungi produce a vast array of secondary metabolites (SMs) and some play a role in agriculture or pharmacology. Sequencing of the rice pathogen Fusarium fujikuroi revealed the presence of far more SM-encoding genes than known products. SM production is energy-consuming and thus tightly regulated, leaving the majority of SM gene clusters silent under laboratory conditions. One important regulatory layer in SM biosynthesis involves histone modifications that render the underlying genes either silent or poised for transcription. Here, we show that the majority of the putative SM gene clusters in F. fujikuroi are located within facultative heterochromatin marked by trimethylated lysine 27 on histone 3 (H3K27me3). Kmt6, the methyltransferase responsible for establishing this histone mark, appears to be essential in this fungus, and knock-down of Kmt6 in the KMT6kd strain shows a drastic phenotype affecting fungal growth and development. Transcription of four so far cryptic and otherwise silent putative SM gene clusters was induced in the KMT6kd strain, in which decreased expression of KMT6 is accompanied by reduced H3K27me3 levels at the respective gene loci and accumulation of novel metabolites. One of the four putative SM gene clusters, named STC5, was analysed in more detail thereby revealing a novel sesquiterpene.

Sound of silence: the beauvericin cluster in Fusarium fujikuroi is controlled by cluster-specific and global regulators mediated by H3K27 modification.

In this study, we compared the secondary metabolite profile of Fusarium fujikuroi and the histone deacetylase mutant ΔHDA1. We identified a novel peak in ΔHDA1, which was identified as beauvericin (BEA). Going in line with a 1000-fold increased BEA production, the respective non-ribosomal peptide synthetase (NRPS)-encoding gene (BEA1), as well as two adjacent genes (BEA2-BEA3), were significantly up-regulated in ΔHDA1 compared to the wild type. A special role was revealed for the ABC transporter Bea3: deletion of the encoding gene resulted in significant up-regulation of BEA1 and BEA2 and drastically elevated product yields. Furthermore, mutation of a conserved sequence motif in the promoter of BEA1 released BEA repression and resulted in elevated product levels. Candidate transcription factors (TFs) that could bind to this motif are the cluster-specific TF Bea4 as well as a homolog of the global mammalian Kruppel-like TF Yin Yang 1 (Yy1), both acting as repressors of BEA biosynthesis. In addition to Hda1, BEA biosynthesis is repressed by the activity of the H3K27 methyltransferase Kmt6. Consistently, Western blot analyses revealed a genome-wide enrichment of H3K27 acetylation (H3K27ac) in the ΔHDA1 and KMT6 knock-down mutants. Subsequent chromatin immunoprecipitation (ChIP) experiments showed elevated H3K27ac modification levels at the BEA cluster.

Two separate key enzymes and two pathway-specific transcription factors are involved in fusaric acid biosynthesis in Fusarium fujikuroi.

Fusaric acid (FSA) is a mycotoxin produced by several fusaria, including the rice pathogen Fusarium fujikuroi. Genes involved in FSA biosynthesis were previously identified as a cluster containing a polyketide synthase (PKS)-encoding (FUB1) and four additional genes (FUB2-FUB5). However, the biosynthetic steps leading to FSA as well as the origin of the nitrogen atom, which is incorporated into the polyketide backbone, remained unknown. In this study, seven additional cluster genes (FUB6-FUB12) were identified via manipulation of the global regulator FfSge1. The extended FUB gene cluster encodes two Zn(II)2 Cys6 transcription factors: Fub10 positively regulates expression of all FUB genes, whereas Fub12 is involved in the formation of the two FSA derivatives, i.e. dehydrofusaric acid and fusarinolic acid, serving as a detoxification mechanism. The major facilitator superfamily transporter Fub11 functions in the export of FSA out of the cell and is essential when FSA levels become critical. Next to Fub1, a second key enzyme was identified, the non-canonical non-ribosomal peptide synthetase Fub8. Chemical analyses of generated mutant strains allowed for the identification of a triketide as PKS product and the proposition of an FSA biosynthetic pathway, thereby unravelling the unique formation of a hybrid metabolite consisting of this triketide and an amino acid moiety.

Interplay between pathway-specific and global regulation of the fumonisin gene cluster in the rice pathogen Fusarium fujikuroi.

The rice pathogenic fungus Fusarium fujikuroi is known to produce a large variety of secondary metabolites. Besides the gibberellins, causing the bakanae effect in infected rice seedlings, the fungus produces several mycotoxins and pigments. Among the 47 putative secondary metabolite gene clusters identified in the genome of F. fujikuroi, the fumonisin gene cluster (FUM) shows very high homology to the FUM cluster of the main fumonisin producer Fusarium verticillioides, a pathogen of maize. Despite the high level of cluster gene conservation, total fumonisin FB1 and FB2 levels (FBx) produced by F. fujikuroi were only 1-10 % compared to F. verticillioides under inducing conditions. Nitrogen repression was found to be relevant for wild-type strains of both species. However, addition of germinated maize kernels activated the FBx production only in F. verticillioides, reflecting the different host specificity of both wild-type strains. Over-expression of the pathway-specific transcription factor Fum21 in F. fujikuroi strongly activated the FUM cluster genes leading to 1000-fold elevated FBx levels. To gain further insights into the nitrogen metabolite repression of FBx biosynthesis, we studied the impact of the global nitrogen regulators AreA and AreB and demonstrated that both GATA-type transcription factors are essential for full activation of the FUM gene cluster. Loss of one of them obstructs the pathway-specific transcription factor Fum21 to fully activate expression of FUM cluster genes.

Mechanistic Characterisation of Two Sesquiterpene Cyclases from the Plant Pathogenic Fungus Fusarium fujikuroi.

Two sesquiterpene cyclases from Fusarium fujikuroi were expressed in Escherichia coli and purified. The first enzyme was inactive because of a critical mutation, but activity was restored by sequence correction through site-directed mutagenesis. The mutated enzyme and two naturally functional homologues from other fusaria converted farnesyl diphosphate into guaia-6,10(14)-diene. The second enzyme produced eremophilene. The absolute configuration of guaia-6,10(14)-diene was elucidated by enantioselective synthesis, while that of eremophilene was evident from the sign of its optical rotation and is opposite to that in plants but the same as in Sorangium cellulosum. The mechanisms of both terpene cyclases were studied with various (13) C- and (2) H-labelled FPP isotopomers.

The VELVET Complex in the Gray Mold Fungus Botrytis cinerea: Impact of BcLAE1 on Differentiation, Secondary Metabolism, and Virulence.

Botrytis cinerea, the gray mold fungus, is an important plant pathogen. Field populations are characterized by variability with regard to morphology, the mode of reproduction (conidiation or sclerotia formation), the spectrum of secondary metabolites (SM), and virulence. Natural variation in bcvel1 encoding the ortholog of Aspergillus nidulans VeA, a member of the VELVET complex, was previously shown to affect light-dependent differentiation, the formation of oxalic acid (OA), and virulence. To gain broader insight into the B. cinerea VELVET complex, an ortholog of A. nidulans LaeA, BcLAE1, a putative interaction partner of BcVEL1, was studied. BcVEL1 but not its truncated versions interacts with BcLAE1 and BcVEL2 (VelB ortholog). In accordance with the expected common as well as specific functions of BcVEL1 and BcLAE1, the deletions of both genes result in similar though not identical phenotypes. Both mutants lost the ability to produce OA, to colonize the host tissue, and to form sclerotia. However, mutants differ with regard to aerial hyphae and conidia formation. Genome-wide expression analyses revealed that BcVEL1 and BcLAE1 have common and distinct target genes. Some of the genes that are underexpressed in both mutants, e.g., those encoding SM-related enzymes, proteases, and carbohydrate-active enzymes, may account for their reduced virulence.

The global regulator FfSge1 is required for expression of secondary metabolite gene clusters but not for pathogenicity in Fusarium fujikuroi.

The plant pathogenic fungus Fusarium fujikuroi is the causal agent of bakanae disease on rice due to its ability to produce gibberellins. Besides these phytohormones, F. fujikuroi is able to produce several other secondary metabolites (SMs). Although much progress has been made in the field of secondary metabolism, the transcriptional regulation of SM biosynthesis is complex and still incompletely understood. Environmental conditions, global as well as pathway-specific regulators and chromatin remodelling have been shown to play major roles. Here, the role of FfSge1, a homologue of the morphological switch regulators Wor1 and Ryp1 in Candida albicans and Histoplasma capsulatum, respectively, is explored with emphasis on secondary metabolism. FfSge1 is not required for formation of conidia and pathogenicity but is involved in vegetative growth. Transcriptome analysis of the mutant Δffsge1 compared with the wild type, as well as comparative chemical analysis between the wild type, Δffsge1 and OE:FfSGE1, revealed that FfSge1 functions as a global activator of secondary metabolism in F. fujikuroi. Double mutants of FfSGE1 and other SM regulatory genes brought insights into the hierarchical regulation of secondary metabolism. In addition, FfSge1 is also required for expression of a yet uncharacterized SM gene cluster containing a non-canonical non-ribosomal peptide synthetase.

Genetic engineering, high resolution mass spectrometry and nuclear magnetic resonance spectroscopy elucidate the bikaverin biosynthetic pathway in Fusarium fujikuroi.

Secondary metabolites of filamentous fungi can be highly bioactive, ranging from antibiotic to cancerogenic properties. In this study we were able to identify a new, yet unknown metabolite produced by Fusarium fujikuroi, an ascomycetous rice pathogen. With the help of genomic engineering and high-performance liquid chromatography (HPLC) coupled to high resolution mass spectrometry (HRMS) followed by isolation and detailed structure elucidation, the new substance could be designated as an unknown bikaverin precursor, missing two methyl- and one hydroxy group, hence named oxo-pre-bikaverin. Though the bikaverin gene cluster has been extensively studied in the past, elucidation of the biosynthetic pathway remained elusive due to a negative feedback loop that regulates the genes within the cluster. To decipher the bikaverin biosynthetic pathway and to overcome these negative regulation circuits, the structural cluster genes BIK2 and BIK3 were overexpressed independently in the ΔΔBIK2/BIK3+OE::BIK1 mutant background by using strong constitutive promoters. Using the software tool MZmine 2, the metabolite profile of the generated mutants obtained by HPLC-HRMS was compared, revealing further intermediates.

An update to polyketide synthase and non-ribosomal synthetase genes and nomenclature in Fusarium.

Members of the genus Fusarium produce a plethora of bioactive secondary metabolites, which can be harmful to humans and animals or have potential in drug development. In this study we have performed comparative analyses of polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) from ten different Fusarium species including F. graminearum (two strains), F. verticillioides, F. solani, F. culmorum, F. pseudograminearum, F. fujikuroi, F. acuminatum, F. avenaceum, F. equiseti, and F. oxysporum (12 strains). This led to identification of 52 NRPS and 52 PKSs orthology groups, respectively, and although not all PKSs and NRPSs are assumed to be intact or functional, the analyses illustrate the huge secondary metabolite potential in Fusarium. In our analyses we identified a core collection of eight NRPSs (NRPS2-4, 6, 10-13) and two PKSs (PKS3 and PKS7) that are conserved in all strains analyzed in this study. The identified PKSs and NRPSs were named based on a previously developed classification system (www.FusariumNRPSPKS.dk). We suggest this system be used when PKSs and NRPSs have to be classified in future sequenced Fusarium strains. This system will facilitate identification of orthologous and non-orthologous NRPSs and PKSs from newly sequenced Fusarium genomes and will aid the scientific community by providing a common nomenclature for these two groups of genes/enzymes.

Deciphering the cryptic genome: genome-wide analyses of the rice pathogen Fusarium fujikuroi reveal complex regulation of secondary metabolism and novel metabolites.

The fungus Fusarium fujikuroi causes "bakanae" disease of rice due to its ability to produce gibberellins (GAs), but it is also known for producing harmful mycotoxins. However, the genetic capacity for the whole arsenal of natural compounds and their role in the fungus' interaction with rice remained unknown. Here, we present a high-quality genome sequence of F. fujikuroi that was assembled into 12 scaffolds corresponding to the 12 chromosomes described for the fungus. We used the genome sequence along with ChIP-seq, transcriptome, proteome, and HPLC-FTMS-based metabolome analyses to identify the potential secondary metabolite biosynthetic gene clusters and to examine their regulation in response to nitrogen availability and plant signals. The results indicate that expression of most but not all gene clusters correlate with proteome and ChIP-seq data. Comparison of the F. fujikuroi genome to those of six other fusaria revealed that only a small number of gene clusters are conserved among these species, thus providing new insights into the divergence of secondary metabolism in the genus Fusarium. Noteworthy, GA biosynthetic genes are present in some related species, but GA biosynthesis is limited to F. fujikuroi, suggesting that this provides a selective advantage during infection of the preferred host plant rice. Among the genome sequences analyzed, one cluster that includes a polyketide synthase gene (PKS19) and another that includes a non-ribosomal peptide synthetase gene (NRPS31) are unique to F. fujikuroi. The metabolites derived from these clusters were identified by HPLC-FTMS-based analyses of engineered F. fujikuroi strains overexpressing cluster genes. In planta expression studies suggest a specific role for the PKS19-derived product during rice infection. Thus, our results indicate that combined comparative genomics and genome-wide experimental analyses identified novel genes and secondary metabolites that contribute to the evolutionary success of F. fujikuroi as a rice pathogen.

Isolation and Structure Elucidation of Fujikurins A-D: Products of the PKS19 Gene Cluster in Fusarium fujikuroi.

Fusarium fujikuroi is a member of the Gibberella fujikuroi species complex and well known for the production of gibberellins and mycotoxins including fusarins and fusaric acid. A recent genome sequencing study revealed that the fungus has the genetic potential to produce many more secondary metabolites than have been reported. This paper describes the structure elucidation of the products of the cryptic and silent PKS19 gene cluster that were recently identified (fujikurins A-D). We present the complete NMR data for the structure elucidation of the main compound fujikurin D, which shows tautomeric 1,3-diketo elements. The different tautomeric structures could be confirmed using quantum chemical calculations. Additionally, the structures of the minor compounds fujikurins A-C were elucidated by high-resolution mass spectrometric fragmentation experiments. It emerged that fujikurin A was identical to the bioactive compound CR377 of the taxonomically unclassified Fusarium strain CR377, while fujikurins B-D have not been reported from other fungi.

Characterization of the fusaric acid gene cluster in Fusarium fujikuroi.

The "bakanae" fungus Fusarium fujikuroi is a common pathogen of rice and produces a variety of mycotoxins, pigments, and phytohormones. Fusaric acid is one of the oldest known secondary metabolites produced by F. fujikuroi and some other Fusarium species. Investigation of its biosynthesis and regulation is of great interest due to its occurrence in cereal-based food and feed. This study describes the identification and characterization of the fusaric acid gene cluster in F. fujikuroi consisting of the PKS-encoding core gene and four co-regulated genes, FUB1-FUB5. Besides fusaric acid, F. fujikuroi produces two fusaric acid-like derivatives: fusarinolic acid and 9,10-dehydrofusaric acid. We provide evidence that these derivatives are not intermediates of the fusaric acid biosynthetic pathway, and that their formation is catalyzed by genes outside of the fusaric acid gene cluster. Target gene deletions of all five cluster genes revealed that not all of them are involved in fusaric acid biosynthesis. We suggest that only two genes, FUB1 and FUB4, are necessary for the biosynthesis. Expression of the FUB genes and production of fusaric acid and the two derivatives are favored under high nitrogen. We show that nitrogen-dependent expression of fusaric acid genes is positively regulated by the nitrogen-responsive GATA transcription factor AreB, and that pH-dependent regulation is mediated by the transcription factor PacC. In addition, fusaric acid production is regulated by two members of the fungal-specific velvet complex: Vel1 and Lae1. In planta expression studies show a higher expression in the favorite host plant rice compared to maize.

Cch1 and Mid1 are functionally required for vegetative growth under low-calcium conditions in the phytopathogenic ascomycete Botrytis cinerea.

In the filamentous phytopathogen Botrytis cinerea, the Ca(2+)/calcineurin signaling cascade has been shown to play an important role in fungal growth, differentiation, and virulence. This study deals with the functional characterization of two components of this pathway, the putative calcium channel proteins Cch1 and Mid1. The cch1 and mid1 genes were deleted, and single and double knockout mutants were analyzed during different stages of the fungal life cycle. Our data indicate that Cch1 and Mid1 are functionally required for vegetative growth under conditions of low extracellular calcium, since the growth of both deletion mutants is strongly impaired when they are exposed to the Ca(2+)-chelating agents EGTA and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). The impact of external Ca(2+) was investigated by supplementing with CaCl(2) and the ionophore A23187, both of which resulted in elevated growth for all mutants. However, deletion of either gene had no impact on germination, sporulation, hyphal morphology, or virulence. By use of the aequorin reporter system to measure intracellular calcium levels, no differences between the mutant strains and the wild type were obtained. Localization studies revealed a subcellular distribution of the Mid1-green fluorescent protein (GFP) fusion protein in network-like filaments, probably the endoplasmic reticulum (ER) membranes, indicating that Mid1 is not a plasma membrane-located calcium channel in B. cinerea.