Fungus Fusarium Fujikuroi Research Articles

The RNAi Machinery in the Fungus Fusarium fujikuroi Is Not Very Active in Synthetic Medium and Is Related to Transposable Elements.

Small RNAS (sRNAs) participate in regulatory RNA interference (RNAi) mechanisms in a wide range of eukaryotic organisms, including fungi. The fungus Fusarium fujikuroi, a model for the study of secondary metabolism, contains a complete set of genes for RNAi pathways. We have analyzed by high-throughput sequencing the content of sRNAs in total RNA samples of F. fujikuroi grown in synthetic medium in the dark or after 1 h of illumination, using libraries below 150 nt, covering sRNAs and their precursors. For comparison, a parallel analysis with Fusarium oxysporum was carried out. The sRNA reads showed a higher proportion of 5' uracil in the RNA samples of the expected sizes in both species, indicating the occurrence of genuine sRNAs, and putative miRNA-like sRNAs (milRNAS) were identified with prediction software. F. fujikuroi carries at least one transcriptionally expressed Ty1/copia-like retrotransposable element, in which sRNAs were found in both sense and antisense DNA strands, while in F. oxysporum skippy-like elements also show sRNA formation. The finding of sRNA in these mobile elements indicates an active sRNA-based RNAi pathway. Targeted deletion of dcl2, the only F. fujikuroi Dicer gene with significant expression under the conditions tested, did not produce appreciable phenotypic or transcriptomic alterations.

Three Genes Involved in Different Signaling Pathways, carS, wcoA, and acyA, Participate in the Regulation of Fusarin Biosynthesis in Fusarium fujikuroi.

The phytopathogenic fungus Fusarium fujikuroi has a rich secondary metabolism which includes the synthesis of very different metabolites in response to diverse environmental cues, such as light or nitrogen. Here, we focused our attention on fusarins, a class of mycotoxins whose synthesis is downregulated by nitrogen starvation. Previous data showed that mutants of genes involved in carotenoid regulation (carS, encoding a RING finger protein repressor), light detection (wcoA, White Collar photoreceptor), and cAMP signaling (AcyA, adenylate cyclase) affect the synthesis of different metabolites. We studied the effect of these mutations on fusarin production and the expression of the fus1 gene, which encodes the key polyketide synthase of the pathway. We found that the three proteins are positive regulators of fusarin synthesis, especially WcoA and AcyA, linking light regulation to cAMP signaling. Genes for two other photoreceptors, the cryptochrome CryD and the Vivid flavoprotein VvdA, were not involved in fusarin regulation. In most cases, there was a correspondence between fusarin production and fus1 mRNA, indicating that regulation is mainly exerted at the transcriptional level. We conclude that fusarin synthesis is subject to a complex control involving regulators from different signaling pathways.

Production of Gibberellins via a Non-Natural Pathway Using Steviol as a Substrate.

Gibberellins (GAs) are plant hormones widely used in agriculture. At present, GAs are produced by fermentation of the fungus Fusarium fujikuroi. However, fungal growth is too slow, resulting in slow fungal fermentation and a low yield. Here, to develop an alternative production source of GAs, an artificial pathway was engineered in Escherichia coli. By selecting and combining enzymes derived from plants and bacteria, a novel 4-enzyme pathway was successfully constructed to produce GAs using steviol, a readily available and less valuable byproduct during enzymatic refining of rebaudioside A, as a feedstock. Whole-cell biotransformation with E. coli strain expressing the novel pathway produced 71.17 ± 2.00 mg/L GA1 and a trace amount of GA3 from steviol in 48 h. This report presents a significant advancement in the fast production of GAs and establishes a method for the metabolism of terpenoids to produce target products in microbial hosts.

HmbC, a Protein of the HMG Family, Participates in the Regulation of Carotenoid Biosynthesis in Fusarium fujikuroi.

In the fungus Fusarium fujikuroi, carotenoid production is up-regulated by light and down-regulated by the CarS RING finger protein, which modulates the mRNA levels of carotenoid pathway genes (car genes). To identify new potential regulators of car genes, we used a biotin-mediated pull-down procedure to detect proteins capable of binding to their promoters. We focused our attention on one of the proteins found in the screening, belonging to the High-Mobility Group (HMG) family that was named HmbC. The deletion of the hmbC gene resulted in increased carotenoid production due to higher mRNA levels of car biosynthetic genes. In addition, the deletion resulted in reduced carS mRNA levels, which could also explain the partial deregulation of the carotenoid pathway. The mutants exhibited other phenotypic traits, such as alterations in development under certain stress conditions, or reduced sensitivity to cell wall degrading enzymes, revealed by less efficient protoplast formation, indicating that HmbC is also involved in other cellular processes. In conclusion, we identified a protein of the HMG family that participates in the regulation of carotenoid biosynthesis. This is probably achieved through an epigenetic mechanism related to chromatin structure, as is frequent in this class of proteins.

Biotransformation of the diterpene 15β-hydroxy-18(4→3)-abeo-ent-kaur-4(19),16-diene by the fungus Fusarium fujikuroi

15β-Hydroxy-18(4 → 3)- abeo - ent -kaur-4(19),16-diene ( 4 ) was biotransformed by the fungus Fusarium fujikuroi into 3α,11β,15β-trihydroxy-18(4 → 3)- abeo - ent -kaur-4(19),16-diene ( 5 ). The hydroxylation at C-3(α) in this diterpene reminds a similar reaction that occurs at C-13 in the biosynthesis of gibberellic acid in this fungus. The presence of the 15β-alcohol in the substrate directs the second hydroxylation at C-11(β), which had been observed in the incubation of ent -kaur-16-ene derivatives with this fungus when the C-19 hydroxylation was inhibited by the existence in the molecule of a 3α-OH or 3-oxo group. We also show that the angelate of the substrate is an undescribed natural product now identified as a component of the plant Distichoselinum tenuifolium . • An undescribed diterpene was identified as a component of Distichoselinum tenuifolium. • Incubation of 15β-hydroxy-18(4→3)- abeo - ent -kaur-4(19),16-diene with Fusarium fujikuroi. • The fermentation afforded 3α,11β,15β-trihydroxy-18(4→3)- abeo - ent -kaur-4(19),16-diene.

Genome-Wide Identification and Functional Analysis of the bZIP Transcription Factor Family in Rice Bakanae Disease Pathogen, Fusarium fujikuroi

Fungal basic leucine zipper (bZIP) proteins play a vital role in biological processes such as growth, biotic/abiotic stress responses, nutrient utilization, and invasion. In this study, genome-wide identification of bZIP genes in the fungus Fusarium fujikuroi, the pathogen of bakanae disease, was carried out. Forty-four genes encoding bZIP transcription factors (TFs) from the genome of F. fujikuroi (FfbZIP) were identified and functionally characterized. Structures, domains, and phylogenetic relationships of the sequences were analyzed by bioinformatic approaches. Based on the phylogenetic relationships with the FfbZIP proteins of eight other fungi, the bZIP genes can be divided into six groups (A–F). The additional conserved motifs have been identified and their possible functions were predicted. To analyze functions of the bZIP genes, 11 FfbZIPs were selected according to different motifs they contained and were knocked out by genetic recombination. Results of the characteristic studies revealed that these FfbZIPs were involved in oxygen stress, osmotic stress, cell wall selection pressure, cellulose utilization, cell wall penetration, and pathogenicity. In conclusion, this study enhanced understandings of the evolution and regulatory mechanism of the FfbZIPs in fungal growth, abiotic/biotic stress resistance, and pathogenicity, which could be the reference for other fungal bZIP studies.

Activity of a SDHI fungicide penflufen and the characterization of natural-resistance in Fusarium fujikuroi

The occurrence of bakanae disease of rice caused by the fungus Fusarium fujikuroi in Zhejiang Province has become increasingly aggravated in recent years, concomitant with the development of resistance to the widely applied fungicides, prochloraz and phenamacril. In this study, the activity of a novel succinate dehydrogenase inhibitor (SDHI) fungicide, penflufen, against different fungi was evaluated in addition to the potential of penflufen in controlling F. fujikuroi infections. Penflufen exhibited good bioactivity against F. fujikuroi, but weak activity against Fusarium spp. and other investigated plant-pathogenic fungi including Colletotrichum spp. In addition to inhibiting mycelial growth, penflufen effectively inhibited F. fujikuroi conidium production. For germination, penflufen could effectively inhibit that of small conidia, but only delay the germination of large conidia. In addition, the sensitivity to penflufen among 100 F. fujikuroi isolates that were collected in areas that were never exposed to SDHIs was determined based on mycelium growth. Sensitivities surprisingly exhibited bimodal distributions, indicating the presence of natural resistance. Cross-resistance was not observed between penflufen in F. fujikuroi and two fungicides that have been extensively applied in field including prochloraz (a DMI) and phenamacril (a 2-cyanoacrylate fungicide), nor with the three SDHIs, fluopyram, benzovindiflupyr, and pydiflumetofen. Additional analysis identified five different point mutations in SDH-A (i.e., at residues 46, 225, 283, 430, and 586) of naturally resistant isolates. These results inform the potential application of the new SDHI fungicide penflufen for managing crop diseases and understanding possible resistance mechanisms among pathogens.

The carP lncRNA Is a carS-Related Regulatory Element with Broad Effects on the Fusarium fujikuroi Transcriptome.

Carotenoid biosynthesis in the fungus Fusarium fujikuroi is regulated by environmental factors, with light being the main stimulating signal. The CarS RING-finger protein plays an important role in the downregulation of structural genes of the carotenoid pathway. A recent transcriptomic analysis on the effect of carS mutation identified a gene for a long non-coding RNA (lncRNA) upstream of carS, called carP, the deletion of which results in increased carS mRNA levels and lack of carotenoid production. We have investigated the function of carP by studying the transcriptomic effect of its deletion and the phenotypes resulting from the reintroduction of carP to a deletion strain. The RNA-seq data showed that the loss of carP affected the mRNA levels of hundreds of genes, especially after illumination. Many of these changes appeared to be cascade effects as a result of changes in carS expression, as suggested by the comparison with differentially expressed genes in a carS mutant. Carotenoid production only recovered when carP was integrated upstream of carS, but not at other genomic locations, indicating a cis-acting mechanism on carS. However, some genes hardly affected by CarS were strongly upregulated in the carP mutant, indicating that carP may have other regulatory functions as an independent regulatory element.

Impact of the White Collar Photoreceptor WcoA on the Fusarium fujikuroi Transcriptome.

The proteins of the White Collar 1 family (WC) constitute a major class of flavin photoreceptors, widely distributed in fungi, that work in cooperation with a WC 2 protein forming a regulatory complex. The WC complex was investigated in great detail in Neurospora crassa, a model fungus in photobiology studies, where it controls all its major photoresponses. The fungus Fusarium fujikuroi, a model system in the production of secondary metabolites, contains a single WC-1 gene called wcoA. The best-known light response in this fungus is the photoinduction of the synthesis of carotenoids, terpenoid pigments with antioxidant properties. Loss of WcoA in F. fujikuroi results in a drastic reduction in the mRNA levels of the carotenoid genes, and a diversity of morphological and metabolic changes, including alterations in the synthesis of several secondary metabolites, suggesting a complex regulatory role. To investigate the function of WcoA, the transcriptome of F. fujikuroi was analyzed in the dark and after 15-, 60- or 240-min illumination in a wild strain and in a formerly investigated wcoA insertional mutant. Using a threshold of four-fold change in transcript levels, 298 genes were activated and 160 were repressed in the wild strain under at least one of the light exposures. Different response patterns were observed among them, with genes exhibiting either fast, intermediate, and slow photoinduction, or intermediate or slow repression. All the fast and intermediate photoresponses, and most of the slow ones, were lost in the wcoA mutant. However, the wcoA mutation altered the expression of a much larger number of genes irrespective of illumination, reaching at least 16% of the annotated genes in this fungus. Such genes include many related to secondary metabolism, as well as others related to photobiology and other cellular functions, including the production of hydrophobins. As judged by the massive transcriptomic changes exhibited by the wcoA mutant in the dark, the results point to WcoA as a master regulatory protein in F. fujikuroi, in addition to a central function as the photoreceptor responsible for most of the transcriptional responses to light in this fungus.

The Current Status of Research on Gibberellin Biosynthesis.

Gibberellins are produced by all vascular plants and several fungal and bacterial species that associate with plants as pathogens or symbionts. In the 60 years since the first experiments on the biosynthesis of gibberellic acid in the fungus Fusarium fujikuroi, research on gibberellin biosynthesis has advanced to provide detailed information on the pathways, biosynthetic enzymes and their genes in all three kingdoms, in which the production of the hormones evolved independently. Gibberellins function as hormones in plants, affecting growth and differentiation in organs in which their concentration is very tightly regulated. Current research in plants is focused particularly on the regulation of gibberellin biosynthesis and inactivation by developmental and environmental cues, and there is now considerable information on the molecular mechanisms involved in these processes. There have also been recent advances in understanding gibberellin transport and distribution and their relevance to plant development. This review describes our current understanding of gibberellin metabolism and its regulation, highlighting the more recent advances in this field.

Different Phenotypes, Similar Genomes: Three Newly Sequenced Fusarium fujikuroi Strains Induce Different Symptoms in Rice Depending on Temperature.

Bakanae, caused by the hemibiotrophic fungus Fusarium fujikuroi, is one of the most important diseases of rice and is attributed to up to 75% of losses, depending on the strain and environmental conditions. Some strains cause elongation and thin leaves, whereas others induce stunting and chlorotic seedlings. Differences in symptoms are attributed to genetic differences in the strains. F. fujikuroi strains Augusto2, CSV1, and I1.3 were sequenced with Illumina MiSeq, and pathogenicity trials were conducted on rice cultivar Galileo, which is susceptible to bakanae. By performing gene prediction, single nucleotide polymorphism (SNP) calling, and structural variant analysis with a reference genome, we show how an extremely limited number of polymorphisms in genes not commonly associated with bakanae disease can cause strong differences in phenotype. CSV1 and Augusto2 were particularly close, with only 21,887 SNPs between them, but they differed in virulence, reaction to temperature, induced symptoms, colony morphology and color, growth speed, fumonisin, and gibberellin production. Genes potentially involved in the shift in phenotype were identified. Furthermore, we show how temperature variation may result in different symptoms even in rice plants inoculated with the same F. fujikuroi strain. Moreover, all of the F. fujikuroi strains became more virulent at higher temperatures. Significant differences were likewise observed in gibberellic acid production and in the expression of both fungal and plant gibberellin biosynthetic genes.

Modulation of Activity of a Carotenoid Pathway Through the Use of the TeT-on Regulatory System: Application in the Fungus Fusarium fujikuroi.

Carotenoids are widespread pigments in photosynthetic species, but they are also found in nonphotosynthetic microorganisms, such as bacteria and fungi. The amenability of fungi to genetic studies have made some fungal species advantageous models in the study of the genetics and biochemistry of carotenoid biosynthesis, while others have been used for biotechnological carotenoid production. The availability of molecular techniques that allow modulating the expression of target genes is a powerful tool in the manipulation of carotenoid synthesis. An example of an adjustable gene expression is based on the tetracycline-controlled transcriptional activation system, known as Tet-on. We describe here the material and protocols for the construction of a Tet-on regulated gene, its introduction in the filamentous fungus F. fujikuroi, and its use to modulate the expression of a negative regulator of carotenoid biosynthesis.

Antifungal Potential and Characterization of Plant Extracts against Fusarium fujikuroi on Rice

Bakanae rice disease caused by fungus Fusarium fujikuroi Nirenberg (F. fujikuroi) and occurs in the most growing rice areas of the world. Transmission of this disease by soil and seed, so seed addressing by different material is useful to control bakanae disease. This study is focused on antifungal activity for some plant extracts i.e. Ammi visnaga (A. visnaga), Eucalyptus globulus (E. globulus), Artemisia judaica (A. judai) and Coriandrum sativum (C. sativum)on the linear growth of F. fujikuroi. Identification of chemical compounds was done for two plant extracts A. visnaga and E. globulus by qualitative and quantitative phytochemical screening, and gas chromatography-mass spectrometry (GC-MS) analysis. Seeds was treatments with plant extracts with different concentrations under greenhouse condition. Changes in seedling content for enzymes such as peroxidase (POX), polyphenol oxidase (PPO) and hydrogen peroxide (H2O2) after 7 and 14 days of treatments by plant extracts has been observed. Management bakanae disease under field condition by seed treatment with plant extracts. The results indicate that A. visnaga was the most effective on the linear growth with concentration 500 ppm. The obtained results from qualitative phytochemical tests for A. visnaga indicates presence of coumarins, tannins, saponin, terpenoids, flavonoids and steroids and absence of phenols. E. globulus found tannins, phenols, saponin, terpenoids, flavonoid and absences of coumarins and steroids. Tannins and terpenoids were the highest quantitative phytochemical constituent determined in A. visnaga. Whilephenols and flavonoids were the most active phytochemical constituents determined in E. globulus. The chemical constituents for GC-MS analysis of A. visnaga were benzene methyl, khellin, visnagin and vitamin E. While, E. globulus haseucalyptol, terpinen, ellagic acid and gallic acid. Under greenhouse condition, A. visnaga was the most effective in reducing the number of death and number of infected seedling, increasing the germination % at different concentrations compared with other treatments. POX, PPO and H2O2 were induced in inoculated seedling compared with the un-inoculated seedling. Seed treatments with A. visnaga and E. globulus produced the highest enzymes increase and decreased H2O2 content in seedling. Under field condition, there are no significant difference between A. visnaga and Rhizolex-T 50% as seed treatment in reduction of disease incidence or disease severity of bakanae in 2017 and 2018 growing seasons. Grain yield was increased in treated plant with A. visnaga and Rhizolex-T 50% in both seasons.

Proteomic dissection of the rice-Fusarium fujikuroi interaction and the correlation between the proteome and transcriptome under disease stress

BackgroundBakanae disease, caused by the fungus Fusarium fujikuroi, occurs widely throughout Asia and Europe and sporadically in other rice production areas. Recent changes in climate and cropping patterns have aggravated this disease. To gain a better understanding of the molecular mechanisms of rice bakanae disease resistance, we employed a 6-plex tandem mass tag approach for relative quantitative proteomic comparison of infected and uninfected rice seedlings 7 days post-inoculation with two genotypes: the resistant genotype 93–11 and the susceptible genotype Nipponbare.ResultsIn total, 123 (77.2% up-regulated, 22.8% down-regulated) and 91 (94.5% up-regulated, 5.5% down-regulated) differentially expressed proteins (DEPs) accumulated in 93–11 and Nipponbare, respectively. Only 11 DEPs were both shared by the two genotypes. Clustering results showed that the protein regulation trends for the two genotypes were highly contrasting, which suggested obviously different interaction mechanisms of the host and the pathogen between 93 and 11 and Nipponbare. Further analysis showed that a noticeable aquaporin, PIP2–2, was sharply upregulated with a fold change (FC) of 109.2 in 93–11, which might be related to pathogen defense and the execution of bakanae disease resistance. Certain antifungal proteins were regulated in both 93–11 and Nipponbare with moderate FCs. These proteins might participate in protecting the cellular integrity required for basic growth of the susceptible genotype. Correlation analysis between the transcriptome and proteome revealed that Pearson correlation coefficients of R = 0.677 (P = 0.0005) and R = − 0.097 (P = 0.702) were obtained for 93–11 and Nipponbare, respectively. Our findings raised an intriguing result that a significant positive correlation only in the resistant genotype, while no correlation was found in the susceptible genotype. The differences in codon usage was hypothesized for the cause of the result.ConclusionsQuantitative proteomic analysis of the rice genotypes 93-11and Nipponbare after F. fujikuroi infection revealed that the aquaporin protein PIP2–2 might execute bakanae disease resistance. The difference in the correlation between the transcriptome and proteome might be due to the differences in codon usage between 93-11and Nipponbare. Overall, the protein regulation trends observed under bakanae disease stress are highly contrasting, and the molecular mechanisms of disease defense are obviously different between 93 and 11 and Nipponbare. In summary, these findings deepen our understanding of the functions of proteins induced by bakanae disease and the mechanisms of rice bakanae disease resistance.

CRISPR/Cas9-Based Genome Editing in the Filamentous Fungus Fusarium fujikuroi and Its Application in Strain Engineering for Gibberellic Acid Production.

The filamentous fungus Fusarium fujikuroi is well-known for its production of natural plant growth hormones: a series of gibberellic acids (GAs). Some GAs, including GA1, GA3, GA4, and GA7, are biologically active and have been widely applied in agriculture. However, the low efficiency of traditional genetic tools limits the further research toward making this fungus more efficient and able to produce tailor-made GAs. Here, we established an efficient CRISPR/Cas9-based genome editing tool for F.fujikuroi. First, we compared three different nuclear localization signals (NLS) and selected an efficient NLS from histone H2B (HTBNLS) to enable the import of the Cas9 protein into the fungal nucleus. Then, different sgRNA expression strategies, both in vitro and different promoter-based in vivo strategies, were explored. The promoters of the U6 small nuclear RNA and 5S rRNA, which were identified in F.fujikuroi, had the highest editing efficiency. The 5S rRNA-promoter-driven genome editing efficiency reached up to 79.2%. What's more, multigene editing was also explored and showed good results. Finally, we used the developed genome editing tool to engineer the metabolic pathways responsible for the accumulation of a series GAs in the filamentous fungus F.fujikuroi, and successfully changed its GA product profile, from GA3 to tailor-made GA4 and GA7 mixtures. Since these mixtures are more efficient for agricultural use, especially for fruit growth, the developed strains will greatly improve industrial GA production.

Fusarium fujikuroi : A novel source of metabolites with herbicidal activity

Abstract This study presents the isolation and identification of a fungus (Fusarium fujikuroi) from the Brazilian Pampa biome that is able to produce phytotoxic secondary metabolites with herbicidal activity. Before selecting such promising fungus, 132 microorganisms were isolated from weed plants with infections symptoms. Each sample of isolated material was used in submerged fermentation to produce a fermented broth. Thereafter, the cells were separated by centrifugation and the supernatant was filtered. Each filtered sample (culture filtrate) was used to evaluate the herbicidal activity in the bioassays with two target plants: Cucumis sativus and Sorghum bicolor. The selection criteria consisted of analyzing germination in pre-emergence, phytotoxicity, plant height and root length in post-emergence, and lesions in detached leaf-punctured assay. The pre-emergence was the primary screening, where 11 samples of culture filtrate provided 100% inhibition of germination of both plants. These samples underwent a secondary screening, which the sample SO210 presented 25% phytotoxicity, 40% reduction of plant height, 28% reduction of root length, and necrosis and chlorosis in 70% of foliar area. The microorganism coded as SO210 was identified as a strain of Fusarium fujikuroi. Up to now, studies that attribute herbicidal activity to this fungus are scarce in the literature. Therefore, this work shows a potential activity of metabolites produced by this fungus toward the inhibition of germination and development of plant weeds.

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.

Nitrate Assimilation in Fusarium fujikuroi Is Controlled by Multiple Levels of Regulation.

Secondary metabolite production of the phytopathogenic ascomycete fungus Fusarium fujikuroi is greatly influenced by the availability of nitrogen. While favored nitrogen sources such as glutamine and ammonium are used preferentially, the uptake and utilization of nitrate is subject to a regulatory mechanism called nitrogen metabolite repression (NMR). In Aspergillus nidulans, the transcriptional control of the nitrate assimilatory system is carried out by the synergistic action of the nitrate-specific transcription factor NirA and the major nitrogen-responsive regulator AreA. In this study, we identified the main components of the nitrate assimilation system in F. fujikuroi and studied the role of each of them regarding the regulation of the remaining components. We analyzed mutants with deletions of the nitrate-specific activator NirA, the nitrate reductase (NR), the nitrite reductase (NiR) and the nitrate transporter NrtA. We show that NirA controls the transcription of the nitrate assimilatory genes NIAD, NIIA, and NRTA in the presence of nitrate, and that the global nitrogen regulator AreA is obligatory for expression of most, but not all NirA target genes (NIAD). By transforming a NirA-GFP fusion construct into the ΔNIAD, ΔNRTA, and ΔAREA mutant backgrounds we revealed that NirA was dispersed in the cytosol when grown in the presence of glutamine, but rapidly sorted to the nucleus when nitrate was added. Interestingly, the rapid and nitrate-induced nuclear translocation of NirA was observed also in the ΔAREA and ΔNRTA mutants, but not in ΔNIAD, suggesting that the fungus is able to directly sense nitrate in an AreA- and NrtA-independent, but NR-dependent manner.

Exploiting a Step in Diterpenoid Biosynthesis by the Fungus Fusarium Fujikuroi

The scope of the microbiological transformation of ent-kaurenoid diterpenes by the fungus Fusarium (Gibberella) fujikuroi which utilise the ent-kaurene and ent-kauren-19-oic acid oxidases and the ring contraction of ring B to gibberellin is reviewed. Constraints arising from the presence of 3α, 15α and 18-hydroxyl groups are noted. The development of a group of potential plant growth regulators which inhibit the ring contraction step in gibberellin biosynthesis is described.