Chromatin-dependent regulation of secondary metabolite biosynthesis in fungi : is the picture complete?

Fungal secondary metabolites are small molecules that exhibit diverse biological activities exploited in medicine, industry and agriculture. Their biosynthesis is governed by co-expressed genes that often co-localize in gene clusters. Most of these secondary metabolite gene clusters are inactive under laboratory conditions, which is due to a tight transcriptional regulation. Modifications of chromatin, the complex of DNA and histone proteins influencing DNA accessibility, play an important role in this regulation. However, tinkering with well-characterised chemical and genetic modifications that affect chromatin alters the expression of only few biosynthetic gene clusters, and thus the regulation of the vast majority of biosynthetic pathways remains enigmatic. In the past, attempts to activate silent gene clusters in fungi mainly focused on histone acetylation and methylation, while in other eukaryotes many other post-translational modifications are involved in transcription regulation. Thus, how chromatin regulates the expression of gene clusters remains a largely unexplored research field. In this review, we argue that focusing on only few well-characterised chromatin modifications is significantly hampering our understanding of the chromatin-based regulation of biosynthetic gene clusters. Research on underexplored chromatin modifications and on the interplay between different modifications is timely to fully explore the largely untapped reservoir of fungal secondary metabolites.</p

Evidence for horizontal transfer of a secondary metabolite gene cluster between fungi

Background Filamentous fungi synthesize many secondary metabolites and are rich in genes encoding proteins involved in their biosynthesis. Genes from the same pathway are often clustered and co-expressed in particular conditions. Such secondary metabolism gene clusters evolve rapidly through multiple rearrangements, duplications and losses. It has long been suspected that clusters can be transferred horizontally between species, but few concrete examples have been described so far. Results In the rice blast fungus Magnaporthe grisea, the avirulence gene ACE1 that codes for a hybrid polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) belongs to a cluster of 15 genes involved in secondary metabolism. Additional related clusters were detected in the ascomycetes Chaetomium globosum, Stagonospora nodorum and Aspergillus clavatus. Gene-by-gene phylogenetic analysis showed that in C. globosum and M. grisea, the evolution of these ACE1-like clusters is characterized by successive complex duplication events including tandem duplication within the M. grisea cluster. The phylogenetic trees also present evidence that at least five of the six genes in the homologous ACE1 gene cluster in A. clavatus originated by horizontal transfer from a donor closely related to M. grisea. Conclusion The ACE1 cluster originally identified in M. grisea is shared by only few fungal species. Its sporadic distribution within euascomycetes is mainly explained by multiple events of duplication and losses. However, because A. clavatus contains an ACE1 cluster of only six genes, we propose that horizontal transfer from a relative of M. grisea into an ancestor of A. clavatus provides a much simpler explanation of the observed data than the alternative of multiple events of duplication and losses of parts of the cluster

Automated alignment-based curation of gene models in filamentous fungi

BACKGROUND: Automated gene-calling is still an error-prone process, particularly for the highly plastic genomes of fungal species. Improvement through quality control and manual curation of gene models is a time-consuming process that requires skilled biologists and is only marginally performed. The wealth of available fungal genomes has not yet been exploited by an automated method that applies quality control of gene models in order to obtain more accurate genome annotations. RESULTS: We provide a novel method named alignment-based fungal gene prediction (ABFGP) that is particularly suitable for plastic genomes like those of fungi. It can assess gene models on a gene-by-gene basis making use of informant gene loci. Its performance was benchmarked on 6,965 gene models confirmed by full-length unigenes from ten different fungi. 79.4% of all gene models were correctly predicted by ABFGP. It improves the output of ab initio gene prediction software due to a higher sensitivity and precision for all gene model components. Applicability of the method was shown by revisiting the annotations of six different fungi, using gene loci from up to 29 fungal genomes as informants. Between 7,231 and 8,337 genes were assessed by ABFGP and for each genome between 1,724 and 3,505 gene model revisions were proposed. The reliability of the proposed gene models is assessed by an a posteriori introspection procedure of each intron and exon in the multiple gene model alignment. The total number and type of proposed gene model revisions in the six fungal genomes is correlated to the quality of the genome assembly, and to sequencing strategies used in the sequencing centre, highlighting different types of errors in different annotation pipelines. The ABFGP method is particularly successful in discovering sequence errors and/or disruptive mutations causing truncated and erroneous gene models. CONCLUSIONS: The ABFGP method is an accurate and fully automated quality control method for fungal gene catalogues that can be easily implemented into existing annotation pipelines. With the exponential release of new genomes, the ABFGP method will help decreasing the number of gene models that require additional manual curation

Novel Introner-Like Elements in fungi are involved in parallel gains of spliceosomal introns

Spliceosomal introns are key components of the eukaryotic gene structure. Although they contributed to the emergence of eukaryotes, their origin remains elusive. In fungi, they might originate from the multiplication of invasive introns named Introner-Like Elements (ILEs). However, so far ILEs have been observed in six fungal species only, including Fulvia fulva and Dothistroma septosporum (Dothideomycetes), arguing against ILE insertion as a general mechanism for intron gain. Here, we identified novel ILEs in eight additional fungal species that are phylogenetically related to F. fulva and D. septosporum using PCR amplification with primers derived from previously identified ILEs. The ILE content appeared unique to each species, suggesting independent multiplication events. Interestingly, we identified four genes each containing two gained ILEs. By analysing intron positions in orthologues of these four genes in Ascomycota, we found that three ILEs had inserted within a 15 bp window that contains regular spliceosomal introns in other fungal species. These three positions are not the result of intron sliding because ILEs are newly gained introns. Furthermore, the alternative hypothesis of an inferred ancestral gain followed by independent losses contradicts the observed degeneration of ILEs. These observations clearly indicate three parallel intron gains in four genes that were randomly identified. Our findings suggest that parallel intron gain is a phenomenon that has been highly underestimated in ILE-containing fungi, and likely in the whole fungal kingdom

Проектування високонапірного робочого колеса багатоступінчатих насосів для об’єктів нафтовидобутку

В статті запропоновано іноваційний метод підвищення напірності ступеня багатоступінчатого насоса до 20% з використанням методу чисельного дослідження.В статье предложен инновационный метод повышения напирности степени многоступенчатого насоса до 20% с использованием метода численного исследования.The paper proposed an innovative method of increasing of the multistage pump head to 20% using the method of numerical investigation

Kinesin-3 and dynein cooperate in long-range retrograde endosome motility along a nonuniform microtubule array

This is the final version. Available from the American Society for Cell Biology via the DOI in this recordThe polarity of microtubules (MTs) determines the motors for intracellular motility, with kinesins moving to plus ends and dynein to minus ends. In elongated cells of Ustilago maydis, dynein is thought to move early endosomes (EEs) toward the septum (retrograde), whereas kinesin-3 transports them to the growing cell tip (anterograde). Occasionally, EEs run up to 90 μm in one direction. The underlying MT array consists of unipolar MTs at both cell ends and antipolar bundles in the middle region of the cell. Cytoplasmic MT-organizing centers, labeled with a γ-tubulin ring complex protein, are distributed along the antipolar MTs but are absent from the unipolar regions. Dynein colocalizes with EEs for 10-20 μm after they have left the cell tip. Inactivation of temperature-sensitive dynein abolishes EE motility within the unipolar MT array, whereas long-range motility is not impaired. In contrast, kinesin-3 is continuously present, and its inactivation stops long-range EE motility. This indicates that both motors participate in EE motility, with dynein transporting the organelles through the unipolar MT array near the cell ends, and kinesin-3 taking over at the beginning of the medial antipolar MT array. The cooperation of both motors mediates EE movements over the length of the entire cell. © 2011 Schuster et al.Biotechnology and Biological Sciences Research Council (BBSRC

The histone code of the fungal genus Aspergillus uncovered by evolutionary and proteomic analyses.

Chemical modifications of DNA and histone proteins impact the organization of chromatin within the nucleus. Changes in these modifications, catalysed by different chromatin-modifying enzymes, influence chromatin organization, which in turn is thought to impact the spatial and temporal regulation of gene expression. While combinations of different histone modifications, the histone code, have been studied in several model species, we know very little about histone modifications in the fungal genus Aspergillus, whose members are generally well studied due to their importance as models in cell and molecular biology as well as their medical and biotechnological relevance. Here, we used phylogenetic analyses in 94 Aspergilli as well as other fungi to uncover the occurrence and evolutionary trajectories of enzymes and protein complexes with roles in chromatin modifications or regulation. We found that these enzymes and complexes are highly conserved in Aspergilli, pointing towards a complex repertoire of chromatin modifications. Nevertheless, we also observed few recent gene duplications or losses, highlighting Aspergillus species to further study the roles of specific chromatin modifications. SET7 (KMT6) and other components of PRC2 (Polycomb Repressive Complex 2), which is responsible for methylation on histone H3 at lysine 27 in many eukaryotes including fungi, are absent in Aspergilli as well as in closely related Penicillium species, suggesting that these lost the capacity for this histone modification. We corroborated our computational predictions by performing untargeted MS analysis of histone post-translational modifications in Aspergillus nidulans. This systematic analysis will pave the way for future research into the complexity of the histone code and its functional implications on genome architecture and gene regulation in fungi

Detection and quantification of the histone code in the fungal genus Aspergillus

In eukaryotes, the combination of different histone post-translational modifications (PTMs) - the histone code - impacts the chromatin organization as compact and transcriptionally silent heterochromatin or accessible and transcriptionally active euchromatin. Although specific histone PTMs have been studied in fungi, an overview of histone PTMs and their relative abundance is still lacking. Here, we used mass spectrometry to detect and quantify histone PTMs in three fungal species belonging to three distinct taxonomic sections of the genus Aspergillus (Aspergillus niger, Aspergillus nidulans (two strains), and Aspergillus fumigatus). We overall detected 23 different histone PTMs, including a majority of lysine methylations and acetylations, and 23 co-occurrence patterns of multiple histone PTMs. Among those, we report for the first time the detection of H3K79me1, H3K79me2, and H4K31ac in Aspergilli. Although all three species harbour the same PTMs, we found significant differences in the relative abundance of H3K9me1/2/3, H3K14ac, H3K36me1 and H3K79me1, as well as the co-occurrence of acetylation on both K18 and K23 of histone H3 in a strain-specific manner. Our results provide novel insights about the underexplored complexity of the histone code in filamentous fungi, and its functional implications on genome architecture and gene regulation

Synergistic action of serine- and metallo-proteases from Fusarium oxysporum f. sp. lycopersici cleaves chitin-binding tomato chitinases, reduces their antifungal activity and enhances fungal virulence

As part of their defence strategy against fungal pathogens, plants secrete chitinases that degrade chitin, the major structural component of fungal cell walls. Some fungi are not sensitive to plant chitinases because they secrete chitin-binding effector proteins that protect their cell wall against these enzymes. However, it is not known how fungal pathogens that lack chitin-binding effectors overcome this plant defence barrier. Here, we investigated the ability of fungal tomato pathogens to cleave chitin-binding domain (CBD)-containing chitinases and its effect on fungal virulence. Four tomato CBD-chitinases were produced in Pichia pastoris and incubated with secreted proteins isolated from seven fungal tomato pathogens. Of these, Fusarium oxysporum f. sp. lycopersici, Verticillium dahliae and Botrytis cinerea were able to cleave the extracellular tomato chitinases SlChi1 and SlChi13. Cleavage by F. oxysporum removed the CBD from the N-terminus, as shown by mass spectrometry, and significantly reduced the chitinase and antifungal activity of both chitinases. Both secreted metallo-protease FoMep1 and serine protease FoSep1 were responsible for this cleavage. Double deletion mutants of FoMep1 and FoSep1 of F. oxysporum lacked chitinase cleavage activity on SlChi1 and SlChi13 and showed reduced virulence on tomato. These results demonstrate the importance of plant chitinase cleavage in fungal virulence

Heterologous expression of the avirulence gene ACE1 from the fungal rice pathogen Magnaporthe oryzae

The ACE1 and RAP1 genes from the avirulence signalling gene cluster of the rice blast fungus Magnaporthe oryzae were expressed in Aspergillus oryzae and M. oryzae itself. Expression of ACE1 alone produced a polyenyl pyrone (magnaporthepyrone), which is regioselectively epoxidised and hydrolysed to give different diols, 6 and 7, in the two host organisms. Analysis of the three introns present in ACE1 determined that A. oryzae does not process intron 2 correctly, while M. oryzae processes all introns correctly in both appressoria and mycelia. Co-expression of ACE1 and RAP1 in A. oryzae produced an amide 8 which is similar to the PKS-NRPS derived backbone of the cytochalasans. Biological testing on rice leaves showed that neither the diols 6 and 7, nor amide 8 was responsible for the observed ACE1 mediated avirulence, however, gene cluster analysis suggests that the true avirulence signalling compound may be a tyrosine-derived cytochalasan compound.Government of Egypt ScholarshipThe School of Chemistry at the University of Bristol and the Mark Evans ScholarshipKano State Government NigeriaMacArthur FoundationBayero UniversityNigerian Petroleum Technology FundMalaysian Govenment ScholarshipEP/F066104/