Search

Coprinopsis cinerea lectin 2 (CCL2) is a fucoside-binding lectin from the basidiomycete C. cinerea that is toxic to the bacterivorous nematode Caenorhabditis elegans as well as animal-parasitic and fungivorous nematodes. We expressed CCL2 in Arabidopsis to assess its protective potential toward plant-parasitic nematodes. Our results demonstrate that expression of CCL2 enhances host resistance against the cyst nematode Heterodera schachtii. Surprisingly, CCL2-expressing plants were also more resistant to fungal pathogens including Botrytis cinerea, and the phytopathogenic bacterium Pseudomonas syringae. In addition, CCL2 expression positively affected plant growth indicating that CCL2 has the potential to improve two important agricultural parameters namely biomass production and general disease resistance. The mechanism of the CCL2-mediated enhancement of plant disease resistance depended on fucoside-binding by CCL2 as transgenic plants expressing a mutant version of CCL2 (Y92A), compromised in fucoside-binding, exhibited wild type (WT) disease susceptibility. The protective effect of CCL2 did not seem to be direct as the lectin showed no growth-inhibition toward B. cinerea in in vitro assays. We detected, however, a significantly enhanced transcriptional induction of plant defense genes in CCL2- but not CCL2-Y92A-expressing lines in response to infection with B. cinerea compared to WT plants. This study demonstrates a potential of fungal defense lectins in plant protection beyond their use as toxins., Frontiers in Plant Science, 12, ISSN:1664-462X

Phytopathogenic Verticillia cause Verticillium wilt on numerous economically important crops. Plant infection begins at the roots, where the fungus is confronted with rhizosphere inhabiting bacteria. The effects of different fluorescent pseudomonads, including some known biocontrol agents of other plant pathogens, on fungal growth of the haploid Verticillium dahliae and/or the amphidiploid Verticillium longisporum were compared on pectin-rich medium, in microfluidic interaction channels, allowing visualization of single hyphae, or on Arabidopsis thaliana roots. We found that the potential for formation of bacterial lipopeptide syringomycin resulted in stronger growth reduction effects on saprophytic Aspergillus nidulans compared to Verticillium spp. A more detailed analyses on bacterial-fungal co-cultivation in narrow interaction channels of microfluidic devices revealed that the strongest inhibitory potential was found for Pseudomonas protegens CHA0, with its inhibitory potential depending on the presence of the GacS/GacA system controlling several bacterial metabolites. Hyphal tip polarity was altered when V. longisporum was confronted with pseudomonads in narrow interaction channels, resulting in a curly morphology instead of straight hyphal tip growth. These results support the hypothesis that the fungus attempts to evade the bacterial confrontation. Alterations due to co-cultivation with bacteria could not only be observed in fungal morphology but also in fungal transcriptome. P. protegens CHA0 alters transcriptional profiles of V. longisporum during 2 h liquid media co-cultivation in pectin-rich medium. Genes required for degradation of and growth on the carbon source pectin were down-regulated, whereas transcripts involved in redox processes were up-regulated. Thus, the secondary metabolite mediated effect of Pseudomonas isolates on Verticillium species results in a complex transcriptional response, leading to decreased growth with precautions for self-protection combined with the initiation of a change in fungal growth direction. This interplay of bacterial effects on the pathogen can be beneficial to protect plants from infection, as shown with A. thaliana root experiments. Treatment of the roots with bacteria prior to infection with V. dahliae resulted in a significant reduction of fungal root colonization. Taken together we demonstrate how pseudomonads interfere with the growth of Verticillium spp. and show that these bacteria could serve in plant protection., Frontiers in Microbiology, 12, ISSN:1664-302X

Bacteria are the main nutritional competitors of saprophytic fungi during colonization of their ecological niches. This competition involves the mutual secretion of antimicrobials that kill or inhibit the growth of the competitor. Over the last years it has been demonstrated that fungi respond to the presence of bacteria with changes of their transcriptome, but the significance of these changes with respect to competition for nutrients is not clear as functional proof of the antibacterial activity of the induced gene products is often lacking. Here, we report the genome-wide transcriptional response of the coprophilous mushroom Coprinopsis cinerea to the bacteria Bacillus subtilis and Escherichia coli. The genes induced upon co-cultivation with each bacterium were highly overlapping, suggesting that the fungus uses a similar arsenal of effectors against Gram-positive and -negative bacteria. Intriguingly, the induced genes appeare to encode predominantly secreted peptides and proteins with predicted antibacterial activities, which was validated by comparative proteomics of the C. cinerea secretome. Induced members of two putative antibacterial peptide and protein families in C. cinerea, the cysteine-stabilized αβ-defensins (Csαβ-defensins) and the GH24-type lysozymes, were purified, and their antibacterial activity was confirmed. These results provide compelling evidence that fungi are able to recognize the presence of bacteria and respond with the expression of an arsenal of secreted antibacterial peptides and proteins., The ISME Journal, 13, ISSN:1751-7362, ISSN:1751-7370

Background It is well known that mushrooms produce defense proteins and secondary metabolites against predators and competitors; however, less is known about the correlation between the tissue-specific expression and the target organism (antagonist) specificity of these molecules. In addition, conserved transcriptional circuitries involved in developing sexual organs in fungi are not characterized, despite the growing number of gene expression datasets available from reproductive and vegetative tissue. The aims of this study were: first, to evaluate the tissue specificity of defense gene expression in the model mushroom Coprinopsis cinerea and, second, to assess the degree of conservation in transcriptional regulation during sexual development in basidiomycetes. Results In order to characterize the regulation in the expression of defense loci and the transcriptional circuitries controlling sexual reproduction in basidiomycetes, we sequenced the poly (A)-positive transcriptome of stage 1 primordia and vegetative mycelium of C. cinerea A43mutB43mut. Our data show that many genes encoding predicted and already characterized defense proteins are differentially expressed in these tissues. The predicted specificity of these proteins with regard to target organisms suggests that their expression pattern correlates with the type of antagonists these tissues are confronted with. Accordingly, we show that the stage 1 primordium-specific protein CC1G_11805 is toxic to insects and nematodes. Comparison of our data to analogous data from Laccaria bicolor and Schizophyllum commune revealed that the transcriptional regulation of nearly 70 loci is conserved and probably subjected to stabilizing selection. A Velvet domain-containing protein was found to be up-regulated in all three fungi, providing preliminary evidence of a possible role of the Velvet protein family in sexual development of basidiomycetes. The PBS-soluble proteome of C. cinerea primordia and mycelium was analyzed by shotgun LC-MS. This proteome data confirmed the presence of intracellular defense proteins in primordia. Conclusions This study shows that the exposure of different tissues in fungi to different types of antagonists shapes the expression pattern of defense loci in a tissue-specific manner. Furthermore, we identify a transcriptional circuitry conserved among basidiomycetes during fruiting body formation that involves, amongst other transcription factors, the up-regulation of a Velvet domain-containing protein., BMC Genomics, 15, ISSN:1471-2164

Bioactive peptide natural products are an important source of therapeutics. Prominent examples are the antibiotic penicillin and the immunosuppressant cyclosporine which are both produced by fungi and have revolutionized modern medicine. Peptide biosynthesis can occur either non-ribosomally via large enzymes referred to as non-ribosomal peptide synthetases (NRPS) or ribosomally. Ribosomal peptides are synthesized as part of a larger precursor peptide where they are posttranslationally modified and subsequently proteolytically released. Such peptide natural products are referred to as ribosomally synthesized and posttranslationally modified peptides (RiPPs). Their biosynthetic pathways have recently received a lot of attention, both from a basic and applied research point of view, due to the discoveries of several novel posttranslational modifications of the peptide backbone. Some of these modifications were so far only known from NRPSs and significantly increase the chemical space covered by this class of peptide natural products. Latter feature, in combination with the promiscuity of the modifying enzymes and the genetic encoding of the peptide sequence, makes RiPP biosynthetic pathways attractive for synthetic biology approaches to identify novel peptide therapeutics via screening of de novo generated peptide libraries and, thus, exploit bioactive peptide natural products beyond their direct use as therapeutics. This review focuses on the recent discovery and characterization of novel RiPP biosynthetic pathways in fungi and their possible application for the development of novel peptide therapeutics., Applied Microbiology and Biotechnology, 103 (14), ISSN:0175-7598

Background It is well known that mushrooms produce defense proteins and secondary metabolites against predators and competitors; however, less is known about the correlation between the tissue-specific expression and the target organism (antagonist) specificity of these molecules. In addition, conserved transcriptional circuitries involved in developing sexual organs in fungi are not characterized, despite the growing number of gene expression datasets available from reproductive and vegetative tissue. The aims of this study were: first, to evaluate the tissue specificity of defense gene expression in the model mushroom Coprinopsis cinerea and, second, to assess the degree of conservation in transcriptional regulation during sexual development in basidiomycetes. Results In order to characterize the regulation in the expression of defense loci and the transcriptional circuitries controlling sexual reproduction in basidiomycetes, we sequenced the poly (A)-positive transcriptome of stage 1 primordia and vegetative mycelium of C. cinerea A43mutB43mut. Our data show that many genes encoding predicted and already characterized defense proteins are differentially expressed in these tissues. The predicted specificity of these proteins with regard to target organisms suggests that their expression pattern correlates with the type of antagonists these tissues are confronted with. Accordingly, we show that the stage 1 primordium-specific protein CC1G_11805 is toxic to insects and nematodes. Comparison of our data to analogous data from Laccaria bicolor and Schizophyllum commune revealed that the transcriptional regulation of nearly 70 loci is conserved and probably subjected to stabilizing selection. A Velvet domain-containing protein was found to be up-regulated in all three fungi, providing preliminary evidence of a possible role of the Velvet protein family in sexual development of basidiomycetes. The PBS-soluble proteome of C. cinerea primordia and mycelium was analyzed by shotgun LC-MS. This proteome data confirmed the presence of intracellular defense proteins in primordia. Conclusions This study shows that the exposure of different tissues in fungi to different types of antagonists shapes the expression pattern of defense loci in a tissue-specific manner. Furthermore, we identify a transcriptional circuitry conserved among basidiomycetes during fruiting body formation that involves, amongst other transcription factors, the up-regulation of a Velvet domain-containing protein. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-492) contains supplementary material, which is available to authorized users.

Fungi are competent producers of a large variety of natural products, including peptides. Peptides can be non-ribosomally encoded, referred to as non-ribosomal peptides (NRPs), or ribosomally encoded, referred to as ribosomally synthesized and posttranslationally modified peptides (RiPPs). While many fungal NRPs have been studied to date, including prominent representatives such as penicillin and cyclosporin, the first fungal RiPP was not discovered until 2007. RiPPs have several properties that make them interesting targets for research and biotechnological applications: Their precursors are genetically encoded and can be genetically modified, they are processed by promiscuous enzymes, and they can adapt a high degree of complexity through various posttranslational modifications. In recent years, another chapter was added to research on RiPPs when it was discovered that a class of peptide-encoding precursor proteins, called KEX2-processed repeat proteins (KEPs), are widely distributed in the fungal kingdom and their functions are largely unknown. The aim of this thesis was to expand our understanding of ribosomally produced fungal peptides, with a special focus on peptides derived from KEPs. Chapter 1 provides an introduction to fungal RiPPs and their biosynthesis. First, the RiPP family of amatoxins/phallotoxins is discussed, which includes famous lethal toxins present in, e.g., the death cap, followed by the most recently discovered RiPP family, the borosins, which carry backbone N-methylations. Finally, the dikaritins are examined. These are functionally diverse cyclic peptides processed from precursor proteins with a KEP-like structure. In addition, the potential of RiPP biosynthetic pathways for the development of peptide therapeutics is discussed, both in terms of the identification of new fungal RiPPs and in terms of a synthetic biology approach using biosynthetic enzymes to generate new-to-nature peptide libraries that could be screened for bioactivity. Chapter 2 describes the structural and functional characterization of KEP-derived peptides in the agaricomycetes Coprinopsis cinerea, Lentinula edodes, Pleurotus ostreatus and Pleurotus eryngii. Genome mining identified several KEPs in C. cinerea. A selection of these proteins was successfully expressed in Pichia pastoris and processed by the yeast’s enzymatic machinery, resulting in an accumulation of KEP-derived peptides in the culture supernatant. This experimental setup was used to establish a protocol for the extraction and detection of KEP-derived peptides in fungal tissues by mass spectrometry. Using this KEP detection protocol, multiple KEP-derived peptides were confirmed in culture supernatant and tissue samples of C. cinerea, L. edodes, P. ostreatus and P. eryngii. In addition, we established a CRISPR-mediated knockout protocol for C. cinerea and generated knockout strains of six different KEPs, three KEX2 homologs and one KEX1 homolog. We found that the kep knockouts resulted in no discernible phenotype, while the kex knockouts revealed defects in mycelial growth and fruiting body formation. These results suggest that the investigated KEP-derived peptides do not have an organism-intrinsic role but rather function in the interaction with the biotic environment. The KEX proteases, on the other hand, apparently target a wide variety of substrates, including some with roles in mycelial growth and differentiation. 6 Chapter 3 presents the new draft genome sequences of four fungal species: Rhizopogon roseolus, Mariannaea elegans, Myrothecium verrucaria and Sphaerostilbella broomeana. The genomes were screened for precursor proteins or gene clusters involved in the synthesis of the peptide macrocycles mariannamides and the backbone N-methylated macrocycles verrucamides and broomeanamides. We performed BLAST searches to find potential RiPP precursor proteins of these peptides and included a general screen for borosin-producing precursor proteins. While this search was unsuccessful, we found non-ribosomal peptide synthase (NRPS) gene clusters in an antiSMASH screening that may be responsible for the biosynthesis of mariannamides, verrucamides and broomeanamides. In addition, we discovered several gene clusters encoding putative peptaibols, an antimicrobial class of peptides. Finally, Chapter 4 summarizes the findings of this thesis and gives an outlook on future experiments