Exploring fungal RiPPs from the perspective of chemical ecology

Since the initial detection, in 2007, of fungal ribosomally synthesised and post-translationally modified peptides (RiPPs), this group of natural products has undergone rapid expansion, with four separate classes now recognised: amatoxins/phallotoxins, borosins, dikaritins, and epichloëcyclins. Largely due to their historically anthropocentric employment in medicine and agriculture, novel fungal proteins and peptides are seldom investigated in relation to the fungus itself. Therefore, although the benefits these compounds confer to humans are often realised, their evolutionary advantage to the fungus, the reason for their continued production, is often obscure or ignored. This review sets out to summarise current knowledge on how these small peptide-derived products influence their producing species and surrounding biotic environment

Rice False Smut: An Increasing Threat to Grain Yield and Quality

Rice false smut (RFS) is the most important grain disease in rice production worldwide. Its epidemics not only lead to yield loss but also reduce grain quality because of multiple mycotoxins generated by the causative pathogen, Villosiclava virens (anamorph: Ustilaginoidea virens). The pathogen infects developing spikelets and specifically converts individual grain into a RFS ball that is established from mycelia covered with powdery chlamydospores, sometimes generating sclerotia. RFS balls seem to be randomly formed in some grains on a panicle of a plant in the paddy field. However, epidemics differ largely among varieties, fields, and seasons. This chapter introduces current understanding on the disease, mycotoxins, the biology of the pathogen, pathogenesis of RFS, rice resistance, the disease cycle, the disease control, and assay

The role of RiPP proteins in plant pathogenic fungi

The ascomycete fungus, Zymoseptoria tritici, has risen in prevalence and significance in the past few decades, overtaking wheat pathogens such as Stagonospora nodorum for the title of most prevalent foliar wheat pathogen in the UK and Europe – as well as several other countries worldwide. Losses to the pathogen can be significant, as such, the fungus and its associated disease, Septoria tritici blotch, presents a huge threat to global wheat production and food security given the dietary importance of wheat grain. Zymoseptoria tritici infection of wheat includes a biotrophic-like latent phase and necrotrophic stage, however, the transition between the two is currently poorly understood, and assumed to involve fungal effectors which trigger the plant hypersensitive response. Equally, fungal ribosomally synthesised and post-translationally modified peptides (RiPPs) are under-researched, despite the RiPP victorin contributing to Cochliobolus victoriae virulence on Vb oat cultivars.This thesis explores a fungal RiPP from Z. tritici, the biosynthetic pathway of which has been characterised bioinformatically with knockout strains produced for future experimental confirmation of the method predicted in this work. Bioinformatic investigation also proved informative regarding RiPP repeat variation between strains of the same species and in identifying novel RiPP producers entirely. Attempts were made to understand the function of the RiPP, to determine whether it was involved in pathogenicity, as with victorin, this however remains elusive. Although the Zymoseptoria RiPP does not have a clear role in virulence given that null mutants were fully virulent, results from this work demonstrated the impact of the environment on the wheat-Zymoseptoria interaction, demonstrating the multiple routes that can be explored to control Z. tritici. Overall, this work has extended our understanding of Zymoseptoria tritici – by examining the environmental conditions conducive or inconducive to infection – and its RiPP, with this also contributing to our knowledge of fungal RiPPs more widely

Doctor of Philosophy

dissertationCyanobactins are peptide natural products that fall under the broad class of the ribosomally synthesized and posttranslationally modified peptides (RiPPs). Since they are synthesized by the ribosome, the biosynthesis of these peptides is genetically encoded. A precursor peptide gene carries the primary amino acid sequence of the natural product. The precursor peptide is surrounded by other genes, which encode posttranslational enzymes that decorate the primary sequence with elaborate structural motifs. Due to the genetically encoded origins of the cyanobactins, simple manipulations at the peptide sequence level are tolerable and lead to the creation of a diversity of natural products. The roots to this tolerance lie in the innate extreme broad-substrate nature of the posttranslational enzymes. Here, we explore the biochemical basis of this promiscuity and in vitro methodologies to create structurally elaborate peptidic motifs. In addition, the cyanobactin biosynthetic machinery is a rich source of enzymes capable of performing a wide array of intriguing chemistry and here we probe into some of these mechanisms. Put together, the broad-substrate nature coupled with the unique enzymology of the cyanobactin biosynthetic machinery provides a toolkit for the creation of designer peptide motifs. This work holds promise in the field of peptide-based drug discovery

Ionic liquids as a versatile tool to study antimicrobial strategies of and against filamentous fungi

"Life threatening microbial infections caused by pathogenic bacteria or fungi are becoming an increasing concern for our society, due to the increasing appearance of antimicrobial (multi-)resistant strains and the lack of appropriate treatment options. In the case of fungal infections, Aspergillus spp. can lead to severe invasive infections with Aspergillus fumigatus being the main cause, especially in severely immunocompromised patients.

Advances in molecular and genomic research to safeguard food and feed supply from aflatoxin contamination

Worldwide recognition that aflatoxin contamination of agricultural commodities by the fungus Aspergillus flavus is a global problem has significantly benefitted from global collaboration for understanding the contaminating fungus, as well as for developing and implementing solutions against the contamination. The effort to address this serious food and feed safety issue has led to a detailed understanding of the taxonomy, ecology, physiology, genomics and evolution of A. flavus, as well as strategies to reduce or control pre-harvest aflatoxin contamination, including (1) biological control, using atoxigenic aspergilli, (2) proteomic and genomic analyses for identifying resistance factors in maize as potential breeding markers to enable development of resistant maize lines, and (3) enhancing host-resistance by bioengineering of susceptible crops, such as cotton, maize, peanut and tree nuts. A post-harvest measure to prevent the occurrence of aflatoxin contamination in storage is also an important component for reducing exposure of populations worldwide to aflatoxins in food and feed supplies. The effect of environmental changes on aflatoxin contamination levels has recently become an important aspect for study to anticipate future contamination levels. The ability of A. flavus to produce dozens of secondary metabolites, in addition to aflatoxins, has created a new avenue of research for understanding the role these metabolites play in the survival and biodiversity of this fungus. The understanding of A. flavus, the aflatoxin contamination problem, and control measures to prevent the contamination has become a unique example for an integrated approach to safeguard global food and feed safety

Lessons on fruiting body morphogenesis from genomes and transcriptomes of Agaricomycetes

Fruiting bodies (sporocarps, sporophores or basidiomata) of mushroom-forming fungi (Agaricomycetes) are among the most complex structures produced by fungi. Unlike vegetative hyphae, fruiting bodies grow determinately and follow a genetically encoded developmental program that orchestrates their growth, tissue differentiation and sexual sporulation. In spite of more than a century of research, our understanding of the molecular details of fruiting body morphogenesis is still limited and a general synthesis on the genetics of this complex process is lacking. In this paper, we aim at a comprehensive identification of conserved genes related to fruiting body morphogenesis and distil novel functional hypotheses for functionally poorly characterised ones. As a result of this analysis, we report 921 conserved developmentally expressed gene families, only a few dozens of which have previously been reported to be involved in fruiting body development. Based on literature data, conserved expression patterns and functional annotations, we provide hypotheses on the potential role of these gene families in fruiting body development, yielding the most complete description of molecular processes in fruiting body morphogenesis to date. We discuss genes related to the initiation of fruiting, differentiation, growth, cell surface and cell wall, defence, transcriptional regulation as well as signal transduction. Based on these data we derive a general model of fruiting body development, which includes an early, proliferative phase that is mostly concerned with laying out the mushroom body plan (via cell division and differentiation), and a second phase of growth via cell expansion as well as meiotic events and sporulation. Altogether, our discussions cover 1 480 genes of Coprinopsis cinerea, and their orthologs in Agaricus bisporus, chrysosporium, Pleurotus ostreatus, and Schizophyllum commune, providing functional hypotheses for similar to 10 % of genes in the genomes of these species. Although experimental evidence for the role of these genes will need to be established in the future, our data provide a roadmap for guiding functional analyses of fruiting related genes in the Agaricomycetes. We anticipate that the gene compendium presented here, combined with developments in functional genomics approaches will contribute to uncovering the genetic bases of one of the most spectacular multicellular developmental processes in fungi

Lessons on fruiting body morphogenesis from genomes and transcriptomes of Agaricomycetes.

Fruiting bodies (sporocarps, sporophores or basidiomata) of mushroom-forming fungi ( Agaricomycetes) are among the most complex structures produced by fungi. Unlike vegetative hyphae, fruiting bodies grow determinately and follow a genetically encoded developmental program that orchestrates their growth, tissue differentiation and sexual sporulation. In spite of more than a century of research, our understanding of the molecular details of fruiting body morphogenesis is still limited and a general synthesis on the genetics of this complex process is lacking. In this paper, we aim at a comprehensive identification of conserved genes related to fruiting body morphogenesis and distil novel functional hypotheses for functionally poorly characterised ones. As a result of this analysis, we report 921 conserved developmentally expressed gene families, only a few dozens of which have previously been reported to be involved in fruiting body development. Based on literature data, conserved expression patterns and functional annotations, we provide hypotheses on the potential role of these gene families in fruiting body development, yielding the most complete description of molecular processes in fruiting body morphogenesis to date. We discuss genes related to the initiation of fruiting, differentiation, growth, cell surface and cell wall, defence, transcriptional regulation as well as signal transduction. Based on these data we derive a general model of fruiting body development, which includes an early, proliferative phase that is mostly concerned with laying out the mushroom body plan (via cell division and differentiation), and a second phase of growth via cell expansion as well as meiotic events and sporulation. Altogether, our discussions cover 1 480 genes of Coprinopsis cinerea, and their orthologs in Agaricus bisporus, Cyclocybe aegerita, Armillaria ostoyae, Auriculariopsis ampla, Laccaria bicolor, Lentinula edodes, Lentinus tigrinus, Mycena kentingensis, Phanerochaete chrysosporium, Pleurotus ostreatus, and Schizophyllum commune, providing functional hypotheses for ~10 % of genes in the genomes of these species. Although experimental evidence for the role of these genes will need to be established in the future, our data provide a roadmap for guiding functional analyses of fruiting related genes in the Agaricomycetes. We anticipate that the gene compendium presented here, combined with developments in functional genomics approaches will contribute to uncovering the genetic bases of one of the most spectacular multicellular developmental processes in fungi. Citation: Nagy LG, Vonk PJ, Künzler M, Földi C, Virágh M, Ohm RA, Hennicke F, Bálint B, Csernetics Á, Hegedüs B, Hou Z, Liu XB, Nan S, M. Pareek M, Sahu N, Szathmári B, Varga T, Wu W, Yang X, Merényi Z (2023). Lessons on fruiting body morphogenesis from genomes and transcriptomes of Agaricomycetes. Studies in Mycology 104: 1-85. doi: 10.3114/sim.2022.104.01