Chitin and chitosan remodeling defines vegetative development and Trichoderma biocontrol.

Fungal parasitism depends on the ability to invade host organisms and mandates adaptive cell wall remodeling to avoid detection and defense reactions by the host. All plant and human pathogens share invasive strategies, which aid to escape the chitin-triggered and chitin-targeted host immune system. Here we describe the full spectrum of the chitin/chitosan-modifying enzymes in the mycoparasite Trichoderma atroviride with a central role in cell wall remodeling. Rapid adaption to a variety of growth conditions, environmental stresses and host defense mechanisms such as oxidative stress depend on the concerted interplay of these enzymes and, ultimately, are necessary for the success of the mycoparasitic attack. To our knowledge, we provide the first in class description of chitin and associated glycopolymer synthesis in a mycoparasite and demonstrate that they are essential for biocontrol. Eight chitin synthases, six chitin deacetylases, additional chitinolytic enzymes, including six chitosanases, transglycosylases as well as accessory proteins are involved in this intricately regulated process. Systematic and biochemical classification, phenotypic characterization and mycoparasitic confrontation assays emphasize the importance of chitin and chitosan assembly in vegetative development and biocontrol in T. atroviride. Our findings critically contribute to understanding the molecular mechanism of chitin synthesis in filamentous fungi and mycoparasites with the overarching goal to selectively exploit the discovered biocontrol strategies.

The N-acetylglucosamine catabolic gene cluster in Trichoderma reesei is controlled by the Ndt80-like transcription factor RON1.

Chitin is an important structural constituent of fungal cell walls composed of N-acetylglucosamine (GlcNAc) monosaccharides, but catabolism of GlcNAc has not been studied in filamentous fungi so far. In the yeast Candida albicans, the genes encoding the three enzymes responsible for stepwise conversion of GlcNAc to fructose-6-phosphate are clustered. In this work, we analysed GlcNAc catabolism in ascomycete filamentous fungi and found that the respective genes are also clustered in these fungi. In contrast to C. albicans, the cluster often contains a gene for an Ndt80-like transcription factor, which we named RON1 (regulator of N-acetylglucosamine catabolism 1). Further, a gene for a glycoside hydrolase 3 protein related to bacterial N-acetylglucosaminidases can be found in the GlcNAc gene cluster in filamentous fungi. Functional analysis in Trichoderma reesei showed that the transcription factor RON1 is a key activator of the GlcNAc gene cluster and essential for GlcNAc catabolism. Furthermore, we present an evolutionary analysis of Ndt80-like proteins in Ascomycota. All GlcNAc cluster genes, as well as the GlcNAc transporter gene ngt1, and an additional transcriptional regulator gene, csp2, encoding the homolog of Neurospora crassa CSP2/GRHL, were functionally characterised by gene expression analysis and phenotypic characterisation of knockout strains in T. reesei.

N-acetylglucosamine, the building block of chitin, inhibits growth of Neurospora crassa.

N-acetylglucosamine (GlcNAc) is the monomer of the polysaccharide chitin, an essential structural component of the fungal cell wall and the arthropod exoskeleton. We recently showed that the genes encoding the enzymes for GlcNAc catabolism are clustered in several ascomycetes. In the present study we tested these fungi for growth on GlcNAc and chitin. All fungi, containing the GlcNAc gene cluster, could grow on GlcNAc with the exception of four independent Neurospora crassa wild-type isolates, which were however able to grow on chitin. GlcNAc even inhibited their growth in the presence of other carbon sources. Genes involved in GlcNAc catabolism were strongly upregulated in the presence of GlcNAc, but during growth on chitin their expression was not increased. Deletion of hxk-3 (encoding the first catabolic enzyme, GlcNAc-hexokinase) and ngt-1 (encoding the GlcNAc transporter) improved growth of N. crassa on GlcNAc in the presence of glycerol. A crucial step in GlcNAc catabolism is enzymatic conversion from glucosamine-6-phosphate to fructose-6-phosphate, catalyzed by the glucosamine-6-phosphate deaminase, DAM-1. To assess, if DAM-1 is compromised in N. crassa, the orthologue from Trichoderma reesei, Trdam1, was expressed in N. crassa. Trdam1 expression partially alleviated the negative effects of GlcNAc in the presence of a second carbon source, but did not fully restore growth on GlcNAc. Our results indicate that the GlcNAc-catabolism pathway is bypassed during growth of N. crassa on chitin by use of an alternative pathway, emphasizing the different strategies that have evolved in the fungal kingdom for chitin utilization.

Molecular and catalytic properties of fungal extracellular cellobiose dehydrogenase produced in prokaryotic and eukaryotic expression systems.

BACKGROUND: Cellobiose dehydrogenase (CDH) is an extracellular enzyme produced by lignocellulolytic fungi. cdh gene expression is high in cellulose containing media, but relatively low CDH concentrations are found in the supernatant of fungal cultures due to strong binding to cellulose. Therefore, heterologous expression of CDH in Pichia pastoris was employed in the last 15 years, but the obtained enzymes were over glycosylated and had a reduced specific activity. RESULTS: We compare the well-established CDH expression host P. pastoris with the less frequently used hosts Escherichia coli, Aspergillus niger, and Trichoderma reesei. The study evaluates the produced quantity and protein homogeneity of Corynascus thermophilus CDH in the culture supernatants, the purification, and finally compares the enzymes in regard to cofactor loading, glycosylation, catalytic constants and thermostability. CONCLUSIONS: Whereas E. coli could only express the catalytic dehydrogenase domain of CDH, all eukaryotic hosts could express full length CDH including the cytochrome domain. The CDH produced by T. reesei was most similar to the CDH originally isolated from the fungus C. thermophilus in regard to glycosylation, cofactor loading and catalytic constants. Under the tested experimental conditions the fungal expression hosts produce CDH of superior quality and uniformity compared to P. pastoris.

The drug diazaborine blocks ribosome biogenesis by inhibiting the AAA-ATPase Drg1.

The drug diazaborine is the only known inhibitor of ribosome biogenesis and specifically blocks large subunit formation in eukaryotic cells. However, the target of this drug and the mechanism of inhibition were unknown. Here we identify the AAA-ATPase Drg1 as a target of diazaborine. Inhibitor binding into the second AAA domain of Drg1 requires ATP loading and results in inhibition of ATP hydrolysis in this site. As a consequence the physiological activity of Drg1, i.e. the release of Rlp24 from pre-60S particles, is blocked, and further progression of cytoplasmic preribosome maturation is prevented. Our results identify the first target of an inhibitor of ribosome biogenesis and provide the mechanism of inhibition of a key step in large ribosomal subunit formation.

Molecular diversity of LysM carbohydrate-binding motifs in fungi.

LysM motifs are carbohydrate-binding modules found in prokaryotes and eukaryotes. They bind to N-acetylglucosamine-containing carbohydrates, such as chitin, chitio-oligosaccharides and peptidoglycan. In this review, we summarize the features of the protein architecture of LysM-containing proteins in fungi and discuss their so far known biochemical properties, transcriptional profiles and biological functions. Further, based on data from evolutionary analyses and consensus pattern profiling of fungal LysM motifs, we show that they can be classified into a fungal-specific group and a fungal/bacterial group. This facilitates the classification and selection of further LysM proteins for detailed analyses and will contribute to widening our understanding of the functional spectrum of this protein family in fungi. Fungal LysM motifs are predominantly found in subgroup C chitinases and in LysM effector proteins, which are secreted proteins with LysM motifs but no catalytic domains. In enzymes, LysM motifs mediate the attachment to insoluble carbon sources. In plants, receptors containing LysM motifs are responsible for the perception of chitin-oligosaccharides and are involved in beneficial symbiotic interactions between plants and bacteria or fungi, as well as plant defence responses. In plant pathogenic fungi, LysM effector proteins have already been shown to have important functions in the dampening of host defence responses as well as protective functions of fungal hyphae against chitinases. However, the large number and diversity of proteins with LysM motifs that are being unravelled in fungal genome sequencing projects suggest that the functional repertoire of LysM effector proteins in fungi is only partially discovered so far.

Quantitative proteomic analysis of plasma membranes from the fish pathogen Saprolegnia parasitica reveals promising targets for disease control.

The phylum Oomycota contains economically important pathogens of animals and plants, including Saprolegnia parasitica, the causal agent of the fish disease saprolegniasis. Due to intense fish farming and banning of the most effective control measures, saprolegniasis has re-emerged as a major challenge for the aquaculture industry. Oomycete cells are surrounded by a polysaccharide-rich cell wall matrix that, in addition to being essential for cell growth, also functions as a protective "armor." Consequently, the enzymes responsible for cell wall synthesis provide potential targets for disease control. Oomycete cell wall biosynthetic enzymes are predicted to be plasma membrane proteins. To identify these proteins, we applied a quantitative (iTRAQ) mass spectrometry-based proteomics approach to the plasma membrane of the hyphal cells of S. parasitica, providing the first complete plasma membrane proteome of an oomycete species. Of significance is the identification of 65 proteins enriched in detergent-resistant microdomains (DRMs). In silico analysis showed that DRM-enriched proteins are mainly involved in molecular transport and ß-1,3-glucan synthesis, potentially contributing to pathogenesis. Moreover, biochemical characterization of the glycosyltransferase activity in these microdomains further supported their role in ß-1,3-glucan synthesis. Altogether, the knowledge gained in this study provides a basis for developing disease control measures targeting specific plasma membrane proteins in S. parasitica.IMPORTANCEThe significance of this research lies in its potential to combat saprolegniasis, a detrimental fish disease, which has resurged due to intensive fish farming and regulatory restrictions. By targeting enzymes responsible for cell wall synthesis in Saprolegnia parasitica, this study uncovers potential avenues for disease control. Particularly noteworthy is the identification of several proteins enriched in membrane microdomains, offering insights into molecular mechanisms potentially involved in pathogenesis. Understanding the role of these proteins provides a foundation for developing targeted disease control measures. Overall, this research holds promise for safeguarding the aquaculture industry against the challenges posed by saprolegniasis.

A comparative cell wall analysis of Trichoderma spp. confirms a conserved polysaccharide scaffold and suggests an important role for chitosan in mycoparasitism.

Fungal cell walls are dynamic extracellular matrices that enable efficient adaptation to changing environments. While the cell wall compositions of yeasts, human, and plant pathogenic fungi have been studied to some extent, the cell walls of mycoparasites remain poorly characterized. Trichoderma species comprise a diverse group of soil fungi with different survival strategies and lifestyles. The comparative study of cell wall carbohydrate-active enzymes in 13 Trichoderma spp. revealed that the types of enzymes involved in chitin and chitosan metabolism are phylogenetically distant between mycoparasitic and saprotrophic species. Here, we compare the carbohydrate composition and function of the cell wall of a saprotrophic strain Trichoderma reesei with that of the mycoparasitic, biological control agent Trichoderma atroviride. Monosaccharide and glycosidic linkage analyses as well as dual in situ interaction assays showed that the cell wall polysaccharide composition is conserved between both species, except for the amounts of chitin detected. The results suggest that the observed accumulation of chitosan during mycoparasitism may prevent host recognition. Remarkably, Trichoderma atroviride undergoes dynamic cell wall adaptations during both vegetative development and mycoparasitism, which appears to be confirmed by an evolutionarily expanded group of specialized enzymes. Overall, our analyses support the notion that habitat specialization is reflected in cell wall architecture and that plastic chitin remodeling may confer an advantage to mycoparasites, ultimately enabling the successful invasion and parasitism of plant pathogens. This information may potentially be exploited for the control of crop diseases using biological agents. IMPORTANCE: Trichoderma species are emerging model fungi for the development of biocontrol agents and are used in industrial biotechnology as efficient enzyme producers. Fungal cell walls are complex structures that differ in carbohydrate, protein, and enzyme composition across taxa. Here, we present a chemical characterization of the cell walls of two Trichoderma spp., namely the predominantly saprotrophic Trichoderma reesei and the mycoparasite Trichoderma atroviride. Chemical profiling revealed that Trichoderma spp. remodel their cell wall to adapt to particular lifestyles, with dynamic changes during vegetative development. Importantly, we found that chitosan accumulation during mycoparasitism of a fungal host emerged as a sophisticated strategy underpinning an effective attack. These insights shed light on the molecular mechanisms that allow mycoparasites to overcome host defenses and can be exploited to improve the application of T. atroviride in biological pest control. Moreover, our results provide valuable information for targeting the fungal cell wall for therapeutic purposes.

The Multilateral Efficacy of Chitosan and Trichoderma on Sugar Beet.

The majority of all fungal formulations contain Trichoderma spp., making them effective biological control agents for agriculture. Chitosan, one of the most effective natural biopolymers, was also reported as a plant resistance enhancer and as a biocide against a variety of plant pathogens. An in vitro three-way interaction assay of T. atroviride, chitosan, and important plant pathogens (such as Cercospora beticola and Fusarium oxysporum) revealed a synergistic effect on fungistasis. Furthermore, chitosan coating on Beta vulgaris ssp. vulgaris seeds positively affected the onset and efficiency of germination. We show that priming with T. atroviride spores or chitosan leads to the induced expression of a pathogenesis-related gene (PR-3), but only supplementation of chitosan led to significant upregulation of phytoalexin synthesis (PAL) and oxidative stress-related genes (GST) as a defense response. Repeated foliar application of either agent promoted growth, triggered defense reactions, and reduced incidence of Cercospora leaf spot (CLS) disease in B. vulgaris. Our data suggest that both agents are excellent candidates to replace or assist common fungicides in use. Chitosan triggered the systemic resistance and had a biocidal effect, while T. atroviride mainly induced stress-related defense genes in B. vulgaris. We assume that both agents act synergistically across different signaling pathways, which could be of high relevance for their combinatorial and thus beneficial application on field.

Cytoplasmic recycling of 60S preribosomal factors depends on the AAA protein Drg1.

Allelic forms of DRG1/AFG2 confer resistance to the drug diazaborine, an inhibitor of ribosome biogenesis in Saccharomyces cerevisiae. Our results show that the AAA-ATPase Drg1 is essential for 60S maturation and associates with 60S precursor particles in the cytoplasm. Functional inactivation of Drg1 leads to an increased cytoplasmic localization of shuttling pre-60S maturation factors like Rlp24, Arx1, and Tif6. Surprisingly, Nog1, a nuclear pre-60S factor, was also relocalized to the cytoplasm under these conditions, suggesting that it is a previously unsuspected shuttling preribosomal factor that is exported with the precursor particles and very rapidly reimported. Proteins that became cytoplasmic under drg1 mutant conditions were blocked on pre-60S particles at a step that precedes the association of Rei1, a later-acting preribosomal factor. A similar cytoplasmic accumulation of Nog1 and Rlp24 in pre-60S-bound form could be seen after overexpression of a dominant-negative Drg1 variant mutated in the D2 ATPase domain. We conclude that the ATPase activity of Drg1 is required for the release of shuttling proteins from the pre-60S particles shortly after their nuclear export. This early cytoplasmic release reaction defines a novel step in eukaryotic ribosome maturation.

A conserved inter-domain communication mechanism regulates the ATPase activity of the AAA-protein Drg1.

AAA-ATPases fulfil essential roles in different cellular pathways and often act in form of hexameric complexes. Interaction with pathway-specific substrate and adaptor proteins recruits them to their targets and modulates their catalytic activity. This substrate dependent regulation of ATP hydrolysis in the AAA-domains is mediated by a non-catalytic N-terminal domain. The exact mechanisms that transmit the signal from the N-domain and coordinate the individual AAA-domains in the hexameric complex are still the topic of intensive research. Here, we present the characterization of a novel mutant variant of the eukaryotic AAA-ATPase Drg1 that shows dysregulation of ATPase activity and altered interaction with Rlp24, its substrate in ribosome biogenesis. This defective regulation is the consequence of amino acid exchanges at the interface between the regulatory N-domain and the adjacent D1 AAA-domain. The effects caused by these mutations strongly resemble those of pathological mutations of the AAA-ATPase p97 which cause the hereditary proteinopathy IBMPFD (inclusion body myopathy associated with Paget's disease of the bone and frontotemporal dementia). Our results therefore suggest well conserved mechanisms of regulation between structurally, but not functionally related members of the AAA-family.

Rlp24 activates the AAA-ATPase Drg1 to initiate cytoplasmic pre-60S maturation.

Formation of eukaryotic ribosomes is driven by energy-consuming enzymes. The AAA-ATPase Drg1 is essential for the release of several shuttling proteins from cytoplasmic pre-60S particles and the loading of late joining proteins. However, its exact role in ribosome biogenesis has been unknown. Here we show that the shuttling protein Rlp24 recruited Drg1 to pre-60S particles and stimulated its ATPase activity. ATP hydrolysis in the second AAA domain of Drg1 was required to release shuttling proteins. In vitro, Drg1 specifically and exclusively extracted Rlp24 from purified pre-60S particles. Rlp24 release required ATP and was promoted by the interaction of Drg1 with the nucleoporin Nup116. Subsequent ATP hydrolysis in the first AAA domain dissociated Drg1 from Rlp24, liberating both proteins for consecutive cycles of activity. Our results show that release of Rlp24 by Drg1 defines a key event in large subunit formation that is a prerequisite for progression of cytoplasmic pre-60S maturation.