Bacterial growth-mediated systems remodelling of Nicotiana benthamiana defines unique signatures of target protein production in molecular pharming.

The need for therapeutics to treat a plethora of medical conditions and diseases is on the rise and the demand for alternative approaches to mammalian-based production systems is increasing. Plant-based strategies provide a safe and effective alternative to produce biological drugs but have yet to enter mainstream manufacturing at a competitive level. Limitations associated with batch consistency and target protein production levels are present; however, strategies to overcome these challenges are underway. In this study, we apply state-of-the-art mass spectrometry-based proteomics to define proteome remodelling of the plant following agroinfiltration with bacteria grown under shake flask or bioreactor conditions. We observed distinct signatures of bacterial protein production corresponding to the different growth conditions that directly influence the plant defence responses and target protein production on a temporal axis. Our integration of proteomic profiling with small molecule detection and quantification reveals the fluctuation of secondary metabolite production over time to provide new insight into the complexities of dual system modulation in molecular pharming. Our findings suggest that bioreactor bacterial growth may promote evasion of early plant defence responses towards Agrobacterium tumefaciens (updated nomenclature to Rhizobium radiobacter). Furthermore, we uncover and explore specific targets for genetic manipulation to suppress host defences and increase recombinant protein production in molecular pharming.

Debunking the Myth of Fusarium poae T-2/HT-2 Toxin Production.

Fusarium poae is commonly detected in field surveys of Fusarium head blight (FHB) of cereal crops and can produce a range of trichothecene mycotoxins. Although experimentally validated reports of F. poae strains producing T-2/HT-2 trichothecenes are rare, F. poae is frequently generalized in the literature as a producer of T-2/HT-2 toxins due to a single study from 2004 in which T-2/HT-2 toxins were detected at low levels from six out of forty-nine F. poae strains examined. To validate/substantiate the observations reported from the 2004 study, the producing strains were acquired and phylogenetically confirmed to be correctly assigned as F. poae; however, no evidence of T-2/HT-2 toxin production was observed from axenic cultures. Moreover, no evidence for a TRI16 ortholog, encoding a key acyltransferase shown to be necessary for T-2 toxin production in other Fusarium species, was observed in any of the de novo assembled genomes of the F. poae strains. Our findings corroborate multiple field-based and in vitro studies on FHB-associated Fusarium populations which also do not support the production of T-2/HT-2 toxins with F. poae and therefore conclude that F. poae should not be generalized as a T-2/HT-2 toxin producing species of Fusarium.

Chromosome-level draft genome sequences of three isolates of the toxigenic fungus Claviceps purpurea showing structural rearrangements.

The whole genomes of three Claviceps purpurea strains were sequenced using Oxford Nanopore Technologies' MinION and assembled into complete, chromosome-level assemblies. The C. purpurea genome consists of eight conserved chromosomes, with evidence of inter-chromosomal structural rearrangements between strains.

CRISPR-Cas9 Gene Editing and Secondary Metabolite Screening Confirm Fusarium graminearum C16 Biosynthetic Gene Cluster Products as Decalin-Containing Diterpenoid Pyrones.

Fusarium graminearum is a causal organism of Fusarium head blight in cereals and maize. Although a few secondary metabolites produced by F. graminearum are considered disease virulence factors, many molecular products of biosynthetic gene clusters expressed by F. graminearum during infection and their associated role in the disease are unknown. In particular, the predicted meroterpenoid products of the biosynthetic gene cluster historically designated as "C16" are likely associated with pathogenicity. Presented here are the results of CRISPR-Cas9 gene-editing experiments disrupting the polyketide synthase and terpene synthase genes associated with the C16 biosynthetic gene cluster in F. graminearum. Culture medium screening experiments using transformant strains were profiled by UHPLC-HRMS and targeted MS2 experiments to confirm the associated secondary metabolite products of the C16 biosynthetic gene cluster as the decalin-containing diterpenoid pyrones, FDDP-D and FDDP-E. Both decalin-containing diterpenoid pyrones were confirmed to be produced in wheat heads challenged with F. graminearum in growth chamber trials. The extent to which the F. graminearum C16 biosynthetic gene cluster is dispersed within the genus Fusarium is discussed along with a proposed role of the FDDPs as pathogen virulence factors.

Multiple Clonostachys rosea UDP-Glycosyltransferases Contribute to the Production of 15-Acetyl-Deoxynivalenol-3-O-Glycoside When Confronted with Fusarium graminearum.

Mycotoxins, derived from toxigenic fungi such as Fusarium, Aspergillus, and Penicillium species have impacted the human food chain for thousands of years. Deoxynivalenol (DON), is a tetracyclic sesquiterpenoid type B trichothecene mycotoxin predominantly produced by F. culmorum and F. graminearum during the infection of corn, wheat, oats, barley, and rice. Glycosylation of DON is a protective detoxification mechanism employed by plants. More recently, DON glycosylating activity has also been detected in fungal microparasitic (biocontrol) fungal organisms. Here we follow up on the reported conversion of 15-acetyl-DON (15-ADON) into 15-ADON-3-O-glycoside (15-ADON-3G) in Clonostachys rosea. Based on the hypothesis that the reaction is likely being carried out by a uridine diphosphate glycosyl transferase (UDP-GTase), we applied a protein structural comparison strategy, leveraging the availability of the crystal structure of rice Os70 to identify a subset of potential C. rosea UDP-GTases that might have activity against 15-ADON. Using CRISPR/Cas9 technology, we knocked out several of the selected UDP-GTases in the C. rosea strain ACM941. Evaluation of the impact of knockouts on the production of 15-ADON-3G in confrontation assays with F. graminearum revealed multiple UDP-GTase enzymes, each contributing partial activities. The relationship between these positive hits and other UDP-GTases in fungal and plant species is discussed.

Untargeted metabolomics screening reveals unique secondary metabolite production from Alternaria section Alternaria.

Alternaria section Alternaria is comprised of many species that infect a broad diversity of important crop plants and cause post-harvest spoilage. Alternaria section Alternaria species, such as A. alternata and A. arborescens, are prolific producers of secondary metabolites that act as virulence factors of disease and are mycotoxins that accumulate in infected tissues-metabolites that can vary in their spectrum of production between individuals from the same fungal species. Untargeted metabolomics profiling of secondary metabolite production using mass spectrometry is an effective means to detect phenotypic anomalies in secondary metabolism within a species. Secondary metabolite phenotypes from 36 Alternaria section Alternaria isolates were constructed to observe frequency of production patterns. A clear and unique mass feature pattern was observed for three of the strains that were linked with the production of the dehydrocurvularin family of toxins and associated detoxification products. Examination of corresponding genomes revealed the presence of the dehydrocurvularin biosynthesis gene cluster associated with a sub-telomeric accessory region. A comparison of sequence similarity and occurrences of the dehydrocurvularin biosynthetic gene cluster within Pleosporalean fungi is presented and discussed.

CRISPR/Cas9 disruption of UGT71L1 in poplar connects salicinoid and salicylic acid metabolism and alters growth and morphology.

Salicinoids are salicyl alcohol-containing phenolic glycosides with strong antiherbivore effects found only in poplars and willows. Their biosynthesis is poorly understood, but recently a UDP-dependent glycosyltransferase, UGT71L1, was shown to be required for salicinoid biosynthesis in poplar tissue cultures. UGT71L1 specifically glycosylates salicyl benzoate, a proposed salicinoid intermediate. Here, we analyzed transgenic CRISPR/Cas9-generated UGT71L1 knockout plants. Metabolomic analyses revealed substantial reductions in the major salicinoids, confirming the central role of the enzyme in salicinoid biosynthesis. Correspondingly, UGT71L1 knockouts were preferred to wild-type by white-marked tussock moth (Orgyia leucostigma) larvae in bioassays. Greenhouse-grown knockout plants showed substantial growth alterations, with decreased internode length and smaller serrated leaves. Reinserting a functional UGT71L1 gene in a transgenic rescue experiment demonstrated that these effects were due only to the loss of UGT71L1. The knockouts contained elevated salicylate (SA) and jasmonate (JA) concentrations, and also had enhanced expression of SA- and JA-related genes. SA is predicted to be released by UGT71L1 disruption, if salicyl salicylate is a pathway intermediate and UGT71L1 substrate. This idea was supported by showing that salicyl salicylate can be glucosylated by recombinant UGT71L1, providing a potential link of salicinoid metabolism to SA and growth impacts. Connecting this pathway with growth could imply that salicinoids are under additional evolutionary constraints beyond selective pressure by herbivores.

Untargeted Metabolomic Profiling of Fungal Species Populations.

This chapter describes protocols for the development of consensus chemical phenotypes or "metabolomes" of fungal populations using ultra-high pressure liquid chromatography coupled to high resolution mass spectrometry (UPLC-HRMS). Isolates are cultured using multiple media conditions to elicit the expression of diverse secondary metabolite biosynthetic gene clusters. The mycelium and spent culture media are extracted using organic solvents and profiled by ultra-high pressure chromatography coupled with a high resolution Thermo Orbitrap XL mass spectrometer with the ability to trap and fragment ions to general MS2 spectra. MS data preprocessing is explained and illustrated using the freely available software MZMine 2. Through data processing, binary matrices of mass features can be generated and then combined into a consensus secondary metabolite phenotype of all isolates grown in all media conditions. The production of consensus chemical phenotypes is useful for screening large fungal populations (both inter and intra-species populations) for isolates potentially expressing novel secondary metabolites or analogs of known secondary metabolites.

Extraction methods for untargeted metabolomics influence enzymatic activity in diverse soils.

Soil organic matter (SOM) is the largest carbon pool in terrestrial ecosystems and underpins the health and productivity of soil. Accurate characterization of its chemical composition will improve our understanding of biotic and abiotic processes regulating its stabilization. Our purpose in this study was to estimate the loss of SOM by microbial and exoenzymatic activity that might occur when soil is extracted for analysis of representative low molecular weight mass features using untargeted metabolomics. Two mined clays (kaolinite, montmorillonite) and three diverse soils (varying in texture, specific surface area and cation exchange capacity) were used to assess the extraction efficiency and loss of three enzymatic activity indicators (2,6-dichloroindophenol sodium salt hydrate [DCIP], 4-methylumbelliferyl phosphate [MUBph] and 3,4-dihydroxy-L-phenylalanine [LDOPA]) during extraction with two different solvents (water and methanol). Losses of the indicators were attributed to extraction method (ultrasonication, shaking, or shaking following chloroform fumigation), physical properties associated with the soil/clay type, and microbial activity. Soil/clay type strongly influenced indicator recovery and hence, SOM recovery. Choice of extraction method strongly influenced the composition and recovery of representative SOM mass features, while the choice of solvent determined whether the soil type or extraction method had a greater influence of compositional differences in the SOM mass features extracted. Extraction following chloroform fumigation had the greatest loss of the indicators, due to enzymatic activity and/or adsorption onto the soil matrix. Minimal variation in composition and loss of SOM mass features occurred during extraction by shaking for the soils tested; we therefore recommend it as the method of choice for untargeted SOM extraction studies.

Evolution of the Ergot Alkaloid Biosynthetic Gene Cluster Results in Divergent Mycotoxin Profiles in Claviceps purpurea Sclerotia.

Research into ergot alkaloid production in major cereal cash crops is crucial for furthering our understanding of the potential toxicological impacts of Claviceps purpurea upon Canadian agriculture and to ensure consumer safety. An untargeted metabolomics approach profiling extracts of C. purpurea sclerotia from four different grain crops separated the C. purpurea strains into two distinct metabolomic classes based on ergot alkaloid content. Variances in C. purpurea alkaloid profiles were correlated to genetic differences within the lpsA gene of the ergot alkaloid biosynthetic gene cluster from previously published genomes and from newly sequenced, long-read genome assemblies of Canadian strains. Based on gene cluster composition and unique polymorphisms, we hypothesize that the alkaloid content of C. purpurea sclerotia is currently undergoing adaptation. The patterns of lpsA gene diversity described in this small subset of Canadian strains provides a remarkable framework for understanding accelerated evolution of ergot alkaloid production in Claviceps purpurea.

A metabolomic study of vegetative incompatibility in Cryphonectria parasitica.

Vegetative incompatibility (VI) is a form of non-self allorecognition in filamentous fungi that restricts conspecific hyphal fusion and the formation of heterokaryons. In the chestnut pathogenic fungus, Cryphonectria parasitica, VI is controlled by six vic loci and has been of particular interest because it impedes the spread of hypoviruses and thus biocontrol strategies. We use nuclear magnetic resonance and high-resolution mass spectrometry to characterize alterations in the metabolome of C. parasitica over an eight-day time course of vic3 incompatibility. Our findings support transcriptomic data that indicated remodeling of secondary metabolite profiles occurs during vic3 -associated VI. VI-associated secondary metabolites include novel forms of calbistrin, decumbenone B, a sulfoxygenated farnesyl S-cysteine analog, lysophosphatidylcholines, and an as-yet unidentified group of lipid disaccharides. The farnesyl S-cysteine analog is structurally similar to pheromones predicted to be produced during VI and is here named 'crypheromonin'. Mass features associated with C. parasitica secondary metabolites skyrin, rugulosin and cryphonectric acid were also detected but were not VI specific. Partitioning of VI-associated secondary metabolites was observed, with crypheromonins and most calbistrins accumulating in the growth medium over time, whereas lysophosphatidylcholines, lipid disaccharide-associated mass features and other calbistrin-associated mass features peaked at distinct time points in the mycelium. Secondary metabolite biosynthetic gene clusters and potential biological roles associated with the detected secondary metabolites are discussed.

Apicidin biosynthesis is linked to accessory chromosomes in Fusarium poae isolates.

BACKGROUND: Fusarium head blight is a disease of global concern that reduces crop yields and renders grains unfit for consumption due to mycotoxin contamination. Fusarium poae is frequently associated with cereal crops showing symptoms of Fusarium head blight. While previous studies have shown F. poae isolates produce a range of known mycotoxins, including type A and B trichothecenes, fusarins and beauvericin, genomic analysis suggests that this species may have lineage-specific accessory chromosomes with secondary metabolite biosynthetic gene clusters awaiting description. METHODS: We examined the biosynthetic potential of 38 F. poae isolates from Eastern Canada using a combination of long-read and short-read genome sequencing and untargeted, high resolution mass spectrometry metabolome analysis of extracts from isolates cultured in multiple media conditions. RESULTS: A high-quality assembly of isolate DAOMC 252244 (Fp157) contained four core chromosomes as well as seven additional contigs with traits associated with accessory chromosomes. One of the predicted accessory contigs harbours a functional biosynthetic gene cluster containing homologs of all genes associated with the production of apicidins. Metabolomic and genomic analyses confirm apicidins are produced in 4 of the 38 isolates investigated and genomic PCR screening detected the apicidin synthetase gene APS1 in approximately 7% of Eastern Canadian isolates surveyed. CONCLUSIONS: Apicidin biosynthesis is linked to isolate-specific putative accessory chromosomes in F. poae. The data produced here are an important resource for furthering our understanding of accessory chromosome evolution and the biosynthetic potential of F. poae.

Accessory Chromosome-Acquired Secondary Metabolism in Plant Pathogenic Fungi: The Evolution of Biotrophs Into Host-Specific Pathogens.

Accessory chromosomes are strain- or pathotype-specific chromosomes that exist in addition to the core chromosomes of a species and are generally not considered essential to the survival of the organism. Among pathogenic fungal species, accessory chromosomes harbor pathogenicity or virulence factor genes, several of which are known to encode for secondary metabolites that are involved in plant tissue invasion. Accessory chromosomes are of particular interest due to their capacity for horizontal transfer between strains and their dynamic "crosstalk" with core chromosomes. This review focuses exclusively on secondary metabolism (including mycotoxin biosynthesis) associated with accessory chromosomes in filamentous fungi and the role accessory chromosomes play in the evolution of secondary metabolite gene clusters. Untargeted metabolomics profiling in conjunction with genome sequencing provides an effective means of linking secondary metabolite products with their respective biosynthetic gene clusters that reside on accessory chromosomes. While the majority of literature describing accessory chromosome-associated toxin biosynthesis comes from studies of Alternaria pathotypes, the recent discovery of accessory chromosome-associated biosynthetic genes in Fusarium species offer fresh insights into the evolution of biosynthetic enzymes such as non-ribosomal peptide synthetases (NRPSs), polyketide synthases (PKSs) and regulatory mechanisms governing their expression.

Transcriptome analysis implicates secondary metabolite production, redox reactions, and programmed cell death during allorecognition in Cryphonectria parasitica.

The underlying molecular mechanisms of programmed cell death associated with fungal allorecognition, a form of innate immunity, remain largely unknown. In this study, transcriptome analysis was used to infer mechanisms activated during barrage formation in vic3-incompatible strains of Cryphonectria parasitica, the chestnut blight fungus. Pronounced differential expression occurred in barraging strains of genes involved in mating pheromone (mf2-1, mf2-2), secondary metabolite production, detoxification (including oxidative stress), apoptosis-related, RNA interference, and HET-domain genes. Evidence for secondary metabolite production and reactive oxygen species (ROS) accumulation is supported through UPLC-HRMS analysis and cytological staining, respectively. Differential expression of mating-related genes and HET-domain genes was further examined by RT-qPCR of incompatible interactions involving each of the six vegetative incompatibility (vic) loci in C. parasitica and revealed distinct recognition process networks. We infer that vegetative incompatibility in C. parasitica activates defence reactions that involve secondary metabolism, resulting in increased toxicity of the extra- and intracellular environment. Accumulation of ROS (and other potential toxins) may result in detoxification failure and activation of apoptosis, sporulation, and the expression of associated pheromone genes. The incompatible reaction leaves abundant traces of a process-specific metabolome as conidiation is initiated.

Four phylogenetic species of ergot from Canada and their characteristics in morphology, alkaloid production, and pathogenicity.

Four ergot species (Claviceps ripicola, C. quebecensis, C. perihumidiphila, and C. occidentalis) were recognized based on analyses of DNA sequences from multiple loci, including two housekeeping genes, RNA polymerase II second largest subunit (RPB2), and translation elongation factor 1-α (TEF1-α), and a single-copy ergot alkaloid synthesis gene (easE) encoding chanoclavine I synthase oxidoreductase. Morphological features, ergot alkaloid production, and pathogenicity on five common cereal crops of each species were evaluated and presented in taxonomic descriptions. A synoptic key was also provided for identification.

Transcriptomic and Exometabolomic Profiling Reveals Antagonistic and Defensive Modes of Clonostachys rosea Action Against Fusarium graminearum.

The mycoparasite Clonostachys rosea ACM941 is under development as a biocontrol organism against Fusarium graminearum, the causative agent of Fusarium head blight in cereals. To identify molecular factors associated with this interaction, the transcriptomic and exometabolomic profiles of C. rosea and F. graminearum GZ3639 were compared during coculture. Prior to physical contact, the antagonistic activity of C. rosea correlated with a response heavily dominated by upregulation of polyketide synthase gene clusters, consistent with the detected accumulation of corresponding secondary metabolite products. Similarly, prior to contact, trichothecene gene clusters were upregulated in F. graminearum, while those responsible for fusarielin and fusarin biosynthesis were downregulated, correlating with an accumulation of trichothecene products in the interaction zone over time. A concomitant increase in 15-acetyl deoxynivalenol-3-glucoside in the interaction zone was also detected, with C. rosea established as the source of this detoxified mycotoxin. After hyphal contact, C. rosea was found to predominantly transcribe genes encoding cell wall-degradation enzymes, major facilitator superfamily sugar transporters, anion:cation symporters, as well as alternative carbon source utilization pathways, together indicative of a transition to necrotropism at this stage. F. graminearum notably activated the transcription of phosphate starvation pathway signature genes at this time. Overall, a number of signature molecular mechanisms likely contributing to antagonistic activity by C. rosea against F. graminearum, as well as its mycotoxin tolerance, are identified in this report, yielding several new testable hypotheses toward understanding the basis of C. rosea as a biocontrol agent for continued agronomic development and application.

A continental scale spatial investigation of lake sediment organic compositions using sedimentomics.

Sedimentomics is a new method used to investigate carbon cycling in sediment organic matter. This untargeted method, based on metabolomics workflows, was used to investigate the molecular composition of sediment organic matter across northern Canada (Nunavut and Northwest Territories). Unique "lake districts" were defined using unsupervised clustering based on changes in sediment organic carbon compositions across space. Supervised machine learning analyses were used to compare the "lake districts" to commonly used regional classification systems like the treeline, ecozones, and/or georegions. Treeline was the best model to explain the compositional variance of sediment organic carbon from lakes across Canada, closely followed by the georegions model. A novel sediment metaphenomics analysis was also applied to determine how well environmental constraints explain the variation of sediment organic matter composition across a continent. We determined that sedimentomics is more informative than traditional measurements (such as total organic carbon) and can be integrated with other "omics" techniques.

Enniatin Production Influences Fusarium avenaceum Virulence on Potato Tubers, but not on Durum Wheat or Peas.

Fusarium avenaceum is a generalist pathogen responsible for diseases in numerous crop species. The fungus produces a series of mycotoxins including the cyclohexadepsipeptide enniatins. Mycotoxins can be pathogenicity and virulence factors in various plant-pathogen interactions, and enniatins have been shown to influence aggressiveness on potato tubers. To determine the role of these mycotoxins in other F. avenaceum-host interactions, enniatin synthase 1 (ESYN1) disruption and overexpression mutants were generated and their ability to infect wheat and peas investigated. As a preliminary study, the transformants were screened for their ability to cause potato tuber necrosis and, consistent with a previous report, enniatin production increased necrotic lesion size on the tubers. By contrast, when the same mutants were assessed in their ability to cause disease in pea roots or durum wheat spikes, no changes in disease symptoms or virulence were observed. While it is known that, at least in the case of wheat, exogenously applied enniatins can cause tissue necrosis, this group of mycotoxins does not appear to be a key factor on its own in disease development on peas or durum wheat.

Terrosamycins A and B, Bioactive Polyether Ionophores from Streptomyces sp. RKND004 from Prince Edward Island Sediment.

Terrosamycins A (1) and B (2), two polycyclic polyether natural products, were purified from the fermentation broth of Streptomyces sp. RKND004 isolated from Prince Edward Island sediment. The one strain-many compounds (OSMAC) approach coupled with UPLC-HRMS-based metabolomics screening led to the identification of these compounds. The structure of 1 was determined from analysis of NMR, HRMS, and X-ray diffraction data. NMR experiments performed on 2 revealed the presence of two methoxy groups replacing two hydroxy groups in 1. Like other polyether ionophores, 1 and 2 exhibited excellent antibiotic activity against Gram-positive pathogens. Interestingly, the terrosamycins also exhibited activity against two breast cancer cell lines.

The Neglected Marine Fungi, Sensu stricto, and Their Isolation for Natural Products' Discovery.

Despite the rapid development of molecular techniques relevant for natural product research, culture isolates remain the primary source from which natural products chemists discover and obtain new molecules from microbial sources. Techniques for obtaining and identifying microbial isolates (such as filamentous fungi) are thus of crucial importance for a successful natural products' discovery program. This review is presented as a "best-practices guide" to the collection and isolation of marine fungi for natural products research. Many of these practices are proven techniques used by mycologists for the isolation of a broad diversity of fungi, while others, such as the construction of marine baiting stations and the collection and processing of sea foam using dilution to extinction plating techniques, are methodological adaptations for specialized use in marine/aquatic environments. To this day, marine fungi, Sensu stricto, remain one of the few underexplored resources of natural products. Cultivability is one of the main limitations hindering the discovery of natural products from marine fungi. Through encouraged collaboration with marine mycologists and the sharing of historically proven mycological practices for the isolation of marine fungi, our goal is to provide natural products chemists with the necessary tools to explore this resource in-depth and discover new and potentially novel natural products.