Gliotoxin-mediated bacterial growth inhibition is caused by specific metal ion depletion.

Overcoming antimicrobial resistance represents a formidable challenge and investigating bacterial growth inhibition by fungal metabolites may yield new strategies. Although the fungal non-ribosomal peptide gliotoxin (GT) is known to exhibit antibacterial activity, the mechanism(s) of action are unknown, although reduced gliotoxin (dithiol gliotoxin; DTG) is a zinc chelator. Furthermore, it has been demonstrated that GT synergises with vancomycin to inhibit growth of Staphylococcus aureus. Here we demonstrate, without precedent, that GT-mediated growth inhibition of both Gram positive and negative bacterial species is reversed by Zn2+ or Cu2+ addition. Both GT, and the known zinc chelator TPEN, mediate growth inhibition of Enterococcus faecalis which is reversed by zinc addition. Moreover, zinc also reverses the synergistic growth inhibition of E. faecalis observed in the presence of both GT and vancomycin (4 µg/ml). As well as zinc chelation, DTG also appears to chelate Cu2+, but not Mn2+ using a 4-(2-pyridylazo)resorcinol assay system and Zn2+ as a positive control. DTG also specifically reacts in Fe3+-containing Siderotec™ assays, most likely by Fe3+ chelation from test reagents. GSH or DTT show no activity in these assays. Confirmatory high resolution mass spectrometry, in negative ion mode, confirmed, for the first time, the presence of both Cu[DTG] and Fe[DTG]2 chelates. Label free quantitative proteomic analysis further revealed major intracellular proteomic remodelling within E. faecalis in response to GT exposure for 30-180 min. Globally, 4.2-7.2% of detectable proteins exhibited evidence of either unique presence/increased abundance or unique absence/decreased abundance (n = 994-1160 total proteins detected), which is the first demonstration that GT affects the bacterial proteome in general, and E. faecalis, specifically. Unique detection of components of the AdcABC and AdcA-II zinc uptake systems was observed, along with apparent ribosomal reprofiling to zinc-free paralogs in the presence of GT. Overall, we hypothesise that GT-mediated bacterial growth inhibition appears to involve intracellular zinc depletion or reduced bioavailability, and based on in vitro chelate formation, may also involve dysregulation of Cu2+ homeostasis.

Recombinant production, characterization and industrial application testing of a novel acidic exo/endo-chitinase from Rasamsonia emersonii.

An acid-active exo/endo-chitinase; comprising a GH18 catalytic domain and substrate insertion domain; originating from the thermophilic filamentous fungus Rasamsonia emersonii, was expressed in Pichia pastoris. In silico analysis including phylogenetic analysis, and recombinant production, purification, biochemical characterisation, and industrial application testing, was carried out. The expressed protein was identified by SDS-PAGE as a smear from 56.3 to 125.1 kDa, which sharpens into bands at 46.0 kDa, 48.4 kDa and a smear above 60 kDa when treated with PNGase F. The acid-active chitinase was primarily a chitobiosidase but displayed some endo-chitinase and acetyl-glucosamidase activity. The enzyme was optimally active at 50 °C, and markedly low pH of 2.8. As far as the authors are aware, this is the lowest pH optima reported for any fungal chitinase. The acid-active chitinase likely plays a role in chitin degradation for cell uptake in its native environment, perhaps in conjunction with a chitin deacetylase. Comparative studies with other R. emersonii chitinases indicate that they may play a synergistic role in this. The acid-active chitinase displayed some efficacy against non-treated substrates; fungal chitin and chitin from shrimp. Thus, it may be suited to industrial chitin hydrolysis reactions for extraction of glucosamine and chitobiose at low pH.

Comparative genomic study of the Penicillium genus elucidates a diverse pangenome and 15 lateral gene transfer events.

The Penicillia are known to produce a wide range natural products-some with devastating outcome for the agricultural industry and others with unexploited potential in different applications. However, a large-scale overview of the biosynthetic potential of different species has been lacking. In this study, we sequenced 93 Penicillium isolates and, together with eleven published genomes that hold similar assembly characteristics, we established a species phylogeny as well as defining a Penicillium pangenome. A total of 5612 genes were shared between ≥ 98 isolates corresponding to approximately half of the average number of genes a Penicillium genome holds. We further identified 15 lateral gene transfer events that have occurred in this collection of Penicillium isolates, which might have played an important role, such as niche adaption, in the evolution of these fungi. The comprehensive characterization of the genomic diversity in the Penicillium genus supersedes single-reference genomes, which do not necessarily capture the entire genetic variation.

F-box receptor mediated control of substrate stability and subcellular location organizes cellular development of Aspergillus nidulans.

Fungal growth and development are coordinated with specific secondary metabolism. This coordination requires 8 of 74 F-box proteins of the filamentous fungus Aspergillus nidulans. F-box proteins recognize primed substrates for ubiquitination by Skp1-Cul1-Fbx (SCF) E3 ubiquitin RING ligases and degradation by the 26S proteasome. 24 F-box proteins are found in the nuclear fraction as part of SCFs during vegetative growth. 43 F-box proteins interact with SCF proteins during growth, development or stress. 45 F-box proteins are associated with more than 700 proteins that have mainly regulatory roles. This corroborates that accurate surveillance of protein stability is prerequisite for organizing multicellular fungal development. Fbx23 combines subcellular location and protein stability control, illustrating the complexity of F-box mediated regulation during fungal development. Fbx23 interacts with epigenetic methyltransferase VipC which interacts with fungal NF-κB-like velvet domain regulator VeA that coordinates fungal development with secondary metabolism. Fbx23 prevents nuclear accumulation of methyltransferase VipC during early development. These results suggest that in addition to their role in protein degradation, F-box proteins also control subcellular accumulations of key regulatory proteins for fungal development.

A Single Aspergillus fumigatus Gene Enables Ergothioneine Biosynthesis and Secretion by Saccharomyces cerevisiae.

The naturally occurring sulphur-containing histidine derivative, ergothioneine (EGT), exhibits potent antioxidant properties and has been proposed to confer human health benefits. Although it is only produced by select fungi and prokaryotes, likely to protect against environmental stress, the GRAS organism Saccharomyces cerevisiae does not produce EGT naturally. Herein, it is demonstrated that the recombinant expression of a single gene, Aspergillus fumigatus egtA, in S. cerevisiae results in EgtA protein presence which unexpectedly confers complete EGT biosynthetic capacity. Both High Performance Liquid Chromatography (HPLC) and LC−mass spectrometry (MS) analysis were deployed to detect and confirm EGT production in S. cerevisiae. The localisation and quantification of the resultant EGT revealed a significantly (p < 0.0001) larger quantity of EGT was extracellularly present in culture supernatants than intracellularly accumulated in 96 h yeast cultures. Methionine addition to cultures improved EGT production. The additional expression of two candidate cysteine desulfurases from A. fumigatus was thought to be required to complete EGT biosynthesis, namely AFUA_2G13295 and AFUA_3G14240, termed egt2a and egt2b in this study. However, the co-expression of egtA and egt2a in S. cerevisiae resulted in a significant decrease in the observed EGT levels (p < 0.05). The AlphaFold prediction of A. fumigatus EgtA 3-Dimensional structure illuminates the bidomain structure of the enzyme and the opposing locations of both active sites. Overall, we clearly show that recombinant S. cerevisiae can biosynthesise and secrete EGT in an EgtA-dependent manner which presents a facile means of producing EGT for biotechnological and biomedical use.

Genome of Pythium myriotylum Uncovers an Extensive Arsenal of Virulence-Related Genes among the Broad-Host-Range Necrotrophic Pythium Plant Pathogens.

The Pythium (Peronosporales, Oomycota) genus includes devastating plant pathogens that cause widespread diseases and severe crop losses. Here, we have uncovered a far greater arsenal of virulence factor-related genes in the necrotrophic Pythium myriotylum than in other Pythium plant pathogens. The genome of a plant-virulent P. myriotylum strain (~70 Mb and 19,878 genes) isolated from a diseased rhizome of ginger (Zingiber officinale) encodes the largest repertoire of putative effectors, proteases, and plant cell wall-degrading enzymes (PCWDEs) among the studied species. P. myriotylum has twice as many predicted secreted proteins than any other Pythium plant pathogen. Arrays of tandem duplications appear to be a key factor of the enrichment of the virulence factor-related genes in P. myriotylum. The transcriptomic analysis performed on two P. myriotylum isolates infecting ginger leaves showed that proteases were a major part of the upregulated genes along with PCWDEs, Nep1-like proteins (NLPs), and elicitin-like proteins. A subset of P. myriotylum NLPs were analyzed and found to have necrosis-inducing ability from agroinfiltration of tobacco (Nicotiana benthamiana) leaves. One of the heterologously produced infection-upregulated putative cutinases found in a tandem array showed esterase activity with preferences for longer-chain-length substrates and neutral to alkaline pH levels. Our results allow the development of science-based targets for the management of P. myriotylum-caused disease, as insights from the genome and transcriptome show that gene expansion of virulence factor-related genes play a bigger role in the plant parasitism of Pythium spp. than previously thought. IMPORTANCE Pythium species are oomycetes, an evolutionarily distinct group of filamentous fungus-like stramenopiles. The Pythium genus includes several pathogens of important crop species, e.g., the spice ginger. Analysis of our genome from the plant pathogen Pythium myriotylum uncovered a far larger arsenal of virulence factor-related genes than found in other Pythium plant pathogens, and these genes contribute to the infection of the plant host. The increase in the number of virulence factor-related genes appears to have occurred through the mechanism of tandem gene duplication events. Genes from particular virulence factor-related categories that were increased in number and switched on during infection of ginger leaves had their activities tested. These genes have toxic activities toward plant cells or activities to hydrolyze polymeric components of the plant. The research suggests targets to better manage diseases caused by P. myriotylum and prompts renewed attention to the genomics of Pythium plant pathogens.

Dual-Transcriptomic, Microscopic, and Biocontrol Analyses of the Interaction Between the Bioeffector Pythium oligandrum and the Pythium Soft-Rot of Ginger Pathogen Pythium myriotylum.

Biological control is a promising approach to suppress diseases caused by Pythium spp. such as Pythium soft rot of ginger caused by P. myriotylum. Unusually for a single genus, it also includes species that can antagonize Pythium plant pathogens, such as Pythium oligandrum. We investigated if a new isolate of P. oligandrum could antagonize P. myriotylum, what changes occurred in gene expression when P. oligandrum (antagonist) and P. myriotylum (host) interacted, and whether P. oligandrum could control soft-rot of ginger caused by P. myriotylum. An isolate of P. oligandrum, GAQ1, recovered from soil could antagonize P. myriotylum in a plate-based confrontation assay whereby P. myriotylum became non-viable. The loss of viability of P. myriotylum coupled with how P. oligandrum hyphae could coil around and penetrate the hyphae of P. myriotylum, indicated a predatory interaction. We investigated the transcriptional responses of P. myriotylum and P. oligandrum using dual-RNAseq at a stage in the confrontation where similar levels of total transcripts were measured from each species. As part of the transcriptional response of P. myriotylum to the presence of P. oligandrum, genes including a subset of putative Kazal-type protease inhibitors were strongly upregulated along with cellulases, elicitin-like proteins and genes involved in the repair of DNA double-strand breaks. In P. oligandrum, proteases, cellulases, and peroxidases featured prominently in the upregulated genes. The upregulation along with constitutive expression of P. oligandrum proteases appeared to be responded to by the upregulation of putative protease inhibitors from P. myriotylum, suggesting a P. myriotylum defensive strategy. Notwithstanding this P. myriotylum defensive strategy, P. oligandrum had a strong disease control effect on soft-rot of ginger caused by P. myriotylum. The newly isolated strain of P. oligandrum is a promising biocontrol agent for suppressing the soft-rot of ginger. The dual-RNAseq approach highlights responses of P. myriotylum that suggests features of a defensive strategy, and are perhaps another factor that may contribute to the variable success and durability of biological attempts to control diseases caused by Pythium spp.

Genomic diversity, chromosomal rearrangements, and interspecies hybridization in the Ogataea polymorpha species complex.

The methylotrophic yeast Ogataea polymorpha has long been a useful system for recombinant protein production, as well as a model system for methanol metabolism, peroxisome biogenesis, thermotolerance, and nitrate assimilation. It has more recently become an important model for the evolution of mating-type switching. Here, we present a population genomics analysis of 47 isolates within the O. polymorpha species complex, including representatives of the species O. polymorpha, Ogataea parapolymorpha, Ogataea haglerorum, and Ogataea angusta. We found low levels of nucleotide sequence diversity within the O. polymorpha species complex and identified chromosomal rearrangements both within and between species. In addition, we found that one isolate is an interspecies hybrid between O. polymorpha and O. parapolymorpha and present evidence for loss of heterozygosity following hybridization.

Generation and characterisation of a semi-synthetic siderophore-immunogen conjugate and a derivative recombinant triacetylfusarinine C-specific monoclonal antibody with fungal diagnostic application.

Invasive pulmonary aspergillosis (IPA) is a severe life-threatening condition. Diagnosis of fungal disease in general, and especially that caused by Aspergillus fumigatus is problematic. A. fumigatus secretes siderophores to acquire iron during infection, which are also essential for virulence. We describe the chemoacetylation of ferrated fusarinine C to diacetylated fusarinine C (DAFC), followed by protein conjugation, which facilitated triacetylfusarinine C (TAFC)-specific monoclonal antibody production with specific recognition of the ferrated form of TAFC. A single monoclonal antibody sequence was ultimately elucidated by a combinatorial strategy involving protein LC-MS/MS, cDNA sequencing and RNAseq. The resultant murine IgG2a monoclonal antibody was secreted in, and purified from, mammalian cell culture (5 mg) and demonstrated to be highly specific for TAFC detection by competitive ELISA (detection limit: 15 nM) and in a lateral flow test system (detection limit: 3 ng), using gold nanoparticle conjugated- DAFC-bovine serum albumin for competition. Overall, this work reveals for the first time a recombinant TAFC-specific monoclonal antibody with diagnostic potential for IPA diagnosis in traditional and emerging patient groups (e.g., COVID-19) and presents a useful strategy for murine Ig sequence determination, and expression in HEK293 cells, to overcome unexpected limitations associated with aberrant or deficient murine monoclonal antibody production.

Transmission of mushroom virus X and the impact of virus infection on the transcriptomes and proteomes of different strains of Agaricus bisporus.

Cultivation of Agaricus bisporus is a large horticultural industry for many countries worldwide, where a single variety is almost grown exclusively. Mushroom virus X (MVX), a complex of multiple positive-sense single stranded RNA (ss(+)RNA) viruses, is a major pathogen of typical A. bisporus crops. MVX can manifest a variety of symptoms in crops and is highly infective and difficult to eradicate once established in host mycelium. Currently our knowledge regarding the molecular response of A. bisporus fruit bodies to MVX infection is limited. In order to study the response of different A. bisporus strains with different susceptibilities to MVX, we designed a model system to evaluate the in-vitro transmission of viruses in A. bisporus hyphae over a time-course, at two crucial phases in the crop cycle. The symptom expression of MVX in these varieties and the transcriptomic and proteomic response of fruit bodies to MVX-infection were examined. Transmission studies revealed the high potential of MVX to spread to uninfected mycelium yet not into the fruit bodies of certain strains in a crop. MVX affected colour and quality of multiple fruit bodies. Gene expression is significantly altered in all strains and between times of inoculation in the crop. Genes related to stress responses displayed differential expression. Proteomic responses revealed restriction of cellular signalling and vesicle transport in infected fruit bodies. This in-depth analysis examining many factors relevant to MVX infection in different A. bisporus strains, will provide key insights into host responses for this commercially important food crop.

Recent advances in oomycete genomics.

The oomycetes are a class of ubiquitous, filamentous microorganisms that include some of the biggest threats to global food security and natural ecosystems. Within the oomycete class are highly diverse species that infect a broad range of animals and plants. Some of the most destructive plant pathogens are oomycetes, such as Phytophthora infestans, the agent of potato late blight and the cause of the Irish famine. Recent years have seen a dramatic increase in the number of sequenced oomycete genomes. Here we review the latest developments in oomycete genomics and some of the important insights that have been gained. Coupled with proteomic and transcriptomic analyses, oomycete genome sequences have revealed tremendous insights into oomycete biology, evolution, genome organization, mechanisms of infection, and metabolism. We also present an updated phylogeny of the oomycete class using a phylogenomic approach based on the 65 oomycete genomes that are currently available.

Comparative Genomic and Proteomic Analyses of Three Widespread Phytophthora Species: Phytophthora chlamydospora, Phytophthora gonapodyides and Phytophthora pseudosyringae.

The Phytophthora genus includes some of the most devastating plant pathogens. Here we report draft genome sequences for three ubiquitous Phytophthora species-Phytophthora chlamydospora, Phytophthora gonapodyides, and Phytophthora pseudosyringae. Phytophthora pseudosyringae is an important forest pathogen that is abundant in Europe and North America. Phytophthora chlamydospora and Ph. gonapodyides are globally widespread species often associated with aquatic habitats. They are both regarded as opportunistic plant pathogens. The three sequenced genomes range in size from 45 Mb to 61 Mb. Similar to other oomycete species, tandem gene duplication appears to have played an important role in the expansion of effector arsenals. Comparative analysis of carbohydrate-active enzymes (CAZymes) across 44 oomycete genomes indicates that oomycete lifestyles may be linked to CAZyme repertoires. The mitochondrial genome sequence of each species was also determined, and their gene content and genome structure were compared. Using mass spectrometry, we characterised the extracellular proteome of each species and identified large numbers of proteins putatively involved in pathogenicity and osmotrophy. The mycelial proteome of each species was also characterised using mass spectrometry. In total, the expression of approximately 3000 genes per species was validated at the protein level. These genome resources will be valuable for future studies to understand the behaviour of these three widespread Phytophthora species.

Proteomic investigation of interhyphal interactions between strains of Agaricus bisporus.

Hyphae of filamentous fungi undergo polar extension, bifurcation and hyphal fusion to form reticulating networks of mycelia. Hyphal fusion or anastomosis, a ubiquitous process among filamentous fungi, is a vital strategy for how fungi expand over their substrate and interact with or recognise self- and non-self hyphae of neighbouring mycelia in their environment. Morphological and genetic characterisation of anastomosis has been studied in many model fungal species, but little is known of the direct proteomic response of two interacting fungal isolates. Agaricus bisporus, the most widely cultivated edible mushroom crop worldwide, was used as an in vitro model to profile the proteomes of interacting cultures. The globally cultivated strain (A15) was paired with two distinct strains; a commercial hybrid strain and a wild isolate strain. Each co-culture presented a different interaction ranging from complete vegetative compatibility (self), lack of interactions, and antagonistic interactions. These incompatible strains are the focus of research into disease-resistance in commercial crops as the spread of intracellular pathogens, namely mycoviruses, is limited by the lack of interhyphal anastomosis. Unique proteomic responses were detected between all co-cultures. An array of cell wall modifying enzymes, plus fungal growth and morphogenesis proteins were found in significantly (P < 0.05) altered abundances. Nitrogen metabolism dominated in the intracellular proteome, with evidence of nitrogen starvation between competing, non-compatible cultures. Changes in key enzymes of A. bisporus morphogenesis were observed, particularly via increased abundance of glucanosyltransferase in competing interactions and certain chitinases in vegetative compatible interactions only. Carbohydrate-active enzyme arsenals are expanded in antagonistic interactions in A. bisporus. Pathways involved in carbohydrate metabolism and genetic information processing were higher in interacting cultures, most notably during self-recognition. New insights into the differential response of interacting strains of A. bisporus will enhance our understanding of potential barriers to viral transmission through vegetative incompatibility. Our results suggest that a differential proteomic response occurs between A. bisporus at strain-level and findings from this work may guide future proteomic investigation of fungal anastomosis.

FISHing in fungi: Visualisation of mushroom virus X in the mycelium of Agaricus bisporus by fluorescence in situ hybridisation.

Agaricus bisporus is a commercial mushroom crop susceptible to a disease caused by a complex of viruses known collectively as mushroom virus X (MVX). Symptoms of MVX include bare patches and mushroom cap discolouration (browning) in the fruiting bodies, phenotypes associated with the viruses AbV6 and AbV16, respectively. Limited understanding exists of the localisation and mobilisation of these viruses within the mycelium of A. bisporus. To this end, a non-destructive fluorescence in situ hybridisation (FISH) method was developed for in situ targeting of AbV6 and AbV16 in A. bisporus mycelium. An MVX strain associated with the bare patch disease phenotype revealed predominantly high signal towards the growing edges of cultures when probed for AbV6, with a 'halo-effect' of high signal intensity around putative vacuoles. An MVX strain associated with the browning disease phenotype showed high signal intensities within reticulating networks of hyphae in a highly compartmentalised manner when probed for AbV16. Localisation of the two viruses in MVX-infected cultures appears independent, as both viruses were found in completely discrete areas of the mycelium in differential patterns. FISH detected low level presence of the two viruses, AbV6 and AbV16 in a number of cultures which had tested negative for the viruses by RT-PCR. This suggests that FISH may be more sensitive at detecting viruses at low levels than molecular methods. This study demonstrates that FISH is a powerful tool in the field of mycovirology.

Characterisation of three novel ß-1,3 glucanases from the medically important house dust mite Dermatophagoides pteronyssinus (airmid).

The European house dust mite, Dermatophagoides pteronyssinus is a major source of airborne allergens worldwide and is found in half of European homes. Interactions between microbes and house dust mites (HDM) are considered important factors that allow them to persist in the home. Laboratory studies indicate the European HDM, D. pteronyssinus is a mycophagous mite, capable of utilising a variety of fungi for nutrients, however specific mycolytic digestive enzymes are unknown. Our previous work identified a number of putative glycosyl hydrolases present in the predicted proteome of D. pteronyssinus airmid and validated the expression of 42 of these. Of note, three GH16 proteins with predicted ß-1,3 glucanase activity were found to be consistently present in the mite body and excretome. Here, we performed an extensive bioinformatic, proteomic and biochemical study to characterize three-novel ß-1,3 glucanases from this medically important house dust mite. The genes encoding novel ß-1,3 glucanases designated Glu1, Glu2 and Glu3 were identified in D. pteronyssinus airmid, each exhibited more than 59% amino acid identity to one another. These enzymes are encoded by Glu genes present in a tri-gene cluster and protein homologs are found in other acari. The patchy phyletic distribution of Glu proteins means their evolutionary history remains elusive, however horizontal gene transfer cannot be completely excluded. Recombinant Glu1 and Glu2 exhibit hydrolytic activity toward laminarin, pachyman and barley glucan. Excreted ß-1,3 glucanase activity was increased in response to D. pteronyssinus airmid feeding on baker's yeast. Active ß-1,3 glucanases are expressed and excreted in the faeces of D. pteronyssinus airmid indicating they are digestive enzymes capable of breaking down ß-1,3 glucans of fungi present in house dust.

Whole Genome Sequence of the Commercially Relevant Mushroom Strain Agaricus bisporus var. bisporus ARP23.

Agaricus bisporus is an extensively cultivated edible mushroom. Demand for cultivation is continuously growing and difficulties associated with breeding programs now means strains are effectively considered monoculture. While commercial growing practices are highly efficient and tightly controlled, the over-use of a single strain has led to a variety of disease outbreaks from a range of pathogens including bacteria, fungi and viruses. To address this, the Agaricus Resource Program (ARP) was set up to collect wild isolates from diverse geographical locations through a bounty-driven scheme to create a repository of wild Agaricus germplasm. One of the strains collected, Agaricus bisporus var. bisporus ARP23, has been crossed extensively with white commercial varieties leading to the generation of a novel hybrid with a dark brown pileus commonly referred to as 'Heirloom'. Heirloom has been successfully implemented into commercial mushroom cultivation. In this study the whole genome of Agaricus bisporus var. bisporus ARP23 was sequenced and assembled with Illumina and PacBio sequencing technology. The final genome was found to be 33.49 Mb in length and have significant levels of synteny to other sequenced Agaricus bisporus strains. Overall, 13,030 putative protein coding genes were located and annotated. Relative to the other A. bisporus genomes that are currently available, Agaricus bisporus var. bisporus ARP23 is the largest A. bisporus strain in terms of gene number and genetic content sequenced to date. Comparative genomic analysis shows that the A. bisporus mating loci in unifactorial and unsurprisingly highly conserved between strains. The lignocellulolytic gene content of all A. bisporus strains compared is also very similar. Our results show that the pangenome structure of A. bisporus is quite diverse with between 60-70% of the total protein coding genes per strain considered as being orthologous and syntenically conserved. These analyses and the genome sequence described herein are the starting point for more detailed molecular analyses into the growth and phenotypical responses of Agaricus bisporus var. bisporus ARP23 when challenged with economically important mycoviruses.

Insights into the transcriptomic response of the plant engineering bacterium Ensifer adhaerens OV14 during transformation.

The ability to engineer plant genomes has been primarily driven by the soil bacterium Agrobacterium tumefaciens but recently the potential of alternative rhizobia such as Rhizobium etli and Ensifer adhaerens OV14, the latter of which supports Ensifer Mediated Transformation (EMT) has been reported. Surprisingly, a knowledge deficit exists in regards to understanding the whole genome processes underway in plant transforming bacteria, irrespective of the species. To begin to address the issue, we undertook a temporal RNAseq-based profiling study of E. adhaerens OV14 in the presence/absence of Arabidopsis thaliana tissues. Following co-cultivation with root tissues, 2333 differentially expressed genes (DEGs) were noted. Meta-analysis of the RNAseq data sets identified a clear shift from plasmid-derived gene expression to chromosomal-based transcription within the early stages of bacterium-plant co-cultivation. During this time, the number of differentially expressed prokaryotic genes increased steadily out to 7 days co-cultivation, a time at which optimum rates of transformation were observed. Gene ontology evaluations indicated a role for both chromosomal and plasmid-based gene families linked specifically with quorum sensing, flagellin production and biofilm formation in the process of EMT. Transcriptional evaluation of vir genes, housed on the pCAMBIA 5105 plasmid in E. adhaerens OV14 confirmed the ability of E. adhaerens OV14 to perceive and activate its transcriptome in response to the presence of 200 µM of acetosyringone. Significantly, this is the first study to characterise the whole transcriptomic response of a plant engineering bacterium in the presence of plant tissues and provides a novel insight into prokaryotic genetic processes that support T-DNA transfer.

Pangloss: A Tool for Pan-Genome Analysis of Microbial Eukaryotes.

Although the pan-genome concept originated in prokaryote genomics, an increasing number of eukaryote species pan-genomes have also been analysed. However, there is a relative lack of software intended for eukaryote pan-genome analysis compared to that available for prokaryotes. In a previous study, we analysed the pan-genomes of four model fungi with a computational pipeline that constructed pan-genomes using the synteny-dependent Pan-genome Ortholog Clustering Tool (PanOCT) approach. Here, we present a modified and improved version of that pipeline which we have called Pangloss. Pangloss can perform gene prediction for a set of genomes from a given species that the user provides, constructs and optionally refines a species pan-genome from that set using PanOCT, and can perform various functional characterisation and visualisation analyses of species pan-genome data. To demonstrate Pangloss's capabilities, we constructed and analysed a species pan-genome for the oleaginous yeast Yarrowialipolytica and also reconstructed a previously-published species pan-genome for the opportunistic respiratory pathogen Aspergillus fumigatus. Pangloss is implemented in Python, Perl and R and is freely available under an open source GPLv3 licence via GitHub.

Control of Development, Secondary Metabolism and Light-Dependent Carotenoid Biosynthesis by the Velvet Complex of Neurospora crassa.

Neurospora crassa is an established reference organism to investigate carotene biosynthesis and light regulation. However, there is little evidence of its capacity to produce secondary metabolites. Here, we report the role of the fungal-specific regulatory velvet complexes in development and secondary metabolism (SM) in N. crassa Three velvet proteins VE-1, VE-2, VOS-1, and a putative methyltransferase LAE-1 show light-independent nucleocytoplasmic localization. Two distinct velvet complexes, a heterotrimeric VE-1/VE-2/LAE-1 and a heterodimeric VE-2/VOS-1 are found in vivo The heterotrimer-complex, which positively regulates sexual development and represses asexual sporulation, suppresses siderophore coprogen production under iron starvation conditions. The VE-1/VE-2 heterodimer controls carotene production. VE-1 regulates the expression of >15% of the whole genome, comprising mainly regulatory and developmental features. We also studied intergenera functions of the velvet complex through complementation of Aspergillus nidulans veA, velB, laeA, vosA mutants with their N. crassa orthologs ve-1, ve-2, lae-1, and vos-1, respectively. Expression of VE-1 and VE-2 in A. nidulans successfully substitutes the developmental and SM functions of VeA and VelB by forming two functional chimeric velvet complexes in vivo, VelB/VE-1/LaeA and VE-2/VeA/LaeA, respectively. Reciprocally, expression of veA restores the phenotypes of the N. crassa ve-1 mutant. All N. crassa velvet proteins heterologously expressed in A. nidulans are localized to the nuclear fraction independent of light. These data highlight the conservation of the complex formation in N. crassa and A. nidulans However, they also underline the intergenera similarities and differences of velvet roles according to different life styles, niches and ontogenetic processes.

Proteome and allergenome of the European house dust mite Dermatophagoides pteronyssinus.

The European house dust mite Dermatophagoides pteronyssinus is of significant medical importance as it is a major elicitor of allergic illnesses. In this analysis we have undertaken comprehensive bioinformatic and proteomic examination of Dermatophagoides pteronyssinus airmid, identified 12,530 predicted proteins and validated the expression of 4,002 proteins. Examination of homology between predicted proteins and allergens from other species revealed as much as 2.6% of the D. pteronyssinus airmid proteins may cause an allergenic response. Many of the potential allergens have evidence for expression (n = 259) and excretion (n = 161) making them interesting targets for future allergen studies. Comparative proteomic analysis of mite body and spent growth medium facilitated qualitative assessment of mite group allergen localisation. Protein extracts from house dust contain a substantial number of uncharacterised D. pteronyssinus proteins in addition to known and putative allergens. Novel D. pteronyssinus proteins were identified to be highly abundant both in house dust and laboratory cultures and included numerous carbohydrate active enzymes that may be involved in cuticle remodelling, bacteriophagy or mycophagy. These data may have clinical applications in the development of allergen-specific immunotherapy that mimic natural exposure. Using a phylogenomic approach utilising a supermatrix and supertree methodologies we also show that D. pteronyssinus is more closely related to Euroglyphus maynei than Dermatophagoides farinae.