Screening the Saccharomyces cerevisiae Nonessential Gene Deletion Library Reveals Diverse Mechanisms of Action for Antifungal Plant Defensins.

Plant defensins are a large family of proteins, most of which have antifungal activity against a broad spectrum of fungi. However, little is known about how they exert their activity. The mechanisms of action of only a few members of the family have been investigated and, in most cases, there are still a number of unknowns. To gain a better understanding of the antifungal mechanisms of a set of four defensins, NaD1, DmAMP1, NbD6, and SBI6, we screened a pooled collection of the nonessential gene deletion set of Saccharomyces cerevisiae Strains with increased or decreased ability to survive defensin treatment were identified based on the relative abundance of the strain-specific barcode as determined by MiSeq next-generation sequencing. Analysis of the functions of genes that are deleted in strains with differential growth in the presence of defensin provides insight into the mechanism of action. The screen identified a novel role for the vacuole in the mechanisms of action for defensins NbD6 and SBI6. The effect of these defensins on vacuoles was further confirmed by using confocal microscopy in both S. cerevisiae and the cereal pathogen Fusarium graminearum These results demonstrate the utility of this screening method to identify novel mechanisms of action for plant defensins.

Evolution of virulence in fungal plant pathogens: exploiting fungal genomics to control plant disease.

The propensity of a fungal pathogen to evolve virulence depends on features of its biology (e.g. mode of reproduction) and of its genome (e.g. amount of repetitive DNA). Populations of Leptosphaeria maculans, a pathogen of Brassica napus (canola), can evolve and overcome disease resistance bred into canola within three years of commercial release of a cultivar. Avirulence effector genes are key fungal genes that are complementary to resistance genes. In L. maculans these genes are embedded within inactivated transposable elements in genomic regions where they are readily mutated or deleted. The risk of resistance breakdown in the field can be minimised by monitoring disease severity of canola cultivars and virulence of fungal populations using high throughput molecular assays and by sowing canola cultivars with different resistance genes in subsequent years. This strategy has been exploited to avert yield losses due to blackleg disease in Australia.

Transposable element-assisted evolution and adaptation to host plant within the Leptosphaeria maculans-Leptosphaeria biglobosa species complex of fungal pathogens.

BACKGROUND: Many plant-pathogenic fungi have a tendency towards genome size expansion, mostly driven by increasing content of transposable elements (TEs). Through comparative and evolutionary genomics, five members of the Leptosphaeria maculans-Leptosphaeria biglobosa species complex (class Dothideomycetes, order Pleosporales), having different host ranges and pathogenic abilities towards cruciferous plants, were studied to infer the role of TEs on genome shaping, speciation, and on the rise of better adapted pathogens. RESULTS: L. maculans 'brassicae', the most damaging species on oilseed rape, is the only member of the species complex to have a TE-invaded genome (32.5%) compared to the other members genomes (<4%). These TEs had an impact at the structural level by creating large TE-rich regions and are suspected to have been instrumental in chromosomal rearrangements possibly leading to speciation. TEs, associated with species-specific genes involved in disease process, also possibly had an incidence on evolution of pathogenicity by promoting translocations of effector genes to highly dynamic regions and thus tuning the regulation of effector gene expression in planta. CONCLUSIONS: Invasion of L. maculans 'brassicae' genome by TEs followed by bursts of TE activity allowed this species to evolve and to better adapt to its host, making this genome species a peculiarity within its own species complex as well as in the Pleosporales lineage.

Acute neuroinflammation promotes a metabolic shift that alters extracellular vesicle cargo in the mouse brain cortex.

Neuroinflammation is initiated through microglial activation and cytokine release which can be induced through lipopolysaccharide treatment (LPS) leading to a transcriptional cascade culminating in the differential expression of target proteins. These differentially expressed proteins can then be packaged into extracellular vesicles (EVs), a form of cellular communication, further propagating the neuroinflammatory response over long distances. Despite this, the EV proteome in the brain, following LPS treatment, has not been investigated. Brain tissue and brain derived EVs (BDEVs) isolated from the cortex of LPS-treated mice underwent thorough characterisation to meet the minimal information for studies of extracellular vesicles guidelines before undergoing mass spectrometry analysis to identify the differentially expressed proteins. Fourteen differentially expressed proteins were identified in the LPS brain tissue samples compared to the controls and 57 were identified in the BDEVs isolated from the LPS treated mice compared to the controls. This included proteins associated with the initiation of the inflammatory response, epigenetic regulation, and metabolism. These results allude to a potential link between small EV cargo and early inflammatory signalling.

Microglial activation induces nitric oxide signalling and alters protein S-nitrosylation patterns in extracellular vesicles.

Neuroinflammation is an underlying feature of neurodegenerative conditions, often appearing early in the aetiology of a disease. Microglial activation, a prominent initiator of neuroinflammation, can be induced through lipopolysaccharide (LPS) treatment resulting in expression of the inducible form of nitric oxide synthase (iNOS), which produces nitric oxide (NO). NO post-translationally modifies cysteine thiols through S-nitrosylation, which can alter function of the target protein. Furthermore, packaging of these NO-modified proteins into extracellular vesicles (EVs) allows for the exertion of NO signalling in distant locations, resulting in further propagation of the neuroinflammatory phenotype. Despite this, the NO-modified proteome of activated microglial EVs has not been investigated. This study aimed to identify the protein post-translational modifications NO signalling induces in neuroinflammation. EVs isolated from LPS-treated microglia underwent mass spectral surface imaging using time of flight-secondary ion mass spectrometry (ToF-SIMS), in addition to iodolabelling and comparative proteomic analysis to identify post-translation S-nitrosylation modifications. ToF-SIMS imaging successfully identified cysteine thiol side chains modified through NO signalling in the LPS treated microglial-derived EV proteins. In addition, the iodolabelling proteomic analysis revealed that the EVs from LPS-treated microglia carried S-nitrosylated proteins indicative of neuroinflammation. These included known NO-modified proteins and those associated with LPS-induced microglial activation that may play an essential role in neuroinflammatory communication. Together, these results show activated microglia can exert broad NO signalling changes through the selective packaging of EVs during neuroinflammation.

Extracellular Vesicles from Fusarium graminearum Contain Protein Effectors Expressed during Infection of Corn.

Fusarium graminearum (Fgr) is a devastating filamentous fungal pathogen that causes diseases in cereals, while producing mycotoxins that are toxic for humans and animals, and render grains unusable. Low efficiency in managing Fgr poses a constant need for identifying novel control mechanisms. Evidence that fungal extracellular vesicles (EVs) from pathogenic yeast have a role in human disease led us to question whether this is also true for fungal plant pathogens. We separated EVs from Fgr and performed a proteomic analysis to determine if EVs carry proteins with potential roles in pathogenesis. We revealed that protein effectors, which are crucial for fungal virulence, were detected in EV preparations and some of them did not contain predicted secretion signals. Furthermore, a transcriptomic analysis of corn (Zea mays) plants infected by Fgr revealed that the genes of some of the effectors were highly expressed in vivo, suggesting that the Fgr EVs are a mechanism for the unconventional secretion of effectors and virulence factors. Our results expand the knowledge on fungal EVs in plant pathogenesis and cross-kingdom communication, and may contribute to the discovery of new antifungals.

A combined ¹H nuclear magnetic resonance and electrospray ionization-mass spectrometry analysis to understand the basal metabolism of plant-pathogenic Fusarium spp.

Many ascomycete Fusarium spp. are plant pathogens that cause disease on both cereal and noncereal hosts. Infection of wheat ears by Fusarium graminearum and F. culmorum typically results in bleaching and a subsequent reduction in grain yield. Also, a large proportion of the harvested grain can be spoiled when the colonizing Fusarium mycelia produce trichothecene mycotoxins, such as deoxynivalenol (DON). In this study, we have explored the intracellular polar metabolome of Fusarium spp. in both toxin-producing and nonproducing conditions in vitro. Four Fusarium spp., including nine well-characterized wild-type field isolates now used routinely in laboratory experimentation, were explored. A metabolic "triple-fingerprint" was recorded using (1)H nuclear magnetic resonance and direct-injection electrospray ionization-mass spectroscopy in both positive- and negative-ionization modes. These combined metabolomic analyses revealed that this technique is sufficient to resolve different wild-type isolates and different growth conditions. Principal components analysis was able to resolve the four species explored-F. graminearum, F. culmorum, F. pseudograminearum, and F. venenatum-as well as individual isolate differences from the same species. The external nutritional environment was found to have a far greater influence on the metabolome than the genotype of the organism. Conserved responses to DON-inducing medium were evident and included increased abundance of key compatible solutes, such as glycerol and mannitol. In addition, the concentration of γ-aminobutyric acid was elevated, indicating that the cellular nitrogen status may be affected by growth on DON-inducing medium.

Histidine-Rich Defensins from the Solanaceae and Brasicaceae Are Antifungal and Metal Binding Proteins.

Plant defensins are best known for their antifungal activity and contribution to the plant immune system. The defining feature of plant defensins is their three-dimensional structure known as the cysteine stabilized alpha-beta motif. This protein fold is remarkably tolerant to sequence variation with only the eight cysteines that contribute to the stabilizing disulfide bonds absolutely conserved across the family. Mature defensins are typically 46-50 amino acids in length and are enriched in lysine and/or arginine residues. Examination of a database of approximately 1200 defensin sequences revealed a subset of defensin sequences that were extended in length and were enriched in histidine residues leading to their classification as histidine-rich defensins (HRDs). Using these initial HRD sequences as a query, a search of the available sequence databases identified over 750 HRDs in solanaceous plants and 20 in brassicas. Histidine residues are known to contribute to metal binding functions in proteins leading to the hypothesis that HRDs would have metal binding properties. A selection of the HRD sequences were recombinantly expressed and purified and their antifungal and metal binding activity was characterized. Of the four HRDs that were successfully expressed all displayed some level of metal binding and two of four had antifungal activity. Structural characterization of the other HRDs identified a novel pattern of disulfide linkages in one of the HRDs that is predicted to also occur in HRDs with similar cysteine spacing. Metal binding by HRDs represents a specialization of the plant defensin fold outside of antifungal activity.

Trehalose biosynthesis is involved in sporulation of Stagonospora nodorum.

Stagonospora nodorum is a necrotrophic fungal pathogen that is the causal agent of leaf and glume blotch on wheat. S. nodorum is a polycyclic pathogen, whereby rain-splashed pycnidiospores attach to and colonise wheat tissue and subsequently sporulate again within 2-3weeks. As several cycles of infection are needed for a damaging infection, asexual sporulation is a critical phase of its infection cycle. A non-targeted metabolomics screen for sporulation-associated metabolites identified that trehalose accumulated significantly in concert with asexual sporulation both in vitro and in planta. A reverse-genetics approach was used to investigate the role of trehalose in asexual sporulation. Trehalose biosynthesis was disrupted by deletion of the gene Tps1, encoding a trehalose 6-phosphate synthase, resulting in almost total loss of trehalose during in vitro growth and in planta. In addition, lesion development and pycnidia formation were also significantly reduced in tps1 mutants. Reintroduction of the Tps1 gene restored trehalose biosynthesis, pathogenicity and sporulation to wild-type levels. Microscopic examination of tps1 infected wheat leaves showed that pycnidial formation often halted at an early stage of development. Further examination of the tps1 phenotype revealed that tps1 pycnidiospores exhibited a reduced germination rate while under heat stress, and tps1 mutants had a reduced growth rate while under oxidative stress. This study confirms a link between trehalose biosynthesis and pathogen fitness in S.nodorum.

Extracellular Vesicles From the Cotton Pathogen Fusarium oxysporum f. sp. vasinfectum Induce a Phytotoxic Response in Plants.

Extracellular vesicles (EVs) represent a system for the coordinated secretion of a variety of molecular cargo including proteins, lipids, nucleic acids, and metabolites. They have an essential role in intercellular communication in multicellular organisms and have more recently been implicated in host-pathogen interactions. Study of the role for EVs in fungal biology has focused on pathogenic yeasts that are major pathogens in humans. In this study we have expanded the investigation of fungal EVs to plant pathogens, specifically the major cotton pathogen Fusarium oxysporum f. sp. vasinfectum. EVs isolated from F. oxysporum f. sp. vasinfectum culture medium have a morphology and size distribution similar to EVs from yeasts such as Candida albicans and Cryptococcus neoformans. A unique feature of the EVs from F. oxysporum f. sp. vasinfectum is their purple color, which is predicted to arise from a napthoquinone pigment being packaged into the EVs. Proteomic analysis of F. oxysporum f. sp. vasinfectum EVs revealed that they are enriched in proteins that function in synthesis of polyketides as well as proteases and proteins that function in basic cellular processes. Infiltration of F. oxysporum f. sp. vasinfectum EVs into the leaves of cotton or N. benthamiana plants led to a phytotoxic response. These observations lead to the hypothesis that F. oxysporum f. sp. vasinfectum EVs are likely to play a crucial role in the infection process.

A metabolomic approach to dissecting osmotic stress in the wheat pathogen Stagonospora nodorum.

A non-targeted metabolomics approach was used to identify significant changes in metabolism upon exposure of the wheat pathogen Stagonospora nodorum to 0.5M NaCl. The polyol arabitol, and to a lesser extent glycerol, was found to accumulate in response to the osmotic stress treatment. Amino acid synthesis was strongly down-regulated whilst mannitol levels were unaffected. A reverse genetic approach was undertaken to dissect the role of arabitol metabolism during salt stress. Strains of S. nodorum lacking a gene encoding an l-arabitol dehydrogenase (abd1), a xylitol dehydrogenase (xdh1) and a double-mutant lacking both genes (abd1xdh1) were exposed to salt and the intracellular metabolites analysed. Arabitol levels were significantly up-regulated upon salt stress in the xdh1 strains but were significantly lower than the wild-type. Arabitol was not significantly different in either the abd1 or the abd1xdh1 strains during osmotic stress but the concentration of glycerol was significantly higher indicating a compensatory mechanism in operation. Genome sequence analysis identified a second possible enzyme capable of synthesizing arabitol explaining the basal level of arabitol present in the abd1xdh1 strains. This study identified that arabitol is the primary compatible solute in S. nodorum but in-built levels of redundancy are present allowing the fungus to tolerate osmotic stress.

Resistance to the Plant Defensin NaD1 Features Modifications to the Cell Wall and Osmo-Regulation Pathways of Yeast.

Over the last few decades, the emergence of resistance to commonly used antifungal molecules has become a major barrier to effective treatment of recurrent life-threatening fungal diseases. Resistance combined with the increased incidence of fungal diseases has created the need for new antifungals, such as the plant defensin NaD1, with different mechanisms of action to broaden treatment options. Antimicrobial peptides produced in plants and animals are promising new molecules in the arsenal of antifungal agents because they have different mechanisms of action to current antifungals and are often targeted specifically to fungal pathogens (van der Weerden et al., 2013). A key step in the development of novel antifungals is an understanding of the potential for the fungus to develop resistance. Here, we have used the prototypic plant defensin NaD1 in serial passages with the model fungus Saccharomyces cerevisiae to examine the evolution of resistance to plant antifungal peptides. The yeast strains did develop tolerance to NaD1, but it occurred more slowly than to the clinically used antifungal caspofungin. Sequencing the genomes of the strains with increased tolerance failed to identify any 'hotspot' mutations associated with increased tolerance to NaD1 and led to the identification of 12 genes that are involved in resistance. Characterization of the strains with increased tolerance to NaD1 also revealed changes in tolerance to abiotic stressors. Resistance developed slowly via an accumulation of single nucleotide mutations and had a fitness penalty associated with it. One of the genes identified FPS1, revealed that there is a common mechanism of resistance to NaD1 that involves the osmotic stress response pathway. These data indicate that it is more difficult to generate resistance to antimicrobial peptides such as NaD1 compared to small molecule antifungals.

Mannitol is required for asexual sporulation in the wheat pathogen Stagonospora nodorum (glume blotch).

The physiological role of the mannitol cycle in the wheat pathogen Stagonospora nodorum (glume blotch) has been investigated by reverse genetics and metabolite profiling. A putative mannitol 2-dehydrogenase gene (Mdh1) was cloned by degenerate PCR and disrupted. The resulting mutated mdh1 strains lacked all detectable NADPH-dependent mannitol dehydrogenase activity. The mdh1 strains were unaffected for mannitol production but, surprisingly, were still able to utilize mannitol as a sole carbon source, suggesting a hitherto unknown mechanism for mannitol catabolism. The mutant strains were not compromised in their ability to cause disease or sporulate. To further our understanding of mannitol metabolism, a previously developed mannitol-1-phosphate dehydrogenase (gene mpd1) disruption construct [Solomon, Tan and Oliver (2005) Mol. Plant-Microbe Interact. 18, 110-115] was introduced into the mutated mdh1 background, resulting in a strain lacking both enzyme activities. The mpd1mdh1 strains were unable to grow on mannitol and produced only trace levels of mannitol. The double-mutant strains were unable to sporulate in vitro when grown on minimal medium for extended periods. Deficiency in sporulation was correlated with the depletion of intracellular mannitol pools. Significantly sporulation could be restored with the addition of mannitol. Pathogenicity of the double mutant was not compromised, although, like the previously characterized mpd1 mutants, the strains were unable to sporulate in planta. These findings not only question the currently hypothesized pathways of mannitol metabolism, but also identify for the first time that mannitol is required for sporulation of a filamentous fungus.

Extracellular peptidases of the cereal pathogen Fusarium graminearum.

The plant pathogenic fungus Fusarium graminearum (Fgr) creates economic and health risks in cereals agriculture. Fgr causes head blight (or scab) of wheat and stalk rot of corn, reducing yield, degrading grain quality, and polluting downstream food products with mycotoxins. Fungal plant pathogens must secrete proteases to access nutrition and to breakdown the structural protein component of the plant cell wall. Research into the proteolytic activity of Fgr is hindered by the complex nature of the suite of proteases secreted. We used a systems biology approach comprising genome analysis, transcriptomics and label-free quantitative proteomics to characterize the peptidases deployed by Fgr during growth. A combined analysis of published microarray transcriptome datasets revealed seven transcriptional groupings of peptidases based on in vitro growth, in planta growth, and sporulation behaviors. A high resolution mass spectrometry-based proteomics analysis defined the extracellular proteases secreted by F. graminearum. A meta-classification based on sequence characters and transcriptional/translational activity in planta and in vitro provides a platform to develop control strategies that target Fgr peptidases.

An avirulence gene, AvrLmJ1, from the blackleg fungus, Leptosphaeria maculans, confers avirulence to Brassica juncea cultivars.

The fungus Leptosphaeria maculans causes blackleg of Brassica species. Here, we report the mapping and subsequent cloning of an avirulence gene from L. maculans. This gene, termed AvrLmJ1, confers avirulence towards all three Brassica juncea cultivars tested. Analysis of RNA-seq data showed that AvrLmJ1 is housed in a region of the L. maculans genome which contains only one gene that is highly expressed in planta. The closest genes are 57 and 33 kb away and, like other avirulence genes of L. maculans, AvrLmJ1 is located within an AT-rich, gene-poor region of the genome. The encoded protein is 141 amino acids, has a predicted signal peptide and is cysteine rich. Two virulent isolates contain a premature stop codon in AvrLmJ1. Complementation of an isolate that forms cotyledonary lesions on B. juncea with the wild-type allele of AvrLmJ1 confers avirulence towards all three B. juncea cultivars tested, suggesting that the gene may confer species-specific avirulence activity.

Genomes and transcriptomes of partners in plant-fungal-interactions between canola (Brassica napus) and two Leptosphaeria species.

Leptosphaeria maculans 'brassicae' is a damaging fungal pathogen of canola (Brassica napus), causing lesions on cotyledons and leaves, and cankers on the lower stem. A related species, L. biglobosa 'canadensis', colonises cotyledons but causes few stem cankers. We describe the complement of genes encoding carbohydrate-active enzymes (CAZys) and peptidases of these fungi, as well as of four related plant pathogens. We also report dual-organism RNA-seq transcriptomes of these two Leptosphaeria species and B. napus during disease. During the first seven days of infection L. biglobosa 'canadensis', a necrotroph, expressed more cell wall degrading genes than L. maculans 'brassicae', a hemi-biotroph. L. maculans 'brassicae' expressed many genes in the Carbohydrate Binding Module class of CAZy, particularly CBM50 genes, with potential roles in the evasion of basal innate immunity in the host plant. At this time, three avirulence genes were amongst the top 20 most highly upregulated L. maculans 'brassicae' genes in planta. The two fungi had a similar number of peptidase genes, and trypsin was transcribed at high levels by both fungi early in infection. L. biglobosa 'canadensis' infection activated the jasmonic acid and salicylic acid defence pathways in B. napus, consistent with defence against necrotrophs. L. maculans 'brassicae' triggered a high level of expression of isochorismate synthase 1, a reporter for salicylic acid signalling. L. biglobosa 'canadensis' infection triggered coordinated shutdown of photosynthesis genes, and a concomitant increase in transcription of cell wall remodelling genes of the host plant. Expression of particular classes of CAZy genes and the triggering of host defence and particular metabolic pathways are consistent with the necrotrophic lifestyle of L. biglobosa 'canadensis', and the hemibiotrophic life style of L. maculans 'brassicae'.

Dothideomycete plant interactions illuminated by genome sequencing and EST analysis of the wheat pathogen Stagonospora nodorum.

Stagonospora nodorum is a major necrotrophic fungal pathogen of wheat (Triticum aestivum) and a member of the Dothideomycetes, a large fungal taxon that includes many important plant pathogens affecting all major crop plant families. Here, we report the acquisition and initial analysis of a draft genome sequence for this fungus. The assembly comprises 37,164,227 bp of nuclear DNA contained in 107 scaffolds. The circular mitochondrial genome comprises 49,761 bp encoding 46 genes, including four that are intron encoded. The nuclear genome assembly contains 26 classes of repetitive DNA, comprising 4.5% of the genome. Some of the repeats show evidence of repeat-induced point mutations consistent with a frequent sexual cycle. ESTs and gene prediction models support a minimum of 10,762 nuclear genes. Extensive orthology was found between the polyketide synthase family in S. nodorum and Cochliobolus heterostrophus, suggesting an ancient origin and conserved functions for these genes. A striking feature of the gene catalog was the large number of genes predicted to encode secreted proteins; the majority has no meaningful similarity to any other known genes. It is likely that genes for host-specific toxins, in addition to ToxA, will be found among this group. ESTs obtained from axenic mycelium grown on oleate (chosen to mimic early infection) and late-stage lesions sporulating on wheat leaves were obtained. Statistical analysis shows that transcripts encoding proteins involved in protein synthesis and in the production of extracellular proteases, cellulases, and xylanases predominate in the infection library. This suggests that the fungus is dependant on the degradation of wheat macromolecular constituents to provide the carbon skeletons and energy for the synthesis of proteins and other components destined for the developing pycnidiospores.

Stagonospora nodorum: cause of stagonospora nodorum blotch of wheat.

UNLABELLED: SUMMARY Stagonospora nodorum is an important pathogen of wheat and related cereals, causing both a leaf and glume blotch. This review summarizes recent advances in our understanding of taxonomy, control and pathogenicity of this species. TAXONOMY: Stagonospora (syn. Septoria) nodorum (Berk.) Castell. and Germano [teleomorph: Phaeosphaeria (syn. Leptosphaeria) nodorum (Müll.) Hedjar.], kingdom Fungi, phylum Ascomycota, subphylum Euascomycota, class Dothideomycetes, order Pleosporales, family Phaeosphaeriaceae, genus Phaeosphaeria, species nodorum. HOST RANGE: Wheat, Triticum aestivum, T. durum, Triticale, are the main hosts but other cereals and wild grasses have been reported to harbour S. nodorum. Disease symptoms are lens-shaped necrotic lesions on leaves, girdling necrosis on stems (especially the nodes, hence 'nodorum') and lesions on glumes. Mature lesions produce pycnidia scattered throughout the lesions, especially as tissue senesces. USEFUL WEBSITES: http://ocid.nacse.org/research/deephyphae/htmls/asco_taxlist_spat.html (taxonomic information), http://ohioline.osu.edu/ac-fact/0002.html (disease information), http://wwwacnfp.murdoch.edu.au/ (ACNFP homepage), http://www.broad.mit.edu/annotation/fungi/stagonospora_nodorum/index.html (genome sequence homepage), http://cogeme.ex.ac.uk/efungi/ (genome sequence annotation and analysis).