GPCR-mediated glucose sensing system regulates light-dependent fungal development and mycotoxin production.

Microorganisms sense environmental fluctuations in nutrients and light, coordinating their growth and development accordingly. Despite their critical roles in fungi, only a few G-protein coupled receptors (GPCRs) have been characterized. The Aspergillus nidulans genome encodes 86 putative GPCRs. Here, we characterise a carbon starvation-induced GPCR-mediated glucose sensing mechanism in A. nidulans. This includes two class V (gprH and gprI) and one class VII (gprM) GPCRs, which in response to glucose promote cAMP signalling, germination and hyphal growth, while negatively regulating sexual development in a light-dependent manner. We demonstrate that GprH regulates sexual development via influencing VeA activity, a key light-dependent regulator of fungal morphogenesis and secondary metabolism. We show that GprH and GprM are light-independent negative regulators of sterigmatocystin biosynthesis. Additionally, we reveal the epistatic interactions between the three GPCRs in regulating sexual development and sterigmatocystin production. In conclusion, GprH, GprM and GprI constitute a novel carbon starvation-induced glucose sensing mechanism that functions upstream of cAMP-PKA signalling to regulate fungal development and mycotoxin production.

Sharing mutants and experimental information prepublication using FgMutantDb (https://scabusa.org/FgMutantDb).

There is no comprehensive storage for generated mutants of Fusarium graminearum or data associated with these mutants. Instead, researchers relied on several independent and non-integrated databases. FgMutantDb was designed as a simple spreadsheet that is accessible globally on the web that will function as a centralized source of information on F. graminearum mutants. FgMutantDb aids in the maintenance and sharing of mutants within a research community. It will serve also as a platform for disseminating prepublication results as well as negative results that often go unreported. Additionally, the highly curated information on mutants in FgMutantDb will be shared with other databases (FungiDB, Ensembl, PhytoPath, and PHI-base) through updating reports. Here we describe the creation and potential usefulness of FgMutantDb to the F. graminearum research community, and provide a tutorial on its use. This type of database could be easily emulated for other fungal species.

Transcriptional profiling of Brazilian Saccharomyces cerevisiae strains selected for semi-continuous fermentation of sugarcane must.

Brazil played a pioneering role in the global establishment of the sugarcane bioethanol industry. The bioethanol fermentation process currently used in Brazil is unique due to the acid wash and recycling of yeast cells. Two, industrially adopted, wild yeast strains, CAT-1 and PE-2, have become the most widely used in Brazil. How these strains respond to the unique fermentation process is poorly understood. The improved performance of CAT-1 and PE-2 is hypothesised to be related to enhanced stress tolerance. This study presents a genome-wide analysis of the CAT-1 and PE-2 transcriptomes during a small-scale fermentation process that mimicked the industrial conditions. The common and unique transcriptional responses of the two strains to the Brazilian fermentation process were identified. Environmental stress response genes were up-regulated postfermenter feeding, demonstrating the impact of the prior acid wash and high glucose environment. Cell wall and oxidative stress tolerance were subsequently demonstrated to be enhanced for the industrial strains. Conversely, numerous genes involved in protein synthesis were down-regulated at the end of fermentation revealing the later impact of ethanol-induced stress. Subsequently, the industrial strains demonstrated a greater tolerance of ethanol and the disruption of endoplasmic reticulum homoeostasis. This increased ethanol tolerance was finally correlated with an increased unfolded protein response and increased HAC1 splicing.

Diverse mycotoxin threats to safe food and feed cereals.

Toxigenic fungi, including Aspergillus and Fusarium species, contaminate our major cereal crops with an array of harmful mycotoxins, which threaten the health of humans and farmed animals. Despite our best efforts to prevent crop diseases, or postharvest spoilage, our cereals are consistently contaminated with aflatoxins and deoxynivalenol, and while established monitoring systems effectively prevent acute exposure, Aspergillus and Fusarium mycotoxins still threaten our food security. This is through the understudied impacts of: (i) our chronic exposure to these mycotoxins, (ii) the underestimated dietary intake of masked mycotoxins, and (iii) the synergistic threat of cocontaminations by multiple mycotoxins. Mycotoxins also have profound economic consequences for cereal and farmed-animal producers, plus their associated food and feed industries, which results in higher food prices for consumers. Climate change and altering agronomic practices are predicted to exacerbate the extent and intensity of mycotoxin contaminations of cereals. Collectively, this review of the diverse threats from Aspergillus and Fusarium mycotoxins highlights the need for renewed and concerted efforts to understand, and mitigate, the increased risks they pose to our food and feed cereals.

Emerging health threat and cost of Fusarium mycotoxins in European wheat.

Mycotoxins harm human and livestock health, while damaging economies. Here we reveal the changing threat of Fusarium head blight (FHB) mycotoxins in European wheat, using data from the European Food Safety Agency and agribusiness (BIOMIN, World Mycotoxin Survey) for ten years (2010-2019). We show persistent, high, single- and multi-mycotoxin contamination alongside changing temporal-geographical distributions, indicative of altering FHB disease pressure and pathogen populations, highlighting the potential synergistic negative health consequences and economic cost.

Incidence of endornaviruses in Phytophthora taxon douglasfir and Phytophthora ramorum.

In this investigation, we show that four Phytophthora taxon douglasfir isolates from the USA, irrespective of their geographical location or host plant, and 20% of a representative cohort of Phytophthora ramorum isolates contain endornavirus dsRNAs. Three endornavirus-specific RT-PCR amplicons were generated by RT-PCR using dsRNA isolated from the four Phytophthora taxon douglasfir isolates and one representative Phytophthora ramorum isolate as template with oligonucleotide primers designed from the sequence of Phytophthora endornavirus 1. The amplified segments showed a very high degree of sequence similarity suggesting that the virus has gone through a population bottleneck during its emergence.

RNAi as an emerging approach to control Fusarium head blight disease and mycotoxin contamination in cereals.

Fusarium graminearum is a major fungal pathogen of cereals worldwide, causing seedling, stem base and floral diseases, including Fusarium head blight (FHB). In addition to yield and quality losses, FHB contaminates cereal grain with mycotoxins, including deoxynivalenol, which are harmful to human, animal and ecosystem health. Currently, FHB control is only partially effective due to several intractable problems. RNA interference (RNAi) is a natural mechanism that regulates gene expression. RNAi has been exploited in the development of new genomic tools that allow the targeted silencing of genes of interest in many eukaryotes. Host-induced gene silencing (HIGS) is a transgenic technology used to silence fungal genes in planta during attempted infection and thereby reduces disease levels. HIGS relies on the host plant's ability to produce mobile small interfering RNA molecules, generated from long double-stranded RNA, which are complementary to targeted fungal genes. These molecules are transferred from the plant to invading fungi via an uncharacterised mechanism, to cause gene silencing. Here, we describe recent advances in RNAi-mediated control of plant pathogenic fungi, highlighting the key advantages and disadvantages. We then discuss the developments and implications of combining HIGS with other methods of disease control. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

A spatial temporal analysis of the Fusarium graminearum transcriptome during symptomless and symptomatic wheat infection.

Fusarium head blight of wheat is one of the most serious and hazardous crop diseases worldwide. Here, a transcriptomic investigation of Fusarium graminearum reveals a new model for symptomless and symptomatic wheat infection. The predicted metabolic state and secretome of F. graminearum were distinct within symptomless and symptomatic wheat tissues. Transcripts for genes involved in the biosynthesis of the mycotoxin, deoxynivalenol, plus other characterized and putative secondary metabolite clusters increased in abundance in symptomless tissue. Transcripts encoding for genes of distinct groups of putative secreted effectors increased within either symptomless or symptomatic tissue. Numerous pathogenicity-associated gene transcripts and transcripts representing PHI-base mutations that impacted on virulence increased in symptomless tissue. In contrast, hydrolytic carbohydrate-active enzyme (CAZyme) and lipase gene transcripts exhibited a different pattern of expression, resulting in elevated transcript abundance during the development of disease symptoms. Genome-wide comparisons with existing datasets confirmed that, within the wheat floral tissue, at a single time point, different phases of infection co-exist, which are spatially separated and reminiscent of both early and late infection. This study provides novel insights into the combined spatial temporal coordination of functionally characterized and hypothesized virulence strategies.

The putative flavin carrier family FlcA-C is important for Aspergillus fumigatus virulence.

Aspergillus fumigatus is an opportunistic fungal pathogen and the most important species causing pulmonary fungal infections. The signaling by calcium is very important for A. fumigatus pathogenicity and it is regulated by the transcription factor CrzA. We have previously used used ChIP-seq (Chromatin Immunoprecipitation DNA sequencing) aiming to identify gene targets regulated by CrzA. We have identified among several genes regulated by calcium stress, the putative flavin transporter, flcA. This transporter belongs to a small protein family composed of FlcA, B, and C. The ΔflcA null mutant showed several phenotypes, such as morphological defects, increased sensitivity to calcium chelating-agent ethylene glycol tetraacetic acid (EGTA), cell wall or oxidative damaging agents and metals, repre-sentative of deficiencies in calcium signaling and iron homeostasis. Increasing calcium concentrations improved significantly the ΔflcA growth and conidiation, indicating that ΔflcA mutant has calcium insufficiency. Finally, ΔflcA-C mutants showed reduced flavin adenine dinucleotide (FAD) and were avirulent in a low dose murine infection model.

The contribution of Aspergillus fumigatus stress responses to virulence and antifungal resistance.

Invasive aspergillosis has emerged as one of the most common life-threatening fungal disease of humans. The emergence of antifungal resistant pathogens represents a current and increasing threat to society. In turn, new strategies to combat fungal infection are urgently required. Fungal adaptations to stresses experienced within the human host are a prerequisite for the survival and virulence strategies of the pathogen. Here, we review the latest information on the signalling pathways in Aspergillus fumigatus that contribute to stress adaptations and virulence, while highlighting their potential as targets for the development of novel combinational antifungal therapies.

The trans-kingdom identification of negative regulators of pathogen hypervirulence.

Modern society and global ecosystems are increasingly under threat from pathogens, which cause a plethora of human, animal, invertebrate and plant diseases. Of increasing concern is the trans-kingdom tendency for increased pathogen virulence that is beginning to emerge in natural, clinical and agricultural settings. The study of pathogenicity has revealed multiple examples of convergently evolved virulence mechanisms. Originally described as rare, but increasingly common, are interactions where a single gene deletion in a pathogenic species causes hypervirulence. This review utilised the pathogen-host interaction database (www.PHI-base.org) to identify 112 hypervirulent mutations from 37 pathogen species, and subsequently interrogates the trans-kingdom, conserved, molecular, biochemical and cellular themes that cause hypervirulence. This study investigates 22 animal and 15 plant pathogens including 17 bacterial and 17 fungal species. Finally, the evolutionary significance and trans-kingdom requirement for negative regulators of hypervirulence and the implication of pathogen hypervirulence and emerging infectious diseases on society are discussed.

An Interspecies Comparative Analysis of the Predicted Secretomes of the Necrotrophic Plant Pathogens Sclerotinia sclerotiorum and Botrytis cinerea.

Phytopathogenic fungi form intimate associations with host plant species and cause disease. To be successful, fungal pathogens communicate with a susceptible host through the secretion of proteinaceous effectors, hydrolytic enzymes and metabolites. Sclerotinia sclerotiorum and Botrytis cinerea are economically important necrotrophic fungal pathogens that cause disease on numerous crop species. Here, a powerful bioinformatics pipeline was used to predict the refined S. sclerotiorum and B. cinerea secretomes, identifying 432 and 499 proteins respectively. Analyses focusing on S. sclerotiorum revealed that 16% of the secretome encoding genes resided in small, sequence heterogeneous, gene clusters that were distributed over 13 of the 16 predicted chromosomes. Functional analyses highlighted the importance of plant cell hydrolysis, oxidation-reduction processes and the redox state to the S. sclerotiorum and B. cinerea secretomes and potentially host infection. Only 8% of the predicted proteins were distinct between the two secretomes. In contrast to S. sclerotiorum, the B. cinerea secretome lacked CFEM- or LysM-containing proteins. The 115 fungal and oomycete genome comparison identified 30 proteins specific to S. sclerotiorum and B. cinerea, plus 11 proteins specific to S. sclerotiorum and 32 proteins specific to B. cinerea. Expressed sequence tag (EST) and proteomic analyses showed that 246 S. sclerotiorum secretome encoding genes had EST support, including 101 which were only expressed in vitro and 49 which were only expressed in planta, whilst 42 predicted proteins were experimentally proven to be secreted. These detailed in silico analyses of two important necrotrophic pathogens will permit informed choices to be made when candidate effector proteins are selected for function analyses in planta.

The predicted secretome of the plant pathogenic fungus Fusarium graminearum: a refined comparative analysis.

The fungus Fusarium graminearum forms an intimate association with the host species wheat whilst infecting the floral tissues at anthesis. During the prolonged latent period of infection, extracellular communication between live pathogen and host cells must occur, implying a role for secreted fungal proteins. The wheat cells in contact with fungal hyphae subsequently die and intracellular hyphal colonisation results in the development of visible disease symptoms. Since the original genome annotation analysis was done in 2007, which predicted the secretome using TargetP, the F. graminearum gene call has changed considerably through the combined efforts of the BROAD and MIPS institutes. As a result of the modifications to the genome and the recent findings that suggested a role for secreted proteins in virulence, the F. graminearum secretome was revisited. In the current study, a refined F. graminearum secretome was predicted by combining several bioinformatic approaches. This strategy increased the probability of identifying truly secreted proteins. A secretome of 574 proteins was predicted of which 99% was supported by transcriptional evidence. The function of the annotated and unannotated secreted proteins was explored. The potential role(s) of the annotated proteins including, putative enzymes, phytotoxins and antifungals are discussed. Characterisation of the unannotated proteins included the analysis of Pfam domains and features associated with known fungal effectors, for example, small size, cysteine-rich and containing internal amino acid repeats. A comprehensive comparative genomic analysis involving 57 fungal and oomycete genomes revealed that only a small number of the predicted F. graminearum secreted proteins can be considered to be either species or sequenced strain specific.

Characterisation of the Fusarium graminearum-Wheat Floral Interaction.

Fusarium Ear Blight is a destructive fungal disease of cereals including wheat and can contaminate the crop with various trichothecene mycotoxins. This investigation has produced a new ß-glucuronidase (GUS) reporter strain that facilitates the quick and easy assessment of plant infection. The constitutively expressed gpdA:GUS strain of Fusarium graminearum was used to quantify the overall colonisation pattern. Histochemical and biochemical approaches confirmed, in susceptible wheat ear infections, the presence of a substantial phase of symptomless fungal growth. Separate analyses demonstrated that there was a reduction in the quantity of physiologically active hyphae as the wheat ear infection proceeded. A simplified linear system of rachis infection was then utilised to evaluate the expression of several TRI genes by RT-qPCR. Fungal gene expression at the advancing front of symptomless infection was compared with the origin of infection in the rachis. This revealed that TRI gene expression was maximal at the advancing front and supports the hypothesis that the mycotoxin deoxynivalenol plays a role in inhibiting plant defences in advance of the invading intercellular hyphae. This study has also demonstrated that there are transcriptional differences between the various phases of fungal infection and that these differences are maintained as the infection proceeds.

The infection biology of Fusarium graminearum: defining the pathways of spikelet to spikelet colonisation in wheat ears.

Fusarium graminearum is one of the main causal agents of Fusarium Ear Blight on wheat. How the pathogen colonises the entire ear is not known. There is controversy over whether this mycotoxin producing pathogenic fungus invades wheat floral tissue using a necrotrophic or another mode of nutrition. A detailed microscopic investigation has revealed how wild-type fungal hyphae, of the sequenced strain PH-1, colonised susceptible wheat ears and spread from spikelet to spikelet. At the advancing infection front, colonisation of the host cortex occurred ahead of any vascular colonisation and the hyphae adapted to the available intercellular space between host cells. Intercellular hyphae then became abundant and host cells lost their entire cellular contents just prior to intracellular colonisation. No host cells died ahead of the infection. However, while these deep cortex infections progressed, just below the surface the highly photosynthetic chlorenchyma cells were observed to have died prior to colonisation. Behind the infection front, hyphae were abundant in the vasculature and the cortex, often growing through the pit fields of thick walled cells. This high level of inter- and intracellular fungal colonisation resulted in the collapse of the non-lignified cell-types. In this middle zone of infection, hyphal diameters were considerably enlarged. Far behind the infection front inter- and intracellular hyphae were devoid of contents and had often collapsed. At later stages of infection, the pathogen switched from predominately vertical to lateral growth and accumulated below the surface of the rachis. Here the lignified host cell walls became heavily degraded and hyphae ruptured the epidermis and produced an aerial mycelium.