Neil Andrew Brown

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.

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.

A Role for Topoisomerase I in Fusarium graminearum and F. culmorum Pathogenesis and Sporulation

Fusarium graminearum and F. culmorum are the causal agents of Fusarium ear blight (FEB) in wheat. A forward genetics approach was taken to discover novel pathogenicity genes in the genome of F. graminearum. A library of transformants created by random plasmid insertional mutagenesis was screened on wheat ears for virulence defects. Plasmid rescue on one of the reduced-virulence mutants revealed a single-copy plasmid insertion in the gene coding for the DNA interacting enzyme, topoisomerase I. Targeted topoisomerase I gene-deletion mutants were created in strains of both F. graminearum and F. culmorum. The top1 mutants of both species exhibited greatly reduced virulence in wheat ear infection assays (GO:0009405 and GO:0044145). Detailed microscopy analyses revealed that top1 hyphal growth was restricted to palea tissue whereas host responses were discernable 1,000 mum further away in the rachis node. Asexual sporulation was reduced in the F. graminearum mutants and was absent from the F. culmorum mutants. The F. graminearum mutant did not develop sexual spores when subjected to an in vitro perithecia production assay. During in vitro growth, the top1 mutants of both species were still able to produce the trichothecene mycotoxin, deoxynivalenol.

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.

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 β-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.

Mitogen activated protein kinases SakA(HOG1) and MpkC collaborate for Aspergillus fumigatus virulence.

Here, we investigated which stress responses were influenced by the MpkC and SakA mitogen‐activated protein kinases of the high‐osmolarity glycerol (HOG) pathway in the fungal pathogen Aspergillus fumigatus. The ΔsakA and the double ΔmpkC ΔsakA mutants were more sensitive to osmotic and oxidative stresses, and to cell wall damaging agents. Both MpkC::GFP and SakA::GFP translocated to the nucleus upon osmotic stress and cell wall damage, with SakA::GFP showing a quicker response. The phosphorylation state of MpkA was determined post exposure to high concentrations of congo red and Sorbitol. In the wild‐type strain, MpkA phosphorylation levels progressively increased in both treatments. In contrast, the ΔsakA mutant had reduced MpkA phosphorylation, and surprisingly, the double ΔmpkC ΔsakA had no detectable MpkA phosphorylation. A. fumigatus ΔsakA and ΔmpkC were virulent in mouse survival experiments, but they had a 40% reduction in fungal burden. In contrast, the ΔmpkC ΔsakA double mutant showed highly attenuated virulence, with approximately 50% mice surviving and a 75% reduction in fungal burden. We propose that both cell wall integrity (CWI) and HOG pathways collaborate, and that MpkC could act by modulating SakA activity upon exposure to several types of stresses and during CW biosynthesis.

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 importance of connections between the cell wall integrity pathway and the unfolded protein response in filamentous fungi.

In the external environment, or within a host organism, filamentous fungi experience sudden changes in nutrient availability, osmolality, pH, temperature and the exposure to toxic compounds. The fungal cell wall represents the first line of defense, while also performing essential roles in morphology, development and virulence. A polarized secretion system is paramount for cell wall biosynthesis, filamentous growth, nutrient acquisition and interactions with the environment. The unique ability of filamentous fungi to secrete has resulted in their industrial adoption as fungal cell factories. Protein maturation and secretion commences in the endoplasmic reticulum (ER). The unfolded protein response (UPR) maintains ER functionality during exposure to secretion and cell wall stress. UPR, therefore, influences secretion and cell wall homeostasis, which in turn impacts upon numerous fungal traits important to pathogenesis and biotechnology. Subsequently, this review describes the relevance of the cell wall and UPR systems to filamentous fungal pathogens or industrial microbes and then highlights interconnections between the two systems. Ultimately, the possible biotechnological applications of an enhanced understanding of such regulatory systems in combating fungal disease, or the removal of natural bottlenecks in protein secretion in an industrial setting, are discussed.

The Aspergillus fumigatus pkcAG579R Mutant Is Defective in the Activation of the Cell Wall Integrity Pathway but Is Dispensable for Virulence in a Neutropenic Mouse Infection Model

Aspergillus fumigatus is an opportunistic human pathogen, which causes the life-threatening disease, invasive pulmonary aspergillosis. In fungi, cell wall homeostasis is controlled by the conserved Cell Wall Integrity (CWI) pathway. In A. fumigatus this signaling cascade is partially characterized, but the mechanisms by which it is activated are not fully elucidated. In this study we investigated the role of protein kinase C (PkcA) in this signaling cascade. Our results suggest that pkcA is an essential gene and is activated in response to cell wall stress. Subsequently, we constructed and analyzed a non-essential A. fumigatus pkcA G579R mutant, carrying a Gly579Arg substitution in the PkcA C1B regulatory domain. The pkcA G579R mutation has a reduced activation of the downstream Mitogen-Activated Protein Kinase, MpkA, resulting in the altered expression of genes encoding cell wall-related proteins, markers of endoplasmic reticulum stress and the unfolded protein response. Furthermore, PkcAG579R is involved in the formation of proper conidial architecture and protection to oxidative damage. The pkcA G579R mutant elicits increased production of TNF-α and phagocytosis but it has no impact on virulence in a murine model of invasive pulmonary aspergillosis. These results highlight the importance of PkcA to the CWI pathway but also indicated that additional regulatory circuits may be involved in the biosynthesis and/or reinforcement of the A. fumigatus cell wall during infection.

G‐protein coupled receptor‐mediated nutrient sensing and developmental control in Aspergillus nidulans

Nutrient sensing and utilisation are fundamental for all life forms. As heterotrophs, fungi have evolved a diverse range of mechanisms for sensing and taking up various nutrients. Despite its importance, only a limited number of nutrient receptors and their corresponding ligands have been identified in fungi. G‐protein coupled receptors (GPCRs) are the largest family of transmembrane receptors. The Aspergillus nidulans genome encodes 16 putative GPCRs, but only a few have been functionally characterised. Our previous study showed the increased expression of an uncharacterised putative GPCR, gprH, during carbon starvation. GprH appears conserved throughout numerous filamentous fungi. Here, we reveal that GprH is a putative receptor involved in glucose and tryptophan sensing. The absence of GprH results in a reduction in cAMP levels and PKA activity upon adding glucose or tryptophan to starved cells. GprH is pre‐formed in conidia and is increasingly active during carbon starvation, where it plays a role in glucose uptake and the recovery of hyphal growth. GprH also represses sexual development under conditions favouring sexual fruiting and during carbon starvation in submerged cultures. In summary, the GprH nutrient‐sensing system functions upstream of the cAMP‐PKA pathway, influences primary metabolism and hyphal growth, while represses sexual development in A. nidulans.