Angus L. Dawe

Genome-wide Transcriptional Profiling of Appressorium Development by the Rice Blast Fungus Magnaporthe oryzae

The rice blast fungus Magnaporthe oryzae is one of the most significant pathogens affecting global food security. To cause rice blast disease the fungus elaborates a specialised infection structure called an appressorium. Here, we report genome wide transcriptional profile analysis of appressorium development using next generation sequencing (NGS). We performed both RNA-Seq and High-Throughput SuperSAGE analysis to compare the utility of these procedures for identifying differential gene expression in M. oryzae. We then analysed global patterns of gene expression during appressorium development. We show evidence for large-scale gene expression changes, highlighting the role of autophagy, lipid metabolism and melanin biosynthesis in appressorium differentiation. We reveal the role of the Pmk1 MAP kinase as a key global regulator of appressorium-associated gene expression. We also provide evidence for differential expression of transporter-encoding gene families and specific high level expression of genes involved in quinate uptake and utilization, consistent with pathogen-mediated perturbation of host metabolism during plant infection. When considered together, these data provide a comprehensive high-resolution analysis of gene expression changes associated with cellular differentiation that will provide a key resource for understanding the biology of rice blast disease.

Use of cDNA Microarrays To Monitor Transcriptional Responses of the Chestnut Blight Fungus Cryphonectria parasitica to Infection by Virulence-Attenuating Hypoviruses

ABSTRACT Hypoviruses are a family of cytoplasmically replicating RNA viruses of the chestnut blight fungus Cryphonectria parasitica. Members of this mycovirus family persistently alter virulence (hypovirulence) and related fungal developmental processes, including asexual and sexual sporulation. In order to gain a better understanding of the molecular basis for these changes, we have developed a C. parasitica cDNA microarray to monitor global transcriptional responses to hypovirus infection. In this report, a spotted DNA microarray representing approximately 2,200 C. parasitica genes was used to monitor changes in the transcriptional profile after infection by the prototypic hypovirus CHV1-EP713. Altered transcript abundance was identified for 295 clones (13.4% of the 2,200 unique cDNAs) as a result of CHV1-EP713 infection—132 up-regulated and 163 down-regulated. In comparison, less than 20 specific C. parasitica genes were previously identified by Northern analysis and mRNA differential display as being responsive to hypovirus infection. A 93% validation rate was achieved between real-time reverse transcription-PCR results and microarray predictions. Differentially expressed genes represented a broad spectrum of biological functions, including stress responses, carbon metabolism, and transcriptional regulation. These findings are consistent with the view that infection by a 12.7-kbp hypovirus RNA results in a persistent reprogramming of a significant portion of the C. parasitica transcriptome. The potential impact of microarray studies on current and future efforts to establish links between hypovirus-mediated changes in cellular gene expression and phenotypes is discussed.

Molecular Characterization of Vegetative Incompatibility Genes that Restrict Hypovirus Transmission in the Chestnut Blight Fungus Cryphonectria parasitica

Genetic nonself recognition systems such as vegetative incompatibility operate in many filamentous fungi to regulate hyphal fusion between genetically dissimilar individuals and to restrict the spread of virulence-attenuating mycoviruses that have potential for biological control of pathogenic fungi. We report here the use of a comparative genomics approach to identify seven candidate polymorphic genes associated with four vegetative incompatibility (vic) loci of the chestnut blight fungus Cryphonectria parasitica. Disruption of candidate alleles in one of two strains that were heteroallelic at vic2, vic6, or vic7 resulted in enhanced virus transmission, but did not prevent barrage formation associated with mycelial incompatibility. Detailed characterization of the vic6 locus revealed the involvement of nonallelic interactions between two tightly linked genes in barrage formation, heterokaryon formation, and asymmetric, gene-specific influences on virus transmission. The combined results establish molecular identities of genes associated with four C. parasitica vic loci and provide insights into how these recognition factors interact to trigger incompatibility and restrict virus transmission.

The Aquaporin Gene Family of the Yellow Fever Mosquito, Aedes aegypti

Background The mosquito, Aedes aegypti, is the principal vector of the Dengue and yellow fever viruses. During feeding, an adult female can take up more than its own body weight in vertebrate blood. After a blood meal females excrete large amounts of urine through their excretion system, the Malpighian tubules (MT). Diuresis starts within seconds after the mosquito starts feeding. Aquaporins (AQPs) are a family of membrane transporters that regulate the flow of water, glycerol and other small molecules across cellular membranes in both prokaryotic and eukaryotic cells. Our aim was to identify aquaporins that function as water channels, mediating transcellular water transport in MTs of adult female Ae. aegypti. Methodology/Principal Findings Using a bioinformatics approach we screened genome databases and identified six putative AQPs in the genome of Ae. aegypti. Phylogenetic analysis showed that five of the six Ae. aegypti AQPs have high similarity to classical water-transporting AQPs of vertebrates. Using microarray, reverse transcription and real time PCR analysis we found that all six AQPs are expressed in distinct patterns in mosquito tissues/body parts. AaAQP1, 4, and 5 are strongly expressed in the adult female MT. RNAi-mediated knockdown of the MT-expressed mosquito AQPs resulted in significantly reduced diuresis. Conclusions/Significance Our results support the notion that AQP1, 4, and 5 function as water transporters in the MTs of adult female Ae. aegypti mosquitoes. Our results demonstrate the importance of these AQPs for mosquito diuresis after blood ingestion and highlight their potential as targets for the development of novel vector control strategies.

Hypovirus molecular biology: from Koch's postulates to host self-recognition genes that restrict virus transmission.

The idea that viruses can be used to control fungal diseases has been a driving force in mycovirus research since the earliest days. Viruses in the family Hypoviridae associated with reduced virulence (hypovirulence) of the chestnut blight fungus, Cryphonectria parasitica, have held a prominent place in this research. This has been due in part to the severity of the chestnut blight epidemics in North America and Europe and early reports of hypovirulence-mediated mitigation of disease in European forests and successful application for control of chestnut blight in chestnut orchards. A more recent contributing factor has been the development of a hypovirus/C. parasitica experimental system that has overcome many of the challenges associated with mycovirus research, stemming primarily from the exclusive intracellular lifestyle shared by all mycoviruses. This chapter will focus on hypovirus molecular biology with an emphasis on the development of the hypovirus/C. parasitica experimental system and its contributions to fundamental and practical advances in mycovirology and the broader understanding of virus-host interactions and fungal pathogenesis.

Vegetative Incompatibility Loci with Dedicated Roles in Allorecognition Restrict Mycovirus Transmission in Chestnut Blight Fungus

Vegetative incompatibility (vic), a form of nonself allorecognition, operates widely in filamentous fungi and restricts transmission of virulence-attenuating hypoviruses in the chestnut blight fungus Cryphonectria parasitica. We report here the use of a polymorphism-based comparative genomics approach to complete the molecular identification of the genetically defined C. parasitica vic loci with the identification of vic1 and vic3. The vic1 locus in the C. parasitica reference strain EP155 consists of a polymorphic HET-domain-containing 771-aa ORF designated vic1a-2, which shares 91% identity with the corresponding vic1a-1 allele, and a small (172 aa) idiomorphic DUF1909-domain-containing ORF designated vic1b-2 that is absent at the vic1-1 locus. Gene disruption of either vic1a-2 or vic1b-2 in strain EP155 eliminated restrictions on virus transmission when paired with a vic1 heteroallelic strain; however, only disruption of vic1a-2 abolished the incompatible programmed cell death (PCD) reaction. The vic3 locus of strain EP155 contains two polymorphic ORFs of 599 aa (vic3a-1) and 102 aa (vic3b-1) that shared 46 and 85% aa identity with the corresponding vic3a-2 and vic3b-2 alleles, respectively. Disruption of either vic3a-1 or vic3b-1 resulted in increased virus transmission. However, elimination of PCD required disruption of both vic3a and vic3b. Additional allelic heterogeneity included a sequence inversion and a 8.5-kb insertion containing a LTR retrotransposon sequence and an adjacent HET-domain gene at the vic1 locus and a 7.7-kb sequence deletion associated with a nonfunctional, pseudo vic locus. Combined gene disruption studies formally confirmed restriction of mycovirus transmission by five C. parasitica vic loci and suggested dedicated roles in allorecognition. The relevance of these results to the acquisition and maintenance of vic genes and the potential for manipulation of vic alleles for enhanced mycovirus transmission are discussed.

Major impacts on the primary metabolism of the plant pathogen Cryphonectria parasitica by the virulence-attenuating virus CHV1-EP713.

Cryphonectria parasitica, the chestnut blight fungus, can be infected by virulence-attenuating mycoviruses of the family Hypoviridae. Previous studies have led to the hypothesis that the hypovirus-infected phenotype is partly due to metabolic changes induced by the viral infection. To investigate this, we measured the metabolic rate and respiration of C. parasitica colonies grown on solid medium. These experiments supported historical observations of other fungal species done in liquid cultures that the metabolic rate steadily declines with age and differentiation of the mycelium. Hypovirus infection increased metabolic rate in the youngest mycelium, but a subsequent decline was also observed as the mycelium aged. By measuring both CO(2) production and O(2) consumption, we also observed that changes occur in carbohydrate metabolism as a result of ageing in both infected and uninfected mycelium. Mycelium on the periphery of the colony exploited fermentation pathways extensively, before transitioning to aerobic carbohydrate metabolism and finally lipid metabolism in the interior regions, despite abundant remaining glucose. However, the hypovirus affected the extent of these changes, with infected mycelium apparently unable to utilize lipid-related metabolic pathways, leading to an increased depletion of glucose. Finally, we used metabolic profi fi ling to determine the changes in accumulation of primary metabolites in wild-type and hypovirus-infected mycelium and found that approximately one-third of the 164 detected metabolites were affected. These results are consistent with those expected from the physiological measurements, with significant alterations noted for compounds related to lipid and carbohydrate metabolism. Additionally, we observed an increase in the accumulation of the polyamine spermidine in the presence of hypovirus. Polyamines have been implicated in antiviral responses of mammalian systems; therefore this may suggest a novel antiviral response mechanism in fungi.

Phosphorylation of phosducin‐like protein BDM‐1 by protein kinase 2 (CK2) is required for virulence and Gβ subunit stability in the fungal plant pathogen Cryphonectria parasitica

Phosducin‐like proteins are conserved regulatory components of G‐protein signalling pathways, which mediate many physiological processes. Identified throughout eukaryotic genomes, they are thought to serve as regulators of Gβγ assembly. Cryphonectria parasitica, a plant pathogen and causative agent of chestnut blight, contains three Gα, one Gβ, one Gγ subunits and phosducin‐like protein BDM‐1 that have important roles in pigmentation, sporulation and virulence. Deletion of either Gβ subunit or BDM‐1 produces identical phenotypes. Additionally, we report that the Gβ subunit is not detectable in absence of BDM‐1. Given that the regulatory role of phosducin‐like proteins may be influenced by protein kinase 2 (CK2), we confirmed that BDM‐1 is a phosphoprotein that can be targeted by CK2 in vitro. Mutagenesis of the five putative CK2 sites revealed that native phosphorylation likely occurs at two locations. Strains bearing a single or double serine to alanine substitutions at those sites were significantly less virulent with only minor phenotypic changes from vegetative colonies. Therefore, CK2 activity appears to mediate key signals that are required for virulence, but not for vegetative growth. Expression of selected CK2 mutants resulted in reduced accumulation of the Gβ subunit, suggesting that phosphorylation of BDM‐1 influences Gβ stability.

Development of a transformation system in the swainsonine producing, slow growing endophytic fungus, Undifilum oxytropis

Undifilum oxytropis (Phylum: Ascomycota; Family: Pleosporaceae) is a slow growing endophytic fungus that produces a toxic alkaloid, swainsonine. This endophyte resides in locoweeds, which are perennial flowering legumes. Consumption of this fungus by grazing animals induces a neurological disorder called locoism. The alkaloid swainsonine, an alpha-mannosidase inhibitor, is responsible for the field toxicity related to locoism. Little is known about the biosynthetic pathway of swainsonine in endophytic fungi. Genetic manipulation of endophytic fungi is important to better understand biochemical pathways involved in alkaloid synthesis, but no transformation system has been available for studying such enzymes in Undifilum. In this study we report the development of protoplast and transformation system for U. oxytropis. Fungal mycelia required for generating protoplasts were grown in liquid culture, then harvested and processed with various enzymes. Protoplasts were transformed with a fungal specific vector driving the expression of Enhanced Green Florescent Protein (EGFP). The quality of transformed protoplasts and transformation efficiency were monitored during the process. In all cases, resistance to antibiotic hygromycin B was maintained. Such manipulation will open avenues for future research to decipher fungal metabolic pathways.

Two-dimensional fractal growth properties of the filamentous fungus Cryphonectria parasitica: the effects of hypovirus infection

Whole‐colony two‐dimensional fractal growth patterns produced by hypovirus‐infected Cryphonectria parasitica (EP155/CHV1‐EP713) were measured and compared with those produced by the isogenic virus‐free strain (EP155) on solid medium. We have quantified statistically significant differences in the rates of expansion and spatial dynamics of colony growth between the two strains and concluded that fractal dimension is affected by the presence of the hypovirus. Therefore, fractal dimension measurement is an effective quantitative tool for testing the effects of mycovirus infection on fungal growth parameters. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)