Adrienne C. Sexton

Genomic analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea.

Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38–39 Mb genomes include 11,860–14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared to <1% of B. cinerea. The arsenal of genes associated with necrotrophic processes is similar between the species, including genes involved in plant cell wall degradation and oxalic acid production. Analysis of secondary metabolism gene clusters revealed an expansion in number and diversity of B. cinerea–specific secondary metabolites relative to S. sclerotiorum. The potential diversity in secondary metabolism might be involved in adaptation to specific ecological niches. Comparative genome analysis revealed the basis of differing sexual mating compatibility systems between S. sclerotiorum and B. cinerea. The organization of the mating-type loci differs, and their structures provide evidence for the evolution of heterothallism from homothallism. These data shed light on the evolutionary and mechanistic bases of the genetically complex traits of necrotrophic pathogenicity and sexual mating. This resource should facilitate the functional studies designed to better understand what makes these fungi such successful and persistent pathogens of agronomic crops.

MyD88 is essential for clearance of Leishmania major: possible role for lipophosphoglycan and Toll-like receptor 2 signaling.

Leishmania major is an obligate intracellular eukaryotic pathogen of mononuclear phagocytes. Invasive promastigotes gain entry into target cells by receptor‐mediated phagocytosis, transform into non‐motile amastigotes and establish in the phagolysosome. Glycosylphosphatidylinositol‐anchored lipophosphoglycan (LPG) is a virulence factor and a major parasite molecule involved in this process. We observed that mice lacking the Toll‐like receptor (TLR) pathway adaptor protein MyD88 were more susceptible to infection with L. major than wild‐type C57BL/6 mice, demonstrating a central role for this innate immune recognition pathway in control of infection, and suggesting that L. major possesses a ligand for TLR. We sought to identify parasite molecules capable of activating the protective Toll pathway, and found that purified Leishmania LPG, but not other surface glycolipids, activate innate immune signaling pathways via TLR2. Activation of cytokine synthesis by LPG required the presence of the lipid anchor and a functional MyD88 adaptor protein. LPG also induced the expression of negative regulatory pathways mediated by members of thesuppressors of cytokine signaling family SOCS‐1 and SOCS‐3. Thus, the Toll pathway is required for resistance to L. major and LPG is a defined TLR agonist from this important human pathogen.

Parallels in fungal pathogenesis on plant and animal hosts.

Fungi are important pathogens of plants and cause more significant yield losses than bacteria or viruses. However, bacteria and viruses are more important than fungi as pathogens of animals; indeed, whether or not a fungus even becomes pathogenic on an animal often depends on the immune status of

Transcriptional Profiling Reveals Suppressed Erythropoiesis, Up-Regulated Glycolysis, and Interferon-Associated Responses in Murine Malaria

The primary pathophysiological events contributing to fatal malaria are the cerebral syndrome, anemia, and lactic acidosis. The molecular basis of each event has been unclear. In the present study, microarray analysis of murine transcriptional responses during the development of severe disease revealed temporal, organ-specific, and pathway-specific patterns. More than 400 genes in the brain and 600 genes in the spleen displayed transcriptional changes. Dominant patterns revealed strongly suppressed erythropoiesis, starting early during infection, and highly up-regulated transcription of genes that control host glycolysis, including lactate dehydrogenase. The latter presents a mechanism that may contribute to metabolic acidosis. No evidence for hypoxia-mediated regulation of these events was observed. Interferon-regulated gene transcripts dominated the inflammatory response to cytokines. These results demonstrate previously unknown transcriptional changes in the host that may underlie the development of malarial syndromes, such as anemia and metabolic dysregulation, and increase the utility of murine models in investigation of basic malarial pathogenesis.

Microsatellite markers reveal genetic differentiation among populations of Sclerotinia sclerotiorum from Australian canola fields.

Eight microsatellite markers were applied to 154 Sclerotinia sclerotiorum isolates from four Australian canola fields, to determine the extent of genetic variation and differentiation in populations of this pathogen. A total of 82 different haplotypes were identified and in each population many haplotypes were unique. Mycelial compatibility grouping, a phenotypic marker system controlled by multiple loci, was often associated with groups of identical or closely related microsatellite haplotypes. Genotypic diversity ranged from 36% to 80% of maximum in the four populations, and gene diversity ranged from 0.23 to 0.79. Genotypic disequilibrium analyses on each of the four populations suggested that both clonal and sexual reproduction contributed to population structure. Analyses based on genetic diversity and fixation indices demonstrated a moderate to high level of differentiation (RST=0.16–0.33, FST=0.18–0.23) between populations from New South Wales and those from Victoria. Despite this genetic diversity, most isolates did not vary in virulence on canola leaves.

Characterisation of a cyanide hydratase gene in the phytopathogenic fungus Leptosphaeria maculans.

Abstract A gene encoding a cyanide hydratase was cloned from an aggressive isolate of Leptosphaeria maculans, the fungus which causes blackleg disease of oilseed Brassica spp. This enzyme catalyses the breakdown of hydrogen cyanide to a less toxic compound, formamide. The predicted amino acid sequence of cyanide hydratase in L. maculans is 77% and 82% identical to cyanide hydratases from two other ascomycetes, Gloeocercospora sorghi and Fusarium lateritium, respectively. The gene is present as a single copy in the L. maculans genome, in both aggressive and non-aggressive isolates, although there is a restriction fragment length polymorphism between these two isolate groups for this gene. The cyanide hydratase promoter contains four putative target sites for GATA transcription factors, proteins that regulate nitrogen metabolism and other processes. Transcription of cyanide hydratase in an aggressive L. maculans isolate is induced strongly by potassium cyanide. Transcription of the gene is detectable in cotyledons of Brassica juncea and B. napus during infection. L. maculans can utilise the reaction product, formamide, as a sole source of nitrogen.

The Natural Killer Complex Regulates Severe Malarial Pathogenesis and Influences Acquired Immune Responses to Plasmodium berghei ANKA

ABSTRACT The natural killer complex (NKC) is a genetic region of highly linked genes encoding several receptors involved in the control of NK cell function. The NKC is highly polymorphic, and allelic variability of various NKC loci has been demonstrated in inbred mice. Making use of BALB.B6-Cmv1r congenic mice, in which the NKC from disease-susceptible C57BL/6 mice has been introduced into the disease-resistant BALB/c background, we show here that during murine malaria infection, the NKC regulates a range of pathophysiological syndromes such as cerebral malaria, pulmonary edema, and severe anemia, which contribute to morbidity and mortality in human malaria. Parasitemia levels were not affected by the NKC genotype, indicating that control of malarial fatalities by the NKC cells does not operate through effects on parasite growth rate. Parasite-specific antibody responses and the proinflammatory gene transcription profile, as well as the TH1/TH2 balance, also appeared to be influenced by NKC genotype, providing evidence that this region, known to control innate immune responses via NK and/or NK T-cell activation, can also significantly regulate acquired immunity to infection. To date, NKC-encoded innate system receptors have been shown mainly to regulate viral infections. Our data provide evidence for critical NKC involvement in the broad immunological responses to a protozoan parasite.

Genes for Glycosylphosphatidylinositol Toxin Biosynthesis in Plasmodium falciparum

ABSTRACT About 2.5 million people die of Plasmodium falciparum malaria every year. Fatalities are associated with systemic and organ-specific inflammation initiated by a parasite toxin. Recent studies show that glycosylphosphatidylinositol (GPI) functions as the dominant parasite toxin in the context of infection. GPIs also serve as membrane anchors for several of the most important surface antigens of parasite invasive stages. GPI anchoring is a complex posttranslational modification produced through the coordinated action of a multicomponent biosynthetic pathway. Here we present eight new genes of P. falciparum selected for encoding homologs of proteins essential for GPI synthesis: PIG-A, PIG-B, PIG-M, PIG-O, GPI1, GPI8, GAA-1, and DPM1. We describe the experimentally verified mRNA and predicted amino acid sequences and in situ localization of the gene products to the parasite endoplasmic reticulum. Moreover, we show preliminary evidence for the PIG-L and PIG-C genes. The biosynthetic pathway of the malaria parasite GPI offers potential targets for drug development and may be useful for studying parasite cell biology and the molecular basis for the pathophysiology of parasitic diseases.

Cloning, purification and characterisation of brassinin glucosyltransferase, a phytoalexin-detoxifying enzyme from the plant pathogen Sclerotinia sclerotiorum

The plant-pathogenic fungus Sclerotinia sclerotiorum can detoxify cruciferous phytoalexins such as brassinin via glucosylation. Here we describe a multifaceted approach including genome mining, transcriptional induction, phytoalexin quantification, protein expression and enzyme purification that led to identification of a S. sclerotiorum glucosyltransferase that detoxifies brassinin. Transcription of this gene, denoted as brassinin glucosyltransferase 1 (SsBGT1), was induced significantly in response to the cruciferous phytoalexins camalexin, cyclobrassinin, brassilexin, brassinin and 3-phenylindole, a camalexin analogue. This gene was also up-regulated during infection of Brassica napus leaves. Levels of brassinin decreased significantly between 48 and 72h post-inoculation, with a concomitant increase in levels of 1-beta-d-glucopyranosylbrassinin, the product of the reaction catalysed by SsBGT1. These findings strongly implicate the involvement of this gene during infection of B. napus. This gene was cloned and expressed in Saccharomyces cerevisiae. The purified recombinant enzyme was able to glucosylate brassinin and two other phytoalexins, albeit much less effectively. This is the first report of a fungal gene involved in detoxification of plant defence molecules via glucosylation.

Fungi have three tetraspanin families with distinct functions.

BackgroundTetraspanins are small membrane proteins that belong to a superfamily encompassing 33 members in human and mouse. These proteins act as organizers of membrane-signalling complexes. So far only two tetraspanin families have been identified in fungi. These are Pls1, which is required for pathogenicity of the plant pathogenic ascomycetes, Magnaporthe grisea, Botrytis cinerea and Colletotrichum lindemuthianum, and Tsp2, whose function is unknown. In this report, we describe a third family of tetraspanins (Tsp3) and a new family of tetraspanin-like proteins (Tpl1) in fungi. We also describe expression of some of these genes in M. grisea and a basidiomycete, Laccaria bicolor, and also their functional analysis in M. grisea.ResultsThe exhaustive search for tetraspanins in fungal genomes reveals that higher fungi (basidiomycetes and ascomycetes) contain three families of tetraspanins (Pls1, Tsp2 and Tsp3) with different distribution amongst phyla. Pls1 is found in ascomycetes and basidiomycetes, whereas Tsp2 is restricted to basidiomycetes and Tsp3 to ascomycetes. A unique copy of each of PLS1 and TSP3 was found in ascomycetes in contrast to TSP2, which has several paralogs in the basidiomycetes, Coprinus cinereus and Laccaria bicolor. A tetraspanin-like family (Tpl1) was also identified in ascomycetes. Transcriptional analyses in various tissues of L. bicolor and M. grisea showed that PLS1 and TSP2 are expressed in all tissues in L. bicolor and that TSP3 and TPL1 are overexpressed in the sexual fruiting bodies (perithecia) and mycelia of M. grisea, suggesting that these genes are not pseudogenes. Phenotypic analysis of gene replacementmutants Δtsp3 and Δtpl1 of M. grisea revealed a reduction of the pathogenicity only on rice, in contrast to Δpls1 mutants, which are completely non-pathogenic on barley and rice.ConclusionA new tetraspanin family (Tsp3) and a tetraspanin-like protein family (Tpl1) have been identified in fungi. Functional analysis by gene replacement showed that these proteins, as well as Pls1, are involved in the infection process of the plant pathogenic fungus M. grisea. The next challenge will be to decipher the role(s) of tetraspanins in a range of symbiotic, saprophytic and human pathogenic fungi.