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Fusarium graminearum is a filamentous fungus that causes devastating diseases on plants of economic importance including maize, wheat, and barley. F. graminearum is able to utilize triglycerides as a carbon source during growth. Extracellular lipases are the preferred enzymes to catalyze the hydrolysis of fats and oils. Lipases are ubiquitous enzymes of considerable physiological significance and industrial potential. Previously, FGL1 was the first described F. graminearum extracellular lipase associated with virulence. We report the biochemical characterization of FGL1 and four new secreted lipases of F. graminearum. The lipase genes of F. graminearum wild-type strain 8/1 were sequenced, cloned and over-expressed in Pichia pastoris. We show that the lipases have their temperature optimum between 30 and 40°C and a pH optimum of ∼7. A broad range of lipase substrates, from C4 to C18 p-nitrophenyl esters, were hydrolyzed efficiently by the lipases, indicating the true lipolytic activity of the enzymes. Expression patterns of these lipases were also analyzed by semiquantitative RT-PCR in F. graminearum cultured in water supplemented with 2% (v/v) wheat germ oil at 28°C. Transcripts of all examined lipases are detectable and the genes are regulated differently under these culture conditions. Our data indicated that F. graminearum possesses a ubiquitous source of secreted lipases, which could be used for industrial intentions. We also provided the foundation of lipase expression in vitro, which is necessary for further characterization., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Candida parapsilosis is an increasingly important human pathogen. To study the interactions of C. parapsilosis with human tissues, we evaluated the effects of the CBS 604 type strain and three different clinical isolates on reconstituted human oral epithelial and epidermal tissues. The newly described species Candida orthopsilosis and Candida metapsilosis were also examined in these models. Microscopy of reconstituted tissues infected with yeast cells revealed severe attenuation, morphological changes and cellular damage. C. orthopsilosis caused damage similar to C. parapsilosis isolates, whereas C. metapsilosis was less virulent. To further quantitate tissue damage, we measured lactate dehydrogenase (LDH) in the culture supernatant. The relative LDH measurements correlated with our histopathological observations. We also examined the effect of the lipase inhibitor Ebelactone B and proteinase inhibitor Pepstatin A, to establish the utility of this model for studying factors of C. parapsilosis virulence. Both Ebelactone B and Pepstatin A reduced the destruction of epidermal and epithelial tissues. Our data show that reconstituted human tissues are extremely useful for modeling host interactions with C. parapsilosis and for studying fungal virulence factors.

SUMMARY Fusarium graminearum is the main causative agent of Fusarium head blight on small grain cereals and of ear rot on maize. The disease leads to dramatic yield losses and to an accumulation of mycotoxins. The most dominant F. graminearum mycotoxins are the trichothecenes, with deoxynivalenol and nivalenol being the most prevalent derivatives. To investigate the involvement of trichothecenes in the virulence of the pathogen, the gene coding for the initial enzyme of the trichothecene pathway was disrupted in three field isolates, differing in chemotype and in virulence. From each isolate three individual disruption mutants were tested for their virulence on wheat, barley and maize. Despite the different initial virulence of the three wild-type progenitor strains on wheat, all disruption mutants caused disease symptoms on the inoculated spikelet, but the symptoms did not spread into other spikelets. On barley, the trichothecene deficient mutants showed no significant difference compared to the wild-type strains: all were equally aggressive. On maize, mutants derived from the NIV-producing strain caused less disease than their wild-type progenitor strain, while mutants derived from DON-producing strains caused the same level of disease as their progenitor strains. These data demonstrate that trichothecenes influence the virulence of F. graminearum in a highly complex manner, which is strongly host as well as moderately chemotype specific.

The red pigmentation of Fusarium graminearum and related species that cause stem and head blight of cereals is due to the deposition of aurofusarin in the cell walls. To determine the importance of this polyketide for fungal physiology and pathogenicity, aurofusarin deficient mutants were produced by random and targeted mutagenesis of F. pseudograminearum and F. graminearum. We show that a gene cluster, including the F. graminearum PKS12 gene, is responsible for the biosynthesis of aurofusarin. Three F. pseudograminearum aurofusarin deficient mutants were disrupted in a region upstream from a gene with sequence homology to the aflatoxin regulatory gene aflR. Comparative PCR analyses of the aurofusarin gene cluster in F. graminearum, F. culmorum, and F. pseudograminearum show conserved organization and expression analyses detected no PKS12 transcripts in any of the mutants. To confirm that PKS12 encodes the precursor for aurofusarin, targeted mutagenesis was carried out in F. graminearum. All disruptants showed an albino phenotype. The DeltaPKS12 mutants have higher growth rate and a 10-fold increase in conidia production compared to the wild type. Aurofusarin does not appear to aid in radiation protection and all the mutants are fully pathogenic on wheat and barley. HPLC analyses of aurofusarin deficient mutants confirm the absence of aurofusarin and show an increase in the level of the mycotoxin zearalenone.

Fusarium graminearum is the causal agent of the Fusarium head blight (FHB) and a destructive pathogen of cereals accounting for high grain yield losses especially on wheat and maize. Like other fungal pathogens, F. graminearum secretes various extracellular enzymes, which are hypothesized to be involved in host infection. Extracellular lipolytic activity of F. graminearum was strongly induced in culture by wheat germ oil; this allowed us to isolate, clone, and characterize a gene (FGL1) encoding a secreted lipase. Expression analysis indicated that FGL1 is induced by lipid-containing substrates and repressed by glucose. In planta, FGL1 transcription was detected 1 day post-infection of wheat spikes. The function of the FGL1 gene product was verified by specifically demonstrating lipase activity after expression in a heterologous host. Ebelactone B, a known lipase inhibitor, repressed the lipolytic activity of the enzyme. Disease severity was strongly reduced when wild-type conidia were supplemented with ebelactone B. Transformation-mediated disruption of FGL1 led to reduced extracellular lipolytic activity in culture and to reduced virulence to both wheat and maize.

Candida parapsilosis is a human pathogenic fungus with increasing importance, particularly in nosocomial infections. For detailed molecular genetic explorations of prototrophic clinical isolates of C. parapsilosis, we developed an efficient transformation system based on a dominant selectable marker. The gene encoding resistance to mycophenolic acid (MPA) was used for selection in yeast transformation. C. parapsilosis cells were transformed with a plasmid vector containing the Candida albicans inosine monophosphate dehydrogenase gene (IMH3) responsible for mycophenolic acid resistance. Transformation was carried out both by electroporation and by the lithium acetate (LiAc) method. The LiAc method resulted in very poor transformation efficiency, while the modified electroporation method yielded a high number of mitotically stable transformants exhibiting unambiguous MPA resistance. Two hundred transformants were analysed for the presence of the C. albicans IMH3(r) gene by polymerase chain reaction. Integration of single or multiple plasmid copies into the genomic DNA of C. parapsilosis was determined by Southern hybridization. To our knowledge, the present study is the first report about a method based on a dominant selectable marker for the transformation of a prototrophic, clinical isolate of C. parapsilosis. The described technique may prove to be an efficient tool for the examination of the biology and virulence of this pathogenic yeast.

During meiosis, homologous recombination occurs between allelic sequences. To evaluate the biological significance of such a pathway in somatic cells, we used transgenic tobacco plants with a restriction site for the rare cutting endonuclease I-SceI within a negative selectable marker gene. These plants were crossed with two tobacco lines containing, in allelic position, either a deletion or an insertion within the marker gene that rendered both marker gene and restriction site inactive. After the double-strand break induction, we selected for repair events resulting in a loss of marker gene function. This loss was mostly due to deletions. We were also able to detect double strand break-induced allelic recombination in which the break was repaired by a faithful copying process from the homologue carrying the shortened transgene. The estimated frequency indicates that homologous recombination in somatic cells between allelic sites appears to occur at the same order of magnitude as between ectopic sites, and is thus far too infrequent to act as major repair pathway. As somatic changes can be transferred to the germ line, the prevalence of intrachromatid rearrangements over allelic recombination might be an indirect prerequisite for the enhanced genome plasticity postulated for plants.