Live-cell imaging of tubulin in the filamentous fungus Magnaporthe grisea treated with anti-microtubule and anti-microfilament agents.

Microtubule dynamics were examined in live cells of the fungal plant pathogen Magnaporthe grisea transformed for constitutive expression of a fusion protein containing enhanced yellow-fluorescent protein and a Neurospora crassa benomyl-resistant allele of beta-tubulin. Transformants retained their ability to differentiate appressoria and cause disease but remained sensitive to benomyl. Linear microtubule arrays and low-level cytoplasmic fluorescence were observed in vegetative hyphae, conidia, germ tubes, and developing appressoria. Fluorescence within nuclei was conspicuously absent during interphase but increased rapidly at the onset of mitosis. Treatment with either benomyl or griseofulvin resulted in the appearance of prominent brightly fluorescent aggregates, including a large aggregate near the apex, with the concomitant disappearance of most cytoplasmic microtubules. Electron microscope imaging of treated cells indicated that the aggregates lacked any obvious profiles of intact microtubules. During these treatments, hyphal tip cells continued to elongate in a nonlinear and aerial fashion at a much slower rate than untreated cells. With subsequent removal of griseofulvin, distal aggregates disappeared rapidly but the apical aggregates persisted longer. Treatment with latrunculin A caused hyphal tip swelling without apparent effect on linear microtubule arrays. Simultaneous treatment with griseofulvin and latrunculin A resulted in depolymerization of microtubules and a cessation of growth, but near-apical fluorescent aggregates were not observed.

Sequence analysis and functional characterization of the dialkylglycine decarboxylase gene DGD1 from Mycosphaerella graminicola.

Dialkylglycine decarboxylase is a pyridoxal phosphate-dependent enzyme in the aminotransferases class III group of enzymes. The enzyme is unique in terms of catalyzing both decarboxylation and transamination. Although the enzymatic activity is present in some bacteria and fungi, the biological role is unclear. We identified and disrupted the dialkylglycine decarboxylase-encoding gene DGD1 in the wheat blotch fungus Mycosphaerella graminicola by transposon-arrayed gene knockout. The DGD1 gene is highly similar to dialkylglycine decarboxylase from the soil bacterium Burkholderia cepacia. Phylogenetic analysis of various class III aminotransferases showed that dialkylglycine decarboxylases from bacteria and fungi are found in a distinct cluster. Functional analysis revealed that dgd1 disruption mutants display wild-type morphology and pathogenicity to wheat. The dgd1 mutants cannot utilize 2-methylalanine as a sole nitrogen source, as assessed by large-scale nutritional utilization analysis. This is the first description of a mutant phenotype of the fungal dialkylglycine decarboxylase gene.

Efficient gene identification and targeted gene disruption in the wheat blotch fungus Mycosphaerella graminicola using TAGKO.

TAGKO ( transposon- arrayed gene knock out) is a highly efficient method for gene discovery and gene function assignment in the rice blast fungus Magnaporthe grisea. Here, we report the application of genome-wide TAGKO to the wheat blotch fungus Mycosphaerella graminicola, including the successful development of electroporation-based transformation for this fungus. A M. graminicola genomic cosmid library was constructed and a pool of 250 cosmid clones was mutagenized by in vitro transposition. Sequence analysis identified 5,110 unique insertion events in the M. graminicola genome. Eleven transposon-tagged cosmid clones (TAGKO clones) were chosen and transformed into the wild-type strain by electroporation. Ten TAGKO clones out of 11 produced gene-specific mutants at a targeting frequency of 15-28%, significantly higher than that of conventional gene-disruption constructs. The remaining clone failed to produce viable mutants, thereby providing indirect evidence for the identification of an essential gene.