Live-cell imaging of tubulin in the filamentous fungus Magnaporthe grisea treated with anti-microtubule and anti-microfilament agents |
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Authors: | Kirk J Czymmek Timothy M Bourett Ying Shao Todd M DeZwaan James A Sweigard Richard J Howard |
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Institution: | (1) Department of Biological Sciences, University of Delaware, Newark, Delaware;(2) DuPont Crop Genetics Research and Development, Experimental Station, Wilmington, Delaware |
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Abstract: | Summary. 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 β-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.
Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s00709-004-0081-3
Correspondence and reprints: Department of Biological Sciences, University of Delaware, Newark, DE 19716, U.S.A.
Present address: Paradigm Genetics Inc., Research Triangle Park, North Carolina, U.S.A. |
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Keywords: | : Benomyl Fluorescence-tagged tubulin Griseofulvin Latrunculin A Magnaporthe grisea Microtubule |
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