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Mycologia 94(2), 2002, pp. 280-289
© 2002 by The Mycological Society of America

Utility of cytoplasmic fluorescent proteins for live-cell imaging of Magnaporthe grisea in planta


Kirk J. Czymmek

     Department of Biological Sciences, University of Delaware, Newark, Delaware 19716

Timothy M. Bourett

     DuPont Crop Genetics, PO Box 80402, Wilmington, Delaware 19880-0402

James A. Sweigard
Anne Carroll

     DuPont Crop Genetics, Delaware Technology Park, Newark, Delaware 19713

Richard J. Howard 1

     DuPont Crop Genetics, PO Box 80402, Wilmington, Delaware 19880-0402

The subcellular expression patterns and fluorescence intensities of cytoplasm-targeted, constitutively expressed blue-, cyano-, green-, yellow- and red-fluorescent protein were assessed in a number of transformants of the blast pathogen, Magnaporthe grisea. All transformants grew normally, remained pathogenic on barley, and, except for those expressing blue fluorescent protein, exhibited significant cytoplasmic fluorescence. The exceptionally intense brightness of some strains proved very useful for laser scanning confocal microscope imaging during invasion of host tissues. Acquisition of three-dimensional data sets from intact, individual, pathogen encounter sites in planta were generated during the time course of pathogenesis using non-invasive optical sectioning methods. Confocal and multiphoton microscopy imaging in conjunction with fluorescent protein expression allowed for the real time documentation of fungal colonization within plant cells and tissues with remarkable ease. These methods constitute valuable new tools for the investigation of plant disease.

Key words: blast disease, confocal microscopy, green fluorescent protein, multiphoton microscopy, plant disease cytology, red fluorescent protein




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