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

The subcellular expression patterns and fluorescence intensities of cytoplasm-targeted, consti- tutively expressed blue-, cyano-, green-, yellow- and red-fluorescent protein were assessed in a number of transformants of the blast pathogen, Magnaporthe gri- sea. All transformants grew normally, remained path- ogenic on barley, and, except for those expressing blue fluorescent protein, exhibited significant cyto- plasmic fluorescence. The exceptionally intense brightness of some strains proved very useful for laser scanning confocal microscope imaging during inva- sion 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 im- aging in conjunction with fluorescent protein ex- pression 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.